Promoter Chromatin Structure Research Articles

Antisense non-coding transcription represses the PHO5 model gene at the level of promoter chromatin structure.

Pervasive transcription of eukaryotic genomes generates non-coding transcripts with regulatory potential. We examined the effects of non-coding antisense transcription on the regulation of expression of the yeast PHO5 gene, a paradigmatic case for gene regulation through promoter chromatin remodeling. A negative role for antisense transcription at the PHO5 gene locus was demonstrated by leveraging the level of overlapping antisense transcription through specific mutant backgrounds, expression from a strong promoter in cis, and use of the CRISPRi system. Furthermore, we showed that enhanced elongation of PHO5 antisense leads to a more repressive chromatin conformation at the PHO5 gene promoter, which is more slowly remodeled upon gene induction. The negative effect of antisense transcription on PHO5 gene transcription is mitigated upon inactivation of the histone deacetylase Rpd3, showing that PHO5 antisense RNA acts via histone deacetylation. This regulatory pathway leads to Rpd3-dependent decreased recruitment of the RSC chromatin remodeling complex to the PHO5 gene promoter upon induction of antisense transcription. Overall, the data in this work reveal an additional level in the complex regulatory mechanism of PHO5 gene expression by showing antisense transcription-mediated repression at the level of promoter chromatin structure remodeling.

SIRT6 regulated nucleosomal occupancy affects Hexokinase 2 expression

To understand the molecular association between inflammation and dysregulated metabolism in glioblastoma, the effect of IL-1β on Hexokinase 2 (HK2) expression was investigated. IL-1β induced HK2 expression was accompanied by heightened SIRT6 and MZF1 levels. IL-1β mediated overall decrease in chromatin compactness on HK2 promoter involved diminished nucleosomal occupancy around the most labile region bearing MZF1 sites. Importantly, SIRT6 and MZF1 served as negative regulators of HK2. Ectopic SIRT6 induced formation and recruitment of MZF1-SIRT6 complex to MZF1 site was concomitant with increased nucleosomal occupancy. The function of SIRT6 as co-repressor of MZF1 was inconspicuous in cells treated with IL-1β alone, as IL-1β-induced HIF-1α prevented SIRT6 availability for interaction with MZF1. Taken together, SIRT6 over-expression establishes a condition whereby reconfiguration of the HK2 promoter chromatin structure makes it receptive to interaction with MZF1/SIRT6 complex, thereby favouring a regulatory state conducive to diminished transcription.

From Structural Variation of Gene Molecules to Chromatin Dynamics and Transcriptional Bursting

Transcriptional activation of eukaryotic genes is accompanied, in general, by a change in the sensitivity of promoter chromatin to endonucleases. The structural basis of this alteration has remained elusive for decades; but the change has been viewed as a transformation of one structure into another, from “closed” to “open” chromatin. In contradistinction to this static and deterministic view of the problem, a dynamical and probabilistic theory of promoter chromatin has emerged as its solution. This theory, which we review here, explains observed variation in promoter chromatin structure at the level of single gene molecules and provides a molecular basis for random bursting in transcription—the conjecture that promoters stochastically transition between transcriptionally conducive and inconducive states. The mechanism of transcriptional regulation may be understood only in probabilistic terms.

PARP1 enhances inflammatory cytokine expression by alteration of promoter chromatin structure in microglia.

BackgroundPoly(ADP-ribose) polymerase 1 (PARP1) is a chromatin-associated enzyme that participates in processes such as transcription and DNA repair through the regulation of chromatin structure. Accumulating evidence suggests an important role for PARP1 enzymatic activity in promoting CNS inflammation by facilitating the expression of inflammatory cytokines in glial cells. However, the molecular mechanisms by which PARP1 enzymatic activity mediates this process are not well understood. In this report we sought to determine the molecular mechanisms by which PARP1 enzymatic activity facilitates the expression of Il1β and TNF in LPS-stimulated BV2 cells.MethodsPARP1 enzymatic activity and histone ADP-ribosylation were measured in LPS-stimulated BV2 cells by radioactive labelling with 32P-NAD+. To assess the effect of histone ADP-ribosylation on nucleosome structure, in vitro nucleosome remodeling, nuclease accessibility and binding assays were performed. These studies were complemented by chromatin immunoprecipitation assays in resting and LPS-stimulated BV2 cells in order to determine the occupancy of PARP1, nucleosomes and the RelA subunit of NF-κB, as well as ADP-ribosylation, at the Il1β and Tnf promoters. Finally, we determined the effect of pharmacological inhibition of PARP1 enzymatic activity on the LPS stimulation-dependent induction of Il1β and Tnf mRNA.ResultsOur results indicate that LPS stimulation induces PARP1 enzymatic activity and histone ADP-ribosylation in the chromatin compartment of BV2 cells. In vitro studies show that nucleosome-bound PARP1 disrupts nucleosome structure histone ADP-ribosylation, increasing the accessibility of nucleosomal DNA. Consistent with this PARP1 is constitutively associated with at the Il1β and Tnf promoters in resting BV2 cells. Upon stimulation with LPS, ADP-ribosylation is observed at these promoters, and this is correlated with increased recruitment of the transcription factor NF-κB, resulting in robust transcription of these inflammatory cytokines. Accordingly, pharmacological inhibition of PARP1 enzymatic activity reduces NF-κB recruitment, and Il1β and Tnf expression in LPS-stimulated microglia.ConclusionsCollectively, our data suggest that PARP1 facilitates inflammatory cytokine expression in microglia by increasing the accessibility of promoter DNA via histone ADP-riboyslation.

Stochastic Fluctuations Link Promoter Chromatin Structure and Gene Expression

Gene expression noise is characterized by random fluctuations in the number of mRNA and protein molecules produced from single genes throughout a cell population. The underlying molecular basis for this random on/off switching of genes was unknown, but hypothesized to arise from stochastic fluctuations in promoter chromatin structure. To address this question, we isolated single PHO5 gene molecules from yeast and mapped their steady-state promoter nucleosome configurations by electron microscopy. We found that the activated PHO5 promoter adopted eight distinct nucleosome configurations including the fully nucleosomal and nucleosome-free states. The probability of observing each of these eight configurations was explained by a stochastic process of nucleosome assembly, disassembly, and sliding and was tuned by the strength of Pho4, the transcriptional activator for PHO5. Downstream PHO5 expression was strongly influenced by promoter nucleosome fluctuations, mechanistically linking gene expression noise to promoter nucleosome dynamics. Further analysis has focused on chromatin remodelers and the role they play in generating promoter nucleosome probability distributions. Our findings suggest that stochastic promoter chromatin remodeling is the basis for transcriptional bursting and that activators, and the remodeling proteins they recruit, play a critical role in generating distinct promoter nucleosome probability distributions.

Major Histocompatibility Complex Class I Core Promoter Elements Are Not Essential for Transcription in vivo

The role of core promoter elements in regulating transcription initiation is largely unknown for genes subject to complex regulation. Major histocompatibility complex class I genes are ubiquitously expressed and governed by tissue-specific and hormonal signals. Transcription initiates at multiple sites within the core promoter, which contains elements homologous to the canonical elements CCAAT, TATAA, Sp1 binding site (Sp1BS), and Initiator (Inr). To determine their functions, expression of class I transgenes with individually mutated elements was assessed. Surprisingly, all mutant promoters supported transcription. However, each mutated core promoter element had a distinct effect on expression: CAAT box mutations modulated constitutive expression in nonlymphoid tissues, whereas TATAA-like element mutations dysregulated transcription in lymphoid tissues. Inr mutations aberrantly elevated expression. Sp1BS element mutations resulted in variegated transgene expression. RNA polymerase II binding and histone H3K4me3 patterns correlated with transgene expression; H3K9me3 marks partially correlated. Whereas the wild-type, TATAA-like, and CAAT mutant promoters were activated by gamma interferon, the Sp1 and Inr mutants were repressed, implicating these elements in regulation of hormonal responses. These results lead to the surprising conclusion that no single element is required for promoter activity. Rather, each plays a distinct role in promoter activity, chromatin structure, tissue-specific expression, and extracellular signaling.

Schistosoma mansoni Mucin Gene (SmPoMuc) Expression: Epigenetic Control to Shape Adaptation to a New Host

The digenetic trematode Schistosoma mansoni is a human parasite that uses the mollusc Biomphalaria glabrata as intermediate host. Specific S. mansoni strains can infect efficiently only certain B. glabrata strains (compatible strain) while others are incompatible. Strain-specific differences in transcription of a conserved family of polymorphic mucins (SmPoMucs) in S. mansoni are the principle determinants for this compatibility. In the present study, we investigated the bases of the control of SmPoMuc expression that evolved to evade B. glabrata diversified antigen recognition molecules. We compared the DNA sequences and chromatin structure of SmPoMuc promoters of two S. mansoni strains that are either compatible (C) or incompatible (IC) with a reference snail host. We reveal that although sequence differences are observed between active promoter regions of SmPoMuc genes, the sequences of the promoters are not diverse and are conserved between IC and C strains, suggesting that genetics alone cannot explain the evolution of compatibility polymorphism. In contrast, promoters carry epigenetic marks that are significantly different between the C and IC strains. Moreover, we show that modifications of the structure of the chromatin of the parasite modify transcription of SmPoMuc in the IC strain compared to the C strain and correlate with the presence of additional combinations of SmPoMuc transcripts only observed in the IC phenotype. Our results indicate that transcription polymorphism of a gene family that is responsible for an important adaptive trait of the parasite is epigenetically encoded. These strain-specific epigenetic marks are heritable, but can change while the underlying genetic information remains stable. This suggests that epigenetic changes may be important for the early steps in the adaptation of pathogens to new hosts, and might be an initial step in adaptive evolution in general.

Genome-wide analysis of chromatin architecture in Oxytricha trifallax: a single-celled eukaryote with 16,000 tiny chromosomes

The pond-dwelling ciliate Oxytricha trifallax is a model system for the study of gene regulatory mechanisms. It possesses a heterochromatin-rich germline micronucleus, and a transcriptionally active somatic macronucleus (MAC). The MAC genome is highly fragmented, consisting of >16,000 unique “nanochromosomes”, with a mean size of 3.2kb. Consequently, only a limited number of nucleosomes can be accommodated in short nanochromosomes. Nanochromosome size does not vary in multiples of ~147bp corresponding to mono-nucleosome-sized DNA, suggesting that nucleosome depleted regions may be especially prevalent. This unusual genome architecture presents novel challenges for the regulation of gene expression. The efficacy and prevalence of chromatin-mediated gene regulation thus remain unclear in Oxytricha. In addition, the paucity of non-coding DNA suggests that promoters in Oxytricha are highly compact, and may be organized differently from other eukaryotes. Our study aims to uncover how the chromatin structure of promoters and coding regions is constrained by chromosome length in the vegetative Oxytricha MAC. To address this, we characterize the chromatin landscape of the MAC through genome-wide mapping of nucleosome positions and DNase I hypersensitive sites. By integrating these studies with existing RNA Polymerase II ChIP-seq and mRNA-seq data, we investigate how chromatin organization varies with nanochromosome length, gene number, and transcriptional output. These data will also be coupled with transcription start site annotations to define promoter architecture in the Oxytricha MAC, including the presence and position of regulatory motifs, the distribution of transcription start sites within a promoter, and local chromatin accessibility. Together, this integrated analysis will test the contribution of chromatin structure to gene expression regulation, and illuminate the variation of eukaryotic gene regulatory mechanisms.

Fluctuations in Chromatin Structure and Gene Expression

Stochastic models of gene expression assume that promoters randomly transition between transcriptionally active and inactive states, a concept that has proved helpful in understanding expression noise and gene regulation. However, the molecular basis of stochastic promoter switching is unknown. By isolation and electron microscopic analysis of single PHO5 gene molecules from yeast, we show that the PHO5 promoter randomly transitions between alternative nucleosome configurations. Mathematical analysis demonstrates that the probability distribution of alternative promoter nucleosome configurations is obtained by a simple stationary Markov process of nucleosome assembly, disassembly and sliding. Our results provide direct evidence for the stochastic transitioning of a eukaryotic promoter between transcriptionally conducive and inconducive nucleosome configurations, supporting a fundamental proposition of stochastic gene expression models. Combination with fluctuation analysis of single cell gene expression provides novel insights into the role of promoter chromatin structure and dynamics for gene expression and regulation.

The Human Imprintome: Regulatory Mechanisms, Methods of Ascertainment, and Roles in Disease Susceptibility

Imprinted genes form a special subset of the genome, exhibiting monoallelic expression in a parent-of-origin-dependent fashion. This monoallelic expression is controlled by parental-specific epigenetic marks, which are established in gametogenesis and early embryonic development and are persistent in all somatic cells throughout life. We define this specific set of cis-acting epigenetic regulatory elements as the imprintome, a distinct and specially tasked subset of the epigenome. Imprintome elements contain DNA methylation and histone modifications that regulate monoallelic expression by affecting promoter accessibility, chromatin structure, and chromatin configuration. Understanding their regulation is critical because a significant proportion of human imprinted genes are implicated in complex diseases. Significant species variation in the repertoire of imprinted genes and their epigenetic regulation, however, will not allow model organisms solely to be used for this crucial purpose. Ultimately, only the human will suffice to accurately define the human imprintome.

Most Primitive Murine Bone Marrow Hematopoietic Stem Cells Express Several Primordial Germline Cells (PGCs) Markers, Including SALL4 - a Proposed Developmental Link Between Hematopoietic and Primordial Germ Cell Lineages

Abstract 4745 Background.Sal-like protein-4 (SALL4), a member of the SALL gene family, is one of the most important transcriptional regulators for primitive stem cells. The SALL4/Oct4/Nanog core transcriptional network governs the self-renewal and pluripotent properties of human and murine ESCs. Sall4 has been demonstrated to be expressed in normal hematopoietic cells (HSCs) and leukemic cells. What is intriguing, in addition to the hematopoietic lineage, Sall4 is expressed in the germline lineage, which includes primordial germ cells (PGCs). Evidence accumulates that hematopoietic stem cells (HSCs) could become specified from a population of migrating PGCs during embryogenesis. In support of this intriguing possibility, HSCs and PGCs are highly migratory populations of stem cells and specification of the first primitive HSCs in yolk sac blood islands as well as the origin of definitive HSCs in the aorta-gonado-mesonephros (AGM) region are chronologically and anatomically correlated with the developmental migration of PGCs. Furthermore, sharing of chromosomal aberrations between germline tumors and leukemias or lymphomas has also been described, which suggests their clonal origin. Our recent work demonstrated the presence of small Oct-4+Nanog+Sca-1+Lin–CD45– stem cells in adult murine BM and small Oct-4+Nanog+CD133+Lin–CD45– stem cells in human umbilical cord blood that can be specified into the hematopoietic lineage (Leukemia 2012;25,1278, Exp. Hematology 2011;39:225). These cells were named very small embryonic-like stem cells (VSELs). Our initial observations suggested that murine VSELs express several markers shared with PGCs (Leukemia 2010;24:1450). Hypothesis.The aim of our study was to test the hypothesis that VSELs are related to PGCs, which would support an alternative explanation of the origin of HSPCs and a potential developmental link between hematopoiesis and the germ line. Experimental strategies.Murine Sca-1+Lin–CD45– VSELs and Sca-1+Lin–CD45+ HSCs were purified by double FACS sorting from murine BM. We employed RQ-PCR analysis, immunohistochemical staining, and promoter methylation analysis to evaluate the expression of genes characteristic of PGC specification and expressed in early PGCs, migrating PGCs, and post-migrating PGCs. In parallel, we also analyzed Sall4 expression. Expression of these genes in BM-purified VSELs and HSCs was studied under steady-state conditions as well as in a model of hematological stress due to prolonged bleeding. Results.We found that murine BM-VSELs highly express Sall4 (700-fold more) compared with BM mononuclear cells. Sall4 expression in VSELs has been subsequently confirmed at the protein level. Sall4 mRNA expression was also detected in murine BM-derived HSCs, but at a much lower level. Of note, the expression of Sall4, like Oct-4 and Nanog, was downregulated in VSELs during their hematopoietic specification in the prolonged-bleeding model. Furthermore, we found that VSELs isolated under steady-state conditions from BM highly express, at the mRNA and protein levels, genes involved in specification of the epiblast (e.g., Stella, Prdm14, Fragilis, Blimp1, Nanos3, and Dnd1). The expression of Stella in VSELs has been evaluated by analysis of histone modifications of this locus by ChIP assay and revealed that the Stella promoter chromatin structure is characteristic of an actively transcribed gene. Next, we focused on the expression of genes specific to various PGC developmental stages and, in addition to genes involved in PGC specification, VSELs highly express Dppa2, Dppa4, and Mvh, which characterize late migratory PGCs. By contrast, however, they do not express Sycp3, Dazl, and LINE1 genes, which are highly expressed in post-migratory PGCs. Conclusions.Our results suggest that VSELs deposited into murine BM express Sall4 and several genes characteristic for epiblast-derived migratory PGCs. In toto, our data support the alternative concept that HSCs could be specified during development from migrating PGCs, and VSELs, as the most primitive population of HSCs in BM, express several germline unique markers, including Sall4. Disclosures:Ratajczak:Neostem Inc: Member of SAB Other.

Chromatin Modulation at the FLO11 Promoter of Saccharomyces cerevisiae by HDAC and Swi/Snf Complexes

Cell adhesion and biofilm formation are critical processes in the pathogenicity of fungi and are mediated through a family of adhesin proteins conserved throughout yeasts and fungi. In Saccharomyces cerevisiae, Flo11 is the main adhesin involved in cell adhesion and biofilm formation, making the study of its function and regulation in this nonpathogenic budding yeast highly relevant. The S. cerevisiae FLO11 gene is driven by a TATA-box-containing promoter that is regulated through one of the longest regulatory upstream regions (3 kb) in yeast. We reported recently that two chromatin cofactor complexes, the Rpd3L deacetylase and the Swi/Snf chromatin-remodeling complexes, contribute significantly to the regulation of FLO11. Here, we analyze directly how these complexes impact on FLO11 promoter chromatin structure and dissect further the interplay between histone deacetylases, chromatin remodeling, and the transcriptional repressor Sfl1. We show that the regulation of chromatin structure represents an important layer of control in the highly complex regulation of the FLO11 promoter.

The Number of Very Small Embryonic Like Stem Cells (VSELs) Decreases During Aging In An IGF-1-Dependent Manner - a Novel Link Between Aging, Caloric Restriction, and the Size of the Stem Cell Pool

AbstractAbstract 4796We have previously demonstrated the presence of very small (smaller than erythrocytes) Oct-4+SSEA-1+Sca-1+Lin-CD45- VSELs in bone marrow (BM) and in several murine adult organs (Leukemia 2006;20:857 and Cytometry 2009;73:1116). These small cells i) have large nuclei that contain primitive open chromatin, ii) express Oct-4 gene (as confirmed by our recent promoter methylation and chromatin structure analysis studies), and iii) posses bivalent domain-marked promoter regions of homeodomain-containing developmental master transcription factors, such as Dlx-, Irx-, Lhx-, Pou-, Pax-, and Six-family proteins. Furthermore, the epigenetic changes in selected somatic-imprinted genes (e.g., Igf2-H19 and RasGrf1) involved in insulin-factor signaling (Igf-1, Igf-2, and Insulin) govern their quiescent state, thus preventing them from unleashing proliferation and spontaneous growth of teratomas (Leukemia 2009;23:2042). O other hand it is well known that i) Igf-1 signaling negatively regulates lifespan in worms, flies, and mammals (Cell 2005;120:449) and that ii) Igf-1 and insulin levels in blood are positively regulated by caloric uptake. Indeed, we found that the pool size and pluripotentiality of VSELs decreases during aging. Accordingly, in our studies performed on young (4-week-old) and old (2-year-old) mice we found that i) the number of VSELs and their pluripotentiality decreases with age, ii) VSELs from old mice show lower expression of Oct-4, Nanog, Sox2, Klf4, and cMyc, iii) the Oct-4 promoter becomes hypermethylated with age and has a closed chromatin structure, iv) VSELs from old mice show somatic methylation in both Igf2-H19 and Rasgrf1 loci, and v), as a result, VSELs from these mice have increased sensitivity to insulin/insulin factors signaling. This suggests that chronic insulin receptor/Igf-1 receptor signaling in VSELs may contribute to age-related depletion of these cells. To explain better the role of insulin signaling in VSELs, we measured by FACS the number of VSELs in murine Laron dwarfs, which exhibit chronic Igf-1 deficiency and, as a result, live 30–40% longer than their normal littermates. We report here, for the first time, that the number of VSELs in the BM of Igf-1–deficient Laron dwarfs is 3–4 fold higher and is maintained at a higher level during aging compared to normal wild type (wt) littermates. Molecular analysis studies will confirm whether or not the molecular signature of VSELs in aging Laron dwarfs is somehow protected from age-related changes (e.g., by the methylation status of the Oct-4 promoter and/or genomic imprinted genes). Based on our data, we postulate novel linkages between Igf-1 level, aging, and the stem cell compartment. According to our hypothesis, early in development a population of VSELs would be deposited in developing organs as a backup for tissue-committed stem cells and play a role in rejuvenation of tissues and organ regeneration after damage. These cells would be protected from uncontrolled proliferation and age-related depletion by changes in imprinted genes that regulate insulin signaling. We further hypothesize that in the adult body the pool of VSEL cells is regulated by the circulating Igf-1 level. An increase in Igf-1 level (e.g., resulting from a chronically high calorie diet) would accelerate an age-dependent decrease in VSELs and their potential to rejuvenate tissues. By contrast, a low Igf-1 level (e.g., as seen in Laron dwarf mutants or due to caloric restriction) would have a protective effect on the overall pool of these cells. Thus, we present for the first time a hypothesis that reconciles aging, longevity, Igf-1 signaling, and caloric uptake and negative effect of Igf-1 and high calorie uptake on number/function of pluripotent VSELs deposited in adult tissues. Disclosures:No relevant conflicts of interest to declare.

Revisiting the Contribution of cis-Elements to Expression Divergence between Duplicated Genes: The Role of Chromatin Structure

Although divergence in expression is thought to be a hallmark of functional dispersal between paralogs postduplication, there is currently a limited understanding of the mechanisms underlying the necessary transcriptional alterations as recent studies have suggested that only a very small proportion of expression variation could be explained by transcriptional variation between paralogs. To further this understanding, we examined comprehensively curated regulatory interactions and genomewide nucleosome occupancy in budding yeast to specifically determine the contribution of cis-elements to expression divergence between extant duplicates. We found that divergence in activation by transcription factors plays a more important role in expression divergence of paralogs than previously appreciated; further, analysis of promoter chromatin structure demonstrated that differential nucleosome organization is coupled with divergent expression of paralogs. By incorporating information of cis-elements encoding transcriptional regulation and chromatin structure, we improved the fraction of expression variation that was previously shown to be explained based on known cis-transcriptional effects by approximately 3-fold. Taken together, our analysis highlights the importance of chromatin divergence involved in expression evolution between paralogs.

Quantitative analysis of the transcription control mechanism

Gene transcription requires a sequence of promoter state transitions, including chromatin remodeling, assembly of the transcription machinery, and clearance of the promoter by RNA polymerase. The rate-limiting steps in this sequence are regulated by transcriptional activators that bind at specific promoter elements. As the transition kinetics of individual promoters cannot be observed, the identity of the activator-controlled steps has remained a matter of speculation. In this study, we investigated promoter chromatin structure, and the intrinsic noise of expression over a wide range of expression values for the PHO5 gene of yeast. Interpretation of our results with regard to a stochastic model of promoter chromatin remodeling and gene expression suggests that the regulatory architecture of the gene expression process is measurably reflected in its intrinsic noise profile. Our chromatin structure and noise analyses indicate that the activator of PHO5 transcription stimulates the rates of promoter nucleosome disassembly, and assembly of the transcription machinery after nucleosome removal, but no other rates of the expression process.

Chromatin structure regulates human cytomegalovirus gene expression during latency, reactivation and lytic infection

Infection of cells with human cytomegalovirus (HCMV) has two potential outcomes. For instance, infection of fibroblasts results in extensive viral gene expression, viral DNA replication and release of progeny virus. In contrast, in undifferentiated myeloid cells, the lytic transcription programme of HCMV is effectively suppressed and cells undergo latent infection. It is now accepted that the suppression of viral lytic gene expression observed during latency in myeloid cells is a result of the inability of undifferentiated cell types to support robust viral immediate early (IE) gene expression — crucial genes responsible for driving the lytic cycle. The repression of IE gene expression in undifferentiated myeloid cells, at least in part, results from specific post-translational modifications of histones associated with the viral major immediate early promoter (MIEP). In cells of the early myeloid lineage, the histone modifications present on the MIEP impart on it a repressive chromatin structure preventing transcriptional activity. Reactivation of HCMV lytic infection is correlated to changes in histone modifications around the MIEP resulting in a chromatin structure conducive to transcriptional activity. These changes are intimately linked with the differentiation of myeloid cells — a phenomenon known to reactivate latent virus in vivo. Chromatin structure of the viral MIEP, therefore, plays a crucial role in latency and reactivation of this persistent human herpesvirus. Whether chromatin-mediated regulation of viral lytic gene expression also occurs, is only beginning to be addressed. However, recent work suggests that all classes of lytic HCMV promoters are subjected to regulation by post-translational modification of their associated histones throughout the time course of infection. Incoming viral genomes appear to be the targets of intrinsic cellular defence mechanisms which attempt to silence viral gene expression through chromatinisation. Viral functions eventually overcome these cellular repression mechanisms permitting high levels of IE gene expression which results in modification of the chromatin structure of early and late gene promoters driving a regulated cascade of viral lytic gene expression and virus production.

Lytic infection of permissive cells with human cytomegalovirus is regulated by an intrinsic ‘pre-immediate-early’ repression of viral gene expression mediated by histone post-translational modification

Human cytomegalovirus (HCMV) lytic gene expression occurs in a regulated cascade, initiated by expression of the viral major immediate-early (IE) proteins. Transcribed from the major IE promoter (MIEP), the major IE genes regulate viral early and late gene expression. This study found that a substantial proportion of infecting viral genomes became associated with histones immediately upon infection of permissive fibroblasts at low m.o.i. and these histones bore markers of repressed chromatin. As infection progressed, however, the viral MIEP became associated with histone marks, which correlate with the known transcriptional activity of the MIEP at IE time points. Interestingly, this chromatin-mediated repression of the MIEP at 'pre-IE' times of infection could be overcome by inhibition of histone deacetylases, as well as by infection at high m.o.i., and resulted in a temporal advance of the infection cycle by inducing premature viral early and late gene expression and DNA replication. As well as the MIEP, and consistent with previous observations, the viral early and late promoters were also initially associated with repressive chromatin. However, changes in histone modifications around these promoters also occurred as infection progressed, and this correlated with the known temporal regulation of the viral early and late gene expression cascade. These data argue that the chromatin structure of all classes of viral genes are initially repressed on infection of permissive cells and that the chromatin structure of HCMV gene promoters plays an important role in regulating the time course of viral gene expression during lytic infection.

Nuclear receptor coactivators: Regulation of turnover and function

Steroid receptor coactivators, such as the SRC‐family of molecules, amplify nuclear receptor induction of gene expression by modifying promoter chromatin structure and implementing all of the downstream subreactions required for efficient transcription. Induced post‐translational modifications such as phosphorylations 'acivate' the SRC‐3 coactivator by allowing formation of active coactivator multimeric complexes with the receptors. Two regulable sets of co‐coactivator molecules control the degradation of SRC‐3 coactivator. E3 ubiquitin ligases control the lifetime of 'transcriptionally activated SRC‐3' by virtue of a 'ubiquitin transcriptional clock', which begins by a monoubiquitin‐dependent activation and ends via a polyubiquitylatin‐dependent degradation. In contrast, the REGγ proteasomal Cap controls the cellular levels of 'inactive SRC‐3 coactivator' via an ubiquitin‐independent and ATP‐independent mechanism. Turnover of coactivators is additionally regulated by specific kinases, such as atypical PKC, which modify the C‐terminal region of SRC‐3 and prevent the entrance of the coactivator into the 20S proteasome core. Since many coactivators are oncogenes (or tumor suppressors), the mechanisms for controlling their levels are crucial for the regulation of all hormone‐mediated physiological and pathological consequences.

062. DEVELOPMENTAL ORIGINS OF TYPE 2 DIABETES: EPIGENETIC MECHANISMS IN BETA CELL FAILURE

The abnormal intrauterine milieu of intrauterine growth retardation (IUGR) permanently alters gene expression and function of pancreatic b -cells leading to the development of diabetes in adulthood. Expression of the pancreatic homeobox transcription factor Pdx1 is permanently reduced in IUGR and epigenetic modifications are responsible for this decrease. Exendin-4 (Ex-4), a long-acting glucagon-like peptide 1 (GLP-1) analog, given on days 1-6 of life increases Pdx1 expression and prevents the development of diabetes in the IUGR rat. Here we show that Ex-4 increases USF-1 and PCAF association at the proximal promoter of Pdx1, thereby increasing histone acetyl transferase (HAT) activity leading to a permanent increase in histone H3 acetylation and H3K4 methylation. Normalization of these histone modifications precludes DNA methylation thereby preventing silencing of Pdx1 in islets of IUGR animals. These studies demonstrate a novel mechanism whereby a short treatment course of Ex-4 in the newborn period prevents diabetes in adulthood by restoring Pdx1 promoter chromatin structure thus preserving Pdx1 transcription.

A SAP30 Complex Inhibits IFN-β Expression in Rift Valley Fever Virus Infected Cells

Rift Valley fever virus (RVFV) nonstructural protein NSs acts as the major determinant of virulence by antagonizing interferon β (IFN-β) gene expression. We demonstrate here that NSs interacts with the host protein SAP30, which belongs to Sin3A/NCoR/HDACs repressor complexes and interacts with the transcription factor YY1 that regulates IFN-β gene expression. Using confocal microscopy and chromatin immunoprecipitation, we show that SAP30, YY1, and Sin3A-associated corepressor factors strongly colocalize with nuclear NSs filaments and that NSs, SAP30 and Sin3A-associated factors are recruited on the IFN-β promoter through YY1, inhibiting CBP recruitment, histone acetylation, and transcriptional activation. To ascertain the role of SAP30, we produced, by reverse genetics, a recombinant RVFV in which the interacting domain in NSs was deleted. The virus was unable to inhibit the IFN response and was avirulent for mice. We discuss here the strategy developed by the highly pathogenic RVFV to evade the host antiviral response, affecting nuclear organization and IFN-β promoter chromatin structure.