The CENP-A nucleosome: where and when it happens during the inner kinetochore's assembly.

CENP-A is an essential histone variant that replaces the canonical H3 at the centromeres and marks these regions epigenetically. The CENP-A nucleosome is the specific building block of centromeric chromatin, and it is recognized by CENP-C and CENP-N, two components of the constitutive centromere-associated network (CCAN), the first protein layer of the kinetochore. Recent proposals of the yeast and human (h)CCAN structures position the assembly on exposed DNA, suggesting an elusive spatiotemporal recognition. We summarize the data on the structural organization of the CENP-A nucleosome and the binding of CENP-C and CENP-N. The latter posits an apparent contradiction in engaging the CENP-A nucleosome versus the CCAN. We propose a reconciliatory model for the assembly of CCAN on centromeric chromatin.

Structure of an H1-Bound 6-Nucleosome Array Reveals an Untwisted Two-Start Chromatin Fiber Conformation.

Chromatin adopts a diversity of regular and irregular fiber structures in vitro and in vivo. However, how an array of nucleosomes folds into and switches between different fiber conformations is poorly understood. We report the 9.7 Å resolution crystal structure of a 6-nucleosome array bound to linker histone H1 determined under ionic conditions that favor incomplete chromatin condensation. The structure reveals a flat two-start helix with uniform nucleosomal stacking interfaces and a nucleosome packing density that is only half that of a twisted 30-nm fiber. Hydroxyl radical footprinting indicates that H1 binds the array in an on-dyad configuration resembling that observed for mononucleosomes. Biophysical, cryo-EM, and crosslinking data validate the crystal structure and reveal that a minor change in ionic environment shifts the conformational landscape to a more compact, twisted form. These findings provide insights into the structural plasticity of chromatin and suggest a possible assembly pathway for a 30-nm fiber.

H2A.Z is involved in premature aging and DSB repair initiation in muscle fibers.

Histone variants are key epigenetic players, but their functional and physiological roles remain poorly understood. Here, we show that depletion of the histone variant H2A.Z in mouse skeletal muscle causes oxidative stress, oxidation of proteins, accumulation of DNA damages, and both neuromuscular junction and mitochondria lesions that consequently lead to premature muscle aging and reduced life span. Investigation of the molecular mechanisms involved shows that H2A.Z is required to initiate DNA double strand break repair by recruiting Ku80 at DNA lesions. This is achieved via specific interactions of Ku80 vWA domain with H2A.Z. Taken as a whole, our data reveal that H2A.Z containing nucleosomes act as a molecular platform to bring together the proteins required to initiate and process DNA double strand break repair.

Structure and Dynamics of a 197 bp Nucleosome in Complex with Linker Histone H1.

Linker histones associate with nucleosomes to promote the formation of higher-order chromatin structure, but the underlying molecular details are unclear. We investigated the structure of a 197 bp nucleosome bearing symmetric 25 bp linker DNA arms in complex with vertebrate linker histone H1. We determined electron cryo-microscopy (cryo-EM) and crystal structures of unbound and H1-bound nucleosomes and validated these structures by site-directed protein cross-linking and hydroxyl radical footprinting experiments. Histone H1 shifts the conformational landscape of the nucleosome by drawing the two linkers together and reducing their flexibility. The H1 C-terminal domain (CTD) localizes primarily to a single linker, while the H1 globular domain contacts the nucleosome dyad and both linkers, associating more closely with the CTD-distal linker. These findings reveal that H1 imparts a strong degree of asymmetry to the nucleosome, which is likely to influence the assembly and architecture of higher-order structures.

The Flexible Ends of CENP-A Nucleosome Are Required for Mitotic Fidelity.

CENP-A is a histone variant, which replaces histone H3 at centromeres and confers unique properties to centromeric chromatin. The crystal structure of CENP-A nucleosome suggests flexible nucleosomal DNA ends, but their dynamics in solution remains elusive and their implication in centromere function is unknown. Using electron cryo-microscopy, we determined the dynamic solution properties of the CENP-A nucleosome. Our biochemical, proteomic, and genetic data reveal that higher flexibility of DNA ends impairs histone H1 binding to the CENP-A nucleosome. Substituting the 2-turn αN-helix of CENP-A with the 3-turn αN-helix of H3 results in compact particles with rigidified DNA ends, able to bind histone H1. In vivo replacement of CENP-A with H3-CENP-A hybrid nucleosomes leads to H1 recruitment, delocalization of kinetochore proteins, and significant mitotic and cytokinesis defects. Our data reveal that the evolutionarily conserved flexible ends of the CENP-A nucleosomes are essential to ensure the fidelity of the mitotic pathway.

Dual role of histone variant H3.3B in spermatogenesis: positive regulation of piRNA transcription and implication in X-chromosome inactivation.

The histone variant H3.3 is encoded by two distinct genes, H3f3a and H3f3b, exhibiting identical amino-acid sequence. H3.3 is required for spermatogenesis, but the molecular mechanism of its spermatogenic function remains obscure. Here, we have studied the role of each one of H3.3A and H3.3B proteins in spermatogenesis. We have generated transgenic conditional knock-out/knock-in (cKO/KI) epitope-tagged FLAG-FLAG-HA-H3.3B (H3.3BHA) and FLAG-FLAG-HA-H3.3A (H3.3AHA) mouse lines. We show that H3.3B, but not H3.3A, is required for spermatogenesis and male fertility. Analysis of the molecular mechanism unveils that the absence of H3.3B led to alterations in the meiotic/post-meiotic transition. Genome-wide RNA-seq reveals that the depletion of H3.3B in meiotic cells is associated with increased expression of the whole sex X and Y chromosomes as well as of both RLTR10B and RLTR10B2 retrotransposons. In contrast, the absence of H3.3B resulted in down-regulation of the expression of piRNA clusters. ChIP-seq experiments uncover that RLTR10B and RLTR10B2 retrotransposons, the whole sex chromosomes and the piRNA clusters are markedly enriched of H3.3. Taken together, our data dissect the molecular mechanism of H3.3B functions during spermatogenesis and demonstrate that H3.3B, depending on its chromatin localization, is involved in either up-regulation or down-regulation of expression of defined large chromatin regions.

Molecular Mechanism of Nucleosome Recognition by the Pioneer Transcription Factor Sox.

Pioneer transcription factors (PTFs) have the remarkable ability to directly bind to chromatin to stimulate vital cellular processes. In this work, we dissect the universal binding mode of Sox PTF by combining extensive molecular simulations and physiochemistry approaches, along with DNA footprinting techniques. As a result, we show that when Sox consensus DNA is located at the solvent-facing DNA strand, Sox binds to the compact nucleosome without imposing any significant conformational changes. We also reveal that the base-specific Sox:DNA interactions (base reading) and Sox-induced DNA changes (shape reading) are concurrently required for sequence-specific nucleosomal DNA recognition. Among three different nucleosome positions located on the positive DNA arm, a sequence-specific reading mechanism is solely satisfied at the superhelical location 2 (SHL2). While SHL2 acts transparently for solvent-facing Sox binding, among the other two positions, SHL4 permits only shape reading. The final position, SHL0 (dyad), on the other hand, allows no reading mechanism. These findings demonstrate that Sox-based nucleosome recognition is essentially guided by intrinsic nucleosome properties, permitting varying degrees of DNA recognition.

iNucs: inter-nucleosome interactions.

MOTIVATION: Deciphering nucleosome-nucleosome interactions is an important step toward mesoscale description of chromatin organization but computational tools to perform such analyses are not publicly available. RESULTS: We developed iNucs, a user-friendly and efficient Python-based bioinformatics tool to compute and visualize nucleosome-resolved interactions using standard pairs format input generated from pairtools. AVAILABILITYAND IMPLEMENTATION: https://github.com/Karimi-Lab/inucs/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Phase-plate cryo-EM structure of the Widom 601 CENP-A nucleosome core particle reveals differential flexibility of the DNA ends.

The histone H3 variant CENP-A marks centromeres epigenetically and is essential for mitotic fidelity. Previous crystallographic studies of the CENP-A nucleosome core particle (NCP) reconstituted with a human α-satellite DNA derivative revealed both DNA ends to be highly flexible, a feature important for CENP-A mitotic functions. However, recent cryo-EM studies of CENP-A NCP complexes comprising primarily Widom 601 DNA reported well-ordered DNA ends. Here, we report the cryo-EM structure of the CENP-A 601 NCP determined by Volta phase-plate imaging. The data reveal that one ('left') 601 DNA end is well ordered whereas the other ('right') end is flexible and partly detached from the histone core, suggesting sequence-dependent dynamics of the DNA termini. Indeed, a molecular dynamics simulation of the CENP-A 601 NCP confirmed the distinct dynamics of the two DNA extremities. Reprocessing the image data using two-fold symmetry yielded a cryo-EM map in which both DNA ends appeared well ordered, indicating that such an artefact may inadvertently arise if NCP asymmetry is lost during image processing. These findings enhance our understanding of the dynamic features that discriminate CENP-A from H3 nucleosomes by revealing that DNA end flexibility can be fine-tuned in a sequence-dependent manner.

H2A.Z is dispensable for both basal and activated transcription in post-mitotic mouse muscles.

While the histone variant H2A.Z is known to be required for mitosis, it is also enriched in nucleosomes surrounding the transcription start site of active promoters, implicating H2A.Z in transcription. However, evidence obtained so far mainly rely on correlational data generated in actively dividing cells. We have exploited a paradigm in which transcription is uncoupled from the cell cycle by developing an in vivo system to inactivate H2A.Z in terminally differentiated post-mitotic muscle cells. ChIP-seq, RNA-seq and ATAC-seq experiments performed on H2A.Z KO post-mitotic muscle cells show that this histone variant is neither required to maintain nor to activate transcription. Altogether, this study provides in vivo evidence that in the absence of mitosis H2A.Z is dispensable for transcription and that the enrichment of H2A.Z on active promoters is a marker but not an active driver of transcription.

Centromeric and ectopic assembly of CENP-A chromatin in health and cancer: old marks and new tracks.

The histone H3 variant CENP-A confers epigenetic identity to the centromere and plays crucial roles in the assembly and function of the kinetochore, thus ensuring proper segregation of our chromosomes. CENP-A containing nucleosomes exhibit unique structural specificities and lack the complex profile of gene expression-associated histone posttranslational modifications found in canonical histone H3 and the H3.3 variant. CENP-A mislocalization into noncentromeric regions resulting from its overexpression leads to chromosomal segregation aberrations and genome instability. Overexpression of CENP-A is a feature of many cancers and is associated with malignant progression and poor outcome. The recent years have seen impressive progress in our understanding of the mechanisms that orchestrate CENP-A deposition at native centromeres and ectopic loci. They have witnessed the description of novel, heterotypic CENP-A/H3.3 nucleosome particles and the exploration of the phenotypes associated with the deregulation of CENP-A and its chaperones in tumor cells. Here, we review the structural specificities of CENP-A nucleosomes, the epigenetic features that characterize the centrochromatin and the mechanisms and factors that orchestrate CENP-A deposition at centromeres. We then review our knowledge of CENP-A ectopic distribution, highlighting experimental strategies that have enabled key discoveries. Finally, we discuss the implications of deregulated CENP-A in cancer.

Combinatorial DNA methylation codes at repetitive elements.

DNA methylation is an essential epigenetic modification, present in both unique DNA sequences and repetitive elements, but its exact function in repetitive elements remains obscure. Here, we describe the genome-wide comparative analysis of the 5mC, 5hmC, 5fC, and 5caC profiles of repetitive elements in mouse embryonic fibroblasts and mouse embryonic stem cells. We provide evidence for distinct and highly specific DNA methylation/oxidation patterns of the repetitive elements in both cell types, which mainly affect CA repeats and evolutionarily conserved mouse-specific transposable elements including IAP-LTRs, SINEs B1m/B2m, and L1Md-LINEs. DNA methylation controls the expression of these retroelements, which are clustered at specific locations in the mouse genome. We show that TDG is implicated in the regulation of their unique DNA methylation/oxidation signatures and their dynamics. Our data suggest the existence of a novel epigenetic code for the most recently acquired evolutionarily conserved repeats that could play a major role in cell differentiation.

ANP32E is a histone chaperone that removes H2A.Z from chromatin.

H2A.Z is an essential histone variant implicated in the regulation of key nuclear events. However, the metazoan chaperones responsible for H2A.Z deposition and its removal from chromatin remain unknown. Here we report the identification and characterization of the human protein ANP32E as a specific H2A.Z chaperone. We show that ANP32E is a member of the presumed H2A.Z histone-exchange complex p400/TIP60. ANP32E interacts with a short region of the docking domain of H2A.Z through a new motif termed H2A.Z interacting domain (ZID). The 1.48 Å resolution crystal structure of the complex formed between the ANP32E-ZID and the H2A.Z/H2B dimer and biochemical data support an underlying molecular mechanism for H2A.Z/H2B eviction from the nucleosome and its stabilization by ANP32E through a specific extension of the H2A.Z carboxy-terminal α-helix. Finally, analysis of H2A.Z localization in ANP32E(-/-) cells by chromatin immunoprecipitation followed by sequencing shows genome-wide enrichment, redistribution and accumulation of H2A.Z at specific chromatin control regions, in particular at enhancers and insulators.

FACT Assists Base Excision Repair by Boosting the Remodeling Activity of RSC.

FACT, in addition to its role in transcription, is likely implicated in both transcription-coupled nucleotide excision repair and DNA double strand break repair. Here, we present evidence that FACT could be directly involved in Base Excision Repair and elucidate the chromatin remodeling mechanisms of FACT during BER. We found that, upon oxidative stress, FACT is released from transcription related protein complexes to get associated with repair proteins and chromatin remodelers from the SWI/SNF family. We also showed the rapid recruitment of FACT to the site of damage, coincident with the glycosylase OGG1, upon the local generation of oxidized DNA. Interestingly, FACT facilitates uracil-DNA glycosylase in the removal of uracil from nucleosomal DNA thanks to an enhancement in the remodeling activity of RSC. This discloses a novel property of FACT wherein it has a co-remodeling activity and strongly enhances the remodeling capacity of the chromatin remodelers. Altogether, our data suggest that FACT may acts in concert with RSC to facilitate excision of DNA lesions during the initial step of BER.

H1-nucleosome interactions and their functional implications.

Linker histones are three domain proteins and consist of a structured (globular) domain, flanked by two likely non-structured NH2- and COOH-termini. The binding of the linker histones to the nucleosome was characterized by different methods in solution. Apparently, the globular domain interacts with the linker DNA and the nucleosome dyad, while the binding of the large and rich in lysines COOH-terminus results in "closing" the linker DNA of the nucleosome and the formation of the "stem" structure. What is the mode of binding of the linker histones within the chromatin fiber remains still elusive. Nonetheless, it is clear that linker histones are essential for both the assembly and maintenance of the condensed chromatin fiber. Interestingly, linker histones are post-translationally modified and how this affects both their binding to chromatin and functions is now beginning to emerge. In addition, linker histones are highly mobile in vivo, but not in vitro. No explanation of this finding is reported for the moment. The higher mobility of the linker histones should, however, have strong impact on their function. Linker histones plays an important role in gene expression regulation and other chromatin related process and their function is predominantly regulated by their posttranslational modifications. However, the detailed mechanism how the linker histones do function remains still not well understood despite numerous efforts. Here we will summarize and analyze the data on the linker histone binding to the nucleosome and the chromatin fiber and will discuss its functional consequences.

Histone H3.3 regulates mitotic progression in mouse embryonic fibroblasts.

H3.3 is a histone variant that marks transcription start sites as well as telomeres and heterochromatic sites on the genome. The presence of H3.3 is thought to positively correlate with the transcriptional status of its target genes. Using a conditional genetic strategy against H3.3B, combined with short hairpin RNAs against H3.3A, we essentially depleted all H3.3 gene expression in mouse embryonic fibroblasts. Following nearly complete loss of H3.3 in the cells, our transcriptomic analyses show very little impact on global gene expression or on the localization of histone variant H2A.Z. Instead, fibroblasts displayed slower cell growth and an increase in cell death, coincident with large-scale chromosome misalignment in mitosis and large polylobed or micronuclei in interphase cells. Thus, we conclude that H3.3 may have an important under-explored additional role in chromosome segregation, nuclear structure, and the maintenance of genome integrity.

Phosphorylation of the CENP-A amino-terminus in mitotic centromeric chromatin is required for kinetochore function.

The role of the mitotic phosphorylation of the amino (NH2) terminus of Centromere Protein A (CENP-A), the histone variant epigenetic centromeric marker, remains elusive. Here, we show that the NH2 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 NH2 terminus, and is independent of the CENP-A NH2 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.

Binding of NF-κB to nucleosomes: effect of translational positioning, nucleosome remodeling and linker histone H1.

NF-κB is a key transcription factor regulating the expression of inflammatory responsive genes. How NF-κB binds to naked DNA templates is well documented, but how it interacts with chromatin is far from being clear. Here we used a combination of UV laser footprinting, hydroxyl footprinting and electrophoretic mobility shift assay to investigate the binding of NF-κB to nucleosomal templates. We show that NF-κB p50 homodimer is able to bind to its recognition sequence, when it is localized at the edge of the core particle, but not when the recognition sequence is at the interior of the nucleosome. Remodeling of the nucleosome by the chromatin remodeling machine RSC was not sufficient to allow binding of NF-κB to its recognition sequence located in the vicinity of the nucleosome dyad, but RSC-induced histone octamer sliding allowed clearly detectable binding of NF-κB with the slid particle. Importantly, nucleosome dilution-driven removal of H2A-H2B dimer led to complete accessibility of the site located close to the dyad to NF-κB. Finally, we found that NF-κB was able to displace histone H1 and prevent its binding to nucleosome. These data provide important insight on the role of chromatin structure in the regulation of transcription of NF-κB dependent genes.

Histone H3 trimethylation at lysine 36 is associated with constitutive and facultative heterochromatin.

The mammalian genome contains numerous regions known as facultative heterochromatin, which contribute to transcriptional silencing during development and cell differentiation. We have analyzed the pattern of histone modifications associated with facultative heterochromatin within the mouse imprinted Snurf-Snrpn cluster, which is homologous to the human Prader-Willi syndrome genomic region. We show here that the maternally inherited Snurf-Snrpn 3-Mb region, which is silenced by a potent transcription repressive mechanism, is uniformly enriched in histone methylation marks usually found in constitutive heterochromatin, such as H4K20me3, H3K9me3, and H3K79me3. Strikingly, we found that trimethylated histone H3 at lysine 36 (H3K36me3), which was previously identified as a hallmark of actively transcribed regions, is deposited onto the silenced, maternally contributed 3-Mb imprinted region. We show that H3K36me3 deposition within this large heterochromatin domain does not correlate with transcription events, suggesting the existence of an alternative pathway for the deposition of this histone modification. In addition, we demonstrate that H3K36me3 is markedly enriched at the level of pericentromeric heterochromatin in mouse embryonic stem cells and fibroblasts. This result indicates that H3K36me3 is associated with both facultative and constitutive heterochromatin. Our data suggest that H3K36me3 function is not restricted to actively transcribed regions only and may contribute to the composition of heterochromatin, in combination with other histone modifications.