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Background Insertion of retroviral genome DNA occurs in the chromatin of the host cell. This step is modulated by chromatin structure as nucleosomes compaction was shown to prevent HIV-1 integration and chromatin remodeling has been reported to affect integration efficiency. LEDGF/p75-mediated targeting of the integration complex toward RNA polymerase II (polII) transcribed regions ensures optimal access to dynamic regions that are suitable for integration. Consequently, we have investigated the involvement of polII-associated factors in the regulation of HIV-1 integration. Results Using a pull down approach coupled with mass spectrometry, we have selected the FACT (FAcilitates Chromatin Transcription) complex as a new potential cofactor of HIV-1 integration. FACT is a histone chaperone complex associated with the polII transcription machinery and recently shown to bind LEDGF/p75. We report here that a tripartite complex can be formed between HIV-1 integrase, LEDGF/p75 and FACT in vitro and in cells. Biochemical analyzes show that FACT-dependent nucleosome disassembly promotes HIV-1 integration into chromatinized templates, and generates highly favored nucleosomal structures in vitro. This effect was found to be amplified by LEDGF/p75. Promotion of this FACT-mediated chromatin remodeling in cells both increases chromatin accessibility and stimulates HIV-1 infectivity and integration. Conclusions Altogether, our data indicate that FACT regulates HIV-1 integration by inducing local nucleosomes dissociation that modulates the functional association between the incoming intasome and the targeted nucleosome. Electronic supplementary material The online version of this article (doi:10.1186/s12977-017-0363-4) contains supplementary material, which is available to authorized users.

The access of Transcription Factors (TFs) to their cognate DNA binding motifs requires a precise control over nucleosome positioning. This is especially important following DNA replication and during mitosis, both resulting in profound changes in nucleosome organization over TF binding regions. Using mouse Embryonic Stem (ES) cells, we show that the TF CTCF displaces nucleosomes from its binding site and locally organizes large and phased nucleosomal arrays, not only in interphase steady-state but also immediately after replication and during mitosis. Correlative analyses suggest this is associated with fast gene reactivation following replication and mitosis. While regions bound by other TFs (Oct4/Sox2), display major rearrangement, the post-replication and mitotic nucleosome positioning activity of CTCF is not unique: Esrrb binding regions are also characterized by persistent nucleosome positioning. Therefore, selected TFs such as CTCF and Esrrb act as resilient TFs governing the inheritance of nucleosome positioning at regulatory regions throughout the cell-cycle., eLife digest A single cell contains several meters of DNA which must be tightly packaged to fit inside. Typically, the DNA is wound around proteins, like a thread around many spools, to form more compact structures called nucleosomes. Before a cell divides in two, however, it needs first to access and replicate its DNA so that each new cell can get a copy of the genetic material. The cell then needs to condense the DNA again so that the two copies can be easily separated via a process called mitosis. These two processes – DNA replication and mitosis – entail major rearrangements of the nucleosomes, which then need to be returned to their original positions. Nucleosomes are also repositioned when cells need to access the coded instructions written in genes. Molecules called transcription factors bind to targets within the DNA to make sure genes are active or inactive at the right times of a cell’s life, but many are evicted from the DNA during its replication and during cell division. Most transcription factors also require nucleosomes to be specifically organized to bind to the DNA, and it remains unclear how the factors re-engage with the DNA and how nucleosomes are managed during and after DNA replication and mitosis. Owens, Papadopoulou et al. set out to understand how nucleosomes are organized immediately after DNA is replicated and while cells divide. Experiments with mouse cells grown in the laboratory showed that certain transcription factors can rebind to their targets within minutes of replication finishing, remain bound to the DNA during cell division, and displace nucleosomes from their binding sites. Owens, Papadopoulou et al. refer to these factors as “resilient transcription factors” and identified two examples, named CTCF and Esrrb. Further experiments showed that, by maintaining the structure of nearby nucleosomes while a cell divides, these resilient transcription factors could quickly reactivate genes immediately after DNA replication and mitosis are complete. These findings show that transcription factors play a fundamental role in maintaining gene regulation from one generation of cells to the next. Further studies on this topic may eventually foster progress in research areas where cell division is paramount, such as regenerative medicine and cancer biology.

International audience; Retrotransposons, the ancestors of retroviruses, have the potential for gene disruption and genomic takeover if not kept in check. Paradoxically, although host cells repress these elements by multiple mechanisms, they are transcribed and are even activated under stress conditions. Here, we describe a new mechanism of retrotransposon regulation through transcription start site (TSS) selection by altered nucleosome occupancy. We show that Fun30 chromatin remodelers cooperate to maintain a high level of nucleosome occupancy at retrotransposon-flanking long terminal repeat (LTR) elements. This enforces the use of a downstream TSS and the production of a truncated RNA incapable of reverse transcription and retrotransposition. However, in stressed cells, nucleosome occupancy at LTR elements is reduced, and the TSS shifts to allow for productive transcription. We propose that controlled retrotransposon transcription from a nonproductive TSS allows for rapid stress-induced activation, while preventing uncontrolled transposon activity in the genome.

International audience; T-box transcription factors play a crucial role in development where they are implicated in patterning and cell fate decisions. Tbx2 and Tbx3 have also been implicated in several cancers including melanoma, and can act as antisenescence factors through their ability to repress p19(ARF) and p21(CIP1) expression. Although several target genes for T-box factors have been identified, it is unknown whether this family of proteins can bind chromatin, a property that would facilitate the epigenetic reprogramming that occurs in both development and cancer progression. Here, we show that Tbx2 has the potential to recognize mitotic chromatin in a DNA-dependent fashion, can interact specifically with the histone H3 N-terminal tail, a property shared with Tbx4, Tbx5 and Tbx6, and can also recognize nucleosomal DNA, with binding to nucleosomes being antagonized by the presence of the histone tails. Strikingly, in vivo Tbx2 co-localization with pericentric heterochromatin appears to be regulated and ectopic expression of Tbx2 leads to severe mitotic defects. Taken together our results suggest that Tbx2, and most likely other members of the T-box family, are able to target chromatin and may indicate a role for the T-box factors in epigenetic reprogramming events.

There is increasing evidence that in systemic lupus erythematosus, nucleosomes, the basic chromatin component, represent both a driving immunogen and a major in vivo target for antibodies. Either a disturbed apoptosis or a reduced clearance of apoptotic cells by phagocytes may lead to an increased exposure of apoptotic nucleosomes to the immune system. These nucleosomes, which have been cleaved and modified during the process of apoptosis, escape normal clearance and encompass epitopes that normally are not encountered by the immune system. This may then lead to tolerance breaking and autoimmunity by the activation of nucleosome-specific autoreactive T cells (that help B cells) and subsequently to the production of anti-nucleosome, anti-histone and anti-DNA autoantibodies. Some anti-nucleosome antibody subsets are pathogenic and are involved in the nephritogenic process in systemic lupus erythematosus. Accordingly, several studies reported: (i) increased plasma circulating nucleosomes that positively correlated with an active disease, (ii) nucleosomes in typical glomerular deposits as well as in the basement membrane of non-lesional skin of systemic lupus erythematosus patients and (iii) a close correlation between nephritis and the presence of anti-nucleosome antibodies. Recent studies reported anti-nucleosome antibodies also in primary anti-phospholipid syndrome and particularly in patients with associated lupus-like disease.

In eukaryotic cells, local chromatin structure and chromatin organization in the nucleus both influence transcriptional regulation. At the local level, the Fun30 chromatin remodeler Fft3 is essential for maintaining proper chromatin structure at centromeres and subtelomeres in fission yeast. Using genome-wide mapping and live cell imaging, we show that this role is linked to controlling nuclear organization of its targets. In fft3∆ cells, subtelomeres lose their association with the LEM domain protein Man1 at the nuclear periphery and move to the interior of the nucleus. Furthermore, genes in these domains are upregulated and active chromatin marks increase. Fft3 is also enriched at retrotransposon-derived long terminal repeat (LTR) elements and at tRNA genes. In cells lacking Fft3, these sites lose their peripheral positioning and show reduced nucleosome occupancy. We propose that Fft3 has a global role in mediating association between specific chromatin domains and the nuclear envelope., Author Summary In the genome of eukaryotic cells, domains of active and repressive chromatin alternate along the chromosome arms. Insulator elements are necessary to shield these different environments from each other. In the fission yeast Schizosaccharomyces pombe, the chromatin remodeler Fft3 is required to maintain the repressed subtelomeric chromatin. Here we show that Fft3 maintains nucleosome structure of insulator elements at the subtelomeric borders. We also observe that subtelomeres and insulator elements move away from the nuclear envelope in cells lacking Fft3. The nuclear periphery is known to harbor repressive chromatin in many eukaryotes and has been implied in insulator function. Our results suggest that chromatin remodeling through Fft3 is required to maintain proper chromatin structure and nuclear organization of insulator elements.

International audience; We have studied the functional and structural properties of nucleosomes reconstituted with H2BFWT, a recently identified putative histone variant of the H2B family with totally unknown function. We show that H2BFWT can replace the conventional histone H2B in the nucleosome. The presence of H2BFWT did not affect the overall structure of the nucleosome, and the H2BFWT nucleosomes exhibited the same stability as conventional nucleosomes. SWI/SNF was able to efficiently remodel and mobilize the H2BFWT nucleosomes. Importantly, H2BFWT, in contrast to conventional H2B, was unable to recruit chromosome condensation factors and to participate in the assembly of mitotic chromosomes. This was determined by the highly divergent (compared to conventional H2B) NH2 tail of H2BFWT. These data, in combination with the observations that H2BFWT was found by others in the sperm nuclei and appeared to be associated with the telomeric chromatin, suggest that H2BFWT could act as a specific epigenetic marker.

Transcriptional activators and coactivators overcome repression by chromatin, but regulation of chromatin disassembly and coactivator binding to promoters is poorly understood. Activation of the yeast HO gene follows the sequential binding of both sequence specific DNA-binding proteins and coactivators during the cell cycle. Here we show that the nucleosome disassembly occurs in waves both along the length of the promoter and during the cell cycle. Different chromatin modifiers are required for chromatin disassembly at different regions of the promoter, with Swi/Snf, the FACT chromatin reorganizer, and the Asf1 histone chaperone each required for nucleosome eviction at distinct promoter regions. FACT and Asf1 both bind to upstream elements of the HO promoter well before the gene is transcribed. The Swi/Snf, SAGA, and Mediator coactivators bind first to the far upstream promoter region and subsequently to a promoter proximal region, and FACT and Asf1 are both required for this coactivator re-recruitment.

International audience; The conversion of male germ cell chromatin to a nucleoprotamine structure is fundamental to the life cycle, yet the underlying molecular details remain obscure. Here we show that an essential step is the genome-wide incorporation of TH2B, a histone H2B variant of hitherto unknown function. Using mouse models in which TH2B is depleted or C-terminally modified, we show that TH2B directs the final transformation of dissociating nucleosomes into protamine-packed structures. Depletion of TH2B induces compensatory mechanisms that permit histone removal by up-regulating H2B and programming nucleosome instability through targeted histone modifications, including lysine crotonylation and arginine methylation. Furthermore, after fertilization, TH2B reassembles onto the male genome during protamine-to-histone exchange. Thus, TH2B is a unique histone variant that plays a key role in the histone-to-protamine packing of the male genome and guides genome-wide chromatin transitions that both precede and follow transmission of the male genome to the egg.

The role of the mitotic phosphorylation of the amino (NH 2 ) terminus of Centromere Protein A (CENP-A), the histone variant epigenetic centromeric marker, remains elusive. Here, we show that the NH 2 terminus of human CENP-A is essential for mitotic progression and that localization of CENP-C, another key centromeric protein, requires only phosphorylation of the CENP-A NH 2 terminus, and is independent of the CENP-A NH 2 terminus length and amino acid sequence. Mitotic CENP-A nucleosomal complexes contain CENP-C and phosphobinding 14-3-3 proteins. In contrast, mitotic nucleosomal complexes carrying nonphosphorylatable CENP-A–S7A contained only low levels of CENP-C and no detectable 14-3-3 proteins. Direct interactions between the phosphorylated form of CENP-A and 14-3-3 proteins as well as between 14-3-3 proteins and CENP-C were demonstrated. Taken together, our results reveal that 14-3-3 proteins could act as specific mitotic “bridges,” linking phosphorylated CENP-A and CENP-C, which are necessary for the platform function of CENP-A centromeric chromatin in the assembly and maintenance of active kinetochores.

International audience; DNA actively interacts with proteins involved in replication, transcription, repair, and regulation processes inside the cell. The base sequence encodes the dynamics of these transformations from the atomic to the nanometre scale length, and over higher spatial scales. In fact, although an important part of the DNA informational content acts locally, it exerts its functions as collective properties of relatively long sequences and manifests as static and dynamic curvature. Physical models that explore different aspects of DNA collective properties associated to such superstructural properties encoded in the sequence will be reviewed. The B-DNA periodicity operates as band-pass-filter; only the local physical-chemical variance associated to the sequence, in phase with the helical periodicity, sums up and reveals at higher scale. In this light, the gel electrophoresis behaviour of DNAs, the nucleosome thermodynamic stability and positioning along genomes were interpreted and discussed. Finally, a part of this review is reserved to describe the ability of some inorganic crystal surfaces to recognize and stabilize certain DNA tracts with peculiar sequences. The collective superstructural properties of DNAs could be involved in the selective interaction between DNA sequence and particular crystal surfaces. It may be conceived that sequences strongly adsorbed on surface could nucleate and expand bits of information in primeval DNA (and/or RNA) chains, early characterized by random sequences, since more protected against the physical-chemical injuries by the environment, and therefore involved in the evolution of their informational content.

Mammalian telomeres stabilize chromosome ends as a result of their assembly into a peculiar form of chromatin comprising a complex of non-histone proteins named shelterin. TRF2, one of the shelterin components, binds to the duplex part of telomeric DNA and is essential to fold the telomeric chromatin into a protective cap. Although most of the human telomeric DNA is organized into tightly spaced nucleosomes, their role in telomere protection and how they interplay with telomere-specific factors in telomere organization is still unclear. In this study we investigated whether TRF2 can regulate nucleosome assembly at telomeres. By means of chromatin immunoprecipitation (ChIP) and Micrococcal Nuclease (MNase) mapping assay, we found that the density of telomeric nucleosomes in human cells was inversely proportional to the dosage of TRF2 at telomeres. This effect was not observed in the G1 phase of the cell cycle but appeared coincident of late or post-replicative events. Moreover, we showed that TRF2 overexpression altered nucleosome spacing at telomeres increasing internucleosomal distance. By means of an in vitro nucleosome assembly system containing purified histones and remodeling factors, we reproduced the short nucleosome spacing found in telomeric chromatin. Importantly, when in vitro assembly was performed in the presence of purified TRF2, nucleosome spacing on a telomeric DNA template increased, in agreement with in vivo MNase mapping. Our results demonstrate that TRF2 negatively regulates the number of nucleosomes at human telomeres by a cell cycle-dependent mechanism that alters internucleosomal distance. These findings raise the intriguing possibility that telomere protection is mediated, at least in part, by the TRF2-dependent regulation of nucleosome organization.

The deposited article is a post-print version and has peer review. The deposited article version contains attached the supplementary materials within the pdf. This publication hasn't any creative commons license associated. Establishment of a proper chromatin landscape is central to genome function. Here, we explain H3 variant distribution by specific targeting and dynamics of deposition involving the CAF-1 and HIRA histone chaperones. Impairing replicative H3.1 incorporation via CAF-1 enables an alternative H3.3 deposition at replication sites via HIRA. Conversely, the H3.3 incorporation throughout the cell cycle via HIRA cannot be replaced by H3.1. ChIP-seq analyses reveal correlation between HIRA-dependent H3.3 accumulation and RNA pol II at transcription sites and specific regulatory elements, further supported by their biochemical association. The HIRA complex shows unique DNA binding properties, and depletion of HIRA increases DNA sensitivity to nucleases. We propose that protective nucleosome gap filling of naked DNA by HIRA leads to a broad distribution of H3.3, and HIRA association with Pol II ensures local H3.3 enrichment at specific sites. We discuss the importance of this H3.3 deposition as a salvage pathway to maintain chromatin integrity. Ligue Nationale contre le Cancer (Equipe labellisée Ligue 2010); PIC Programs; the European Commission Network of Excellence EpiGeneSys (HEALTH-F4-2010-257082), the European Commission ITN FP7-PEOPLE-2007-215148 “Image DDR” and FP7-PEOPLE-2008-238176 “Nucleosome 4D,” ERC Advanced Grant 2009-AdG_20090506 “Eccentric,” the European Commission large-scale integrating project FP7_HEALTH-2010-259743 “MODHEP,” ANR “ECenS” ANR-09-BLAN-0257-01, ANR “ChromaTin” ANR-10-BLAN-1326-03, and INCa “GepiG.” The lab of P.D.A. is funded by NIA program project P01 AG031862. L.E.T.J. was supported by the Fundação Calouste Gulbenkian, Fundação para a Ciência e a Tecnologia (FCT) grant BIA-PRO/100537/2008, the European Commission FP7 programme, and an EMBO installation grant. info:eu-repo/semantics/publishedVersion

International audience; The interaction of histone H1 with linker DNA results in the formation of the nucleosomal stem structure, with considerable influence on chromatin organization. In a recent paper [Syed,S.H., Goutte-Gattat,D., Becker,N., Meyer,S., Shukla,M.S., Hayes,J.J., Everaers,R., Angelov,D., Bednar,J. and Dimitrov,S. (2010) Single-base resolution mapping of H1-nucleosome interactions and 3D organization of the nucleosome. Proc. Natl Acad. Sci. USA, 107, 9620-9625], we published results of biochemical footprinting and cryo-electron-micrographs of reconstituted mono-, di- and tri-nucleosomes, for H1 variants with different lengths of the cationic C-terminus. Here, we present a detailed account of the analysis of the experimental data and we include thermal fluctuations into our nano-scale model of the stem structure. By combining (i) crystal and NMR structures of the nucleosome core particle and H1, (ii) the known nano-scale structure and elasticity of DNA, (iii) footprinting information on the location of protected sites on the DNA backbone and (iv) cryo-electron micrographs of reconstituted tri-nucleosomes, we arrive at a description of a polymorphic, hierarchically organized stem with a typical length of 20 ± 2 base pairs. A comparison to linker conformations inferred for poly-601 fibers with different linker lengths suggests, that intra-stem interactions stabilize and facilitate the formation of dense chromatin fibers.

Meiotic recombination predominantly occurs at discrete genomic loci called recombination hotspots, but the features defining these areas are still largely unknown (reviewed in1-5). To enable a comprehensive analysis of hotspot-associated DNA and chromatin characteristics we developed a direct molecular approach for mapping meiotic DNA double stranded breaks that initiate recombination. Here, we present the genome-wide distribution of recombination initiation sites in the mouse genome, constituting the first physical map of recombination hotspots in a multi-cellular organism. Hotspot centres are mapped with approximately 200-nucleotide precision that enables analysis of the fine structural details of the preferred recombination sites. We determine that hotspots share a centrally distributed consensus motif, possess a nucleotide skew that changes polarity at the centre of hotspots, and have an intrinsic preference to be occupied by a nucleosome. Furthermore, we find that the vast majority of recombination initiation sites in mouse males are associated with testis-specific trimethylation of lysine 4 on histone H3 that is distinct from histone H3 lysine 4 trimethylation marks associated with transcription. The recombination map presented here has been derived from a homogeneous mouse population with a defined genetic background and therefore, lends itself to extensive future experimental exploration. Importantly, the mapping technique developed here does not depend on availability of genetic markers and hence can be easily adapted for other species with complex genomes. Our findings uncover several fundamental features of mammalian recombination hotspots and underline the power of the new recombination map for future studies of genetic recombination, genome stability and evolution.

International audience; Histone variants within the H2A family show high divergences in their C-terminal regions. In this work, we have studied how these divergences and in particular, how a part of the H2A COOH-terminus, the docking domain, is implicated in both structural and functional properties of the nucleosome. Using biochemical methods in combination with Atomic Force Microscopy and Electron Cryo-Microscopy, we show that the H2A-docking domain is a key structural feature within the nucleosome. Deletion of this domain or replacement with the incomplete docking domain from the variant H2A.Bbd results in significant structural alterations in the nucleosome, including an increase in overall accessibility to nucleases, un-wrapping of ∼10 bp of DNA from each end of the nucleosome and associated changes in the entry/exit angle of DNA ends. These structural alterations are associated with a reduced ability of the chromatin remodeler RSC to both remodel and mobilize the nucleosomes. Linker histone H1 binding is also abrogated in nucleosomes containing the incomplete docking domain of H2A.Bbd. Our data illustrate the unique role of the H2A-docking domain in coordinating the structural-functional aspects of the nucleosome properties. Moreover, our data suggest that incorporation of a 'defective' docking domain may be a primary structural role of H2A.Bbd in chromatin.

Pontvianne, Frédéric et al. 13 p.-8 fig., In eukaryotes, 45S rRNA genes are arranged in tandem arrays in copy numbers ranging from several hundred to several thousand in plants. Although it is clear that not all copies are transcribed under normal growth conditions, the molecular basis controlling the expression of specific sets of rRNA genes remains unclear. Here, we report four major rRNA gene variants in Arabidopsis thaliana. Interestingly, while transcription of one of these rRNA variants is induced, the others are either repressed or remain unaltered in A. thaliana plants with a disrupted nucleolin-like protein gene (Atnuc-L1). Remarkably, the most highly represented rRNA gene variant, which is inactive in WT plants, is reactivated in Atnuc-L1 mutants. We show that accumulated pre–rRNAs originate from RNA Pol I transcription and are processed accurately. Moreover, we show that disruption of the AtNUC-L1 gene induces loss of symmetrical DNA methylation without affecting histone epigenetic marks at rRNA genes.Collectively, these data reveal a novel mechanism for rRNA gene transcriptional regulation in which the nucleolin protein plays a major role in controlling active and repressed rRNA gene variants in Arabidopsis., This work was supported by the Centre National de la Recherche Scientifique(CNRS). Work performed in FJM laboratory was financed by a grant of the Spanish National Plan for R&D, N u ESP2006-13600-C02-02.

International audience; Despite the key role of the linker histone H1 in chromatin structure and dynamics, its location and interactions with nucleosomal DNA have not been elucidated. In this work we have used a combination of electron cryomicroscopy, hydroxyl radical footprinting, and nanoscale modeling to analyze the structure of precisely positioned mono-, di-, and trinucleosomes containing physiologically assembled full-length histone H1 or truncated mutants of this protein. Single-base resolution *OH footprinting shows that the globular domain of histone H1 (GH1) interacts with the DNA minor groove located at the center of the nucleosome and contacts a 10-bp region of DNA localized symmetrically with respect to the nucleosomal dyad. In addition, GH1 interacts with and organizes about one helical turn of DNA in each linker region of the nucleosome. We also find that a seven amino acid residue region (121-127) in the COOH terminus of histone H1 was required for the formation of the stem structure of the linker DNA. A molecular model on the basis of these data and coarse-grain DNA mechanics provides novel insights on how the different domains of H1 interact with the nucleosome and predicts a specific H1-mediated stem structure within linker DNA.

In their recent paper in Cell, Furuyama and Henikoff (2009) report that nucleosomes in centromeres may be right-handed, that is, they wrap DNA in a right-handed manner and induce positive supercoils. This raises intriguing new questions, such as how centromeric histone variants may be assembled into right-handed particles, and why chromatin would retain negative supercoiling in chromosome arms but positive supercoiling in centromeres. We wish to comment on these new findings in the context of topological insights that we have gained from recent in vitro experiments with centromeric nucleosomes and single chromatin fibers submitted to torsional constraints and from 3D modeling of chromatin dynamics.

International audience; Histone modifications are central to the regulation of all DNA-dependent processes. Lys64 of histone H3 (H3K64) lies within the globular domain at a structurally important position. We identify trimethylation of H3K64 (H3K64me3) as a modification that is enriched at pericentric heterochromatin and associated with repeat sequences and transcriptionally inactive genomic regions. We show that this new mark is dynamic during the two main epigenetic reprogramming events in mammals. In primordial germ cells, H3K64me3 is present at the time of specification, but it disappears transiently during reprogramming. In early mouse embryos, it is inherited exclusively maternally; subsequently, the modification is rapidly removed, suggesting an important role for H3K64me3 turnover in development. Taken together, our findings establish H3K64me3 as a previously uncharacterized histone modification that is preferentially localized to repressive chromatin. We hypothesize that H3K64me3 helps to 'secure' nucleosomes, and perhaps the surrounding chromatin, in an appropriately repressed state during development.

The organization of eukaryotic genomes is characterized by the presence of distinct euchromatic and heterochromatic sub-nuclear compartments. In Saccharomyces cerevisiae heterochromatic loci, including telomeres and silent mating type loci, form clusters at the nuclear periphery. We have employed live cell 3-D imaging and chromosome conformation capture (3C) to determine the contribution of nuclear positioning and heterochromatic factors in mediating associations of the silent mating type loci. We identify specific long-range interactions between HML and HMR that are dependent upon silencing proteins Sir2p, Sir3p, and Sir4p as well as Sir1p and Esc2p, two proteins involved in establishment of silencing. Although clustering of these loci frequently occurs near the nuclear periphery, colocalization can occur equally at more internal positions and is not affected in strains deleted for membrane anchoring proteins yKu70p and Esc1p. In addition, appropriate nucleosome assembly plays a role, as deletion of ASF1 or combined disruption of the CAF-1 and HIR complexes abolishes the HML-HMR interaction. Further, silencer proteins are required for clustering, but complete loss of clustering in asf1 and esc2 mutants had only minor effects on silencing. Our results indicate that formation of heterochromatic clusters depends on correctly assembled heterochromatin at the silent loci and, in addition, identify an Asf1p-, Esc2p-, and Sir1p-dependent step in heterochromatin formation that is not essential for gene silencing but is required for long-range interactions., Author Summary Chromosomes are non-randomly positioned inside cells, and this organization is relevant for genome regulation. Spatial clustering of heterochromatic loci provides a striking example of nuclear compartmentalization. In S. cerevisiae, the presence of heterochromatic sub-nuclear domains has been well established, but their mechanisms of formation are not fully understood. Here, we analyzed the DNA elements and protein complexes that are critical for formation of heterochromatic clusters. We focused on heterochromatic regions on chromosome III—the two telomeres, as well as the silent mating type loci HML and HMR, located on the left and right end of the chromosome, respectively. We employed live cell 3-D imaging and chromosome conformation capture (3C) and found that these loci specifically interact most prominently near silencer elements that flank the loci. Analysis of a panel of mutants showed that complexes involved in silencing are also involved in long-range interactions. Interestingly, we find that heterochromatic interactions are mechanistically distinct from silencing and independent of tethering to the nuclear periphery. Our results indicate that formation of heterochromatic clusters depends on correctly assembled heterochromatin, and point to a step in heterochromatin formation that is not essential for gene silencing but is required for long-range interactions between heterochromatic loci.

Epigenetic refers to a range of heritable chromatin modifications including DNA methylation, histone modifications, remodeling of nucleosomes and higher order chromatin modifications. In the framework of chromatin remodeling activities, the poly(ADP-ribosyl)ation of nuclear proteins catalyzed by PARPs, particularly PARP-1 and PARP-2, plays a fundamental role and as such have the potential to orchestrate various chromatin-based biological tasks including transcription, DNA repair and differentiation. In this review, we propose a short overview of the more recent experimental data that shed light on the role of poly(ADP-ribosyl)ation in the translation of the histone code. We will essentially focus on the different mechanisms by which PARP activity regulates the global chromatin environment and how this affects cellular pathways.

The persistence of transcriptionally silent but replication-competent HIV-1 reservoirs in Highly Active Anti-Retroviral Therapy (HAART)-treated infected individuals, represents a major hurdle to virus eradication. Activation of HIV-1 gene expression in these cells together with an efficient HAART has been proposed as an adjuvant therapy aimed at decreasing the pool of latent viral reservoirs. Using the latently-infected U1 monocytic cell line and latently-infected J-Lat T-cell clones, we here demonstrated a strong synergistic activation of HIV-1 production by clinically used histone deacetylase inhibitors (HDACIs) combined with prostratin, a non-tumor-promoting nuclear factor (NF)- kappaB inducer. In J-Lat cells, we showed that this synergism was due, at least partially, to the synergistic recruitment of unresponsive cells into the expressing cell population. A combination of prostratin+HDACI synergistically activated the 5' Long Terminal Repeat (5'LTR) from HIV-1 Major group subtypes representing the most prevalent viral genetic forms, as shown by transient transfection reporter assays. Mechanistically, HDACIs increased prostratin-induced DNA-binding activity of nuclear NF-kappaB and degradation of cytoplasmic NF-kappaB inhibitor, IkappaBalpha .Moreover, the combined treatment prostratin+HDACI caused a more pronounced nucleosomal remodeling in the U1 viral promoter region than the treatments with the compounds alone. This more pronounced remodeling correlated with a synergistic reactivation of HIV-1 transcription following the combined treatment prostratin+HDACI, as demonstrated by measuring recruitment of RNA polymerase II to the 5'LTR and both initiated and elongated transcripts. The physiological relevance of the prostratin+HDACI synergism was shown in CD8(+)-depleted peripheral blood mononuclear cells from HAART-treated patients with undetectable viral load. Moreover, this combined treatment reactivated viral replication in resting CD4(+) T cells isolated from similar patients. Our results suggest that combinations of different kinds of proviral activators may have important implications for reducing the size of latent HIV-1 reservoirs in HAART-treated patients., Journal Article, Research Support, Non-U.S. Gov't, info:eu-repo/semantics/published

Monitoring Editor: Yixian Zheng Heterochromatic chromosomal regions undergo large-scale reorganization and progressively aggregate, forming chromocenters. These are dynamic structures that rapidly adapt to various stimuli that influence gene expression patterns, cell cycle progression, and differentiation. Np95-ICBP90 (m- and h-UHRF1) is a histone binding protein expressed only in proliferating cells. During pericentromeric heterochromatin (PH) replication, Np95 specifically relocalizes to chromocenters where it highly concentrates in the replication factories that correspond to less compacted DNA. Np95 recruits HDAC and DNMT1 to PH and depletion of Np95 impairs PH replication. Here we show that Np95 causes large-scale modifications of chromocenters independently from the H3:K9 and H4:K20 trimethylation pathways, from the expression levels of HP1, from DNA methylation and from the cell cycle. The PHD domain is essential to induce this effect. The PHD domain is also required in vitro to increase access of a restriction enzyme to DNA packaged into nucleosomal arrays. We propose that the PHD domain of Np95-ICBP90 contributes to the opening and/or stabilization of dense chromocenter structures to support the recruitment of modifying enzymes - like HDAC and DNMT1 - required for the replication and formation of PH.

Epigénome er paléogénomie; International audience; Nucleosome positioning plays an essential role in cellular processes by modulating accessibility of DNA to proteins. Here, using only sequence-dependent DNA flexibility and intrinsic curvature, we predict the nucleosome occupancy along the genomes of Saccharomyces cerevisiae and Drosophila melanogaster and demonstrate the predictive power and universality of our model through its correlation with experimentally determined nucleosome occupancy data. In yeast promoter regions, the computed average nucleosome occupancy closely superimposes with experimental data, exhibiting a

International audience; Nucleosomes were discovered more than thirty years ago as the basic repeating units of chromatin. Since then, nucleosomes have progressively revealed their taste to come in many appearances, upon either adjunction of other proteins (e.g., a fifth histone or a nonhistone protein, HMG-N), histone substitution for isoforms (histone variants), depletion of one or the two H2A-H2B dimers (sub-nucleosomes), intimate two-particle association, or isomeric structural alterations. The resulting entities, some of them are only transient, acquire new properties useful for their specific roles in chromatin function. These structures are presented here in the chronological order of their identification, from the chromatosome to the sub-nucleosomal hexasome and tetrasome, and from the dinucleosomal altosome and nucleodisome to the nucleosome variants and altered forms: the old lexosome and the most recent reversome.

Using magnetic tweezers to investigate the mechanical response of single chromatin fibers, we show that fibers submitted to large positive torsion transiently trap positive turns, at a rate of one turn per nucleosome. A comparison with the response of fibers of tetrasomes (the (H3-H4)2 tetramer bound with ~50 bp of DNA) obtained by depletion of H2A-H2B dimers, suggests that the trapping reflects a nucleosome chiral transition to a metastable form built on the previously documented righthanded tetrasome. In view of its low energy, 33 pages (double spacing), 7 figures

International audience; The histone variant H2A.Bbd appeared to be associated with active chromatin, but how it functions is unknown. We have dissected the properties of nucleosome containing H2A.Bbd. Atomic force microscopy (AFM) and electron cryo-microscopy (cryo-EM) showed that the H2A.Bbd histone octamer organizes only approximately 130 bp of DNA, suggesting that 10 bp of each end of nucleosomal DNA are released from the octamer. In agreement with this, the entry/exit angle of the nucleosomal DNA ends formed an angle close to 180 degrees and the physico-chemical analysis pointed to a lower stability of the variant particle. Reconstitution of nucleosomes with swapped-tail mutants demonstrated that the N-terminus of H2A.Bbd has no impact on the nucleosome properties. AFM, cryo-EM and chromatin remodeling experiments showed that the overall structure and stability of the particle, but not its property to interfere with the SWI/SNF induced remodeling, were determined to a considerable extent by the H2A.Bbd docking domain. These data show that the whole H2A.Bbd histone fold domain is responsible for the unusual properties of the H2A.Bbd nucleosome.

Histone deacetylase (HDAC) inhibitors are a promising class of anticancer agents for the treatment of solid and hematological malignancies. The precise mechanism by which HDAC inhibitors mediate their effects on tumor cell growth, differentiation, and/or apoptosis is the subject of intense research. Previously we described a family of multiprotein complexes that contain histone deacetylase 1/2 (HDAC1/2) and the histone demethylase BHC110 (LSD1). Here we show that HDAC inhibitors diminish histone H3 lysine 4 (H3K4) demethylation by BHC110 in vitro. In vivo analysis revealed an increased H3K4 methylation concomitant with inhibition of nucleosomal deacetylation by HDAC inhibitors. Reconstitution of recombinant complexes revealed a functional connection between HDAC1 and BHC110 only when nucleosomal substrates were used. Importantly, while the enzymatic activity of BHC110 is required to achieve optimal deacetylation in vitro, in vivo analysis following ectopic expression of an enzymatically dead mutant of BHC110 (K661A) confirmed the functional cross talk between the demethylase and deacetylase enzymes. Our studies not only reveal an intimate link between the histone demethylase and deacetylase enzymes but also identify histone demethylation as a secondary target of HDAC inhibitors.

The Cyclin E1 gene ( CCNE1 ) is an ideal model to explore the mechanisms that control the transcription of cell cycle-regulated genes whose expression rises transiently before entry into S phase. E2F-dependent regulation of the CCNE1 promoter was shown to correlate with changes in the level of H3-K9 acetylation/methylation of nucleosomal histones positioned at the transcriptional start site region. Here we show that, upon growth stimulation, the same region is subject to variations of H3-R17 and H3-R26 methylation that correlate with the recruitment of coactivator-associated arginine methyltransferase 1 (CARM1) onto the CCNE1 and DHFR promoters. Accordingly, CARM1-deficient cells lack these modifications and present lowered levels and altered kinetics of CCNE1 and DHFR mRNA expression. Consistently, reporter gene assays demonstrate that CARM1 functions as a transcriptional coactivator for their E2F1/DP1-stimulated expression. CARM1 recruitment at the CCNE1 gene requires activator E2Fs and ACTR, a member of the p160 coactivator family that is frequently overexpressed in human breast cancer. Finally, we show that grade-3 breast tumors present coelevated mRNA levels of ACTR and CARM1 , along with their transcriptional target CCNE1 . All together, our results indicate that CARM1 is an important regulator of the CCNE1 gene.

Minireview/Minisynthèse; Chromatin modifications play a crucial role in regulating DNA metabolism. Chromatin structures can be remodeled by covalently modifying histones, by shifting nucleosomes along the DNA, and by changing the histone composition of nucleosomes. Lately, nucleosome displacement has been extensively described within transcribed genes and DNA breaks. This review focuses on recently published work that describes the relationships between histone modification/exchange and nucleosome displacement.

The understanding of the long-range correlations (LRC) observed in DNA sequences is still an open and very challenging problem. In this paper, we start reviewing recent results obtained when exploring the scaling properties of eucaryotic, eubacterial and archaeal genomic sequences using the space-scale decomposition provided by the wavelet transform (WT). These results suggest that the existence of LRC up to distances approximately 20-30 kbp is the signature of the nucleosomal structure and dynamics of the chromatin fiber. Actually the LRC are mainly observed in the DNA bending profiles obtained when using some structural coding of the DNA sequences that accounts for the fluctuations of the local double-helix curvature within the nucleosome complex. Because of the approximate planarity of nucleosomal DNA loops, we then study the influence of the LRC structural disorder on the thermodynamical properties of 2D elastic chains submitted locally to mechanical/topological constraint as loops. The equilibrium properties of the one-loop system are derived numerically and analytically in the quite realistic weak-disorder limit. The LRC are shown to favor the spontaneous formation of small loops, the larger the LRC, the smaller the size of the loop. We further investigate the dynamical behavior of such a loop using the mean first passage time (MFPT) formalism. We show that the typical short-time loop dynamics is superdiffusive in the presence of LRC. For displacements larger than the loop size, we use large-deviation theory to derive a LRC-dependent anomalous-diffusion rule that accounts for the lack of disorder self-averaging. Potential biological implications on DNA loops involved in nucleosome positioning and dynamics in eucaryotic chromatin are discussed.

The histone variant H3.3 can be incorporated in chromatin independently of DNA synthesis. By imaging using green fluorescent protein-tagged histones, H3.3 deposition has been found to be linked with transcriptional activation. Here, we investigated H3.3 incorporation during G1 progression on cell-cycle-regulated E2F-dependent genes and on some control loci. We transiently transfected resting cells with an expression vector for tagged H3.3 and we analysed its presence by chromatin immunoprecipitation. We found that replication-independent H3.3 deposition occurred on actively transcribed genes, but not on silent loci, thereby confirming its link with transcription. Interestingly, we observed similar levels of H3.3 occupancy on promoters and on the coding regions of the corresponding genes, indicating that H3.3 deposition is not restricted to promoters. Finally, H3.3 occupancy correlated with the presence of transcription-competent RNA polymerase II. Taken together, our results support the hypothesis that H3.3 is incorporated after disruption of nucleosomes mediated by transcription elongation.

International audience; Remodeling machines play an essential role in the control of gene expression, but how their activity is regulated is not known. Here we report that the nuclear protein nucleolin possesses a histone chaperone activity and that this factor greatly enhances the activity of the chromatin remodeling machineries SWI/SNF and ACF. Interestingly, nucleolin is able to induce the remodeling by SWI/SNF of macroH2A, but not of H2ABbd nucleosomes, which are otherwise resistant to remodeling. This new histone chaperone promotes the destabilization of the histone octamer, helping the dissociation of a H2A-H2B dimer, and stimulates the SWI/SNF-mediated transfer of H2A-H2B dimers. Furthermore, nucleolin facilitates transcription through the nucleosome, which is reminiscent of the activity of the FACT complex. This work defines new functions for histone chaperones in chromatin remodeling and regulation of transcription and explains how nucleolin could act on transcription.

macroH2A (mH2A) is an unusual histone variant consisting of a histone H2A-like domain fused to a large nonhistone region. In this work, we show that histone mH2A represses p300- and Gal4-VP16-dependent polymerase II transcription, and we have dissected the mechanism by which this repression is realized. The repressive effect of mH2A is observed at the level of initiation but not at elongation of transcription, and mH2A interferes with p300-dependent histone acetylation. The nonhistone region of mH2A is responsible for both the repression of initiation of transcription and the inhibition of histone acetylation. In addition, the presence of this domain of mH2A within the nucleosome is able to block nucleosome remodeling and sliding of the histone octamer to neighboring DNA segments by the remodelers SWI/SNF and ACF. These data unambiguously identify mH2A as a strong transcriptional repressor and show that the repressive effect of mH2A is realized on at least two different transcription activation chromatin-dependent pathways: histone acetylation and nucleosome remodeling.

International audience; In Drosophila and several other metazoan organisms, there are two genes that encode related but distinct homologs of ADA2-type transcriptional adaptors. Here we describe mutations of the two Ada2 genes of Drosophila melanogaster. By using mutant Drosophila lines, which allow the functional study of individual ADA2s, we demonstrate that both Drosophila Ada2 genes are essential. Ada2a and Ada2b null homozygotes are late-larva and late-pupa lethal, respectively. Double mutants have a phenotype identical to that of the Ada2a mutant. The overproduction of ADA2a protein from transgenes cannot rescue the defects resulting from the loss of Ada2b, nor does complementation work vice versa, indicating that the two Ada2 genes of Drosophila have different functions. An analysis of germ line mosaics generated by pole-cell transplantation revealed that the Ada2a function (similar to that reported for Ada2b) is required in the female germ line. A loss of the function of either of the Ada2 genes interferes with cell proliferation. Interestingly, the Ada2b null mutation reduces histone H3 K14 and H3 K9 acetylation and changes TAF10 localization, while the Ada2a null mutation does not. Moreover, the two ADA2s are differently required for the expression of the rosy gene, involved in eye pigment production, and for Dmp53-mediated apoptosis. The data presented here demonstrate that the two genes encoding homologous transcriptional adaptor ADA2 proteins in Drosophila are both essential but are functionally distinct.

This research was originally published in The Journal of Biological Chemistry. © the American Society for Biochemistry and Molecular Biology; International audience; We have studied the sample concentration-dependent and external stress-dependent stability of native and reconstituted nucleosomal arrays. Whereas upon stretching a single chromatin fiber in a solution of very low chromatin concentration the statistical distribution of DNA length released upon nucleosome unfolding shows only one population centered around approximately 25 nm, in nucleosome stabilizing conditions a second population with average length of approximately 50 nm was observed. Using radioactively labeled histone H3 and H2B, we demonstrate that upon lowering the chromatin concentration to very low values, first the linker histones are released, followed by the H2A-H2B dimer, whereas the H3-H4 tetramer remains stably attached to DNA even at the lowest concentration studied. The nucleosomal arrays reconstituted on a 5 S rDNA tandem repeat exhibited similar behavior. This suggests that the 25-nm disruption length is a consequence of the histone H2A-H2B dimer dissociation from the histone octamer. In nucleosome stabilizing conditions, a full approximately 145 bp is constrained in the nucleosome. Our data demonstrate that the nucleosome stability and histone octamer integrity can be severely degraded in experiments where the sample concentration is low.

This research was originally published in The Journal of Biological Chemistry. © the American Society for Biochemistry and Molecular Biology; International audience; We have studied the sample concentration-dependent and external stress-dependent stability of native and reconstituted nucleosomal arrays. Whereas upon stretching a single chromatin fiber in a solution of very low chromatin concentration the statistical distribution of DNA length released upon nucleosome unfolding shows only one population centered around approximately 25 nm, in nucleosome stabilizing conditions a second population with average length of approximately 50 nm was observed. Using radioactively labeled histone H3 and H2B, we demonstrate that upon lowering the chromatin concentration to very low values, first the linker histones are released, followed by the H2A-H2B dimer, whereas the H3-H4 tetramer remains stably attached to DNA even at the lowest concentration studied. The nucleosomal arrays reconstituted on a 5 S rDNA tandem repeat exhibited similar behavior. This suggests that the 25-nm disruption length is a consequence of the histone H2A-H2B dimer dissociation from the histone octamer. In nucleosome stabilizing conditions, a full approximately 145 bp is constrained in the nucleosome. Our data demonstrate that the nucleosome stability and histone octamer integrity can be severely degraded in experiments where the sample concentration is low.

International audience; The histone H2ABbd is a novel histone variant of H2A with a totally unknown function. We have investigated the behaviour of the H2ABbd nucleosomes. Nucleosomes were reconstituted with recombinant histone H2ABbd and changes in their conformations at different salt concentrations were studied by analytical centrifugation. The data are in agreement with H2ABbd being less tightly bound compared with conventional H2A in the nucleosome. In addition, stable cell lines expressing either green fluorescent protein (GFP)-H2A or GFP-H2ABbd were established and the mobility of both fusions was measured by fluorescence recovery after photobleaching. We show that GFP-H2ABbd exchanges much more rapidly than GFP-H2A within the nucleosome. The reported data are compatible with a lower stability of the variant H2ABbd nucleosome compared with the conventional H2A particle.

International audience; A histone variant H2ABbd was recently identified, but its function is totally unknown. Here we have studied the structural and functional properties of nucleosome and nucleosomal arrays reconstituted with this histone variant. We show that H2ABbd can replace the conventional H2A in the nucleosome, but this replacement results in alterations of the nucleosomal structure. The remodeling complexes SWI/SNF and ACF are unable to mobilize the variant H2ABbd nucleosome. However, SWI/SNF was able to increase restriction enzyme access to the variant nucleosome and assist the transfer of variant H2ABbd-H2B dimer to a tetrameric histone H3-H4 particle. In addition, the p300- and Gal4-VP16-activated transcription appeared to be more efficient for H2ABbd nucleosomal arrays than for conventional H2A arrays. The intriguing mechanisms by which H2ABbd affects both nucleosome remodeling and transcription are discussed.

International audience; The histones H2A, H2B, H3 and H4 are very conserved basic proteins that wrap almost two turns of DNA to form the nucleosome core. Conventional histones can be replaced with histone variants that are found in all eukaryotic organisms. Together with other nucleosome modification pathways, histone variants participate in the functional specialization of chromatin. In this review, we focus on three major H2A histone variants: H2A.X, H2A.Z and macroH2A. Recent discoveries highlight their involvement in crucial events such as DNA repair and transcriptional regulation.

International audience; In eukaryotes cell division is accompanied by phosphorylation of histone H3 at serine 10. In this work we have studied the kinase activity responsible for this histone H3 modification by using cell-free extracts prepared from Xenopus eggs. We have found that the Xenopus aurora-A kinase pEg2, immunoprecipitated from the extract, is able to phosphorylate specifically histone H3 at serine 10. The enzyme is incorporated into chromatin during in vitro chromosome assembly, and the kinetics of this incorporation parallels that of histone H3 phosphorylation. Recombinant pEg2 phosphorylates efficiently histone H3 at serine 10 in reconstituted nucleosomes and in sperm nuclei decondensed in heated extracts. These data identify pEg2 as a good candidate for mitotic histone H3 kinase. However, immunodepletion of pEg2 does not interfere with the chromosome assembly properties of the extract nor with the pattern of H3 phosphorylation, suggesting the existence of multiple kinases involved in this H3 modification in Xenopus eggs. This hypothesis is supported by in gel activity assay experiments using extracts from Saccharomyces cerevisiae.

Broad spectrum caspase inhibitors have been found to reduce neurodegeneration caused by cerebral ischemia. We studied whether blockade of group I caspases, mainly caspase-1, using the inhibitor Ac-YVAD.cmk reduced infarct volume and produced prolonged neuroprotection. Ac-YVAD.cmk (300 ng/rat) was injected intracerebroventricularly 10 min after permanent middle cerebral artery occlusion in the rat. Drug treatment induced a significant reduction of infarct volume not only 24 hr after ischemia (total damage, percentage of hemisphere volume: control, 41.1 ± 2.3%; treated, 26.5 ± 2.1%;p< 0.05) but also 6 d later (total damage: control, 30.6 ± 2.2%; treated, 23.0 ± 2.2%;p< 0.05). Ac-YVAD.cmk treatment resulted in a reduction not only of caspase-1 (control, 100 ± 20.3%; treated, 3.4 ± 10.4%;p< 0.01) but also of caspase-3 (control, 100 ± 30.3%; treated, 13.2 ± 9.5%;p< 0.05) activity at 24 hr and led to a parallel decrease of apoptosis as measured by nucleosome quantitation (control, 100 ± 11.8%; treated, 47 ± 5.9%;p< 0.05). Six days after treatment no differences in these parameters could be detected between control and treated animals. Likewise, brain levels of the proinflammatory cytokines IL-1β and TNF-α were reduced at 24 hr (39.5 ± 23.7 and 51.9 ± 10.3% of control, respectively) but not at 6 d. Other cytokines, IL-10, MCP-1, MIP-2, and the gaseous mediator nitric oxide, were not modified by the treatment. These findings indicate that blockade of caspase-1-like activity induces a long-lasting neuroprotective effect that, in our experimental conditions, takes place in the early stages of damage progression. Finally, this effect is achieved by interfering with both apoptotic and inflammatory mechanisms.

Euglena chromatin, spread according to the Dubochet technique, displays the nucleosomal organization with the typical zig-zag conformation following short periods of digestion with micrococcal nuclease. After comparative kinetic experiments on vitamin B12 starved and control Euglena nuclei, a larger percentage of monosomes appears after 2 mm. in the starved chromatin (86%) compared to the control (59%). No difference is found for longer period of incubation.

Biotin-labeled deoxyuridine triphosphate (BiodUTP) has the potential to be a useful affinity probe for studies on DNA repair, if it can be incorporated into DNA repair patches and does not inhibit subsequent steps in the excision repair pathway. We have synthesized BiodUTP by an improved procedure and have used permeable normal human fibroblasts to determine the effect of substituting BiodUTP for thymidine triphosphate on several steps in the excision repair pathway: incision, polymerization, ligation, and nucleosome rearrangement. The results demonstrate that BiodUTP is efficiently incorporated into repair patches and has little or no effect on the repair process. The presence of BiodUMP in ligated repair patches has been used to visualize the repair patches by electron microscopy following incubation with ferritin-labeled avidin. This approach has been used to estimate the maximum size of repair patches induced by ultraviolet radiation.

The effect of poly(ADP-ribose) synthesis on chromatin structure was investigated by velocity sedimentation and electron microscopy. We demonstrate that locally relaxed regions can be generated within polynucleosome chains by the activity of their intrinsic poly(ADP-ribose)polymerase. This relaxation phenomenon is also shown to be NAD dependent and to be correlated with the formation of hyper(ADP-ribosyl)ated forms of histone H1. Evidence is also presented which suggests that hyper(ADP-ribosyl)ated histone H1 is neither released from the relaxed chromatin, nor does it seem to participate in polynucleosomal aggregation.

International audience; Epigenetic mechanisms regulate expression of the genome to generate various cell types during development or orchestrate cellular responses to external stimuli. Recent studies highlight that bacteria can affect the chromatin structure and transcriptional program of host cells by influencing diverse epigenetic factors (i.e., histone modifications, DNA methylation, chromatin-associated complexes, noncoding RNAs, and RNA splicing factors). In this article, we first review the molecular bases of the epigenetic language and then describe the current state of research regarding how bacteria can alter epigenetic marks and machineries. Bacterial-induced epigenetic deregulations may affect host cell function either to promote host defense or to allow pathogen persistence. Thus, pathogenic bacteria can be considered as potential epimutagens able to reshape the epigenome. Their effects might generate specific, long-lasting imprints on host cells, leading to a memory of infection that influences immunity and might be at the origin of unexplained diseases.

Human genes contain a 10 bp repeat of RR dinucleotides focused around the first nucleosome position suggesting a role in transcriptional control., Background The position of a nucleosome, both translational along the DNA molecule and rotational between the histone core and the DNA, is controlled by many factors, including the regular occurrence of specific dinucleotides with a period of approximately 10 bp, important for the rotational setting of the DNA around the histone octamer. Results We show that such a 10 bp periodic signal of purine-purine dinucleotides occurs in phase with the transcription start site (TSS) of human genes and is centered on the position of the first (+1) nucleosome downstream of the TSS. These data support a direct link between transcription and the rotational setting of the nucleosome. The periodic signal is most prevalent in genes that contain CpG islands that are expressed at low levels in a tissue-specific manner and are involved in the control of transcription. Conclusions These results, together with several lines of evidence from the recent literature, support a new model whereby the +1 nucleosome could be more efficiently disassembled from gene promoters by H3K56 acetylation marks if the periodic signal specifies an optimal rotational setting.