ALBERT

Description: Ultradian glucocorticoid rhythms are highly conserved across mammalian species, however, their functional significance is not yet fully understood. Here we demonstrate that pulsatile corticosterone replacement in adrenalectomised rats induces a dynamic pattern of glucocorticoid receptor (GR) binding at ~3,000 genomic sites in liver at the pulse peak, subsequently not found during the pulse nadir. In contrast, constant corticosterone replacement induced prolonged binding at the majority of these sites. Additionally, each pattern further induced markedly different transcriptional responses. During pulsatile treatment, intragenic occupancy by active RNA polymerase II exhibited pulsatile dynamics with transient changes in enrichment, either decreased or increased depending on the gene, which mostly returned to baseline during the inter-pulse interval. In contrast, constant corticosterone exposure induced prolonged effects on RNA polymerase II occupancy at the majority of gene targets, thus acting as a sustained regulatory signal for both transactivation and repression of glucocorticoid target genes. The nett effect of these differences were consequently seen in the liver transcriptome as RNA-seq analysis indicated that despite the same overall amount of corticosterone infused, twice the number of transcripts were regulated by constant corticosterone infusion, when compared to pulsatile. Target genes that were found to be differentially regulated in a pattern-dependent manner were enriched in functional pathways including carbohydrate, cholesterol, glucose and fat metabolism as well as inflammation, suggesting a functional role for dysregulated glucocorticoid rhythms in the development of metabolic dysfunction.

Description: The prospect of utilizing CRISPR-based gene-drive technology for controlling populations has generated much excitement. However, the potential for spillovers of gene-drive alleles from the target population to non-target populations has raised concerns. Here, using mathematical models, we investigate the possibility of limiting spillovers to non-target populations by designing differential-targeting gene drives, in which the expected equilibrium gene-drive allele frequencies are high in the target population but low in the non-target population. We find that achieving differential targeting is possible with certain configurations of gene drive parameters, but, in most cases, only under relatively low migration rates between populations. Under high migration, differential targeting is possible only in a narrow region of the parameter space. Because fixation of the gene drive in the non-target population could severely disrupt ecosystems, we outline possible ways to avoid this outcome. We apply our model to two potential applications of gene drives—field trials for malaria-vector gene drives and control of invasive species on islands. We discuss theoretical predictions of key requirements for differential targeting and their practical implications.

Description: Genome-wide association studies (GWASs) have identified multiple susceptibility loci for Alzheimer’s disease (AD), which is characterized by early and progressive damage to the hippocampus. However, the association of hippocampal gene expression with AD and the underlying neurobiological pathways remain largely unknown. Based on the genomic and transcriptomic data of 111 hippocampal samples and the summary data of two large-scale meta-analyses of GWASs, a transcriptome-wide association study (TWAS) was performed to identify genes with significant associations between hippocampal expression and AD. We identified 54 significantly associated genes using an AD-GWAS meta-analysis of 455,258 individuals; 36 of the genes were confirmed in another AD-GWAS meta-analysis of 63,926 individuals. Fine-mapping models further prioritized 24 AD-related genes whose effects on AD were mediated by hippocampal expression, including APOE and two novel genes (PTPN9 and PCDHA4). These genes are functionally related to amyloid-beta formation, phosphorylation/dephosphorylation, neuronal apoptosis, neurogenesis and telomerase-related processes. By integrating the predicted hippocampal expression and neuroimaging data, we found that the hippocampal expression of QPCTL and ERCC2 showed significant difference between AD patients and cognitively normal elderly individuals as well as correlated with hippocampal volume. Mediation analysis further demonstrated that hippocampal volume mediated the effect of hippocampal gene expression (QPCTL and ERCC2) on AD. This study identifies two novel genes associated with AD by integrating hippocampal gene expression and genome-wide association data and reveals candidate hippocampus-mediated neurobiological pathways from gene expression to AD.

Description: Here we show that multiple modes of Notch signaling activation specify the complexity of spatial cellular interactions necessary for stem cell niche assembly. In particular, we studied the formation of the germline stem cell niche in Drosophila ovaries, which is a two-step process whereby terminal filaments are formed first. Then, terminal filaments signal to the adjacent cap cell precursors, resulting in Notch signaling activation, which is necessary for the lifelong acquisition of stem cell niche cell fate. The genetic data suggest that in order to initiate the process of stem cell niche assembly, Notch signaling is activated among non-equipotent cells via distant induction, where germline Delta is delivered to somatic cells located several diameters away via cellular projections generated by primordial germ cells. At the same time, to ensure the robustness of niche formation, terminal filament cell fate can also be induced by somatic Delta via cis- or trans-inhibition. This exemplifies a double security mechanism that guarantees that the germline stem cell niche is formed, since it is indispensable for the adjacent germline precursor cells to acquire and maintain stemness necessary for successful reproduction. These findings contribute to our understanding of the formation of stem cell niches in their natural environment, which is important for stem cell biology and regenerative medicine.

Description: Expansion of the hexanucleotide repeat (HR) in the first intron of the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) in Caucasians. All C9orf72-ALS/FTD patients share a common risk (R) haplotype. To study C9orf72 expression and splicing from the mutant R allele compared to the complementary normal allele in ALS/FTD patients, we initially created a detailed molecular map of the single nucleotide polymorphism (SNP) signature and the HR length of the various C9orf72 haplotypes in Caucasians. We leveraged this map to determine the allelic origin of transcripts per patient, and decipher the effects of pathological and normal HR lengths on C9orf72 expression and splicing. In C9orf72 ALS patients’ cells, the HR expanded allele, compared to non-R allele, was associated with decreased levels of a downstream initiated transcript variant and increased levels of transcripts initiated upstream of the HR. HR expanded R alleles correlated with high levels of unspliced intron 1 and activation of cryptic donor splice sites along intron 1. Retention of intron 1 was associated with sequential intron 2 retention. The SNP signature of C9orf72 haplotypes described here enables allele-specific analysis of transcriptional products and may pave the way to allele-specific therapeutic strategies.

Description: Planarians are flatworms and can perform whole-body regeneration. This ability involves a mechanism to distinguish between anterior-facing wounds that require head regeneration and posterior-facing wounds that require tail regeneration. How this head-tail regeneration polarity decision is made is studied to identify principles underlying tissue-identity specification in regeneration. We report that inhibition of activin-2, which encodes an Activin-like signaling ligand, resulted in the regeneration of ectopic posterior-facing heads following amputation. During tissue turnover in uninjured planarians, positional information is constitutively expressed in muscle to maintain proper patterning. Positional information includes Wnts expressed in the posterior and Wnt antagonists expressed in the anterior. Upon amputation, several wound-induced genes promote re-establishment of positional information. The head-versus-tail regeneration decision involves preferential wound induction of the Wnt antagonist notum at anterior-facing over posterior-facing wounds. Asymmetric activation of notum represents the earliest known molecular distinction between head and tail regeneration, yet how it occurs is unknown. activin-2 RNAi animals displayed symmetric wound-induced activation of notum at anterior- and posterior-facing wounds, providing a molecular explanation for their ectopic posterior-head phenotype. activin-2 RNAi animals also displayed anterior-posterior (AP) axis splitting, with two heads appearing in anterior blastemas, and various combinations of heads and tails appearing in posterior blastemas. This was associated with ectopic nucleation of anterior poles, which are head-tip muscle cells that facilitate AP and medial-lateral (ML) pattern, at posterior-facing wounds. These findings reveal a role for Activin signaling in determining the outcome of AP-axis-patterning events that are specific to regeneration.

Description: Mitochondria are essential for maintaining skeletal muscle metabolic homeostasis during adaptive response to a myriad of physiologic or pathophysiological stresses. The mechanisms by which mitochondrial function and contractile fiber type are concordantly regulated to ensure muscle function remain poorly understood. Evidence is emerging that the Folliculin interacting protein 1 (Fnip1) is involved in skeletal muscle fiber type specification, function, and disease. In this study, Fnip1 was specifically expressed in skeletal muscle in Fnip1-transgenic (Fnip1Tg) mice. Fnip1Tg mice were crossed with Fnip1-knockout (Fnip1KO) mice to generate Fnip1TgKO mice expressing Fnip1 only in skeletal muscle but not in other tissues. Our results indicate that, in addition to the known role in type I fiber program, FNIP1 exerts control upon muscle mitochondrial oxidative program through AMPK signaling. Indeed, basal levels of FNIP1 are sufficient to inhibit AMPK but not mTORC1 activity in skeletal muscle cells. Gain-of-function and loss-of-function strategies in mice, together with assessment of primary muscle cells, demonstrated that skeletal muscle mitochondrial program is suppressed via the inhibitory actions of FNIP1 on AMPK. Surprisingly, the FNIP1 actions on type I fiber program is independent of AMPK and its downstream PGC-1α. These studies provide a vital framework for understanding the intrinsic role of FNIP1 as a crucial factor in the concerted regulation of mitochondrial function and muscle fiber type that determine muscle fitness.

Description: Neurogenesis in the developing neocortex begins with the generation of the preplate, which consists of early-born neurons including Cajal-Retzius (CR) cells and subplate neurons. Here, utilizing the Ebf2-EGFP transgenic mouse in which EGFP initially labels the preplate neurons then persists in CR cells, we reveal the dynamic transcriptome profiles of early neurogenesis and CR cell differentiation. Genome-wide RNA-seq and ChIP-seq analyses at multiple early neurogenic stages have revealed the temporal gene expression dynamics of early neurogenesis and distinct histone modification patterns in early differentiating neurons. We have identified a new set of coding genes and lncRNAs involved in early neuronal differentiation and validated with functional assays in vitro and in vivo. In addition, at E15.5 when Ebf2-EGFP+ cells are mostly CR neurons, single-cell sequencing analysis of purified Ebf2-EGFP+ cells uncovers molecular heterogeneities in CR neurons, but without apparent clustering of cells with distinct regional origins. Along a pseudotemporal trajectory these cells are classified into three different developing states, revealing genetic cascades from early generic neuronal differentiation to late fate specification during the establishment of CR neuron identity and function. Our findings shed light on the molecular mechanisms governing the early differentiation steps during cortical development, especially CR neuron differentiation.

Description: The BAF complex plays an important role in the development of a wide range of tissues by modulating gene expression programs at the chromatin level. However, its role in neural crest development has remained unclear. To determine the role of the BAF complex, we deleted BAF155/BAF170, the core subunits required for the assembly, stability, and functions of the BAF complex in neural crest cells (NCCs). Neural crest-specific deletion of BAF155/BAF170 leads to embryonic lethality due to a wide range of developmental defects including craniofacial, pharyngeal arch artery, and OFT defects. RNAseq and transcription factor enrichment analysis revealed that the BAF complex modulates the expression of multiple signaling pathway genes including Hippo and Notch, essential for the migration, proliferation, and differentiation of the NCCs. Furthermore, we demonstrated that the BAF complex is essential for the Brg1-Yap-Tead-dependent transcription of target genes in NCCs. Together, our results demonstrate an important role of the BAF complex in modulating the gene regulatory network essential for neural crest development.

Description: Polymerase theta-mediated end joining (TMEJ) is a chromosome break repair pathway that is able to rescue the lethality associated with the loss of proteins involved in early steps in homologous recombination (e.g., BRCA1/2). This is due to the ability of polymerase theta (Pol θ) to use resected, 3’ single stranded DNA tails to repair chromosome breaks. These resected DNA tails are also the starting substrate for homologous recombination. However, it remains unknown if TMEJ can compensate for the loss of proteins involved in more downstream steps during homologous recombination. Here we show that the Holliday junction resolvases SLX4 and GEN1 are required for viability in the absence of Pol θ in Drosophila melanogaster, and lack of all three proteins results in high levels of apoptosis. Flies deficient in Pol θ and SLX4 are extremely sensitive to DNA damaging agents, and mammalian cells require either Pol θ or SLX4 to survive. Our results suggest that TMEJ and Holliday junction formation/resolution share a common DNA substrate, likely a homologous recombination intermediate, that when left unrepaired leads to cell death. One major consequence of Holliday junction resolution by SLX4 and GEN1 is cancer-causing loss of heterozygosity due to mitotic crossing over. We measured mitotic crossovers in flies after a Cas9-induced chromosome break, and observed that this mutagenic form of repair is increased in the absence of Pol θ. This demonstrates that TMEJ can function upstream of the Holiday junction resolvases to protect cells from loss of heterozygosity. Our work argues that Pol θ can thus compensate for the loss of the Holliday junction resolvases by using homologous recombination intermediates, suppressing mitotic crossing over and preserving the genomic stability of cells.

Description: XIST establishes inactivation across its chromosome of origin, even when expressed from autosomal transgenes. To identify the regions of human XIST essential for recruiting heterochromatic marks we generated a series of overlapping deletions in an autosomal inducible XIST transgene present in 8p of the HT1080 male fibrosarcoma cell line. We examined the ability of each construct to enrich its unified XIST territory with the histone marks established by PRC1 and PRC2 as well as the heterochromatin factors MacroH2A and SMCHD1. Chromatin enrichment of ubH2A by PRC1 required four distinct regions of XIST, and these were completely distinct from the two domains crucial for enrichment of H3K27me3 by PRC2. Both the domains required, as well as the impact of PRC1 and PRC2 inhibitors, suggest that PRC1 is required for SMCHD1 while PRC2 function is necessary for MacroH2A recruitment, although incomplete overlap of regions implicates roles for additional factors. This cooperativity between factors contributes to the requirement for multiple separate domains being required for each feature examined. The independence of the PRC1/PRC2 pathways was observed when XIST was expressed both autosomally or from the X chromosome suggesting that these observations are not purely a result of the context in which XIST operates. Although independent domains were required for the PRC1 and PRC2 pathways overall all regions tested were important for some aspect of XIST functionality, demonstrating both modularity and cooperativity across the XIST lncRNA.

Description: Heat shock protein 47 (HSP47), encoded by the SERPINH1 gene, is a molecular chaperone essential for correct folding of collagens. We report a homozygous p.(R222S) substitution in HSP47 in a child with severe osteogenesis imperfecta leading to early demise. p.R222 is a highly conserved residue located within the collagen interacting surface of HSP47. Binding assays show a significantly reduced affinity of HSP47-R222S for type I collagen. This altered interaction leads to posttranslational overmodification of type I procollagen produced by dermal fibroblasts, with increased glycosylation and/or hydroxylation of lysine and proline residues as shown by mass spectrometry. Since we also observed a normal intracellular folding and secretion rate of type I procollagen, this overmodification cannot be explained by prolonged exposure of the procollagen molecules to the modifying hydroxyl- and glycosyltransferases, as is commonly observed in other types of OI. We found significant upregulation of several molecular chaperones and enzymes involved in procollagen modification and folding on Western blot and RT-qPCR. In addition, we showed that an imbalance in binding of HSP47-R222S to unfolded type I collagen chains in a gelatin sepharose pulldown assay results in increased binding of other chaperones and modifying enzymes. The elevated expression and binding of this molecular ensemble to type I procollagen suggests a compensatory mechanism for the aberrant binding of HSP47-R222S, eventually leading to overmodification of type I procollagen chains. Together, these results illustrate the importance of HSP47 for proper posttranslational modification and provide insights into the molecular pathomechanisms of the p.(R222S) alteration in HSP47, which leads to a severe OI phenotype.

Description: Altered patterns of recombination on 21q have long been associated withthe nondisjunction chromosome21within oocytes and the increased risk of having a child with Down syndrome. Unfortunately the genetic etiology of these altered patterns of recombination have yet to be elucidated. We for the first time genotyped the gene MCM9, a candidate gene for recombination regulation and DNA repair in mothers with or without children with Down syndrome. In our approach, we identified the location of recombination on the maternal chromosome 21 using short tandem repeat markers, then stratified our population by the origin of meiotic error and age at conception. We observed that twenty-five out of forty-one single nucleotide polymorphic sites within MCM9 exhibited an association with meiosis I error (N = 700), but not with meiosis II error (N = 125). This association was maternal age-independent. Several variants exhibited aprotective association with MI error, some were neutral. Maternal age stratified characterization of cases revealed that MCM9 risk variants were associated with an increased chance of reduced recombination on 21q within oocytes. The spatial distribution of single observed recombination events revealed no significant change in the location of recombination among women harbouring MCM9 risk, protective, or neutral variant. Additionally, we identified a total of six novel polymorphic variants and two novel alleles that were either risk imparting or protective against meiosis I nondisjunction. In silico analyses using five different programs suggest the risk variants either cause a change in protein function or may alter the splicing pattern of transcripts and disrupt the proportion of different isoforms of MCM9 products within oocytes. These observations bring us a significant step closer to understanding the molecular basis of recombination errors in chromosome 21 nondisjunction within oocytes that leads to birth of child with Down syndrome.

Description: DNA methylation is found throughout all domains of life, yet the extent and function of DNA methylation differ among eukaryotes. Strains of the plant pathogenic fungus Zymoseptoria tritici appeared to lack cytosine DNA methylation (5mC) because gene amplification followed by Repeat-Induced Point mutation (RIP) resulted in the inactivation of the dim2 DNA methyltransferase gene. 5mC is, however, present in closely related sister species. We demonstrate that inactivation of dim2 occurred recently as some Z. tritici isolates carry a functional dim2 gene. Moreover, we show that dim2 inactivation occurred by a different path than previously hypothesized. We mapped the genome-wide distribution of 5mC in strains with or without functional dim2 alleles. Presence of functional dim2 correlates with high levels of 5mC in transposable elements (TEs), suggesting a role in genome defense. We identified low levels of 5mC in strains carrying non-functional dim2 alleles, suggesting that 5mC is maintained over time, presumably by an active Dnmt5 DNA methyltransferase. Integration of a functional dim2 allele in strains with mutated dim2 restored normal 5mC levels, demonstrating de novo cytosine methylation activity of Dim2. To assess the importance of 5mC for genome evolution, we performed an evolution experiment, comparing genomes of strains with high levels of 5mC to genomes of strains lacking functional dim2. We found that presence of a functional dim2 allele alters nucleotide composition by promoting C to T transitions (C→T) specifically at CpA (CA) sites during mitosis, likely contributing to TE inactivation. Our results show that 5mC density at TEs is a polymorphic trait in Z. tritici populations that can impact genome evolution.

Description: Synthetic gene drive constructs could, in principle, provide the basis for highly efficient interventions to control disease vectors and other pest species. This efficiency derives in part from leveraging natural processes of dispersal and gene flow to spread the construct and its impacts from one population to another. However, sometimes (for example, with invasive species) only specific populations are in need of control, and impacts on non-target populations would be undesirable. Many gene drive designs use nucleases that recognise and cleave specific genomic sequences, and one way to restrict their spread would be to exploit sequence differences between target and non-target populations. In this paper we propose and model a series of low threshold double drive designs for population suppression, each consisting of two constructs, one imposing a reproductive load on the population and the other inserted into a differentiated locus and controlling the drive of the first. Simple deterministic, discrete-generation computer simulations are used to assess the alternative designs. We find that the simplest double drive designs are significantly more robust to pre-existing cleavage resistance at the differentiated locus than single drive designs, and that more complex designs incorporating sex ratio distortion can be more efficient still, even allowing for successful control when the differentiated locus is neutral and there is up to 50% pre-existing resistance in the target population. Similar designs can also be used for population replacement, with similar benefits. A population genomic analysis of CRISPR PAM sites in island and mainland populations of the malaria mosquito Anopheles gambiae indicates that the differentiation needed for our methods to work can exist in nature. Double drives should be considered when efficient but localised population genetic control is needed and there is some genetic differentiation between target and non-target populations.

Description: Impaired formation of the intrahepatic biliary network leads to cholestatic liver diseases, which are frequently associated with autoimmune disorders. Using a chemical mutagenesis strategy in zebrafish combined with computational network analysis, we screened for novel genes involved in intrahepatic biliary network formation. We positionally cloned a mutation in the nckap1l gene, which encodes a cytoplasmic adaptor protein for the WAVE regulatory complex. The mutation is located in the last exon after the stop codon of the primary splice isoform, only disrupting a previously unannotated minor splice isoform, which indicates that the minor splice isoform is responsible for the intrahepatic biliary network phenotype. CRISPR/Cas9-mediated nckap1l deletion, which disrupts both the primary and minor isoforms, showed the same defects. In the liver of nckap1l mutant larvae, WAVE regulatory complex component proteins are degraded specifically in biliary epithelial cells, which line the intrahepatic biliary network, thus disrupting the actin organization of these cells. We further show that nckap1l genetically interacts with the Cdk5 pathway in biliary epithelial cells. These data together indicate that although nckap1l was previously considered to be a hematopoietic cell lineage-specific protein, its minor splice isoform acts in biliary epithelial cells to regulate intrahepatic biliary network formation.

Description: The cohesin complex spatially organizes interphase chromatin by bringing distal genomic loci into close physical proximity, looping out the intervening DNA. Mutation of cohesin complex subunits is observed in cancer and developmental disorders, but the mechanisms through which these mutations may contribute to disease remain poorly understood. Here, we investigate a recurrent missense mutation to the hinge domain of the cohesin subunit SMC1A, observed in acute myeloid leukemia. Engineering this mutation into murine embryonic stem cells caused widespread changes in gene expression, including dysregulation of the pluripotency gene expression program. This mutation reduced cohesin levels at promoters and enhancers, decreased DNA loops and interactions across short genomic distances, and weakened insulation at CTCF-mediated DNA loops. These findings provide insight into how altered cohesin function contributes to disease and identify a requirement for the cohesin hinge domain in three-dimensional chromatin structure.

Description: DNA double-strand breaks arise in vivo when a dicentric chromosome (two centromeres on one chromosome) goes through mitosis with the two centromeres attached to opposite spindle pole bodies. Repair of the DSBs generates phenotypic diversity due to the range of monocentric derivative chromosomes that arise. To explore whether DSBs may be differentially repaired as a function of their spatial position in the chromosome, we have examined the structure of monocentric derivative chromosomes from cells containing a suite of dicentric chromosomes in which the distance between the two centromeres ranges from 6.5 kb to 57.7 kb. Two major classes of repair products, homology-based (homologous recombination (HR) and single-strand annealing (SSA)) and end-joining (non-homologous (NHEJ) and micro-homology mediated (MMEJ)) were identified. The distribution of repair products varies as a function of distance between the two centromeres. Genetic dependencies on double strand break repair (Rad52), DNA ligase (Lif1), and S phase checkpoint (Mrc1) are indicative of distinct repair pathway choices for DNA breaks in the pericentromeric chromatin versus the arms.

Description: The genetic basis of general plant vigor is of major interest to food producers, yet the trait is recalcitrant to genetic mapping because of the number of loci involved, their small effects, and linkage. Observations of heterosis in many crops suggests that recessive, malfunctioning versions of genes are a major cause of poor performance, yet we have little information on the mutational spectrum underlying these disruptions. To address this question, we generated a long-read assembly of a tropical japonica rice (Oryza sativa) variety, Carolina Gold, which allowed us to identify structural mutations (〉50 bp) and orient them with respect to their ancestral state using the outgroup, Oryza glaberrima. Supporting prior work, we find substantial genome expansion in the sativa branch. While transposable elements (TEs) account for the largest share of size variation, the majority of events are not directly TE-mediated. Tandem duplications are the most common source of insertions and are highly enriched among 50-200bp mutations. To explore the relative impact of various mutational classes on crop fitness, we then track these structural events over the last century of US rice improvement using 101 resequenced varieties. Within this material, a pattern of temporary hybridization between medium and long-grain varieties was followed by recent divergence. During this long-term selection, structural mutations that impact gene exons have been removed at a greater rate than intronic indels and single-nucleotide mutations. These results support the use of ab initio estimates of mutational burden, based on structural data, as an orthogonal predictor in genomic selection.

Description: The winged insects of the order Diptera are colloquially named for their most recognizable phenotype: flight. These insects rely on flight for a number of important life history traits, such as dispersal, foraging, and courtship. Despite the importance of flight, relatively little is known about the genetic architecture of flight performance. Accordingly, we sought to uncover the genetic modifiers of flight using a measure of flies’ reaction and response to an abrupt drop in a vertical flight column. We conducted a genome wide association study (GWAS) using 197 of the Drosophila Genetic Reference Panel (DGRP) lines, and identified a combination of additive and marginal variants, epistatic interactions, whole genes, and enrichment across interaction networks. Egfr, a highly pleiotropic developmental gene, was among the most significant additive variants identified. We functionally validated 13 of the additive candidate genes’ (Adgf-A/Adgf-A2/CG32181, bru1, CadN, flapper (CG11073), CG15236, flippy (CG9766), CREG, Dscam4, form3, fry, Lasp/CG9692, Pde6, Snoo), and introduce a novel approach to whole gene significance screens: PEGASUS_flies. Additionally, we identified ppk23, an Acid Sensing Ion Channel (ASIC) homolog, as an important hub for epistatic interactions. We propose a model that suggests genetic modifiers of wing and muscle morphology, nervous system development and function, BMP signaling, sexually dimorphic neural wiring, and gene regulation are all important for the observed differences flight performance in a natural population. Additionally, these results represent a snapshot of the genetic modifiers affecting drop-response flight performance in Drosophila, with implications for other insects.

Description: Transcriptional rewiring is the regulation of different target genes by orthologous regulators in different organisms. While this phenomenon has been observed, it has not been extensively studied, particularly in core regulatory systems. Several global cell cycle regulators are conserved in the Alphaproteobacteria, providing an excellent model to study this phenomenon. First characterized in Caulobacter crescentus, GcrA and CcrM compose a DNA methylation-based regulatory system that helps coordinate the complex life cycle of this organism. These regulators are well-conserved across Alphaproteobacteria, but the extent to which their regulatory targets are conserved is not known. In this study, the regulatory targets of GcrA and CcrM were analyzed by SMRT-seq, RNA-seq, and ChIP-seq technologies in the Alphaproteobacterium Brevundimonas subvibrioides, and then compared to those of its close relative C. crescentus that inhabits the same environment. Although the regulators themselves are highly conserved, the genes they regulate are vastly different. GcrA directly regulates 204 genes in C. crescentus, and though B. subvibrioides has orthologs to 147 of those genes, only 48 genes retained GcrA binding in their promoter regions. Additionally, only 12 of those 48 genes demonstrated significant transcriptional change in a gcrA mutant, suggesting extensive transcriptional rewiring between these organisms. Similarly, out of hundreds of genes CcrM regulates in each of these organisms, only 2 genes were found in common. When multiple Alphaproteobacterial genomes were analyzed bioinformatically for potential GcrA regulatory targets, the regulation of genes involved in DNA replication and cell division was well conserved across the Caulobacterales but not outside this order. This work suggests that significant transcriptional rewiring can occur in cell cycle regulatory systems even over short evolutionary distances.

Description: Previous research on risk factors for obstructive heart defects (OHDs) focused on maternal and infant genetic variants, prenatal environmental exposures, and their potential interaction effects. Less is known about the role of paternal genetic variants or environmental exposures and risk of OHDs. We examined parent-of-origin effects in transmission of alleles in the folate, homocysteine, or transsulfuration pathway genes on OHD occurrence in offspring. We used data on 569 families of liveborn infants with OHDs born between October 1997 and August 2008 from the National Birth Defects Prevention Study to conduct a family-based case-only study. Maternal, paternal, and infant DNA were genotyped using an Illumina Golden Gate custom single nucleotide polymorphism (SNP) panel. Relative risks (RR), 95% confidence interval (CI), and likelihood ratio tests from log-linear models were used to estimate the parent-of-origin effect of 877 SNPs in 60 candidate genes in the folate, homocysteine, and transsulfuration pathways on the risk of OHDs. Bonferroni correction was applied for multiple testing. We identified 3 SNPs in the transsulfuration pathway and 1 SNP in the folate pathway that were statistically significant after Bonferroni correction. Among infants who inherited paternally-derived copies of the G allele for rs6812588 in the RFC1 gene, the G allele for rs1762430 in the MGMT gene, and the A allele for rs9296695 and rs4712023 in the GSTA3 gene, RRs for OHD were 0.11 (95% CI: 0.04, 0.29, P = 9.16x10-7), 0.30 (95% CI: 0.17, 0.53, P = 9.80x10-6), 0.34 (95% CI: 0.20, 0.57, P = 2.28x10-5), and 0.34 (95% CI: 0.20, 0.58, P = 3.77x10-5), respectively, compared to infants who inherited maternally-derived copies of the same alleles. We observed statistically significant decreased risk of OHDs among infants who inherited paternal gene variants involved in folate and transsulfuration pathways.

Description: Traditional predictive models for transcriptome-wide association studies (TWAS) consider only single nucleotide polymorphisms (SNPs) local to genes of interest and perform parameter shrinkage with a regularization process. These approaches ignore the effect of distal-SNPs or other molecular effects underlying the SNP-gene association. Here, we outline multi-omics strategies for transcriptome imputation from germline genetics to allow more powerful testing of gene-trait associations by prioritizing distal-SNPs to the gene of interest. In one extension, we identify mediating biomarkers (CpG sites, microRNAs, and transcription factors) highly associated with gene expression and train predictive models for these mediators using their local SNPs. Imputed values for mediators are then incorporated into the final predictive model of gene expression, along with local SNPs. In the second extension, we assess distal-eQTLs (SNPs associated with genes not in a local window around it) for their mediation effect through mediating biomarkers local to these distal-eSNPs. Distal-eSNPs with large indirect mediation effects are then included in the transcriptomic prediction model with the local SNPs around the gene of interest. Using simulations and real data from ROS/MAP brain tissue and TCGA breast tumors, we show considerable gains of percent variance explained (1–2% additive increase) of gene expression and TWAS power to detect gene-trait associations. This integrative approach to transcriptome-wide imputation and association studies aids in identifying the complex interactions underlying genetic regulation within a tissue and important risk genes for various traits and disorders.

Description: Complex age-associated phenotypes are caused, in part, by an interaction between an individual’s genotype and age. The mechanisms governing such interactions are however not entirely understood. Here, we provide a novel transcriptional mechanism-based framework–SNiPage, to investigate such interactions, whereby a transcription factor (TF) whose expression changes with age (age-associated TF), binds to a polymorphic regulatory element in an allele-dependent fashion, rendering the target gene’s expression dependent on both, the age and the genotype. Applying SNiPage to GTEx, we detected ~637 significant TF-SNP-Gene triplets on average across 25 tissues, where the TF binds to a regulatory SNP in the gene’s promoter or putative enhancer and potentially regulates its expression in an age- and allele-dependent fashion. The detected SNPs are enriched for epigenomic marks indicative of regulatory activity, exhibit allele-specific chromatin accessibility, and spatial proximity to their putative gene targets. Furthermore, the TF-SNP interaction-dependent target genes have established links to aging and to age-associated diseases. In six hypertension-implicated tissues, detected interactions significantly inform hypertension state of an individual. Lastly, the age-interacting SNPs exhibit a greater proximity to the reported phenotype/diseases-associated SNPs than eSNPs identified in an interaction-independent fashion. Overall, we present a novel mechanism-based model, and a novel framework SNiPage, to identify functionally relevant SNP-age interactions in transcriptional control and illustrate their potential utility in understanding complex age-associated phenotypes.

Description: Loading of the MCM replicative helicase at origins of replication is a highly regulated process that precedes DNA replication in all eukaryotes. The stoichiometry of MCM loaded at origins has been proposed to be a key determinant of when those origins initiate replication during S phase. Nevertheless, the genome-wide regulation of MCM loading stoichiometry and its direct effect on replication timing remain unclear. In order to investigate why some origins load more MCM than others, we perturbed MCM levels in budding yeast cells and, for the first time, directly measured MCM levels and replication timing in the same experiment. Reduction of MCM levels through degradation of Mcm4, one of the six obligate components of the MCM complex, slowed progression through S phase and increased sensitivity to replication stress. Reduction of MCM levels also led to differential loading at origins during G1, revealing origins that are sensitive to reductions in MCM and others that are not. Sensitive origins loaded less MCM under normal conditions and correlated with a weak ability to recruit the origin recognition complex (ORC). Moreover, reduction of MCM loading at specific origins of replication led to a delay in their replication during S phase. In contrast, overexpression of MCM had no effects on cell cycle progression, relative MCM levels at origins, or replication timing, suggesting that, under optimal growth conditions, cellular MCM levels are not limiting for MCM loading. Our results support a model in which the loading capacity of origins is the primary determinant of MCM stoichiometry in wild-type cells, but that stoichiometry is controlled by origins’ ability to recruit ORC and compete for MCM when MCM becomes limiting.

Description: Saltatorial locomotion is a type of hopping gait that in mammals can be found in rabbits, hares, kangaroos, and some species of rodents. The molecular mechanisms that control and fine-tune the formation of this type of gait are unknown. Here, we take advantage of one strain of domesticated rabbits, the sauteur d’Alfort, that exhibits an abnormal locomotion behavior defined by the loss of the typical jumping that characterizes wild-type rabbits. Strikingly, individuals from this strain frequently adopt a bipedal gait using their front legs. Using a combination of experimental crosses and whole genome sequencing, we show that a single locus containing the RAR related orphan receptor B gene (RORB) explains the atypical gait of these rabbits. We found that a splice-site mutation in an evolutionary conserved site of RORB results in several aberrant transcript isoforms incorporating intronic sequence. This mutation leads to a drastic reduction of RORB-positive neurons in the spinal cord, as well as defects in differentiation of populations of spinal cord interneurons. Our results show that RORB function is required for the performance of saltatorial locomotion in rabbits.

Description: While linkage disequilibrium (LD) is an important parameter in genetics and evolutionary biology, the drivers of LD remain elusive. Using whole-genome sequences from across a species’ range, we assessed the impact of demographic history and mating system on LD. Both range expansion and a shift from outcrossing to selfing in North American Arabidopsis lyrata were associated with increased average genome-wide LD. Our results indicate that range expansion increases short-distance LD at the farthest range edges by about the same amount as a shift to selfing. However, the extent over which LD in genic regions unfolds was shorter for range expansion compared to selfing. Linkage among putatively neutral variants and between neutral and deleterious variants increased to a similar degree with range expansion, providing support that genome-wide LD was positively associated with mutational load. As a consequence, LD combined with mutational load may decelerate range expansions and set range limits. Finally, a small number of genes were identified as LD outliers, suggesting that they experience selection by either of the two demographic processes. These included genes involved in flowering and photoperiod for range expansion, and the self-incompatibility locus for mating system.

Description: The tumor suppressor IKAROS binds and represses multiple NOTCH target genes. For their induction upon NOTCH signaling, IKAROS is removed and replaced by NOTCH Intracellular Domain (NICD)-associated proteins. However, IKAROS remains associated to other NOTCH activated genes upon signaling and induction. Whether IKAROS could participate to the induction of this second group of NOTCH activated genes is unknown. We analyzed the combined effect of IKAROS abrogation and NOTCH signaling on the expression of NOTCH activated genes in erythroid cells. In IKAROS-deleted cells, we observed that many of these genes were either overexpressed or no longer responsive to NOTCH signaling. IKAROS is then required for the organization of bivalent chromatin and poised transcription of NOTCH activated genes belonging to either of the aforementioned groups. Furthermore, we show that IKAROS-dependent poised organization of the NOTCH target Cdkn1a is also required for its adequate induction upon genotoxic insults. These results highlight the critical role played by IKAROS in establishing bivalent chromatin and transcriptional poised state at target genes for their activation by NOTCH or other stress signals.

Description: Allelic expression imbalance (AEI), quantified by the relative expression of two alleles of a gene in a diploid organism, can help explain phenotypic variations among individuals. Traditional methods detect AEI using bulk RNA sequencing (RNA-seq) data, a data type that averages out cell-to-cell heterogeneity in gene expression across cell types. Since the patterns of AEI may vary across different cell types, it is desirable to study AEI in a cell-type-specific manner. Although this can be achieved by single-cell RNA sequencing (scRNA-seq), it requires full-length transcript to be sequenced in single cells of a large number of individuals, which are still cost prohibitive to generate. To overcome this limitation and utilize the vast amount of existing disease relevant bulk tissue RNA-seq data, we developed BSCET, which enables the characterization of cell-type-specific AEI in bulk RNA-seq data by integrating cell type composition information inferred from a small set of scRNA-seq samples, possibly obtained from an external dataset. By modeling covariate effect, BSCET can also detect genes whose cell-type-specific AEI are associated with clinical factors. Through extensive benchmark evaluations, we show that BSCET correctly detected genes with cell-type-specific AEI and differential AEI between healthy and diseased samples using bulk RNA-seq data. BSCET also uncovered cell-type-specific AEIs that were missed in bulk data analysis when the directions of AEI are opposite in different cell types. We further applied BSCET to two pancreatic islet bulk RNA-seq datasets, and detected genes showing cell-type-specific AEI that are related to the progression of type 2 diabetes. Since bulk RNA-seq data are easily accessible, BSCET provides a convenient tool to integrate information from scRNA-seq data to gain insight on AEI with cell type resolution. Results from such analysis will advance our understanding of cell type contributions in human diseases.

Description: Many complex genomic rearrangements arise through template switch errors, which occur in DNA replication when there is a transient polymerase switch to an alternate template nearby in three-dimensional space. While typically investigated at kilobase-to-megabase scales, the genomic and evolutionary consequences of this mutational process are not well characterised at smaller scales, where they are often interpreted as clusters of independent substitutions, insertions and deletions. Here we present an improved statistical approach using pair hidden Markov models, and use it to detect and describe short-range template switches underlying clusters of mutations in the multi-way alignment of hominid genomes. Using robust statistics derived from evolutionary genomic simulations, we show that template switch events have been widespread in the evolution of the great apes’ genomes and provide a parsimonious explanation for the presence of many complex mutation clusters in their phylogenetic context. Larger-scale mechanisms of genome rearrangement are typically associated with structural features around breakpoints, and accordingly we show that atypical patterns of secondary structure formation and DNA bending are present at the initial template switch loci. Our methods improve on previous non-probabilistic approaches for computational detection of template switch mutations, allowing the statistical significance of events to be assessed. By specifying realistic evolutionary parameters based on the genomes and taxa involved, our methods can be readily adapted to other intra- or inter-species comparisons.

Description: The telomere-bound shelterin complex is essential for chromosome-end protection and genomic stability. Little is known on the regulation of shelterin components by extracellular signals including developmental and environmental cues. Here, we show that human TRF1 is subjected to AKT-dependent regulation. To study the importance of this modification in vivo, we generate knock-in human cell lines carrying non-phosphorylatable mutants of the AKT-dependent TRF1 phosphorylation sites by CRISPR-Cas9. We find that TRF1 mutant cells show decreased TRF1 binding to telomeres and increased global and telomeric DNA damage. Human cells carrying non-phosphorylatable mutant TRF1 alleles show accelerated telomere shortening, demonstrating that AKT-dependent TRF1 phosphorylation regulates telomere maintenance in vivo. TRF1 mutant cells show an impaired response to proliferative extracellular signals as well as a decreased tumorigenesis potential. These findings indicate that telomere protection and telomere length can be regulated by extracellular signals upstream of PI3K/AKT activation, such as growth factors, nutrients or immune regulators, and this has an impact on tumorigenesis potential.

Description: Paramutation is a form of non-Mendelian inheritance in which the expression of a paramutable allele changes when it encounters a paramutagenic allele. This change in expression of the paramutable alleles is stably inherited even after segregation of both alleles. While the discovery of paramutation and studies of its underlying mechanism were made with alleles that change plant pigmentation, paramutation-like phenomena are known to modulate the expression of other traits and in other eukaryotes, and many cases have probably gone undetected. It is likely that epigenetic mechanisms are responsible for the phenomenon, as paramutation forms epialleles, genes with identical sequences but different expression states. This could account for the intergenerational inheritance of the paramutated allele, providing profound evidence that triggered epigenetic changes can be maintained over generations. Here, we use a case of paramutation that affects a transgenic selection reporter gene in tetraploid Arabidopsis thaliana. Our data suggest that different types of small RNA are derived from paramutable and paramutagenic epialleles. In addition, deletion of a repeat within the epiallele changes its paramutability. Further, the temperature during the growth of the epiallelic hybrids determines the degree and timing of the allelic interaction. The data further make it plausible why paramutation in this system becomes evident only in the segregating F2 population of tetraploid plants containing both epialleles. In summary, the results support a model for polyploidy-associated paramutation, with similarities as well as distinctions from other cases of paramutation.

Description: The evolution of insect resistance to insecticides is frequently associated with overexpression of one or more cytochrome P450 enzyme genes. Although overexpression of CYP450 genes is a well-known mechanism of insecticide resistance, the underlying regulatory mechanisms are poorly understood. Here we uncovered the mechanisms of overexpression of the P450 gene, CYP321A8 in a major pest insect, Spodoptera exigua that is resistant to multiple insecticides. CYP321A8 confers resistance to organophosphate (chlorpyrifos) and pyrethroid (cypermethrin and deltamethrin) insecticides in this insect. Constitutive upregulation of transcription factors CncC/Maf are partially responsible for upregulated expression of CYP321A8 in the resistant strain. Reporter gene assays and site-directed mutagenesis analyses demonstrated that CncC/Maf enhanced the expression of CYP321A8 by binding to specific sites in the promoter. Additional cis-regulatory elements resulting from a mutation in the CYP321A8 promoter in the resistant strain facilitates the binding of the orphan nuclear receptor, Knirps, and enhances the promoter activity. These results demonstrate that two independent mechanisms; overexpression of transcription factors and mutations in the promoter region resulting in a new cis-regulatory element that facilitates binding of the orphan nuclear receptor are involved in overexpression of CYP321A8 in insecticide-resistant S. exigua.

Description: Fish oil supplementation is widely used for reducing serum triglycerides (TAGs) but has mixed effects on other circulating cardiovascular biomarkers. Many genetic polymorphisms have been associated with blood lipids, including high- and low-density-lipoprotein cholesterol (HDL-C, LDL-C), total cholesterol, and TAGs. Here, the gene-diet interaction effects of fish oil supplementation on these lipids were analyzed in a discovery cohort of up to 73,962 UK Biobank participants, using a 1-degree-of-freedom (1df) test for interaction effects and a 2-degrees-of-freedom (2df) test to jointly analyze interaction and main effects. Associations with P 〈 1×10−6 in either test (26,157; 18,300 unique variants) were advanced to replication in up to 7,284 participants from the Atherosclerosis Risk in Communities (ARIC) Study. Replicated associations reaching 1df P 〈 0.05 (2,175; 1,763 unique variants) were used in meta-analyses. We found 13 replicated and 159 non-replicated (UK Biobank only) loci with significant 2df joint tests that were predominantly driven by main effects and have been previously reported. Four novel interaction loci were identified with 1df P 〈 5×10−8 in meta-analysis. The lead variant in the GJB6-GJB2-GJA3 gene cluster, rs112803755 (A〉G; minor allele frequency = 0.041), shows exclusively interaction effects. The minor allele is significantly associated with decreased TAGs in individuals with fish oil supplementation, but with increased TAGs in those without supplementation. This locus is significantly associated with higher GJB2 expression of connexin 26 in adipose tissue; connexin activity is known to change upon exposure to omega-3 fatty acids. Significant interaction effects were also found in three other loci in the genes SLC12A3 (HDL-C), ABCA6 (LDL-C), and MLXIPL (LDL-C), but highly significant main effects are also present. Our study identifies novel gene-diet interaction effects for four genetic loci, whose effects on blood lipids are modified by fish oil supplementation. These findings highlight the need and possibility for personalized nutrition.

Description: Ciliary beating requires the coordinated activity of numerous axonemal complexes. The protein composition and role of radial spokes (RS), nexin links (N-DRC) and dyneins (ODAs and IDAs) is well established. However, how information is transmitted from the central apparatus to the RS and across other ciliary structures remains unclear. Here, we identify a complex comprising the evolutionarily conserved proteins Ccdc96 and Ccdc113, positioned parallel to N-DRC and forming a connection between RS3, dynein g, and N-DRC. Although Ccdc96 and Ccdc113 can be transported to cilia independently, their stable docking and function requires the presence of both proteins. Deletion of either CCDC113 or CCDC96 alters cilia beating frequency, amplitude and waveform. We propose that the Ccdc113/Ccdc96 complex transmits signals from RS3 and N-DRC to dynein g and thus regulates its activity and the ciliary beat pattern.

Description: Precise regulation of ocular size is a critical determinant of normal visual acuity. Although it is generally accepted that ocular growth relies on a cascade of signaling events transmitted from the retina to the sclera, the factors and mechanism(s) involved are poorly understood. Recent studies have highlighted the importance of the retinal secreted serine protease PRSS56 and transmembrane glycoprotein MFRP, a factor predominantly expressed in the retinal pigment epithelium (RPE), in ocular size determination. Mutations in PRSS56 and MFRP constitute a major cause of nanophthalmos, a condition characterized by severe reduction in ocular axial length/extreme hyperopia. Interestingly, common variants of these genes have been implicated in myopia, a condition associated with ocular elongation. Consistent with these findings, mice with loss of function mutation in PRSS56 or MFRP exhibit a reduction in ocular axial length. However, the molecular network and cellular processes involved in PRSS56- and MFRP-mediated ocular axial growth remain elusive. Here, we show that Adamts19 expression is significantly upregulated in the retina of mice lacking either Prss56 or Mfrp. Importantly, using genetic mouse models, we demonstrate that while ADAMTS19 is not required for ocular growth during normal development, its inactivation exacerbates ocular axial length reduction in Prss56 and Mfrp mutant mice. These results suggest that the upregulation of retinal Adamts19 is part of an adaptive molecular response to counteract impaired ocular growth. Using a complementary genetic approach, we show that loss of PRSS56 or MFRP function prevents excessive ocular axial growth in a mouse model of early-onset myopia caused by a null mutation in Irbp, thus, demonstrating that PRSS56 and MFRP are also required for pathological ocular elongation. Collectively, our findings provide new insights into the molecular network involved in ocular axial growth and support a role for molecular crosstalk between the retina and RPE involved in refractive development.

Description: Most epigenome-wide association studies (EWAS) quantify DNA methylation (DNAm) in peripheral tissues such as whole blood to identify positions in the genome where variation is statistically associated with a trait or exposure. As whole blood comprises a mix of cell types, it is unclear whether trait-associated DNAm variation is specific to an individual cellular population. We collected three peripheral tissues (whole blood, buccal epithelial and nasal epithelial cells) from thirty individuals. Whole blood samples were subsequently processed using fluorescence-activated cell sorting (FACS) to purify five constituent cell-types (monocytes, granulocytes, CD4+ T cells, CD8+ T cells, and B cells). DNAm was profiled in all eight sample-types from each individual using the Illumina EPIC array. We identified significant differences in both the level and variability of DNAm between different sample types, and DNAm data-derived estimates of age and smoking were found to differ dramatically across sample types from the same individual. We found that for the majority of loci variation in DNAm in individual blood cell types was only weakly predictive of variance in DNAm measured in whole blood, although the proportion of variance explained was greater than that explained by either buccal or nasal epithelial samples. Covariation across sample types was much higher for DNAm sites influenced by genetic factors. Overall, we observe that DNAm variation in whole blood is additively influenced by a combination of the major blood cell types. For a subset of sites, however, variable DNAm detected in whole blood can be attributed to variation in a single blood cell type providing potential mechanistic insight about EWAS findings. Our results suggest that associations between whole blood DNAm and traits or exposures reflect differences in multiple cell types and our data will facilitate the interpretation of findings in epigenetic epidemiology.

Description: Chromosomal inversions contribute widely to adaptation and speciation, yet they present a unique evolutionary puzzle as both their allelic content and frequency evolve in a feedback loop. In this simulation study, we quantified the role of the allelic content in determining the long-term fate of the inversion. Recessive deleterious mutations accumulated on both arrangements with most of them being private to a given arrangement. This led to increasing overdominance, allowing for the maintenance of the inversion polymorphism and generating strong non-adaptive divergence between arrangements. The accumulation of mutations was mitigated by gene conversion but nevertheless led to the fitness decline of at least one homokaryotype under all considered conditions. Surprisingly, this fitness degradation could be permanently halted by the branching of an arrangement into multiple highly divergent haplotypes. Our results highlight the dynamic features of inversions by showing how the non-adaptive evolution of allelic content can play a major role in the fate of the inversion.

Description: The natural history of tuberculosis (TB) is characterized by a large inter-individual outcome variability after exposure to Mycobacterium tuberculosis. Specifically, some highly exposed individuals remain resistant to M. tuberculosis infection, as inferred by tuberculin skin test (TST) or interferon-gamma release assays (IGRAs). We performed a genome-wide association study of resistance to M. tuberculosis infection in an endemic region of Southern Vietnam. We enrolled household contacts (HHC) of pulmonary TB cases and compared subjects who were negative for both TST and IGRA (n = 185) with infected individuals (n = 353) who were either positive for both TST and IGRA or had a diagnosis of TB. We found a genome-wide significant locus on chromosome 10q26.2 with a cluster of variants associated with strong protection against M. tuberculosis infection (OR = 0.42, 95%CI 0.35–0.49, P = 3.71×10−8, for the genotyped variant rs17155120). The locus was replicated in a French multi-ethnic HHC cohort and a familial admixed cohort from a hyper-endemic area of South Africa, with an overall OR for rs17155120 estimated at 0.50 (95%CI 0.45–0.55, P = 1.26×10−9). The variants are located in intronic regions and upstream of C10orf90, a tumor suppressor gene which encodes an ubiquitin ligase activating the transcription factor p53. In silico analysis showed that the protective alleles were associated with a decreased expression in monocytes of the nearby gene ADAM12 which could lead to an enhanced response of Th17 lymphocytes. Our results reveal a novel locus controlling resistance to M. tuberculosis infection across different populations.

Description: Beyond the haploid genome, mammalian sperm carry a payload of epigenetic information with the potential to modulate offspring phenotypes. Recent studies show that the small RNA repertoire of sperm is remodeled during post-testicular maturation in the epididymis. Epididymal maturation has also been linked to changes in the sperm methylome, suggesting that the epididymis might play a broader role in shaping the sperm epigenome. Here, we characterize the genome-wide methylation landscape in seven germ cell populations from throughout the male reproductive tract. We find very few changes in the cytosine methylation landscape between testicular germ cell populations and cauda epididymal sperm, demonstrating that the sperm methylome is stable throughout post-testicular maturation. Although our sequencing data suggested that caput epididymal sperm exhibit a highly unusual methylome, follow-up studies revealed that this resulted from contamination of caput sperm by extracellular DNA. Extracellular DNA formed web-like structures that ensnared sperm, and was present only in sperm samples obtained from the caput epididymis and vas deferens of virgin males. Curiously, contaminating extracellular DNA was associated with citrullinated histone H3, potentially resulting from a PAD-driven genome decondensation process. Taken together, our data emphasize the stability of cytosine methylation in mammalian sperm, and identify a surprising, albeit transient, period during which sperm are associated with extracellular DNA.

Description: Fission yeast Cleavage and Polyadenylation Factor (CPF), a 13-subunit complex, executes the cotranscriptional 3’ processing of RNA polymerase II (Pol2) transcripts that precedes transcription termination. The three-subunit DPS sub-complex of CPF, consisting of a PP1-type phosphoprotein phosphatase Dis2, a WD-repeat protein Swd22, and a putative phosphatase regulatory factor Ppn1, associates with the CPF core to form the holo-CPF assembly. Here we probed the functional, physical, and genetic interactions of DPS by focusing on the Ppn1 subunit, which mediates association of DPS with the core. Transcriptional profiling by RNA-seq defined limited but highly concordant sets of protein-coding genes that were dysregulated in ppn1Δ, swd22Δ and dis2Δ cells, which included the DPSΔ down-regulated phosphate homeostasis genes pho1 and pho84 that are controlled by lncRNA-mediated transcriptional interference. Essential and inessential modules of the 710-aa Ppn1 protein were defined by testing the effects of Ppn1 truncations in multiple genetic backgrounds in which Ppn1 is required for growth. An N-terminal 172-aa disordered region was dispensable and its deletion alleviated hypomorphic phenotypes caused by deleting C-terminal aa 640–710. A TFIIS-like domain (aa 173–330) was not required for viability but was important for Ppn1 activity in phosphate homeostasis. Distinct sites within Ppn1 for binding to Dis2 (spanning Ppn1 aa 506 to 532) and Swd22 (from Ppn1 aa 533 to 578) were demarcated by yeast two-hybrid assays. Dis2 interaction-defective missense mutants of full-length Ppn1 (that retained Swd22 interaction) were employed to show that binding to Dis2 (or its paralog Sds21) was necessary for Ppn1 biological activity. Ppn1 function was severely compromised by missense mutations that selectively affected its binding to Swd22.

Description: Indole-3-acetic acid (IAA) is the most common, naturally occurring phytohormone that regulates cell division, differentiation, and senescence in plants. The capacity to synthesize IAA is also widespread among plant-associated bacterial and fungal species, which may use IAA as an effector molecule to define their relationships with plants or to coordinate their physiological behavior through cell-cell communication. Fungi, including many species that do not entertain a plant-associated life style, are also able to synthesize IAA, but the physiological role of IAA in these fungi has largely remained enigmatic. Interestingly, in this context, growth of the budding yeast Saccharomyces cerevisiae is sensitive to extracellular IAA. Here, we use a combination of various genetic approaches including chemical-genetic profiling, SAturated Transposon Analysis in Yeast (SATAY), and genetic epistasis analyses to identify the mode-of-action by which IAA inhibits growth in yeast. Surprisingly, these analyses pinpointed the target of rapamycin complex 1 (TORC1), a central regulator of eukaryotic cell growth, as the major growth-limiting target of IAA. Our biochemical analyses further demonstrate that IAA inhibits TORC1 both in vivo and in vitro. Intriguingly, we also show that yeast cells are able to synthesize IAA and specifically accumulate IAA upon entry into stationary phase. Our data therefore suggest that IAA contributes to proper entry of yeast cells into a quiescent state by acting as a metabolic inhibitor of TORC1.

Description: Throughout human history, large-scale migrations have facilitated the formation of populations with ancestry from multiple previously separated populations. This process leads to subsequent shuffling of genetic ancestry through recombination, producing variation in ancestry between populations, among individuals in a population, and along the genome within an individual. Recent methodological and empirical developments have elucidated the genomic signatures of this admixture process, bringing previously understudied admixed populations to the forefront of population and medical genetics. Under this theme, we present a collection of recent PLOS Genetics publications that exemplify recent progress in human genetic admixture studies, and we discuss potential areas for future work.

Description: Environmental factors challenge the physiological homeostasis in animals, thereby evoking stress responses. Various mechanisms have evolved to counter stress at the organism level, including regulation by neuropeptides. In recent years, much progress has been made on the mechanisms and neuropeptides that regulate responses to metabolic/nutritional stress, as well as those involved in countering osmotic and ionic stresses. Here, we identified a peptidergic pathway that links these types of regulatory functions. We uncover the neuropeptide Corazonin (Crz), previously implicated in responses to metabolic stress, as a neuroendocrine factor that inhibits the release of a diuretic hormone, CAPA, and thereby modulates the tolerance to osmotic and ionic stress. Both knockdown of Crz and acute injections of Crz peptide impact desiccation tolerance and recovery from chill-coma. Mapping of the Crz receptor (CrzR) expression identified three pairs of Capa-expressing neurons (Va neurons) in the ventral nerve cord that mediate these effects of Crz. We show that Crz acts to restore water/ion homeostasis by inhibiting release of CAPA neuropeptides via inhibition of cAMP production in Va neurons. Knockdown of CrzR in Va neurons affects CAPA signaling, and consequently increases tolerance for desiccation, ionic stress and starvation, but delays chill-coma recovery. Optogenetic activation of Va neurons stimulates excretion and simultaneous activation of Crz and CAPA-expressing neurons reduces this response, supporting the inhibitory action of Crz. Thus, Crz inhibits Va neurons to maintain osmotic and ionic homeostasis, which in turn affects stress tolerance. Earlier work demonstrated that systemic Crz signaling restores nutrient levels by promoting food search and feeding. Here we additionally propose that Crz signaling also ensures osmotic homeostasis by inhibiting release of CAPA neuropeptides and suppressing diuresis. Thus, Crz ameliorates stress-associated physiology through systemic modulation of both peptidergic neurosecretory cells and the fat body in Drosophila.

Description: Within the glioblastoma cellular niche, glioma stem cells (GSCs) can give rise to differentiated glioma cells (DGCs) and, when necessary, DGCs can reciprocally give rise to GSCs to maintain the cellular equilibrium necessary for optimal tumor growth. Here, using ribosome profiling, transcriptome and m6A RNA sequencing, we show that GSCs from patients with different subtypes of glioblastoma share a set of transcripts, which exhibit a pattern of m6A loss and increased protein translation during differentiation. The target sequences of a group of miRNAs overlap the canonical RRACH m6A motifs of these transcripts, many of which confer a survival advantage in glioblastoma. Ectopic expression of the RRACH-binding miR-145 induces loss of m6A, formation of FTO/AGO1/ILF3/miR-145 complexes on a clinically relevant tumor suppressor gene (CLIP3) and significant increase in its nascent translation. Inhibition of miR-145 maintains RRACH m6A levels of CLIP3 and inhibits its nascent translation. This study highlights a critical role of miRNAs in assembling complexes for m6A demethylation and induction of protein translation during GSC state transition.

Description: Eukaryotic gene expression is extensively regulated by cellular stress and pathogen infections. We have previously shown that herpes simplex virus 1 (HSV-1) and several cellular stresses cause widespread disruption of transcription termination (DoTT) of RNA polymerase II (RNAPII) in host genes and that the viral immediate early factor ICP27 plays an important role in HSV-1-induced DoTT. Here, we show that HSV-1 infection also leads to widespread changes in alternative polyadenylation (APA) of host mRNAs. In the majority of cases, polyadenylation shifts to upstream poly(A) sites (PAS), including many intronic PAS. Mechanistically, ICP27 contributes to HSV-1-mediated APA regulation. HSV-1- and ICP27-induced activation of intronic PAS is sequence-dependent and does not involve general inhibition of U1 snRNP. HSV1-induced intronic polyadenylation is accompanied by early termination of RNAPII. HSV-1-induced mRNAs polyadenylated at intronic PAS (IPA) are exported into the cytoplasm while APA isoforms with extended 3’ UTRs are sequestered in the nuclei, both preventing the expression of the full-length gene products. Finally we provide evidence that HSV-induced IPA isoforms are translated. Together with other recent studies, our results suggest that viral infection and cellular stresses induce a multi-faceted host response that includes DoTT and changes in APA profiles.

Description: Conventionally viewed as male hormone, androgens play a critical role in female fertility. Although androgen receptors (AR) are transcription factors, to date very few direct transcriptional targets of ARs have been identified in the ovary. Using mouse models, this study provides three critical insights about androgen-induced gene regulation in the ovary and its impact on female fertility. First, RNA-sequencing reveals a number of genes and biological processes that were previously not known to be directly regulated by androgens in the ovary. Second, androgens can also influence gene expression by decreasing the tri-methyl mark on lysine 27 of histone3 (H3K27me3), a gene silencing epigenetic mark. ChIP-seq analyses highlight that androgen-induced modulation of H3K27me3 mark within gene bodies, promoters or distal enhancers have a much broader impact on ovarian function than the direct genomic effects of androgens. Third, androgen-induced decrease of H3K27me3 is mediated through (a) inhibiting the expression and activity of Enhancer of Zeste Homologue 2 (EZH2), a histone methyltransferase that promotes tri-methylation of K27 and (b) by inducing the expression of a histone demethylase called Jumonji domain containing protein-3 (JMJD3/KDM6B), responsible for removing the H3K27me3 mark. Androgens through the PI3K/Akt pathway, in a transcription-independent fashion, increase hypoxia-inducible factor 1 alpha (HIF1α) protein levels, which in turn induce JMJD3 expression. Furthermore, proof of concept studies involving in vivo knockdown of Ar in the ovary and ovarian (granulosa) cell-specific Ar knockout mouse model show that ARs regulate the expression of key ovarian genes through modulation of H3K27me3.

Description: Drosophila larvae and pupae are at high risk of parasitoid infection in nature. To circumvent parasitic stress, fruit flies have developed various survival strategies, including cellular and behavioral defenses. We show that adult Drosophila females exposed to the parasitic wasps, Leptopilina boulardi, decrease their total egg-lay by deploying at least two strategies: Retention of fully developed follicles reduces the number of eggs laid, while induction of caspase-mediated apoptosis eliminates the vitellogenic follicles. These reproductive defense strategies require both visual and olfactory cues, but not the MB247-positive mushroom body neuronal function, suggesting a novel mode of sensory integration mediates reduced egg-laying in the presence of a parasitoid. We further show that neuropeptide F (NPF) signaling is necessary for both retaining matured follicles and activating apoptosis in vitellogenic follicles. Whereas previous studies have found that gut-derived NPF controls germ stem cell proliferation, we show that sensory-induced changes in germ cell development specifically require brain-derived NPF signaling, which recruits a subset of NPFR-expressing cell-types that control follicle development and retention. Importantly, we found that reduced egg-lay behavior is specific to parasitic wasps that infect the developing Drosophila larvae, but not the pupae. Our findings demonstrate that female fruit flies use multimodal sensory integration and neuroendocrine signaling via NPF to engage in parasite-specific cellular and behavioral survival strategies.

Description: Neurodegenerative diseases are characterized by neuron loss and accumulation of undegraded protein aggregates. These phenotypes are partially due to defective protein degradation in neuronal cells. Autophagic clearance of aggregated proteins is critical to protein quality control, but the underlying mechanisms are still poorly understood. Here we report the essential role of WDR81 in autophagic clearance of protein aggregates in models of Huntington’s disease (HD), Parkinson’s disease (PD) and Alzheimer’s disease (AD). In hippocampus and cortex of patients with HD, PD and AD, protein level of endogenous WDR81 is decreased but autophagic receptor p62 accumulates significantly. WDR81 facilitates the recruitment of autophagic proteins onto Htt polyQ aggregates and promotes autophagic clearance of Htt polyQ subsequently. The BEACH and MFS domains of WDR81 are sufficient for its recruitment onto Htt polyQ aggregates, and its WD40 repeats are essential for WDR81 interaction with covalent bound ATG5-ATG12. Reduction of WDR81 impairs the viability of mouse primary neurons, while overexpression of WDR81 restores the viability of fibroblasts from HD patients. Notably, in Caenorhabditis elegans, deletion of the WDR81 homolog (SORF-2) causes accumulation of p62 bodies and exacerbates neuron loss induced by overexpressed α-synuclein. As expected, overexpression of SORF-2 or human WDR81 restores neuron viability in worms. These results demonstrate that WDR81 has crucial evolutionarily conserved roles in autophagic clearance of protein aggregates and maintenance of cell viability under pathological conditions, and its reduction provides mechanistic insights into the pathogenesis of HD, PD, AD and brain disorders related to WDR81 mutations.

Description: During sexual reproduction the parental homologous chromosomes find each other (pair) and align along their lengths by integrating local sequence homology with large-scale contiguity, thereby allowing for precise exchange of genetic information. The Synaptonemal Complex (SC) is a conserved zipper-like structure that assembles between the homologous chromosomes, bringing them together and regulating exchanges between them. However, the molecular mechanisms by which the SC carries out these functions remain poorly understood. Here we isolated and characterized two mutations in the dimerization interface in the middle of the SC zipper in C. elegans. The mutations perturb both chromosome alignment and the regulation of genetic exchanges. Underlying the chromosome-scale phenotypes are distinct alterations to the way SC subunits interact with one another. We propose a model whereby the SC brings homologous chromosomes together through two activities: obligate zipping that prevents assembly on unpaired chromosomes; and a tendency to extend pairing interactions along the entire length of the chromosomes.

Description: The conserved fungal velvet family regulatory proteins link development and secondary metabolite production. The velvet domain for DNA binding and dimerization is similar to the structure of the Rel homology domain of the mammalian NF-κB transcription factor. A comprehensive study addressed the functions of all four homologs of velvet domain encoding genes in the fungal life cycle of the soil-borne plant pathogenic fungus Verticillium dahliae. Genetic, cell biological, proteomic and metabolomic analyses of Vel1, Vel2, Vel3 and Vos1 were combined with plant pathogenicity experiments. Different phases of fungal growth, development and pathogenicity require V. dahliae velvet proteins, including Vel1-Vel2, Vel2-Vos1 and Vel3-Vos1 heterodimers, which are already present during vegetative hyphal growth. The major novel finding of this study is that Vel1 is necessary for initial plant root colonization and together with Vel3 for propagation in planta by conidiation. Vel1 is needed for disease symptom induction in tomato. Vel1, Vel2, and Vel3 control the formation of microsclerotia in senescent plants. Vel1 is the most important among all four V. dahliae velvet proteins with a wide variety of functions during all phases of the fungal life cycle in as well as ex planta.

Description: Haplolethals (HL) are regions of diploid genomes that in one dose are fatal for the organism. Their biological meaning is obscure because heterozygous loss-of-function mutations result in dominant lethality (DL) and, consequently, should be under strong negative selection. We report an in depth study of the HL associated to the gene wings up A (wupA). It encodes 13 transcripts (A-M) that yield 11 protein isoforms (A-K) of Troponin I (TnI). They are functionally diverse in their control of muscle contraction, cell polarity and cell proliferation. Isoform K transfers to the nucleus where it increases transcription of the cell proliferation related genes CDK2, CDK4, Rap and Rab5. The nuclear translocation of isoform K is prevented by the co-expression of A or B isoforms, which illustrates isoform interactions. The corresponding DL mutations are, either DNA rearrangements clustered towards the gene 3’ end, thus affecting the genomic organization of all transcripts, or CRISPR-induced mutations in one of the two ATG sites which eliminate a subset of wupA products. The joint elimination of isoforms C, F, G and H, however, do not cause DL phenotypes. Genetically driven expression of single isoforms rescue neither DL nor any of the mutants known in the gene, suggesting that normal function requires properly regulated expression of specific combinations, rather than single, TnI isoforms. We conclude that the wupA associated HL results from the combined haploinsufficiency of a large set of TnI isoforms. The qualitative and quantitative normal expression of which, requires the chromosomal integrity of the wupA genomic region. Since all fly TnI isoforms are encoded in the same gene, its HL condition becomes unavoidable. These wupA features are comparable to those of dpp, the only other HL studied to some extent, and reveal a scenario of strict dosage dependence with implications for gene expression regulation and splitting.

Description: Long noncoding RNAs (lncRNAs) are emerging as important regulators in plant development, but few of them have been functionally characterized in fruit ripening. Here, we have identified 25,613 lncRNAs from strawberry ripening fruits based on RNA-seq data from poly(A)-depleted libraries and rRNA-depleted libraries, most of which exhibited distinct temporal expression patterns. A novel lncRNA, FRILAIR harbours the miR397 binding site that is highly conserved in diverse strawberry species. FRILAIR overexpression promoted fruit maturation in the Falandi strawberry, which was consistent with the finding from knocking down miR397, which can guide the mRNA cleavage of both FRILAIR and LAC11a (encoding a putative laccase-11-like protein). Moreover, LAC11a mRNA levels were increased in both FRILAIR overexpressing and miR397 knockdown fruits, and accelerated fruit maturation was also found in LAC11a overexpressing fruits. Overall, our study demonstrates that FRILAIR can act as a noncanonical target mimic of miR397 to modulate the expression of LAC11a in the strawberry fruit ripening process.

Description: The generation of lineage-specific gene expression programmes that alter proliferation capacity, metabolic profile and cell type-specific functions during differentiation from multipotent stem cells to specialised cell types is crucial for development. During differentiation gene expression programmes are dynamically modulated by a complex interplay between sequence-specific transcription factors, associated cofactors and epigenetic regulators. Here, we study U-shaped (Ush), a multi-zinc finger protein that maintains the multipotency of stem cell-like hemocyte progenitors during Drosophila hematopoiesis. Using genomewide approaches we reveal that Ush binds to promoters and enhancers and that it controls the expression of three gene classes that encode proteins relevant to stem cell-like functions and differentiation: cell cycle regulators, key metabolic enzymes and proteins conferring specific functions of differentiated hemocytes. We employ complementary biochemical approaches to characterise the molecular mechanisms of Ush-mediated gene regulation. We uncover distinct Ush isoforms one of which binds the Nucleosome Remodeling and Deacetylation (NuRD) complex using an evolutionary conserved peptide motif. Remarkably, the Ush/NuRD complex specifically contributes to the repression of lineage-specific genes but does not impact the expression of cell cycle regulators or metabolic genes. This reveals a mechanism that enables specific and concerted modulation of functionally related portions of a wider gene expression programme. Finally, we use genetic assays to demonstrate that Ush and NuRD regulate enhancer activity during hemocyte differentiation in vivo and that both cooperate to suppress the differentiation of lamellocytes, a highly specialised blood cell type. Our findings reveal that Ush coordinates proliferation, metabolism and cell type-specific activities by isoform-specific cooperation with an epigenetic regulator.

Description: Mutations in RanBP2 (also known as Nup358), one of the main components of the cytoplasmic filaments of the nuclear pore complex, contribute to the overproduction of acute necrotizing encephalopathy (ANE1)-associated cytokines. Here we report that RanBP2 represses the translation of the interleukin 6 (IL6) mRNA, which encodes a cytokine that is aberrantly up-regulated in ANE1. Our data indicates that soon after its production, the IL6 messenger ribonucleoprotein (mRNP) recruits Argonautes bound to let-7 microRNA. After this mRNP is exported to the cytosol, RanBP2 sumoylates mRNP-associated Argonautes, thereby stabilizing them and enforcing mRNA silencing. Collectively, these results support a model whereby RanBP2 promotes an mRNP remodelling event that is critical for the miRNA-mediated suppression of clinically relevant mRNAs, such as IL6.

Description: The exogenous light signal and endogenous auxin are two critical factors that antagonistically regulate hypocotyl growth. However, the regulatory mechanisms integrating light and auxin signaling pathways need further investigation. In this study, we identified a direct link between the light and auxin signaling pathways mediated by the auxin transcriptional repressor IAA3 and light-controlled PIF transcription factors in Arabidopsis. The gain-of-function mutation in IAA3 caused hyposensitivity to light, whereas disruption of IAA3 led to an elongated hypocotyl under different light intensity conditions, indicating that IAA3 is required in light regulated hypocotyl growth. Genetic studies showed that the function of IAA3 in hypocotyl elongation is dependent on PIFs. Our data further demonstrated that IAA3 interacts with PIFs in vitro and in vivo, and it attenuates the DNA binding activities of PIFs to the target genes. Moreover, IAA3 negatively regulates the expression of PIFs-dependent genes. Collectively, our study reveals an interplay mechanism of light and auxin on the regulation of hypocotyl growth, coordinated by the IAA3 and PIFs transcriptional regulatory module.

Description: During DNA replication newly synthesized histones are incorporated into the chromatin of the replicating sister chromatids. In the yeast Saccharomyces cerevisiae new histone H3 molecules are acetylated at lysine 56. This modification is carefully regulated during the cell cycle, and any disruption of this process is a source of genomic instability. Here we show that the protein kinase Dun1 is necessary in order to maintain viability in the absence of the histone deacetylases Hst3 and Hst4, which remove the acetyl moiety from histone H3. This lethality is not due to the well-characterized role of Dun1 in upregulating dNTPs, but rather because Dun1 is needed in order to counteract the checkpoint kinase Rad53 (human CHK2) that represses the activity of late firing origins. Deletion of CTF18, encoding the large subunit of an alternative RFC-like complex (RLC), but not of components of the Elg1 or Rad24 RLCs, is enough to overcome the dependency of cells with hyper-acetylated histones on Dun1. We show that the detrimental function of Ctf18 depends on its interaction with the leading strand polymerase, Polε. Our results thus show that the main problem of cells with hyper-acetylated histones is the regulation of their temporal and replication programs, and uncover novel functions for the Dun1 protein kinase and the Ctf18 clamp loader.

Description: Preemptive pharmacogenetic testing has the potential to improve drug dosing by providing point-of-care patient genotype information. Nonetheless, its implementation in the Chinese population is limited by the lack of population-wide data. In this study, secondary analysis of exome sequencing data was conducted to study pharmacogenomics in 1116 Hong Kong Chinese. We aimed to identify the spectrum of actionable pharmacogenetic variants and rare, predicted deleterious variants that are potentially actionable in Hong Kong Chinese, and to estimate the proportion of dispensed drugs that may potentially benefit from genotype-guided prescription. The projected preemptive pharmacogenetic testing prescription impact was evaluated based on the patient prescription data of the public healthcare system in 2019, serving 7.5 million people. Twenty-nine actionable pharmacogenetic variants/ alleles were identified in our cohort. Nearly all (99.6%) subjects carried at least one actionable pharmacogenetic variant, whereas 93.5% of subjects harbored at least one rare deleterious pharmacogenetic variant. Based on the prescription data in 2019, 13.4% of the Hong Kong population was prescribed with drugs with pharmacogenetic clinical practice guideline recommendations. The total expenditure on actionable drugs was 33,520,000 USD, and it was estimated that 8,219,000 USD (24.5%) worth of drugs were prescribed to patients with an implicated actionable phenotype. Secondary use of exome sequencing data for pharmacogenetic analysis is feasible, and preemptive pharmacogenetic testing has the potential to support prescription decisions in the Hong Kong Chinese population.

Description: Although most organisms synthesize methionine from homocysteine and methyl folates, some have “core” methionine synthases that lack folate-binding domains and use other methyl donors. In vitro, the characterized core synthases use methylcobalamin as a methyl donor, but in vivo, they probably rely on corrinoid (vitamin B12-binding) proteins. We identified four families of core methionine synthases that are distantly related to each other (under 30% pairwise amino acid identity). From the characterized enzymes, we identified the families MesA, which is found in methanogens, and MesB, which is found in anaerobic bacteria and archaea with the Wood-Ljungdahl pathway. A third uncharacterized family, MesC, is found in anaerobic archaea that have the Wood-Ljungdahl pathway and lack known forms of methionine synthase. We predict that most members of the MesB and MesC families accept methyl groups from the iron-sulfur corrinoid protein of that pathway. The fourth family, MesD, is found only in aerobic bacteria. Using transposon mutants and complementation, we show that MesD does not require 5-methyltetrahydrofolate or cobalamin. Instead, MesD requires an uncharacterized protein family (DUF1852) and oxygen for activity.

Description: Transcriptional dynamic in response to environmental and developmental cues are fundamental to biology, yet many mechanistic aspects are poorly understood. One such example is fungal plant pathogens, which use secreted proteins and small molecules, termed effectors, to suppress host immunity and promote colonization. Effectors are highly expressed in planta but remain transcriptionally repressed ex planta, but our mechanistic understanding of these transcriptional dynamics remains limited. We tested the hypothesis that repressive histone modification at H3-Lys27 underlies transcriptional silencing ex planta, and that exchange for an active chemical modification contributes to transcription of in planta induced genes. Using genetics, chromatin immunoprecipitation and sequencing and RNA-sequencing, we determined that H3K27me3 provides significant local transcriptional repression. We detail how regions that lose H3K27me3 gain H3K27ac, and these changes are associated with increased transcription. Importantly, we observed that many in planta induced genes were marked by H3K27me3 during axenic growth, and detail how altered H3K27 modification influences transcription. ChIP-qPCR during in planta growth suggests that H3K27 modifications are generally stable, but can undergo dynamics at specific genomic locations. Our results support the hypothesis that dynamic histone modifications at H3K27 contributes to fungal genome regulation and specifically contributes to regulation of genes important during host infection.

Description: DNA lesions inflicted by activation-induced deaminase (AID) instrumentally initiate the processes reshaping immunoglobulin genes in mature B-cells, from local somatic hypermutation (SHM) to junctions of distant breaks during class switch recombination (CSR). It remains incompletely understood how these divergent outcomes of AID attacks are differentially and temporally focused, with CSR strictly occurring in the Ig heavy chain (IgH) locus while SHM concentrates on rearranged V(D)J regions in the IgH and Ig light chain loci. In the IgH locus, disruption of either the 3’Regulatory Region (3’RR) super-enhancer or of switch (S) regions preceding constant genes, profoundly affects CSR. Reciprocally, we now examined if these elements are sufficient to induce CSR in a synthetic locus based on the Igκ locus backbone. Addition of a surrogate “core 3’RR” (c3’RR) and of a pair of transcribed and spliced Switch regions, together with a reporter system for “κ-CSR” yielded a switchable Igκ locus. While the c3’RR stimulated SHM at S regions, it also lowered the local SHM threshold necessary for switch recombination to occur. The 3’RR thus both helps recruit AID to initiate DNA lesions, but then also promotes their resolution through long-distance synapses and recombination following double-strand breaks.

Description: In order to boost the number and diversity of neurons generated from neural stem cells, intermediate neural progenitors (INPs) need to maintain their homeostasis by avoiding both dedifferentiation and premature differentiation. Elucidating how INPs maintain homeostasis is critical for understanding the generation of brain complexity and various neurological diseases resulting from defects in INP development. Here we report that Six4 expressed in Drosophila type II neuroblast (NB) lineages prevents the generation of supernumerary type II NBs and premature differentiation of INPs. We show that loss of Six4 leads to supernumerary type II NBs likely due to dedifferentiation of immature INPs (imINPs). We provide data to further demonstrate that Six4 inhibits the expression and activity of PntP1 in imINPs in part by forming a trimeric complex with Earmuff and PntP1. Furthermore, knockdown of Six4 exacerbates the loss of INPs resulting from the loss of PntP1 by enhancing ectopic Prospero expression in imINPs, suggesting that Six4 is also required for preventing premature differentiation of INPs. Taken together, our work identified a novel transcription factor that likely plays important roles in maintaining INP homeostasis.

Description: Cancer is driven by somatic mutations that result in a cellular fitness advantage. This selective advantage is expected to be counterbalanced by the immune system when these driver mutations simultaneously lead to the generation of neoantigens, novel peptides that are presented at the cancer cell membrane via HLA molecules from the MHC complex. The presentability of these peptides is determined by a patient’s MHC genotype and it has been suggested that this results in MHC genotype-specific restrictions of the oncogenic mutational landscape. Here, we generated a set of virtual patients, each with an identical and prototypical MHC genotype, and show that the earlier reported HLA affinity differences between observed and unobserved mutations are unrelated to MHC genotype variation. We demonstrate how these differences are secondary to high frequencies of 13 hot spot driver mutations in 6 different genes. Several oncogenic mechanisms were identified that lower the peptides’ HLA affinity, including phospho-mimicking substitutions in BRAF, destabilizing tyrosine mutations in TP53 and glycine-rich mutational contexts in the GTP-binding KRAS domain. In line with our earlier findings, our results emphasize that HLA affinity predictions are easily misinterpreted when studying immunogenic selection processes.

Description: Wing polymorphism is an evolutionary feature found in a wide variety of insects, which offers a model system for studying the evolutionary significance of dispersal. In the wing-dimorphic planthopper Nilaparvata lugens, the insulin/insulin-like growth factor signaling (IIS) pathway acts as a ‘master signal’ that directs the development of either long-winged (LW) or short-winged (SW) morphs via regulation of the activity of Forkhead transcription factor subgroup O (NlFoxO). However, downstream effectors of the IIS–FoxO signaling cascade that mediate alternative wing morphs are unclear. Here we found that vestigial (Nlvg), a key wing-patterning gene, is selectively and temporally regulated by the IIS–FoxO signaling cascade during the wing-morph decision stage (fifth-instar stage). RNA interference (RNAi)-mediated silencing of Nlfoxo increase Nlvg expression in the fifth-instar stage (the last nymphal stage), thereby inducing LW development. Conversely, silencing of Nlvg can antagonize the effects of IIS activity on LW development, redirecting wing commitment from LW to the morph with intermediate wing size. In vitro and in vivo binding assays indicated that NlFoxO protein may suppress Nlvg expression by directly binding to the first intron region of the Nlvg locus. Our findings provide a first glimpse of the link connecting the IIS pathway to the wing-patterning network on the developmental plasticity of wings in insects, and help us understanding how phenotypic diversity is generated by the modification of a common set of pattern elements.

Description: Several intracellular trafficking pathways contribute to the regulation of AMPA receptor (AMPAR) levels at synapses and the control of synaptic strength. While much has been learned about these intracellular trafficking pathways, a major challenge is to understand how extracellular factors, such as growth factors, neuropeptides and hormones, impinge on specific AMPAR trafficking pathways to alter synaptic function and behavior. Here, we identify the secreted ligand PVF-1 and its cognate VEGF receptor homologs, VER-1 and VER-4, as regulators of glutamate signaling in C. elegans. Loss of function mutations in ver-1, ver-4, or pvf-1, result in decreased cell surface levels of the AMPAR GLR-1 and defects in glutamatergic behavior. Rescue experiments indicate that PVF-1 is expressed and released from muscle, whereas the VERs function in GLR-1-expressing neurons to regulate surface levels of GLR-1 and glutamatergic behavior. Additionally, ver-4 is unable to rescue glutamatergic behavior in the absence of pvf-1, suggesting that VER function requires endogenous PVF-1. Inducible expression of a pvf-1 rescuing transgene suggests that PVF-1 can function in the mature nervous system to regulate GLR-1 signaling. Genetic double mutant analysis suggests that the VERs act together with the VPS-35/retromer recycling complex to promote cell surface levels of GLR-1. Our data support a genetic model whereby PVF-1/VER signaling acts with retromer to promote recycling and cell surface levels of GLR-1 to control behavior.

Description: Vesicular trafficking defects, particularly those in the autophagolysosomal system, have been strongly implicated in the pathogenesis of Parkinson’s disease and related α-synucleinopathies. However, mechanisms mediating dysfunction of membrane trafficking remain incompletely understood. Using a Drosophila model of α-synuclein neurotoxicity with widespread and robust pathology, we find that human α-synuclein expression impairs autophagic flux in aging adult neurons. Genetic destabilization of the actin cytoskeleton rescues F-actin accumulation, promotes autophagosome clearance, normalizes the autophagolysosomal system, and rescues neurotoxicity in α-synuclein transgenic animals through an Arp2/3 dependent mechanism. Similarly, mitophagosomes accumulate in human α-synuclein-expressing neurons, and reversal of excessive actin stabilization promotes both clearance of these abnormal mitochondria-containing organelles and rescue of mitochondrial dysfunction. These results suggest that Arp2/3 dependent actin cytoskeleton stabilization mediates autophagic and mitophagic dysfunction and implicate failure of autophagosome maturation as a pathological mechanism in Parkinson’s disease and related α-synucleinopathies.

Description: During the development of a holometabolous insect such as Drosophila, specific group of cells in the larva survive during metamorphosis, unlike the other larval cells, and finally give rise to the differentiated adult structures. These cells, also known as Adult Progenitor Cells (APCs), maintain their multipotent capacity, differentially respond to hormonal and nutritional signals, survive the intrinsic and environmental stress and respond to the final differentiation cues. However, not much is known about the specific molecular mechanisms that account for their unique characteristics. Here we show that a specific Drosophila APC gene, headcase (hdc), has a dual role in the normal development of these cells. It acts at a systemic level by controlling the hormone ecdysone in the prothoracic gland and at the same time it acts locally as a tissue growth suppressor in the APC clusters, where it modulates the activity of the TOR pathway and promotes their survival by contributing in the regulation of the Unfolded Protein Response. We also show that hdc provides protection against stress in the APCs and that its ectopic expression in cells that do not usually express hdc can confer these cells with an additional stress protection. Hdc is the founding member of a group of homolog proteins identified from C. elegans to humans, where has been found associated with cancer progression. The finding that the Drosophila hdc is specifically expressed in progenitor cells and that it provides protection against stress opens up a new hypothesis to be explored regarding the role of the human Heca and its contribution to carcinogenesis.

Description: Epidemiological studies of obesity, Type-2 diabetes (T2D), cardiovascular diseases and several common cancers have revealed an increased risk in Native Hawaiians compared to European- or Asian-Americans living in the Hawaiian islands. However, there remains a gap in our understanding of the genetic factors that affect the health of Native Hawaiians. To fill this gap, we studied the genetic risk factors at both the chromosomal and sub-chromosomal scales using genome-wide SNP array data on ~4,000 Native Hawaiians from the Multiethnic Cohort. We estimated the genomic proportion of Native Hawaiian ancestry (“global ancestry,” which we presumed to be Polynesian in origin), as well as this ancestral component along each chromosome (“local ancestry”) and tested their respective association with binary and quantitative cardiometabolic traits. After attempting to adjust for non-genetic covariates evaluated through questionnaires, we found that per 10% increase in global Polynesian genetic ancestry, there is a respective 8.6%, and 11.0% increase in the odds of being diabetic (P = 1.65×10−4) and having heart failure (P = 2.18×10−4), as well as a 0.059 s.d. increase in BMI (P = 1.04×10−10). When testing the association of local Polynesian ancestry with risk of disease or biomarkers, we identified a chr6 region associated with T2D. This association was driven by an uniquely prevalent variant in Polynesian ancestry individuals. However, we could not replicate this finding in an independent Polynesian cohort from Samoa due to the small sample size of the replication cohort. In conclusion, we showed that Polynesian ancestry, which likely capture both genetic and lifestyle risk factors, is associated with an increased risk of obesity, Type-2 diabetes, and heart failure, and that larger cohorts of Polynesian ancestry individuals will be needed to replicate the putative association on chr6 with T2D.

Description: Intracellular Ca2+ level is under strict regulation through calcium channels and storage pools including the endoplasmic reticulum (ER). Mutations in certain ion channel subunits, which cause mis-regulated Ca2+ influx, induce the excitotoxic necrosis of neurons. In the nematode Caenorhabditis elegans, dominant mutations in the DEG/ENaC sodium channel subunit MEC-4 induce six mechanosensory (touch) neurons to undergo excitotoxic necrosis. These necrotic neurons are subsequently engulfed and digested by neighboring hypodermal cells. We previously reported that necrotic touch neurons actively expose phosphatidylserine (PS), an “eat-me” signal, to attract engulfing cells. However, the upstream signal that triggers PS externalization remained elusive. Here we report that a robust and transient increase of cytoplasmic Ca2+ level occurs prior to the exposure of PS on necrotic touch neurons. Inhibiting the release of Ca2+ from the ER, either pharmacologically or genetically, specifically impairs PS exposure on necrotic but not apoptotic cells. On the contrary, inhibiting the reuptake of cytoplasmic Ca2+ into the ER induces ectopic necrosis and PS exposure. Remarkably, PS exposure occurs independently of other necrosis events. Furthermore, unlike in mutants of DEG/ENaC channels, in dominant mutants of deg-3 and trp-4, which encode Ca2+ channels, PS exposure on necrotic neurons does not rely on the ER Ca2+ pool. Our findings indicate that high levels of cytoplasmic Ca2+ are necessary and sufficient for PS exposure. They further reveal two Ca2+-dependent, necrosis-specific pathways that promote PS exposure, a “two-step” pathway initiated by a modest influx of Ca2+ and further boosted by the release of Ca2+ from the ER, and another, ER-independent, pathway. Moreover, we found that ANOH-1, the worm homolog of mammalian phospholipid scramblase TMEM16F, is necessary for efficient PS exposure in thapsgargin-treated worms and trp-4 mutants, like in mec-4 mutants. We propose that both the ER-mediated and ER-independent Ca2+ pathways promote PS externalization through activating ANOH-1.

Description: Cholesterol-dependent cytolysins (CDCs), of which intermedilysin (ILY) is an archetypal member, are a group of pore-forming toxins secreted by a large variety of pathogenic bacteria. These toxins, secreted as soluble monomers, oligomerize upon interaction with cholesterol in the target membrane and transect it as pores of diameters of up to 100 to 300 Å. These pores disrupt cell membranes and result in cell lysis. The immune receptor CD59 is a well-established cellular factor required for intermedilysin pore formation. In this study, we applied genome-wide CRISPR-Cas9 knock-out screening to reveal additional cellular co-factors essential for ILY-mediated cell lysis. We discovered a plethora of genes previously not associated with ILY, many of which are important for membrane constitution. We show that heparan sulfates facilitate ILY activity, which can be inhibited by heparin. Furthermore, we identified hits in both protein and lipid glycosylation pathways and show a role for glucosylceramide, demonstrating that membrane organization is important for ILY activity. We also cross-validated identified genes with vaginolysin and pneumolysin and found that pneumolysin’s cytolytic activity strongly depends on the asymmetric distribution of membrane phospholipids. This study shows that membrane-targeting toxins combined with genetic screening can identify genes involved in biological membrane composition and metabolism.

Description: Supernumerary mini-chromosomes–a unique type of genomic structural variation–have been implicated in the emergence of virulence traits in plant pathogenic fungi. However, the mechanisms that facilitate the emergence and maintenance of mini-chromosomes across fungi remain poorly understood. In the blast fungus Magnaporthe oryzae (Syn. Pyricularia oryzae), mini-chromosomes have been first described in the early 1990s but, until very recently, have been overlooked in genomic studies. Here we investigated structural variation in four isolates of the blast fungus M. oryzae from different grass hosts and analyzed the sequences of mini-chromosomes in the rice, foxtail millet and goosegrass isolates. The mini-chromosomes of these isolates turned out to be highly diverse with distinct sequence composition. They are enriched in repetitive elements and have lower gene density than core-chromosomes. We identified several virulence-related genes in the mini-chromosome of the rice isolate, including the virulence-related polyketide synthase Ace1 and two variants of the effector gene AVR-Pik. Macrosynteny analyses around these loci revealed structural rearrangements, including inter-chromosomal translocations between core- and mini-chromosomes. Our findings provide evidence that mini-chromosomes emerge from structural rearrangements and segmental duplication of core-chromosomes and might contribute to adaptive evolution of the blast fungus.

Description: The conserved zona pellucida (ZP) domain is found in hundreds of extracellular proteins that are expressed in various organs and play a variety of roles as structural components, receptors and tumor suppressors. A liver-specific zona pellucida domain-containing protein (LZP), also named OIT3, has been shown to be mainly expressed in human and mouse hepatocytes; however, the physiological function of LZP in the liver remains unclear. Here, we show that Lzp deletion inhibited very low-density lipoprotein (VLDL) secretion, leading to hepatic TG accumulation and lower serum TG levels in mice. The apolipoprotein B (apoB) levels were significantly decreased in the liver, serum, and VLDL particles of LZP-deficient mice. In the presence of LZP, which is localized to the endoplasmic reticulum (ER) and Golgi apparatus, the ER-associated degradation (ERAD) of apoB was attenuated; in contrast, in the absence of LZP, apoB was ubiquitinated by AMFR, a known E3 ubiquitin ligase specific for apoB, and was subsequently degraded, leading to lower hepatic apoB levels and inhibited VLDL secretion. Interestingly, hepatic LZP levels were elevated in mice challenged with a high-fat diet and humans with simple hepatic steatosis, suggesting that LZP contributes to the physiological regulation of hepatic TG homeostasis. In general, our data establish an essential role for LZP in hepatic TG transportation and VLDL secretion by preventing the AMFR-mediated ubiquitination and degradation of apoB and therefore provide insight into the molecular function of LZP in hepatic lipid metabolism.

Description: The RB1 tumor suppressor is recurrently mutated in a variety of cancers including retinoblastomas, small cell lung cancers, triple-negative breast cancers, prostate cancers, and osteosarcomas. Finding new synthetic lethal (SL) interactions with RB1 could lead to new approaches to treating cancers with inactivated RB1. We identified 95 SL partners of RB1 based on a Drosophila screen for genetic modifiers of the eye phenotype caused by defects in the RB1 ortholog, Rbf1. We validated 38 mammalian orthologs of Rbf1 modifiers as RB1 SL partners in human cancer cell lines with defective RB1 alleles. We further show that for many of the RB1 SL genes validated in human cancer cell lines, low activity of the SL gene in human tumors, when concurrent with low levels of RB1 was associated with improved patient survival. We investigated higher order combinatorial gene interactions by creating a novel Drosophila cancer model with co-occurring Rbf1, Pten and Ras mutations, and found that targeting RB1 SL genes in this background suppressed the dramatic tumor growth and rescued fly survival whilst having minimal effects on wild-type cells. Finally, we found that drugs targeting the identified RB1 interacting genes/pathways, such as UNC3230, PYR-41, TAK-243, isoginkgetin, madrasin, and celastrol also elicit SL in human cancer cell lines. In summary, we identified several high confidence, evolutionarily conserved, novel targets for RB1-deficient cells that may be further adapted for the treatment of human cancer.

Description: We have gained considerable insight into the mechanisms which recognize and repair DNA damage, but how they adapt to extreme environmental challenges remains poorly understood. Cavefish have proven to be fascinating models for exploring the evolution of DNA repair in the complete absence of UV-induced DNA damage and light. We have previously revealed that the Somalian cavefish Phreatichthys andruzzii, lacks photoreactivation repair via the loss of light, UV and ROS-induced photolyase gene transcription mediated by D-box enhancer elements. Here, we explore whether other systems repairing UV-induced DNA damage have been similarly affected in this cavefish model. By performing a comparative study using P. andruzzii and the surface-dwelling zebrafish, we provide evidence for a conservation of sunlight-regulated Nucleotide Excision Repair (NER). Specifically, the expression of the ddb2 gene which encodes a key NER recognition factor is robustly induced following exposure to light, UV and oxidative stress in both species. As in the case of the photolyase genes, D-boxes in the ddb2 promoter are sufficient to induce transcription in zebrafish. Interestingly, despite the loss of D-box-regulated photolyase gene expression in P. andruzzii, the D-box is required for ddb2 induction by visible light and oxidative stress in cavefish. However, in the cavefish ddb2 gene this D-box-mediated induction requires cooperation with an adjacent, highly conserved E2F element. Furthermore, while in zebrafish UV-induced ddb2 expression results from transcriptional activation accompanied by stabilization of the ddb2 mRNA, in P. andruzzii UV induces ddb2 expression exclusively via an increase in mRNA stability. Thus, we reveal plasticity in the transcriptional and post transcriptional mechanisms regulating the repair of sunlight-induced DNA damage under long-term environmental challenges.

Description: Whether hard sweeps or soft sweeps dominate adaptation has been a matter of much debate. Recently, we developed haplotype homozygosity statistics that (i) can detect both hard and soft sweeps with similar power and (ii) can classify the detected sweeps as hard or soft. The application of our method to population genomic data from a natural population of Drosophila melanogaster (DGRP) allowed us to rediscover three known cases of adaptation at the loci Ace, Cyp6g1, and CHKov1 known to be driven by soft sweeps, and detected additional candidate loci for recent and strong sweeps. Surprisingly, all of the top 50 candidates showed patterns much more consistent with soft rather than hard sweeps. Recently, Harris et al. 2018 criticized this work, suggesting that all the candidate loci detected by our haplotype statistics, including the positive controls, are unlikely to be sweeps at all and that instead these haplotype patterns can be more easily explained by complex neutral demographic models. They also claim that these neutral non-sweeps are likely to be hard instead of soft sweeps. Here, we reanalyze the DGRP data using a range of complex admixture demographic models and reconfirm our original published results suggesting that the majority of recent and strong sweeps in D. melanogaster are first likely to be true sweeps, and second, that they do appear to be soft. Furthermore, we discuss ways to take this work forward given that most demographic models employed in such analyses are necessarily too simple to capture the full demographic complexity, while more realistic models are unlikely to be inferred correctly because they require a large number of free parameters.

Description: Gene drive elements promote the spread of linked traits, providing methods for changing the composition or fate of wild populations. Drive mechanisms that are self-limiting are attractive because they allow control over the duration and extent of trait spread in time and space, and are reversible through natural selection as drive wanes. Self-sustaining Cleave and Rescue (ClvR) elements include a DNA sequence-modifying enzyme such as Cas9/gRNAs that disrupts endogenous versions of an essential gene, a tightly linked recoded version of the essential gene resistant to cleavage (the Rescue), and a Cargo. ClvR spreads by creating loss-of-function (LOF) conditions in which those without ClvR die because they lack functional copies of the essential gene. We use modeling to show that when the Rescue-Cargo and one or both components required for LOF allele creation (Cas9 and gRNA) reside at different locations (split ClvR), drive of Rescue-Cargo is self-limiting due to a progressive decrease in Cas9 frequency, and thus opportunities for creation of LOF alleles, as spread occurs. Importantly, drive strength and duration can be extended in a measured manner—which is still self-limiting—by moving the two components close enough to each other that they experience some degree of linkage. With linkage, Cas9 transiently experiences drive by hitchhiking with Rescue-Cargo until linkage disequilibrium between the two disappears, a function of recombination frequency and number of generations, creating a novel point of control. We implement split ClvR in Drosophila, with key elements on different chromosomes. Cargo/Rescue/gRNAs spreads to high frequency in a Cas9-dependent manner, while the frequency of Cas9 decreases. These observations show that measured, transient drive, coupled with a loss of future drive potential, can be achieved using the simple toolkit that make up ClvR elements—Cas9 and gRNAs and a Rescue/Cargo.

Description: Post-transcriptional regulation of gene expression is crucial during the oocyte-to-embryo transition, a highly dynamic process characterized by the absence of nuclear transcription. Thus, changes to the RNA content are solely dependent on RNA degradation. Although several mechanisms that promote RNA decay during embryogenesis have been identified, it remains unclear which machineries contribute to remodeling the maternal transcriptome. Here, we focused on the degradation factor Ski7 in zebrafish. Homozygous ski7 mutant fish had higher proportions of both poor quality eggs and eggs that were unable to develop beyond the one-cell stage. Consistent with the idea that Ski7 participates in remodeling the maternal RNA content, transcriptome profiling identified hundreds of misregulated mRNAs in the absence of Ski7. Furthermore, upregulated genes were generally lowly expressed in wild type, suggesting that Ski7 maintains low transcript levels for this subset of genes. Finally, GO enrichment and proteomic analyses of misregulated factors implicated Ski7 in the regulation of redox processes. This was confirmed experimentally by an increased resistance of ski7 mutant embryos to reductive stress. Our results provide first insights into the physiological role of vertebrate Ski7 as a post-transcriptional regulator during the oocyte-to-embryo transition.

Description: A recent genome-wide association study (GWAS) in African descent populations identified novel loci associated with skin pigmentation. However, how genomic variations affect skin pigmentation and how these skin pigmentation gene variants affect serum 25(OH) vitamin D variation has not been explored in African Americans (AAs). In order to further understand genetic factors that affect human skin pigmentation and serum 25(OH)D variation, we performed a GWAS for skin pigmentation with 395 AAs and a replication study with 681 AAs. Then, we tested if the identified variants are associated with serum 25(OH) D concentrations in a subset of AAs (n = 591). Skin pigmentation, Melanin Index (M-Index), was measured using a narrow-band reflectometer. Multiple regression analysis was performed to identify variants associated with M-Index and to assess their role in serum 25(OH)D variation adjusting for population stratification and relevant confounding variables. A variant near the SLC24A5 gene (rs2675345) showed the strongest signal of association with M-Index (P = 4.0 x 10−30 in the pooled dataset). Variants in SLC24A5, SLC45A2 and OCA2 together account for a large proportion of skin pigmentation variance (11%). The effects of these variants on M-Index was modified by sex (P for interaction = 0.009). However, West African Ancestry (WAA) also accounts for a large proportion of M-Index variance (23%). M-Index also varies among AAs with high WAA and high Genetic Score calculated from top variants associated with M-Index, suggesting that other unknown genomic factors related to WAA are likely contributing to skin pigmentation variation. M-Index was not associated with serum 25(OH)D concentrations, but the Genetic Score was significantly associated with vitamin D deficiency (serum 25(OH)D levels less than 12 ng/mL) (OR, 1.30; 95% CI, 1.04–1.64). The findings support the hypothesis suggesting that skin pigmentation evolved responding to increased demand for subcutaneous vitamin D synthesis in high latitude environments.

Description: In the fruit fly Drosophila melanogaster, male-specific splicing and translation of the Fruitless transcription factor (FruM) alters the presence, anatomy, and/or connectivity of 〉60 types of central brain neurons that interconnect to generate male-typical behaviors. While the indispensable function of FruM in sex-specific behavior has been understood for decades, the molecular mechanisms underlying its activity remain unknown. Here, we take a genome-wide, brain-wide approach to identifying regulatory elements whose activity depends on the presence of FruM. We identify 436 high-confidence genomic regions differentially accessible in male fruitless neurons, validate candidate regions as bona-fide, differentially regulated enhancers, and describe the particular cell types in which these enhancers are active. We find that individual enhancers are not activated universally but are dedicated to specific fru+ cell types. Aside from fru itself, genes are not dedicated to or common across the fru circuit; rather, FruM appears to masculinize each cell type differently, by tweaking expression of the same effector genes used in other circuits. Finally, we find FruM motifs enriched among regulatory elements that are open in the female but closed in the male. Together, these results suggest that FruM acts cell-type-specifically to decommission regulatory elements in male fruitless neurons.

Description: Hundreds of mutations in a single gene result in rare diseases, but why mutations induce severe or attenuated states remains poorly understood. Defect in glycine decarboxylase (GLDC) causes Non-ketotic Hyperglycinemia (NKH), a neurological disease associated with elevation of plasma glycine. We unified a human multiparametric NKH mutation scale that separates severe from attenuated neurological disease with new in silico tools for murine and human genome level-analyses, gathered in vivo evidence from mice engineered with top-ranking attenuated and a highly pathogenic mutation, and integrated the data in a model of pre- and post-natal disease outcomes, relevant for over a hundred major and minor neurogenic mutations. Our findings suggest that highly severe neurogenic mutations predict fatal, prenatal disease that can be remedied by metabolic supplementation of dams, without amelioration of persistent plasma glycine. The work also provides a systems approach to identify functional consequences of mutations across hundreds of genetic diseases. Our studies provide a new framework for a large scale understanding of mutation functions and the prediction that severity of a neurogenic mutation is a direct measure of pre-natal disease in neurometabolic NKH mouse models. This framework can be extended to analyses of hundreds of monogenetic rare disorders where the underlying genes are known but understanding of the vast majority of mutations and why and how they cause disease, has yet to be realized.

Description: Mammalian tooth crown formation has long served as a model for investigating how patterning and morphogenesis are orchestrated during development. However, the mechanism underlying root patterning and morphogenesis remains poorly understood. In this study, we find that Lhx6 labels a subpopulation of root progenitor cells in the apical dental mesenchyme, which is closely associated with furcation development. Loss of Lhx6 leads to furcation and root number defects, indicating that Lhx6 is a key root patterning regulator. Among the multiple cellular events regulated by Lhx6 is the odontoblast fate commitment of progenitor cells, which it controls in a cell-autonomous manner. Specifically, Lhx6 loss leads to elevated expression of the Wnt antagonist Sfrp2 and down-regulation of Wnt signaling in the furcation region, while overactivation of Wnt signaling in Lhx6+ progenitor cells partially restore the furcation defects in Lhx6-/- mice. Collectively, our findings have important implications for understanding organ morphogenesis and future strategies for tooth root regeneration.

Description: Diapause, a programmed developmental arrest primarily induced by seasonal environmental changes, is very common in the animal kingdom, and found in vertebrates and invertebrates alike. Diapause provides an adaptive advantage to animals, as it increases the odds of surviving adverse conditions. In insects, individuals perceive photoperiodic cues and modify endocrine signaling to direct reproductive diapause traits, such as ovary arrest and increased fat accumulation. However, it remains unclear as to which endocrine factors are involved in this process and how they regulate the onset of reproductive diapause. Here, we found that the long day-mediated drop in the concentration of the steroid hormone ecdysone is essential for the preparation of photoperiodic reproductive diapause in Colaphellus bowringi, an economically important cabbage beetle. The diapause-inducing long-day condition reduced the expression of ecdysone biosynthetic genes, explaining the drop in the titer of 20-hydroxyecdysone (20E, the active form of ecdysone) in female adults. Application of exogenous 20E induced vitellogenesis and ovarian development but reduced fat accumulation in the diapause-destined females. Knocking down the ecdysone receptor (EcR) in females destined for reproduction blocked reproductive development and induced diapause traits. RNA-seq and hormone measurements indicated that 20E stimulates the production of juvenile hormone (JH), a key endocrine factor in reproductive diapause. To verify this, we depleted three ecdysone biosynthetic enzymes via RNAi, which confirmed that 20E is critical for JH biosynthesis and reproductive diapause. Importantly, impairing Met function, a component of the JH intracellular receptor, partially blocked the 20E-regulated reproductive diapause preparation, indicating that 20E regulates reproductive diapause in both JH-dependent and -independent manners. Finally, we found that 20E deficiency decreased ecdysis-triggering hormone signaling and reduced JH production, thereby inducing diapause. Together, these results suggest that 20E signaling is a pivotal regulator that coordinates reproductive plasticity in response to environmental inputs.

Description: Neurons are highly specialized cells with polarized cellular processes and subcellular domains. As vital organelles for neuronal functions, mitochondria are distributed by microtubule-based transport systems. Although the essential components of mitochondrial transport including motors and cargo adaptors are identified, it is less clear how mitochondrial distribution among somato-dendritic and axonal compartment is regulated. Here, we systematically study mitochondrial motors, including four kinesins, KIF5, KIF17, KIF1, KLP-6, and dynein, and transport regulators in C. elegans PVD neurons. Among all these motors, we found that mitochondrial export from soma to neurites is mainly mediated by KIF5/UNC-116. Interestingly, UNC-116 is especially important for axonal mitochondria, while dynein removes mitochondria from all plus-end dendrites and the axon. We surprisingly found one mitochondrial transport regulator for minus-end dendritic compartment, TRAK-1, and two mitochondrial transport regulators for axonal compartment, CRMP/UNC-33 and JIP3/UNC-16. While JIP3/UNC-16 suppresses axonal mitochondria, CRMP/UNC-33 is critical for axonal mitochondria; nearly no axonal mitochondria present in unc-33 mutants. We showed that UNC-33 is essential for organizing the population of UNC-116-associated microtubule bundles, which are tracks for mitochondrial trafficking. Disarrangement of these tracks impedes mitochondrial transport to the axon. In summary, we identified a compartment-specific transport regulation of mitochondria by UNC-33 through organizing microtubule tracks for different kinesin motors other than microtubule polarity.

Description: Individual differences in early-life vocabulary measures are heritable and associated with subsequent reading and cognitive abilities, although the underlying mechanisms are little understood. Here, we (i) investigate the developmental genetic architecture of expressive and receptive vocabulary in early-life and (ii) assess timing of emerging genetic associations with mid-childhood verbal and non-verbal skills. We studied longitudinally assessed early-life vocabulary measures (15–38 months) and later-life verbal and non-verbal skills (7–8 years) in up to 6,524 unrelated children from the population-based Avon Longitudinal Study of Parents and Children (ALSPAC) cohort. We dissected the phenotypic variance of rank-transformed scores into genetic and residual components by fitting multivariate structural equation models to genome-wide genetic-relationship matrices. Our findings show that the genetic architecture of early-life vocabulary involves multiple distinct genetic factors. Two of these genetic factors are developmentally stable and also contribute to genetic variation in mid-childhood skills: One genetic factor emerging with expressive vocabulary at 24 months (path coefficient: 0.32(SE = 0.06)) was also related to later-life reading (path coefficient: 0.25(SE = 0.12)) and verbal intelligence (path coefficient: 0.42(SE = 0.13)), explaining up to 17.9% of the phenotypic variation. A second, independent genetic factor emerging with receptive vocabulary at 38 months (path coefficient: 0.15(SE = 0.07)), was more generally linked to verbal and non-verbal cognitive abilities in mid-childhood (reading path coefficient: 0.57(SE = 0.07); verbal intelligence path coefficient: 0.60(0.10); performance intelligence path coefficient: 0.50(SE = 0.08)), accounting for up to 36.1% of the phenotypic variation and the majority of genetic variance in these later-life traits (≥66.4%). Thus, the genetic foundations of mid-childhood reading and cognitive abilities are diverse. They involve at least two independent genetic factors that emerge at different developmental stages during early language development and may implicate differences in cognitive processes that are already detectable during toddlerhood.

Description: Hybridization of closely related plant species is frequently connected to endosperm arrest and seed failure, for reasons that remain to be identified. In this study, we investigated the molecular events accompanying seed failure in hybrids of the closely related species pair Capsella rubella and C. grandiflora. Mapping of QTL for the underlying cause of hybrid incompatibility in Capsella identified three QTL that were close to pericentromeric regions. We investigated whether there are specific changes in heterochromatin associated with interspecific hybridizations and found a strong reduction of chromatin condensation in the endosperm, connected with a strong loss of CHG and CHH methylation and random loss of a single chromosome. Consistent with reduced DNA methylation in the hybrid endosperm, we found a disproportionate deregulation of genes located close to pericentromeric regions, suggesting that reduced DNA methylation allows access of transcription factors to targets located in heterochromatic regions. Since the identified QTL were also associated with pericentromeric regions, we propose that relaxation of heterochromatin in response to interspecies hybridization exposes and activates loci leading to hybrid seed failure.

Description: In mammals, cellular identity is defined through strict regulation of chromatin modifications and DNA methylation that control gene expression. Methylation of cytosines at CpG sites in the genome is mainly associated with suppression; however, the reason for enhancer-specific methylation is not fully understood. We used sequential ChIP-bisulfite-sequencing for H3K4me1 and H3K27ac histone marks. By collecting data from the same genomic region, we identified enhancers differentially methylated between these two marks. We observed a global gain of CpG methylation primarily in H3K4me1-marked nucleosomes during mouse embryonic stem cell differentiation. This gain occurred largely in enhancer regions that regulate genes critical for differentiation. The higher levels of DNA methylation in H3K4me1- versus H3K27ac-marked enhancers, despite it being the same genomic region, indicates cellular heterogeneity of enhancer states. Analysis of single-cell RNA-seq profiles demonstrated that this heterogeneity correlates with gene expression during differentiation. Furthermore, heterogeneity of enhancer methylation correlates with transcription start site methylation. Our results provide insights into enhancer-based functional variation in complex biological systems.

Description: Karyotype, including the chromosome and arm numbers, is a fundamental genetic characteristic of all organisms and has long been used as a species-diagnostic character. Additionally, karyotype evolution plays an important role in divergent adaptation and speciation. Centric fusion and fission change chromosome numbers, whereas the intra-chromosomal movement of the centromere, such as pericentric inversion, changes arm numbers. A probabilistic model simultaneously incorporating both chromosome and arm numbers has not been established. Here, we built a probabilistic model of karyotype evolution based on the “karyograph”, which treats karyotype evolution as a walk on the two-dimensional space representing the chromosome and arm numbers. This model enables analysis of the stationary distribution with a stable karyotype for any given parameter. After evaluating their performance using simulated data, we applied our model to two large taxonomic groups of fish, Eurypterygii and series Otophysi, to perform maximum likelihood estimation of the transition rates and reconstruct the evolutionary history of karyotypes. The two taxa significantly differed in the evolution of arm number. The inclusion of speciation and extinction rates demonstrated possibly high extinction rates in species with karyotypes other than the most typical karyotype in both groups. Finally, we made a model including polyploidization rates and applied it to a small plant group. Thus, the use of this probabilistic model can contribute to a better understanding of tempo and mode in karyotype evolution and its possible role in speciation and extinction.

Description: An important prelude to bacterial infection is the ability of a pathogen to survive independently of the host and to withstand environmental stress. The compatible solute trehalose has previously been connected with diverse abiotic stress tolerances, particularly osmotic shock. In this study, we combine molecular biology and biochemistry to dissect the trehalose metabolic network in the opportunistic human pathogen Pseudomonas aeruginosa PAO1 and define its role in abiotic stress protection. We show that trehalose metabolism in PAO1 is integrated with the biosynthesis of branched α-glucan (glycogen), with mutants in either biosynthetic pathway significantly compromised for survival on abiotic surfaces. While both trehalose and α-glucan are important for abiotic stress tolerance, we show they counter distinct stresses. Trehalose is important for the PAO1 osmotic stress response, with trehalose synthesis mutants displaying severely compromised growth in elevated salt conditions. However, trehalose does not contribute directly to the PAO1 desiccation response. Rather, desiccation tolerance is mediated directly by GlgE-derived α-glucan, with deletion of the glgE synthase gene compromising PAO1 survival in low humidity but having little effect on osmotic sensitivity. Desiccation tolerance is independent of trehalose concentration, marking a clear distinction between the roles of these two molecules in mediating responses to abiotic stress.

Description: The RNA-binding protein Mrn1 in Saccharomyces cerevisiae targets over 300 messenger RNAs, including many involved in cell wall biogenesis. The impact of Mrn1 on these target transcripts is not known, however, nor is the cellular role for this regulation. We have shown that Mrn1 represses target mRNAs through the action of its disordered, asparagine-rich amino-terminus. Its endogenous targets include the paralogous SUN domain proteins Nca3 and Uth1, which affect mitochondrial and cell wall structure and function. While loss of MRN1 has no effect on fermentative growth, we found that mrn1Δ yeast adapt more quickly to respiratory conditions. These cells also have enlarged mitochondria in fermentative conditions, mediated in part by dysregulation of NCA3, and this may explain their faster switch to respiration. Our analyses indicated that Mrn1 acts as a hub for integrating cell wall integrity and mitochondrial biosynthesis in a carbon-source responsive manner.

Description: How temperature determines sex remains unknown. A recent hypothesis proposes that conserved cellular mechanisms (calcium and redox; ‘CaRe’ status) sense temperature and identify genes and regulatory pathways likely to be involved in driving sexual development. We take advantage of the unique sex determining system of the model organism, Pogona vitticeps, to assess predictions of this hypothesis. P. vitticeps has ZZ male: ZW female sex chromosomes whose influence can be overridden in genetic males by high temperatures, causing male-to-female sex reversal. We compare a developmental transcriptome series of ZWf females and temperature sex reversed ZZf females. We demonstrate that early developmental cascades differ dramatically between genetically driven and thermally driven females, later converging to produce a common outcome (ovaries). We show that genes proposed as regulators of thermosensitive sex determination play a role in temperature sex reversal. Our study greatly advances the search for the mechanisms by which temperature determines sex.

Description: Optogenetics has been harnessed to shed new mechanistic light on current and future therapeutic strategies. This has been to date achieved by the regulation of ion flow and electrical signals in neuronal cells and neural circuits that are known to be affected by disease. In contrast, the optogenetic delivery of trophic biochemical signals, which support cell survival and are implicated in degenerative disorders, has never been demonstrated in an animal model of disease. Here, we reengineered the human and Drosophila melanogaster REarranged during Transfection (hRET and dRET) receptors to be activated by light, creating one-component optogenetic tools termed Opto-hRET and Opto-dRET. Upon blue light stimulation, these receptors robustly induced the MAPK/ERK proliferative signaling pathway in cultured cells. In PINK1B9 flies that exhibit loss of PTEN-induced putative kinase 1 (PINK1), a kinase associated with familial Parkinson’s disease (PD), light activation of Opto-dRET suppressed mitochondrial defects, tissue degeneration and behavioral deficits. In human cells with PINK1 loss-of-function, mitochondrial fragmentation was rescued using Opto-dRET via the PI3K/NF-кB pathway. Our results demonstrate that a light-activated receptor can ameliorate disease hallmarks in a genetic model of PD. The optogenetic delivery of trophic signals is cell type-specific and reversible and thus has the potential to inspire novel strategies towards a spatio-temporal regulation of tissue repair.

Description: The comorbid association of autoimmune diseases with cancers has been a major obstacle to successful anti-cancer treatment. Cancer survival rate decreases significantly in patients with preexisting autoimmunity. However, to date, the molecular and cellular profiles of such comorbidities are poorly understood. We used Aicardi-Goutières syndrome (AGS) as a model autoimmune disease and explored the underlying mechanisms of genome instability in AGS-associated-gene-deficient patient cells. We found that R-loops are highly enriched at transcription-replication conflict regions of the genome in fibroblast of patients bearing SAMHD1 mutation, which is the AGS-associated-gene mutation most frequently reported with tumor and malignancies. In SAMHD1-depleted cells, R-loops accumulated with the concomitant activation of DNA damage responses. Removal of R-loops in SAMHD1 deficiency reduced cellular responses to genome instability. Furthermore, downregulation of SAMHD1 expression is associated with various types of cancer and poor survival rate. Our findings suggest that SAMHD1 functions as a tumor suppressor by resolving R-loops, and thus, SAMHD1 and R-loop may be novel diagnostic markers and targets for patient stratification in anti-cancer therapy.