Danièle Roche

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.

HJURP is a cell-cycle-dependent maintenance and deposition factor of CENP-A at centromeres.

The histone H3 variant CenH3, called CENP-A in humans, is central in centromeric chromatin to ensure proper chromosome segregation. In the absence of an underlying DNA sequence, it is still unclear how CENP-A deposition at centromeres is determined. Here, we purified non-nucleosomal CENP-A complexes to identify direct CENP-A partners involved in such a mechanism and identified HJURP. HJURP was not detected in H3.1- or H3.3-containing complexes, indicating its specificity for CENP-A. HJURP centromeric localization is cell cycle regulated, and its transient appearance at the centromere coincides precisely with the proposed time window for new CENP-A deposition. Furthermore, HJURP downregulation leads to a major reduction in CENP-A at centromeres and impairs deposition of newly synthesized CENP-A, causing mitotic defects. We conclude that HJURP is a key factor for CENP-A deposition and maintenance at centromeres.

Reversible disruption of pericentric heterochromatin and centromere function by inhibiting deacetylases

Histone modifications might act to mark and maintain functional chromatin domains during both interphase and mitosis. Here we show that pericentric heterochromatin in mammalian cells is specifically responsive to prolonged treatment with deacetylase inhibitors. These defined regions relocate at the nuclear periphery and lose their properties of retaining HP1 (heterochromatin protein 1) proteins. Subsequent defects in chromosome segregation arise in mitosis. All these changes can reverse rapidly after drug removal. Our data point to a crucial role of histone underacetylation within pericentric heterochromatin regions for their association with HP1 proteins, their nuclear compartmentalization and their contribution to centromere function.

New Histone Incorporation Marks Sites of UV Repair in Human Cells

Chromatin organization is compromised during the repair of DNA damage. It remains unknown how and to what extent epigenetic information is preserved in vivo. A central question is whether chromatin reorganization involves recycling of parental histones or new histone incorporation. Here, we devise an approach to follow new histone deposition upon UV irradiation in human cells. We show that new H3.1 histones get incorporated in vivo at repair sites. Remarkably we find that H3.1, which is deposited during S phase, is also incorporated outside of S phase. Histone deposition is dependent on nucleotide excision repair (NER), indicating that it occurs at a postrepair stage. The histone chaperone chromatin assembly factor 1 (CAF-1) is directly involved in the histone deposition process in vivo. We conclude that chromatin restoration after damage cannot rely simply on histone recycling. New histone incorporation at repair sites both challenges epigenetic stability and possibly contributes to damage memory.

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.

The HP1α-CAF1-SetDB1-containing complex provides H3K9me1 for Suv39-mediated K9me3 in pericentric heterochromatin

Trimethylation of lysine 9 in histone H3 (H3K9me3) enrichment is a characteristic of pericentric heterochromatin. The hypothesis of a stepwise mechanism to establish and maintain this mark during DNA replication suggests that newly synthesized histone H3 goes through an intermediate methylation state to become a substrate for the histone methyltransferase Suppressor of variegation 39 (Suv39H1/H2). How this intermediate methylation state is achieved and how it is targeted to the correct place at the right time is not yet known. Here, we show that the histone H3K9 methyltransferase SetDB1 associates with the specific heterochromatin protein 1α (HP1α)–chromatin assembly factor 1 (CAF1) chaperone complex. This complex monomethylates K9 on non‐nucleosomal histone H3. Therefore, the heterochromatic HP1α–CAF1–SetDB1 complex probably provides H3K9me1 for subsequent trimethylation by Suv39H1/H2 in pericentric regions. The connection of CAF1 with DNA replication, HP1α with heterochromatin formation and SetDB1 for H3K9me1 suggests a highly coordinated mechanism to ensure the propagation of H3K9me3 in pericentric heterochromatin during DNA replication.

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.

The effects of histone deacetylase inhibitors on heterochromatin: implications for anticancer therapy?

Histone acetylation regulates many chromosome functions, such as gene expression and chromosome segregation. Histone deacetylase inhibitors (HDACIs) induce growth arrest, differentiation and apoptosis of cancer cells ex vivo, as well as in vivo in tumour‐bearing animal models, and are now undergoing clinical trials as anti‐tumour agents. However, little attention has been paid to how HDACIs function in these biological settings and why different cells respond in different ways. Here, we discuss the consequences of inhibiting histone deacetylases in cycling versus non‐cycling cells, in light of the dynamics of histone acetylation patterns with a specific emphasis on heterochromatic regions of the genome.

Initiation and bidirectional propagation of chromatin assembly from a target site for nucleotide excision repair

To restore full genomic integrity in a eukaryotic cell, DNA repair processes have to be coordinated with the resetting of nucleosomal organization. We have established a cell‐free system using Drosophila embryo extracts to investigate the mechanism linking de novo nucleosome formation to nucleotide excision repair (NER). Closed‐circular DNA containing a uniquely placed cisplatin–DNA adduct was used to follow chromatin assembly specifically from a site of NER. Nucleosome formation was initiated from a target site for NER. The assembly of nucleosomes propagated bidirectionally, creating a regular nucleosomal array extending beyond the initiation site. Furthermore, this chromatin assembly was still effective when the repair synthesis step in the NER process was inhibited.

UVC irradiation, DNA repair and chromatin assembly

The beginning of embryonic life is characterized by a period oi transcriptional silence. In the mouse, polymerase&dependent RNA synthesis resumes at the end of the first cell cycle (Bouniol C., Nguyen E., Debey P. (1995), Exp. Cell Res., 218, 57-62) in a nonregulated manner, the need of enhancer appearing at the Z-cell stage (Majumder S., Miranda M., DePamphiis M.L. (1993), EMBO I., 12, 1131-1140). During the same period, important chromatin remodelling and nuclear reorganization occur. Similar modulation of chromatin structure and activity arti also observed in nuclear transfer experiments. We focused our study on the chromosomal non-histone HMGI protein which interacts with the minor groove of AT-rich double stranded DNA and is suspected to play the general role of a “chromatin architectural factor” (Bustin M., Reeves R. (1996), Prog. Nucleic Acid Res. Mol. Biol., 54, 35-100). We analyzed the possible implicdtion of this protein in transcriptional activity of normal embryos and after reconstitution by nuclear transfers. Cell specific methylation patterns and ievels are mainlained stat& in ihc soma. At the inverse, dramatic changes of DNA-methylation have beea reportedboth in embryo proper and in germ cells. Most of the studies devoted to this subject used mole&u techniques in order to detect chan4ees occuring in DNA of house keeping, tissue specific or imprinted genes. WI& those tbchniques it is impossible to get a synthetic view of the methytation state at the ch&mosomal ltvel, lack of information damaging to further understand phenomenons occurlrrg during gametogpesis, In this aim, we a&ii an in situ approach using-monoclonal anti&dies raised against 5merhyicytosine (5-mC) in order to follow these changes during the initial phase of mouse sp&qi&ogenosis. In this purpose, cytogeneric prepantiiolls were successively stair& by giemsa, DAPI and anti-5mC an&&li~