Jean-Pierre Quivy

Higher-order structure in pericentric heterochromatin involves a distinct pattern of histone modification and an RNA component

Post-translational modification of histone tails is thought to modulate higher-order chromatin structure. Combinations of modifications including acetylation, phosphorylation and methylation have been proposed to provide marks recognized by specific proteins. This is exemplified, in both mammalian cells and fission yeast, by transcriptionally silent constitutive pericentric heterochromatin. Such heterochromatin contains histones that are generally hypoacetylated and methylated by Suv39h methyltransferases at lysine 9 of histone H3 (H3-K9). Each of these modification states has been implicated in the maintenance of HP1 protein–binding at pericentric heterochromatin, in transcriptional silencing and in centromere function. In particular, H3-K9 methylation is thought to provide a marking system for the establishment and maintenance of stably repressed regions and heterochromatin subdomains. To address the question of how these two types of modifications, as well as other unidentified parameters, function to maintain pericentric heterochromatin, we used a combination of histone deacetylase inhibitors, RNAse treatments and an antibody raised against methylated branched H3-K9 peptides. Our results show that both H3-K9 acetylation and methylation can occur on independent sets of H3 molecules in pericentric heterochromatin. In addition, we identify an RNA- and histone modification–dependent structure that brings methylated H3-K9 tails together in a specific configuration required for the accumulation of HP1 proteins in these domains.

HIRA is critical for a nucleosome assembly pathway independent of DNA synthesis.

The mammalian HIRA gene encodes a histone-interacting protein whose homolog in Xenopus laevis is characterized here. In vitro, recombinant Xenopus HIRA bound purified core histones and promoted their deposition onto plasmid DNA. The Xenopus HIRA protein, tightly associated with nuclear structures in somatic cells, was found in a soluble maternal pool in early embryos. Xenopus egg extracts, known for their chromatin assembly efficiency, were specifically immunodepleted for HIRA. These depleted extracts were severely impaired in their ability to assemble nucleosomes on nonreplicated DNA, although nucleosome formation associated with DNA synthesis remained efficient. Furthermore, this defect was largely corrected by reintroduction of HIRA along with (H3-H4)(2) tetramers. We thus delineate a nucleosome assembly pathway that depends on HIRA.

A CAF-1-PCNA-mediated chromatin assembly pathway triggered by sensing DNA damage.

ABSTRACT Sensing DNA damage is crucial for the maintenance of genomic integrity and cell cycle progression. The participation of chromatin in these events is becoming of increasing interest. We show that the presence of single-strand breaks and gaps, formed either directly or during DNA damage processing, can trigger the propagation of nucleosomal arrays. This nucleosome assembly pathway involves the histone chaperone chromatin assembly factor 1 (CAF-1). The largest subunit (p150) of this factor interacts directly with proliferating cell nuclear antigen (PCNA), and critical regions for this interaction on both proteins have been mapped. To isolate proteins specifically recruited during DNA repair, damaged DNA linked to magnetic beads was used. The binding of both PCNA and CAF-1 to this damaged DNA was dependent on the number of DNA lesions and required ATP. Chromatin assembly linked to the repair of single-strand breaks was disrupted by depletion of PCNA from a cell-free system. This defect was rescued by complementation with recombinant PCNA, arguing for role of PCNA in mediating chromatin assembly linked to DNA repair. We discuss the importance of the PCNA–CAF-1 interaction in the context of DNA damage processing and checkpoint control.

A CAF-1 dependent pool of HP1 during heterochromatin duplication

To investigate how the complex organization of heterochromatin is reproduced at each replication cycle, we examined the fate of HP1‐rich pericentric domains in mouse cells. We find that replication occurs mainly at the surface of these domains where both PCNA and chromatin assembly factor 1 (CAF‐1) are located. Pulse–chase experiments combined with high‐resolution analysis and 3D modeling show that within 90 min newly replicated DNA become internalized inside the domain. Remarkably, during this time period, a specific subset of HP1 molecules (α and γ) coinciding with CAF‐1 and replicative sites is resistant to RNase treatment. Furthermore, these replication‐associated HP1 molecules are detected in Suv39 knockout cells, which otherwise lack stable HP1 staining at pericentric heterochromatin. This replicative pool of HP1 molecules disappears completely following p150CAF‐1 siRNA treatment. We conclude that during replication, the interaction of HP1 with p150CAF‐1 is essential to promote delivery of HP1 molecules to heterochromatic sites, where they are subsequently retained by further interactions with methylated H3‐K9 and RNA.

SUMOylation promotes de novo targeting of HP1α to pericentric heterochromatin

HP1 enrichment at pericentric heterochromatin is considered important for centromere function. Although HP1 binding to H3K9me3 can explain its accumulation at pericentric heterochromatin, how it is initially targeted there remains unclear. Here, in mouse cells, we reveal the presence of long nuclear noncoding transcripts corresponding to major satellite repeats at the periphery of pericentric heterochromatin. Furthermore, we find that major transcripts in the forward orientation specifically associate with SUMO-modified HP1 proteins. We identified this modification as SUMO-1 and mapped it in the hinge domain of HP1α. Notably, the hinge domain and its SUMOylation proved critical to promote the initial targeting of HP1α to pericentric domains using de novo localization assays, whereas they are dispensable for maintenance of HP1 domains. We propose that SUMO-HP1, through a specific association with major forward transcript, is guided at the pericentric heterochromatin domain to seed further HP1 localization.

Histone Chaperones: Assisting Histone Traffic and Nucleosome Dynamics

The functional organization of eukaryotic DNA into chromatin uses histones as components of its building block, the nucleosome. Histone chaperones, which are proteins that escort histones throughout their cellular life, are key actors in all facets of histone metabolism; they regulate the supply and dynamics of histones at chromatin for its assembly and disassembly. Histone chaperones can also participate in the distribution of histone variants, thereby defining distinct chromatin landscapes of importance for genome function, stability, and cell identity. Here, we discuss our current knowledge of the known histone chaperones and their histone partners, focusing on histone H3 and its variants. We then place them into an escort network that distributes these histones in various deposition pathways. Through their distinct interfaces, we show how they affect dynamics during DNA replication, DNA damage, and transcription, and how they maintain genome integrity. Finally, we discuss the importance of histone chaperones during development and describe how misregulation of the histone flow can link to disease.

An epigenetic silencing pathway controlling T helper 2 cell lineage commitment

During immune responses, naive CD4+ T cells differentiate into several T helper (TH) cell subsets under the control of lineage-specifying genes. These subsets (TH1, TH2 and TH17 cells and regulatory T cells) secrete distinct cytokines and are involved in protection against different types of infection. Epigenetic mechanisms are involved in the regulation of these developmental programs, and correlations have been drawn between the levels of particular epigenetic marks and the activity or silencing of specifying genes during differentiation. Nevertheless, the functional relevance of the epigenetic pathways involved in TH cell subset differentiation and commitment is still unclear. Here we explore the role of the SUV39H1–H3K9me3–HP1α silencing pathway in the control of TH2 lineage stability. This pathway involves the histone methylase SUV39H1, which participates in the trimethylation of histone H3 on lysine 9 (H3K9me3), a modification that provides binding sites for heterochromatin protein 1α (HP1α) and promotes transcriptional silencing. This pathway was initially associated with heterochromatin formation and maintenance but can also contribute to the regulation of euchromatic genes. We now propose that the SUV39H1–H3K9me3–HP1α pathway participates in maintaining the silencing of TH1 loci, ensuring TH2 lineage stability. In TH2 cells that are deficient in SUV39H1, the ratio between trimethylated and acetylated H3K9 is impaired, and the binding of HP1α at the promoters of silenced TH1 genes is reduced. Despite showing normal differentiation, both SUV39H1-deficient TH2 cells and HP1α-deficient TH2 cells, in contrast to wild-type cells, expressed TH1 genes when recultured under conditions that drive differentiation into TH1 cells. In a mouse model of TH2-driven allergic asthma, the chemical inhibition or loss of SUV39H1 skewed T-cell responses towards TH1 responses and decreased the lung pathology. These results establish a link between the SUV39H1–H3K9me3–HP1α pathway and the stability of TH2 cells, and they identify potential targets for therapeutic intervention in TH2-cell-mediated inflammatory diseases.

The HP1-p150/CAF-1 interaction is required for pericentric heterochromatin replication and S-phase progression in mouse cells.

The heterochromatin protein 1 (HP1)-rich heterochromatin domains next to centromeres are crucial for chromosome segregation during mitosis. This mitotic function requires their faithful reproduction during the preceding S phase, a process whose mechanism and regulation are current puzzles. Here we show that p150, a subunit of chromatin assembly factor 1, has a key role in the replication of pericentric heterochromatin and S-phase progression in mouse cells, independently of its known function in histone deposition. By a combination of depletion and complementation assays in vivo, we link this unique function of p150 to its ability to interact with HP1. Absence of this functional interaction triggers S-phase arrest at the time of replication of pericentromeric heterochromatin, without eliciting known DNA-based checkpoint pathways. Notably, in cells lacking the histone methylases Suv39h, in which pericentric domains do not show HP1 accumulation, p150 is dispensable for S-phase progression.

CAF-1 Is Essential for Heterochromatin Organization in Pluripotent Embryonic Cells

During mammalian development, chromatin dynamics and epigenetic marking are important for genome reprogramming. Recent data suggest an important role for the chromatin assembly machinery in this process. To analyze the role of chromatin assembly factor 1 (CAF-1) during pre-implantation development, we generated a mouse line carrying a targeted mutation in the gene encoding its large subunit, p150CAF-1. Loss of p150CAF-1 in homozygous mutants leads to developmental arrest at the 16-cell stage. Absence of p150CAF-1 in these embryos results in severe alterations in the nuclear organization of constitutive heterochromatin. We provide evidence that in wild-type embryos, heterochromatin domains are extensively reorganized between the two-cell and blastocyst stages. In p150CAF-1 mutant 16-cell stage embryos, the altered organization of heterochromatin displays similarities to the structure of heterochromatin in two- to four-cell stage wild-type embryos, suggesting that CAF-1 is required for the maturation of heterochromatin during preimplantation development. In embryonic stem cells, depletion of p150CAF-1 using RNA interference results in the mislocalization, loss of clustering, and decondensation of pericentric heterochromatin domains. Furthermore, loss of CAF-1 in these cells results in the alteration of epigenetic histone methylation marks at the level of pericentric heterochromatin. These alterations of heterochromatin are not found in p150CAF-1-depleted mouse embryonic fibroblasts, which are cells that are already lineage committed, suggesting that CAF-1 is specifically required for heterochromatin organization in pluripotent embryonic cells. Our findings underline the role of the chromatin assembly machinery in controlling the spatial organization and epigenetic marking of the genome in early embryos and embryonic stem cells.

Dimerization of the largest subunit of chromatin assembly factor 1: importance in vitro and during Xenopus early development

To date, the in vivo importance of chromatin assembly factors during development in vertebrates is unknown. Chromatin assembly factor 1 (CAF‐1) represents the best biochemically characterized factor promoting chromatin assembly during DNA replication or repair in human cell‐free systems. Here, we identify a Xenopus homologue of the largest subunit of CAF‐1 (p150). Novel dimerization properties are found conserved in both Xenopus and human p150. A region of 36 amino acids required for p150 dimerization was identified. Deletion of this domain abolishes the ability of p150 to promote chromatin assembly in vitro. A dominant‐negative interference based on these dimerization properties occurs both in vitro and in vivo. In the embryo, nuclear organization was severely affected and cell cycle progression was impaired during the rapid early cleaving stages of Xenopus development. We propose that the rapid proliferation at early developmental stages necessitates the unique properties of an assembly factor that can ensure a tight coupling between DNA replication or repair and chromatin assembly.