ALBERT

Description: Associations between cells and the basement membrane are critical for a variety of biological events including cell proliferation, cell migration, cell differentiation and the maintenance of tissue integrity. Dystroglycan is a highly glycosylated basement membrane receptor, and is involved in physiological processes that maintain integrity of the skeletal muscle, as well as development and function of the central nervous system. Aberrant O-glycosylation of the α subunit of this protein, and a concomitant loss of dystroglycan's ability to function as a receptor for extracellular matrix (ECM) ligands that bear laminin globular (LG) domains, occurs in several congenital/limb-girdle muscular dystrophies (also referred to as dystroglycanopathies). Recent genetic studies revealed that mutations in DAG1 (which encodes dystroglycan) and at least 17 other genes disrupt the ECM receptor function of dystroglycan and cause disease. Here, we summarize recent advances in our understanding of the enzymatic functions of two of these disease genes: the like-glycosyltransferase (LARGE) and protein O-mannose kinase (POMK, previously referred to as SGK196). In addition, we discuss the structure of the glycan that directly binds the ECM ligands and the mechanisms by which this functional motif is linked to dystroglycan. In light of the fact that dystroglycan functions as a matrix receptor and the polysaccharide synthesized by LARGE is the binding motif for matrix proteins, we propose to name this novel polysaccharide structure matriglycan.

Description: Mutations in the gene for telomerase reverse transcriptase ( hTERT ) are associated with diseases including dyskeratosis congenita, aplastic anemia, pulmonary fibrosis and cancer. Understanding the molecular basis of these telomerase-associated diseases requires dependable quantitative measurements of telomerase enzyme activity. Furthermore, recent findings that the human POT1-TPP1 chromosome end-binding protein complex stimulates telomerase activity and processivity provide incentive for testing variant telomerases in the presence of these factors. In the present work, we compare multiple disease-associated hTERT variants reconstituted with the RNA subunit hTR in two systems (rabbit reticulocyte lysates and human cell lines) with respect to telomerase enzymatic activity, processivity and activation by telomere proteins. Surprisingly, many of the previously reported disease-associated hTERT alleles give near-normal telomerase enzyme activity. It is possible that a small deficit in telomerase activity is sufficient to cause telomere shortening over many years. Alternatively, mutations may perturb functions such as the recruitment of telomerase to telomeres, which are essential in vivo but not revealed by simple enzyme assays.

Description: Agnathans (jawless vertebrates) occupy a key phylogenetic position for illuminating the evolution of vertebrate anatomy and physiology. Evaluation of the agnathan globin gene repertoire can thus aid efforts to reconstruct the origin and evolution of the globin genes of vertebrates, a superfamily that includes the well-known model proteins hemoglobin and myoglobin. Here, we report a comprehensive analysis of the genome of the sea lamprey ( Petromyzon marinus ) which revealed 23 intact globin genes and two hemoglobin pseudogenes. Analyses of the genome of the Arctic lamprey ( Lethenteron camtschaticum ) identified 18 full length and five partial globin gene sequences. The majority of the globin genes in both lamprey species correspond to the known agnathan hemoglobins. Both genomes harbor two copies of globin X, an ancient globin gene that has a broad phylogenetic distribution in the animal kingdom. Surprisingly, we found no evidence for an ortholog of neuroglobin in the lamprey genomes. Expression and phylogenetic analyses identified an ortholog of cytoglobin in the lampreys; in fact, our results indicate that cytoglobin is the only orthologous vertebrate-specific globin that has been retained in both gnathostomes and agnathans. Notably, we also found two globins that are highly expressed in the heart of P. marinus , thus representing functional myoglobins. Both genes have orthologs in L. camtschaticum. Phylogenetic analyses indicate that these heart-expressed globins are not orthologous to the myoglobins of jawed vertebrates (Gnathostomata), but originated independently within the agnathans. The agnathan myoglobin and hemoglobin proteins form a monophyletic group to the exclusion of functionally analogous myoglobins and hemoglobins of gnathostomes, indicating that specialized respiratory proteins for O 2 transport in the blood and O 2 storage in the striated muscles evolved independently in both lineages. This dual convergence of O 2 -transport and O 2 -storage proteins in agnathans and gnathostomes involved the convergent co-option of different precursor proteins in the ancestral globin repertoire of vertebrates.

Description: Muscular dystrophy is characterized by progressive skeletal muscle weakness and dystrophic muscle exhibits degeneration and regeneration of muscle cells, inflammation and fibrosis. Skeletal muscle fibrosis is an excessive deposition of components of the extracellular matrix including an accumulation of Collagen VI. We hypothesized that a reduction of Collagen VI in a muscular dystrophy model that presents with fibrosis would result in reduced muscle pathology and improved muscle function. To test this hypothesis, we crossed -sarcoglycan-null mice, a model of limb-girdle muscular dystrophy type 2C, with a Col6a2-deficient mouse model. We found that the resulting -sarcoglycan-null/Col6a2ex5 mice indeed exhibit reduced muscle pathology compared with -sarcoglycan-null mice. Specifically, fewer muscle fibers are degenerating, fiber size varies less, Evans blue dye uptake is reduced and serum creatine kinase levels are lower. Surprisingly, in spite of this reduction in muscle pathology, muscle function is not significantly improved. In fact, grip strength and maximum isometric tetanic force are even lower in -sarcoglycan-null/Col6a2ex5 mice than in -sarcoglycan-null mice. In conclusion, our results reveal that Collagen VI-mediated fibrosis contributes to skeletal muscle pathology in -sarcoglycan-null mice. Importantly, however, our data also demonstrate that a reduction in skeletal muscle pathology does not necessarily lead to an improvement of skeletal muscle function, and this should be considered in future translational studies.

Description: A selective sweep is the result of strong positive selection driving newly occurring or standing genetic variants to fixation, and can dramatically alter the pattern and distribution of allelic diversity in a population. Population-level sequencing data have enabled discoveries of selective sweeps associated with genes involved in recent adaptations in many species. In contrast, much debate but little evidence addresses whether "selfish" genes are capable of fixation—thereby leaving signatures identical to classical selective sweeps—despite being neutral or deleterious to organismal fitness. We previously described R2d2 , a large copy-number variant that causes nonrandom segregation of mouse Chromosome 2 in females due to meiotic drive. Here we show population-genetic data consistent with a selfish sweep driven by alleles of R2d2 with high copy number ( R2d2 HC ) in natural populations. We replicate this finding in multiple closed breeding populations from six outbred backgrounds segregating for R2d2 alleles. We find that R2d2 HC rapidly increases in frequency, and in most cases becomes fixed in significantly fewer generations than can be explained by genetic drift. R2d2 HC is also associated with significantly reduced litter sizes in heterozygous mothers, making it a true selfish allele. Our data provide direct evidence of populations actively undergoing selfish sweeps, and demonstrate that meiotic drive can rapidly alter the genomic landscape in favor of mutations with neutral or even negative effects on overall Darwinian fitness. Further study will reveal the incidence of selfish sweeps, and will elucidate the relative contributions of selfish genes, adaptation and genetic drift to evolution.

Description: The evolutionary fate of chimeric fusion genes may be strongly influenced by their recombinational mode of origin and the nature of functional divergence between the parental genes. In the β-globin gene family of placental mammals, the two postnatally expressed - and β-globin genes ( HBD and HBB , respectively) have a propensity for recombinational exchange via gene conversion and unequal crossing-over. In the latter case, there are good reasons to expect differences in retention rates for the reciprocal HBB / HBD and HBD / HBB fusion genes due to thalassemia pathologies associated with the HBD / HBB "Lepore" deletion mutant in humans. Here, we report a comparative genomic analysis of the mammalian β-globin gene cluster, which revealed that chimeric HBB / HBD fusion genes originated independently in four separate lineages of laurasiatherian mammals: Eulipotyphlans (shrews, moles, and hedgehogs), carnivores, microchiropteran bats, and cetaceans. In cases where an independently derived "anti-Lepore" duplication mutant has become fixed, the parental HBD and/or HBB genes have typically been inactivated or deleted, so that the newly created HBB / HBD fusion gene is primarily responsible for synthesizing the β-type subunits of adult and fetal hemoglobin (Hb). Contrary to conventional wisdom that the HBD gene is a vestigial relict that is typically inactivated or expressed at negligible levels, we show that HBD -like genes often encode a substantial fraction (20–100%) of β-chain Hbs in laurasiatherian taxa. Our results indicate that the ascendancy or resuscitation of genes with HBD -like coding sequence requires the secondary acquisition of HBB -like promoter sequence via unequal crossing-over or interparalog gene conversion.

Description: LARGE-dependent modification enables α-dystroglycan (α-DG) to bind to its extracellular matrix ligands. Mutations in the LARGE gene and several others involved in O -mannosyl glycan synthesis have been identified in congenital and limb-girdle muscular dystrophies that are characterized by perturbed glycosylation and reduced ligand-binding affinity of α-DG. LARGE is a bifunctional glycosyltransferase that alternately transfers xylose and glucuronic acid, thereby generating the heteropolysaccharides on α-DG that confer its ligand binding. Although the LARGE paralog LARGE2 (also referred to as GYLTL1B) has likewise been shown to enhance the functional modification of α-DG in cultured cells, its enzymatic activities have not been identified. Here, we report that LARGE2 is also a bifunctional glycosyltransferase and compare its properties with those of LARGE. By means of a high-performance liquid chromatography-based enzymatic assay, we demonstrate that like LARGE, LARGE2 has xylosyltransferase (Xyl-T) and glucuronyltransferase (GlcA-T) activities, as well as polymerizing activity. Notably, however, the pH optima of the Xyl-T and GlcA-T of LARGE2 are distinct from one another and also from those of LARGE. Our results suggest that LARGE and LARGE2 catalyze the same glycosylation reactions for the functional modification of α-DG, but that they have different biochemical properties.

Description: The Drug–Gene Interaction Database (DGIdb, www.dgidb.org ) is a web resource that consolidates disparate data sources describing drug–gene interactions and gene druggability. It provides an intuitive graphical user interface and a documented application programming interface (API) for querying these data. DGIdb was assembled through an extensive manual curation effort, reflecting the combined information of twenty-seven sources. For DGIdb 2.0, substantial updates have been made to increase content and improve its usefulness as a resource for mining clinically actionable drug targets. Specifically, nine new sources of drug–gene interactions have been added, including seven resources specifically focused on interactions linked to clinical trials. These additions have more than doubled the overall count of drug–gene interactions. The total number of druggable gene claims has also increased by 30%. Importantly, a majority of the unrestricted, publicly-accessible sources used in DGIdb are now automatically updated on a weekly basis, providing the most current information for these sources. Finally, a new web view and API have been developed to allow searching for interactions by drug identifiers to complement existing gene-based search functionality. With these updates, DGIdb represents a comprehensive and user friendly tool for mining the druggable genome for precision medicine hypothesis generation.

Description: Both LARGE1 (formerly LARGE) and its paralog LARGE2 are bifunctional glycosyltransferases with xylosy- and glucuronyltransferase activities, and are capable of synthesizing polymers composed of a repeating disaccharide [-3Xylα1,3GlcAβ1-]. Post-translational modification of the O -mannosyl glycan of α-dystroglycan (α-DG) with the polysaccharide is essential for it to act as a receptor for ligands in the extracellular matrix (ECM), and both LARGE paralogs contribute to the modification in vivo. LARGE1 and LARGE2 have different tissue distribution profiles and enzymatic properties; however, the functional difference of the homologs remains to be determined, and α-DG is the only known substrate for the modification by LARGE1 or LARGE2. Here we show that LARGE2 can modify proteoglycans (PGs) with the laminin-binding glycan. We found that overexpression of LARGE2, but not LARGE1, mediates the functional modification on the surface of DG –/– , Pomt1 –/– and Fktn –/– embryonic stem cells. We identified a heparan sulfate-PG glypican-4 as a substrate for the LARGE2-dependent modification by affinity purification and subsequent mass spectrometric analysis. Furthermore, we showed that LARGE2 could modify several additional PGs with the laminin-binding glycan, most likely within the glycosaminoglycan (GAG)-protein linkage region. Our results indicate that LARGE2 can modify PGs with the GAG-like polysaccharide composed of xylose and glucuronic acid to confer laminin binding. Thus, LARGE2 may play a differential role in stabilizing the basement membrane and modifying its functions by augmenting the interactions between laminin globular domain-containing ECM proteins and PGs.

Description: : Visualizing and summarizing data from genomic studies continues to be a challenge. Here, we introduce the GenVisR package to addresses this challenge by providing highly customizable, publication-quality graphics focused on cohort level genome analyses. GenVisR provides a rapid and easy-to-use suite of genomic visualization tools, while maintaining a high degree of flexibility by leveraging the abilities of ggplot2 and Bioconductor. Availability and Implementation: GenVisR is an R package available via Bioconductor ( https://bioconductor.org/packages/GenVisR ) under GPLv3. Support is available via GitHub ( https://github.com/griffithlab/GenVisR/issues ) and the Bioconductor support website. Contacts: obigriffith@wustl.edu or mgriffit@wustl.edu Supplementary information: Supplementary data are available at Bioinformatics online.