Alexey A. Soshnev

Every amino acid matters: essential contributions of histone variants to mammalian development and disease

Despite a conserved role for histones as general DNA packaging agents, it is now clear that another key function of these proteins is to confer variations in chromatin structure to ensure dynamic patterns of transcriptional regulation in eukaryotes. The incorporation of histone variants is particularly important to this process. Recent knockdown and knockout studies in various cellular systems, as well as direct mutational evidence from human cancers, now suggest a crucial role for histone variant regulation in processes as diverse as differentiation and proliferation, meiosis and nuclear reprogramming. In this Review, we provide an overview of histone variants in the context of their unique functions during mammalian germ cell and embryonic development, and examine the consequences of aberrant histone variant regulation in human disease.

Networking in the nucleus: a spotlight on LEM-domain proteins

Proteins resident in the inner nuclear membrane and underlying nuclear lamina form a network that regulates nuclear functions. This review highlights a prominent family of nuclear lamina proteins that carries the LAP2-emerin-MAN1-domain (LEM-D). LEM-D proteins share an ability to bind lamins and tether repressive chromatin at the nuclear periphery. The importance of this family is underscored by findings that loss of individual LEM-D proteins causes progressive, tissue-restricted diseases, known as laminopathies. Diverse functions of LEM-D proteins are linked to interactions with unique and overlapping partners including signal transduction effectors, transcription factors and architectural proteins. Recent investigations suggest that LEM-D proteins form hubs within the nuclear lamina that integrate external signals important for tissue homeostasis and maintenance of progenitor cell populations.

A Conserved Long Noncoding RNA Affects Sleep Behavior in Drosophila

Metazoan genomes encode an abundant collection of mRNA-like, long noncoding (lnc)RNAs. Although lncRNAs greatly expand the transcriptional repertoire, we have a limited understanding of how these RNAs contribute to developmental regulation. Here, we investigate the function of the Drosophila lncRNA called yellow-achaete intergenic RNA (yar). Comparative sequence analyses show that the yar gene is conserved in Drosophila species representing 40–60 million years of evolution, with one of the conserved sequence motifs encompassing the yar promoter. Further, the timing of yar expression in Drosophila virilis parallels that in D. melanogaster, suggesting that transcriptional regulation of yar is conserved. The function of yar was defined by generating null alleles. Flies lacking yar RNAs are viable and show no overt morphological defects, consistent with maintained transcriptional regulation of the adjacent yellow (y) and achaete (ac) genes. The location of yar within a neural gene cluster led to the investigation of effects of yar in behavioral assays. These studies demonstrated that loss of yar alters sleep regulation in the context of a normal circadian rhythm. Nighttime sleep was reduced and fragmented, with yar mutants displaying diminished sleep rebound following sleep deprivation. Importantly, these defects were rescued by a yar transgene. These data provide the first example of a lncRNA gene involved in Drosophila sleep regulation. We find that yar is a cytoplasmic lncRNA, suggesting that yar may regulate sleep by affecting stabilization or translational regulation of mRNAs. Such functions of lncRNAs may extend to vertebrates, as lncRNAs are abundant in neural tissues.

Greater Than the Sum of Parts: Complexity of the Dynamic Epigenome

Information encoded in DNA is interpreted, modified, and propagated as chromatin. The diversity of inputs encountered by eukaryotic genomes demands a matching capacity for transcriptional outcomes provided by the combinatorial and dynamic nature of epigenetic processes. Advances in genome editing, visualization technology, and genome-wide analyses have revealed unprecedented complexity of chromatin pathways, offering explanations to long-standing questions and presenting new challenges. Here, we review recent findings, exemplified by the emerging understanding of crossregulatory interactions within chromatin, and emphasize the pathologic outcomes of epigenetic misregulation in cancer.

Genome-wide studies of the multi-zinc finger Drosophila Suppressor of Hairy-wing protein in the ovary

The Drosophila Suppressor of Hairy-wing [Su(Hw)] protein is a globally expressed, multi-zinc finger (ZnF) DNA-binding protein. Su(Hw) forms a classic insulator when bound to the gypsy retrotransposon and is essential for female germline development. These functions are genetically separable, as exemplified by Su(Hw)f that carries a defective ZnF10, causing a loss of insulator but not germline function. Here, we completed the first genome-wide analysis of Su(Hw)-binding sites (SBSs) in the ovary, showing that tissue-specific binding is not responsible for the restricted developmental requirements for Su(Hw). Mapping of ovary Su(Hw)f SBSs revealed that female fertility requires binding to only one third of the wild-type sites. We demonstrate that Su(Hw)f retention correlates with binding site affinity and partnership with Modifier of (mdg4) 67.2 protein. Finally, we identify clusters of co-regulated ovary genes flanked by Su(Hw)f bound sites and show that loss of Su(Hw) has limited effects on transcription of these genes. These data imply that the fertility function of Su(Hw) may not depend upon the demarcation of transcriptional domains. Our studies establish a framework for understanding the germline Su(Hw) function and provide insights into how chromatin occupancy is achieved by multi-ZnF proteins, the most common transcription factor class in metazoans.

Context Differences Reveal Insulator and Activator Functions of a Su(Hw) Binding Region

Insulators are DNA elements that divide chromosomes into independent transcriptional domains. The Drosophila genome contains hundreds of binding sites for the Suppressor of Hairy-wing [Su(Hw)] insulator protein, corresponding to locations of the retroviral gypsy insulator and non-gypsy binding regions (BRs). The first non-gypsy BR identified, 1A-2, resides in cytological region 1A. Using a quantitative transgene system, we show that 1A-2 is a composite insulator containing enhancer blocking and facilitator elements. We discovered that 1A-2 separates the yellow (y) gene from a previously unannotated, non-coding RNA gene, named yar for y-achaete (ac) intergenic RNA. The role of 1A-2 was elucidated using homologous recombination to excise these sequences from the natural location, representing the first deletion of any Su(Hw) BR in the genome. Loss of 1A-2 reduced yar RNA accumulation, without affecting mRNA levels from the neighboring y and ac genes. These data indicate that within the 1A region, 1A-2 acts an activator of yar transcription. Taken together, these studies reveal that the properties of 1A-2 are context-dependent, as this element has both insulator and enhancer activities. These findings imply that the function of non-gypsy Su(Hw) BRs depends on the genomic environment, predicting that Su(Hw) BRs represent a diverse collection of genomic regulatory elements.

The role of the Suppressor of Hairy-wing insulator protein in Drosophila oogenesis

The Drosophila Suppressor of Hairy wing [Su(Hw)] insulator protein has an essential role in the development of the female germline. Here we investigate the function of Su(Hw) in the ovary. We show that Su(Hw) is universally expressed in somatic cells, while germ cell expression is dynamic. Robust levels accumulate in post-mitotic germ cells, where Su(Hw) localization is limited to chromosomes within nurse cells, the specialized cells that support oocyte growth. Although loss of Su(Hw) causes global defects in nurse cell chromosome structure, we demonstrate that these architectural changes are not responsible for the block in oogenesis. Connections between the fertility and insulator functions of Su(Hw) were investigated through studies of the two gypsy insulator proteins, Modifier of (mdg4)67.2 (Mod67.2) and Centrosomal Protein of 190kDa (CP190). Accumulation of these proteins is distinct from Su(Hw), with Mod67.2 and CP190 showing uniform expression in all cells during early stages of oogenesis that diminishes in later stages. Although Mod67.2 and CP190 extensively co-localize with Su(Hw) on nurse cell chromosomes, neither protein is required for nurse cell chromosome development or oocyte production. These data indicate that while the gypsy insulator function requires both Mod67.2 and CP190, these proteins are not essential for oogenesis. These studies represent the first molecular investigations of Su(Hw) function in the germline, which uncover distinct requirements for Su(Hw) insulator and ovary functions.

The insulator protein Suppressor of Hairy wing is an essential transcriptional repressor in the Drosophila ovary

Suppressor of Hairy-wing [Su(Hw)] is a DNA-binding factor required for gypsy insulator function and female germline development in Drosophila. The insulator function of the gypsy retrotransposon depends on Su(Hw) binding to clustered Su(Hw) binding sites (SBSs) and recruitment of the insulator proteins Centrosomal Protein 190 kD (CP190) and Modifier of mdg4 67.2 kD (Mod67.2). By contrast, the Su(Hw) germline function involves binding to non-clustered SBSs and does not require CP190 or Mod67.2. Here, we identify Su(Hw) target genes, using genome-wide analyses in the ovary to uncover genes with an ovary-bound SBS that are misregulated upon Su(Hw) loss. Most Su(Hw) target genes demonstrate enriched expression in the wild-type CNS. Loss of Su(Hw) leads to increased expression of these CNS-enriched target genes in the ovary and other tissues, suggesting that Su(Hw) is a repressor of neural genes in non-neural tissues. Among the Su(Hw) target genes is RNA-binding protein 9 (Rbp9), a member of the ELAV/Hu gene family. Su(Hw) regulation of Rbp9 appears to be insulator independent, as Rbp9 expression is unchanged in a genetic background that compromises the functions of the CP190 and Mod67.2 insulator proteins, even though both localize to Rbp9 SBSs. Rbp9 misregulation is central to su(Hw)-/- sterility, as Rbp9+/-, su(Hw)-/- females are fertile. Eggs produced by Rbp9+/-, su(Hw)-/- females show patterning defects, revealing a somatic requirement for Su(Hw) in the ovary. Our studies demonstrate that Su(Hw) is a versatile transcriptional regulatory protein with an essential developmental function involving transcriptional repression.

Restoration of Topoisomerase 2 Function by Complementation of Defective Monomers in Drosophila

Type II topoisomerases are essential ATP-dependent homodimeric enzymes required for transcription, replication, and chromosome segregation. These proteins alter DNA topology by generating transient enzyme-linked double-strand breaks for passage of one DNA strand through another. The central role of type II topoisomerases in DNA metabolism has made these enzymes targets for anticancer drugs. Here, we describe a genetic screen that generated novel alleles of Drosophila Topoisomerase 2 (Top2). Fifteen alleles were obtained, resulting from nonsense and missense mutations. Among these, 14 demonstrated recessive lethality, with one displaying temperature-sensitive lethality. Several newly generated missense alleles carry amino acid substitutions in conserved residues within the ATPase, Topoisomerase/Primase, and Winged helix domains, including four that encode proteins with alterations in residues associated with resistance to cancer chemotherapeutics. Animals lacking zygotic Top2 function can survive to pupation and display reduced cell division and altered polytene chromosome structure. Inter se crosses between six strains carrying Top2 missense alleles generated morphologically normal trans-heterozygous adults, which showed delayed development and were female sterile. Complementation occurred between alleles encoding Top2 proteins with amino acid substitutions in the same functional domain and between alleles encoding proteins with substitutions in different functional domains. Two complementing alleles encode proteins with amino acid substitutions associated with drug resistance. These observations suggest that dimerization of mutant Top2 monomers can restore enzymatic function. Our studies establish the first series of Top2 alleles in a multicellular organism. Future analyses of these alleles will enhance our knowledge about the contributions made by type II topoisomerases to development.

Diabetes-linked transcription factor HNF4α regulates metabolism of endogenous methylarginines and β-aminoisobutyric acid by controlling expression of alanine-glyoxylate aminotransferase 2

Elevated levels of circulating asymmetric and symmetric dimethylarginines (ADMA and SDMA) predict and potentially contribute to end organ damage in cardiovascular diseases. Alanine-glyoxylate aminotransferase 2 (AGXT2) regulates systemic levels of ADMA and SDMA, and also of beta-aminoisobutyric acid (BAIB)-a modulator of lipid metabolism. We identified a putative binding site for hepatic nuclear factor 4 α (HNF4α) in AGXT2 promoter sequence. In a luciferase reporter assay we found a 75% decrease in activity of Agxt2 core promoter after disruption of the HNF4α binding site. Direct binding of HNF4α to Agxt2 promoter was confirmed by chromatin immunoprecipitation assay. siRNA-mediated knockdown of Hnf4a led to an almost 50% reduction in Agxt2 mRNA levels in Hepa 1–6 cells. Liver-specific Hnf4a knockout mice exhibited a 90% decrease in liver Agxt2 expression and activity, and elevated plasma levels of ADMA, SDMA and BAIB, compared to wild-type littermates. Thus we identified HNF4α as a major regulator of Agxt2 expression. Considering a strong association between human HNF4A polymorphisms and increased risk of type 2 diabetes our current findings suggest that downregulation of AGXT2 and subsequent impairment in metabolism of dimethylarginines and BAIB caused by HNF4α deficiency might contribute to development of cardiovascular complications in diabetic patients.