ALBERT

Description: Identification of cell-type specific enhancers is important for understanding the regulation of programs controlling cellular development and differentiation. Recent studies have shown that enhancers are frequently associated with biologically relevant and disease-associated genetic variants. We hypothesized that unique sets of enhancers and super enhancers regulate gene expression in erythroid cells, a specialized cell type evolved to carry oxygen, and associated variants influence erythroid phenotypic variability. Active enhancers are part of a chromatin landscape marked by histone H3 lysine 4 monomethylation (H3K4me1) and histone H3 lysine 27 acetylation (H3K27Ac). A subset of enhancers, called super enhancers, important for regulating genes critical for cell-type specific identify, have been described. Super enhancers span large regions of chromatin, have domains of transcription factors (TF), significant amounts of H3K4me1 and H3K27Ac modification, and significant amounts of Mediator (MED1) occupancy, frequently with the transcriptional activator BRD4. Using ChIP-seq, genome wide maps of enhancers were constructed for H3K4me1, H3K27Ac, MED1, and BRD4 using primary human erythroid cell chromatin. These data were combined with parallel gene expression analyses determined via RNA-seq and enhancers and super enhancers identified. Cell and tissue-type specific enhancers act over distances of tens to hundreds of kilobases, thus bona fide erythroid enhancers are expected to be enriched in the genomic vicinity of genes expressed and functional in erythroid cells. Sites of occupancy of H3K4me1 were correlated with levels of gene expression in erythroid cells. To exclude gene promoters, H3K4me1 within 1kb of annotated transcriptional start sites (TSS) were excluded from analyses. Consistent with their predicted function, there was significantly higher levels of erythroid transcription for genes with H3K4me1 occupancy within 1-50kb of the TSS of genes cf. genes with H3K4me1 occupancy 〉50kb of a TSS (p value

Description: The Mds1 and Evi1 complex locus (Mecom) gives rise to several alternative transcripts implicated in leukemogenesis. However, the contribution that Mecom-derived gene products make to normal hematopoiesis remains largely unexplored. To investigate the role of the upstream transcription start site of Mecom in adult hematopoiesis, we created a mouse model with a lacZ knock-in at this site, termed MEm1, which eliminates Mds1-Evi1 (ME), the longer, PR-domain–containing isoform produced by the gene (also known as PRDM3). β-galactosidase–marking studies revealed that, within hematopoietic cells, ME is exclusively expressed in the stem cell compartment. ME deficiency leads to a reduction in the number of HSCs and a complete loss of long-term repopulation capacity, whereas the stem cell compartment is shifted from quiescence to active cycling. Genetic exploration of the relative roles of endogenous ME and EVI1 isoforms revealed that ME preferentially rescues long-term HSC defects. RNA-seq analysis in Lin−Sca-1+c-Kit+ cells (LSKs) of MEm1 documents near complete silencing of Cdkn1c, encoding negative cell-cycle regulator p57-Kip2. Reintroduction of ME into MEm1 LSKs leads to normalization of both p57-Kip2 expression and growth control. Our results clearly demonstrate a critical role of PR-domain–containing ME in linking p57-kip2 regulation to long-term HSC function.

Description: Key Points PK deficiency manifests a broad spectrum in anemia severity that moderately improves after splenectomy. Close attention to monitoring for iron overload, gallstones, and other complications is recommended in all patients with PK deficiency.

Description: Maturation of erythroid progenitors is associated with significant changes in gene expression in the context of a nucleus that dramatically decreases in size in preparation for enucleation, and is regulated by the coordinated action of transcriptional regulators and epigenetic modifiers. In eukaryotes, all DNA is bound by histone proteins into chromatin. Posttranslational modifications of the N-terminal "tails" of these proteins are key regulators of chromatin structure and gene expression. We hypothesized that terminal erythroid maturation is associated with changes in the abundance of specific histone posttranslational modifications. To address this hypothesis, we utilized mass spectrometry to perform an unbiased assessment of the abundance histone post translational modifications in maturing erythroblasts. We cultured peripheral blood CD34+ hematopoietic stem and progenitor cells (HSPCs) down the erythroid lineage using a semi-synchronous culture system (as outlined in Gautier et al. Cell Reports 2016), and sent cells for mass spectrometry on day 7 of erythroid maturation, when the cells are predominately basophilic erythroblasts, and on day 12 of erythroid maturation, when they are predominately poly- and ortho- chromatic erythroblasts. The maturation stage of the cells was confirmed by both cytospins and imaging flow cytometric analyses. Two independent replicates were performed and key results confirmed by western blotting. Terminal erythroid maturation was associated with a dramatic decline in the abundance of multiple histone marks associated with active transcription elongation, including Histone H3 lysine 36 di- and tri-methylation (H3K36me2, H3K36me3), and Histone H3 Lysine 79 di-methylation (H3K79me2). Surprisingly, this was not accompanied by an increase in the abundance of repressive heterochromatin marks (H3K27me3, H3K9me3, and H4K20me3) or a global decline in histone acetylation. Histone H4 lysine 16 acetylation (H4K16Ac), associated with RNA polymerase II pause release (Kapoor-Vazirani MCB 2011) significantly declined, but multiple acetylation marks including H3K36Ac and H3K23Ac increased in abundance. As expected, the abundance histone H4 lysine 20 mono-methylation (H4K20me1), which is implicated both in erythroblast chromatin condensation (Malik Cell Reports 2017) and the regulation of RNA Polymerase II pausing (Kapoor-Vazirani MCB 2011) also significantly increased. Consistent with these data, integration of RNA-seq and ChIP-seq data identified 3,058 genes whose expression decreased from basophilic erythroblast to orthochromatic erythroblasts, which lost enrichment for H3K36me3 (mark of active elongation) without accumulating H3K27me3 (heterochromatin mark). Based on these data, we hypothesized that RNA polymerase II pausing is a critical regulator of gene expression in maturing erythroblasts. RNA Polymerase II (Pol II) pausing is a highly regulated mechanism of transcriptional regulation, whereby transcription is initiated, but pauses 30-60bp downstream of the transcription start site. For paused Pol II to be released into active elongation, pTEFb must hyper-phosphorylate Serine 2 of the Pol II c-terminal domain (CTD). Importantly, pTEFb can be directed to specific loci through interaction with transcription factors, including GATA1 (Elagib Blood 2008; Bottardi NAR 2011). Hexim1 is a key regulator of Pol II pausing that sequesters pTEFb and inhibits its action. Consistent with a central role for Pol II pausing dynamics in the regulation of terminal erythroid maturation, Hexim1 is highly expressed in erythroid cells compared to most other cell types and its expression increases during terminal erythroid maturation. Conversely, the expression of CCNT1 and CKD9, the components of pTEFb, decline during terminal maturation, and the level of elongation competent (Ser2 and Ser2/Ser5 CTD phosphorylated) Pol II also decreases dramatically. To gain insights into the function of Pol II pausing in maturing erythroblasts, we induced Hexim1 expression in HUDEP2 cells (Kurita PLoS One 2013) using hexamethane bisacetamide (HMBA). HMBA treatment increased Hexim1 levels a dose dependent manner and was associated with gene expression and phenotypic changes suggestive of accelerated erythroid maturation. Together, these data suggest that RNA Pol II pausing dynamics are an important regulator of terminal erythroid maturation. Disclosures No relevant conflicts of interest to declare.

Description: Key Points There is heterogeneity in the clinical, laboratory, and genetic bases of HX. Alterations in PIEZO1 channel kinetics, response to osmotic stress, and membrane trafficking may contribute to channel dysfunction in HX.

Description: Key Points Mutations in the Gardos channel, encoded by the KCNN4 gene, were identified in individuals from 2 hereditary xerocytosis kindreds. These findings support recent data indicating the Gardos channel plays a role in normal erythrocyte volume homeostasis.

Description: The EVI1 gene at chromosome 3q26 is associated with acute myeloid leukemogenesis, due to both chromosomal rearrangement and to overexpression in the absence of rearrangement. Some rearrangements such as t(3;3) and inv(3) result in overexpression of EVI1 protein, while translocation t(3;21) yields an AML1-MDS1-EVI1 (AME) fusion protein. EVI1 possesses two zinc finger domains, an N-terminal domain with fingers 1–7, which binds to GACAAGATA, and a C-terminal domain (fingers 8–10) which binds GAAGATGAG. Inhibition of EVI1 function with a small molecule compound may provide a targeted therapy for EVI1-expressing leukemias. As a first step towards inhibiting the leukemogenic function of EVI1, we performed structure-function studies on both EVI1 and AME protein to determine what domains are critical for malignant transformation activity. Assays were Rat1 fibroblasts in a soft agar colony forming assay for EVI1; primary bone marrow cells in a serial replating assay for AME. Both assays revealed that mutation of arginine 205 in zinc finger 6 of EVI1, which completely abrogates sequencespecific DNA binding via the N-terminal zinc finger domain, resulted in complete loss of transforming activity; mutations in other domains, such as the C-terminal zinc finger domain, CtBP binding domain, and the domains of AML1 had less of an effect or no effect on transforming activity. In an effort to inhibit EVI1 leukemogenic function, we developed a polyamide, DH-IV-298, designed to block zinc fingers 1–7 binding to the GACAAGATA motif. DNAseI footprinting revealed a specific interaction between DH-IV-298 and the GACAAGATA motif; no significant interaction was observed elsewhere; a mismatch polyamide failed to footprint at equivalent concentrations; and DH-IV-298 failed to bind to a control DNA lacking the GACAAGATA motif. Electromobility shift assay showed that, at a 1:1 polyamide:DNA ratio, DH-IV-298 lowered EVI1:DNA affinity by over 98%, while mismatch was significantly less effective (74% reduction). To assess the effect of DH-IV-298 on EVI1 binding to DNA in vivo, we performed CAT reporter assays in a NIH-3T3-derived cell line with a chromosome-embedded tet-inducible EVI1-VP16 as well as a EVI1-responsive CAT reporter. Removal of tetracycline resulted in a four-fold increase in CAT activity that was completely blocked by DH-IV-298. The mismatch polyamide was significantly less effective than DH-IV-298. Further studies are being performed to assess the effect on endogenous gene expression, and on growth of leukemic cells that express EVI1. These studies provide evidence that a cell permeable small molecule compound may effectively block the activity of a leukemogenic transcription factor.

Description: Hereditary spherocytosis (HS), the most common inherited hemolytic anemia in Northern Europeans, is dominantly inherited in ~two third of cases. Clinically, patients with nondominant HS (ndHS) are more severely affected than those with typical, dominant HS. Biochemical and genetic studies implicate defects of α-spectrin in most ndHS patients and in patients with the related disorder hereditary pyropoikilocytosis (HPP). However, in most cases, neither the precise genetic basis nor the mechanism of disease are known. We studied individuals from 23 kindreds: 10 ndHS kindreds, 3 HPP kindreds, and 10 kindreds with transfusion-dependent (TD) anemia, using whole exome sequencing. A variety of novel mutant SPTA1 alleles were identified, including nonsense, splicing, and insertion/deletion mutations, frequently in trans to missense mutations. One patient had no SPTA1 mutations and 14 patients had only one SPTA1 mutation. Patients with 0 or 1 mutation all carried the common ndHS-linked α-spectrinBug Hill allele. We hypothesized that a production-defective SPTA1 allele is shared by these patients and is in linkage disequilibrium with the αBug Hill allele. Whole genome sequencing was performed on 2 ndHS patients heterozygous for the αBug Hill variant. Data were compared to samples in the 1000 Genomes database with the αBug Hill variant. A series of genetic analyses revealed a single common SPTA1 variant, the αLEPRA allele. This variant, described in a ndHS patient in trans to an SPTA1 nonsense mutation (JCI 98:2300, 1996), was associated with an elongated α-spectrin mRNA transcript. The αLEPRA allele was only present on 4 of 4610 alleles in 1000 Genomes. In all 4 cases, it was heterozygous and in cis to the αBugHill allele. Analysis of αBugHill haplotypes revealed 3 predominant patterns with the haplotype of the ndHS patients identical to the 4 heterozygous αLEPRA individuals. Genotyping the mutation-negative patient alleles revealed all carried the αLEPRA mutation. In the original report, RT-PCR of reticulocyte RNA demonstrated the αLEPRA allele was associated with an elongated α-spectrin mRNA transcript originating 70nt from the 3' end of intron 30. It was unclear if or how the αLEPRA mutation influenced α-spectrin mRNA splicing, as identical elongated α-spectrin mRNA transcripts are observed in erythroid cells from patients who do not carry the αLEPRA allele. Splicing analysis of intron 30 using SCROOGLE predicted: 1) a branch point at the expected location 31bp 5' of exon 31 (branch point 1, BP1); 2) an alternate upstream branch point centered on an "A" 2bp 3' of the αLEPRA mutation (BP2) 98bp 5' of exon 31; 3) identified a novel alternate 3' acceptor site downstream of BP2; and 4) the αLEPRA mutation significantly improves BP2. To determine if BP2 is a functional BP, we studied SPTA1 splicing using minigene assays in K562 cells. Wild type (WT) minigenes produced a small amount of elongated α-spectrin mRNA, while mutation of the obligate "A" of BP2 completely eliminated elongated transcript production. Mutation of the BP2 "A" using CRISPRCas9-based gene editing on a WT background in K562 cells eliminated the elongated α-spectrin mRNA transcripts. Minigene assays also revealed deletion of the alternate 3' acceptor splice site or improvement of BP1 to a U2 consensus site both eliminated the elongated transcript. Thus the αLEPRA mutation in intron 30, located upstream of an alternate 3' acceptor site, changes a weak alternate BP to a strong BP in the context of a poor primary BP. These changes lead to increased utilization of an alternate 3'acceptor site, creating an elongated transcript that leads to frameshift with a novel termination codon. This termination codon is in a position predicted to activate nonsense mediated decay (NMD). To address whether NMD of the elongated transcript is the mechanism of α-spectrin deficiency, we created K562 cells homozygous for the αLEPRA allele using gene editing and treated them with emetine or cycloheximide, NMD inhibitors. In both WT and homozygous αLEPRA cells, the total amount of elongated transcript increased relative to WT. These studies resolve an important unanswered question by demonstrating a novel mechanism of genetic disease is responsible for many cases of ndHS, HPP, and TD anemia. These data will facilitate disease diagnosis, as most current diagnostic gene panels do not include this intronic region, and they identify a new target for therapeutic gene manipulation. Disclosures No relevant conflicts of interest to declare.

Description: Key Points Transcriptomes and enhancers of human CD4+ Tfh and non-Tfh T effector cells reveal cell type–specific differences. These data are a significant resource for understanding mechanisms of normal and perturbed Tfh cell function.