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Histone H3 lysine 9 methylation is associated with long‐term transcriptional repression through recruitment of heterochromatin protein 1 (HP1) proteins. These proteins are believed to promote the formation of dense chromatin structures interfering with DNA accessibility. During the G2 phase of the cell cycle, HP1 proteins are delocalized from foci of pericentromeric heterochromatin, while a wave of H3 serine 10 phosphorylation is initiated within these regions. Here, we show that in vivo phosphorylation of serine 10 in G2 can occur on histone tails methylated on lysine 9. Unexpectedly, this modification favours rather than prevents HP1 binding to chromatin. Dissociation of HP1 from the methylated histone H3 tails is observed only after a third modification by acetylation of lysine 14, which occurs in prophase. We propose that phosphoacetylation of histone H3 could be a general mechanism allowing the cell to overcome HP1‐mediated transcriptional repression.

Multiple Sclerosis (MS) is an autoimmune disease associated with abnormal expression of a subset of cytokines, resulting in inappropriate T-lymphocyte activation and uncontrolled immune response. A key issue in the field is the need to understand why these cytokines are transcriptionally activated in the patients. Here, we have examined several transcription units subject to pathological reactivation in MS, including the TNFα and IL8 cytokine genes and also several Human Endogenous RetroViruses (HERVs). We find that both the immune genes and the HERVs require the heterochromatin protein HP1α for their transcriptional repression. We further show that the Peptidylarginine Deiminase 4 (PADI4), an enzyme with a suspected role in MS, weakens the binding of HP1α to tri-methylated histone H3 lysine 9 by citrullinating histone H3 arginine 8. The resulting de-repression of both cytokines and HERVs can be reversed with the PADI-inhibitor Cl-amidine. Finally, we show that in peripheral blood mononuclear cells (PBMCs) from MS patients, the promoters of TNFα, and several HERVs share a deficit in HP1α recruitment and an augmented accumulation of histone H3 with a double citrulline 8 tri-methyl lysine 9 modifications. Thus, our study provides compelling evidence that HP1α and PADI4 are regulators of both immune genes and HERVs, and that multiple events of transcriptional reactivation in MS patients can be explained by the deficiency of a single mechanism of gene silencing. In patients suffering from Multiple Sclerosis (MS), T lymphocytes express abnormally high levels of a subset of cytokines. The same cells also transcribe a series of vestigial retroviral sequences normally silenced by chromatin factors. In this study, we have searched for regulatory mechanisms shared between the cytokines and the retroviral sequences. We find that the repressor protein HP1α is present on the promoter of both types of transcription units in normal cells and that the recruitment of this protein to these promoters is decreased in MS patients. Furthermore, we show that the delocalization of HP1α from these promoters can be caused by citrullination of histone H3, and we provide evidence indicating that levels of this histone modification is augmented in MS patients. Together our data provide a possible explanation for the simultaneously increased transcriptional activity of cytokines and endogenous retroviruses in MS-patient T lymphocytes and suggest that inhibitors of the enzyme responsible for the increased citrullination of histone H3 could help restore normal levels of cytokine activity in the patients.

The heterochromatin-enriched HP1 proteins play a critical role in regulation of transcription. These proteins contain two related domains known as the chromo- and the chromoshadow-domain. The chromo-domain binds histone H3 tails methylated on lysine 9. However, in vivo and in vitro experiments have shown that the affinity of HP1 proteins to native methylated chromatin is relatively poor and that the opening of chromatin occurring during DNA replication facilitates their binding to nucleosomes. These observations prompted us to investigate whether HP1 proteins have additional histone binding activities, envisioning also affinity for regions potentially occluded by the nucleosome structure. We find that the chromoshadow-domain interacts with histone H3 in a region located partially inside the nucleosomal barrel at the entry/exit point of the nucleosome. Interestingly, this region is also contacted by the catalytic subunits of the human SWI/SNF complex. In vitro, efficient SWI/SNF remodeling requires this contact and is inhibited in the presence of HP1 proteins. The antagonism between SWI/SNF and HP1 proteins is also observed in vivo on a series of interferon-regulated genes. Finally, we show that SWI/SNF activity favors loading of HP1 proteins to chromatin both in vivo and in vitro. Altogether, our data suggest that HP1 chromoshadow-domains can benefit from the opening of nucleosomal structures to bind chromatin and that HP1 proteins use this property to detect and arrest unwanted chromatin remodeling. HP1 proteins are transcriptional regulators frequently associated with gene silencing, a phenomenon involving masking of promoter DNA by dense chromatin. Owing to their chromo-domain, these proteins can read and bind an epigenetic mark that on many non-expressed genes is present on histone H3 at the surface of the nucleosome (the fundamental packing unit of chromatin). However, the binding to this mark does not explain the repressing activity of HP1 proteins. Here, we show that these proteins can establish a second contact with histone H3, independently of the epigenetic mark. This second contact site is located inside the nucleosome, in a position likely to be inaccessible. Interestingly, this site is also contacted by a subunit of the SWI/SNF complex and this contact is required for the ATP-dependent chromatin remodeling catalyzed by SWI/SNF. We provide evidence suggesting that HP1 proteins use the SWI/SNF chromatin remodeling to gain access to the contact site inside the nucleosome and to prevent further remodeling by competing with SWI/SNF for binding at this position. These observations lead us to suggest that HP1 proteins function as gatekeepers on promoters, detecting and stopping unwanted exposure of internal nucleosomal sites.

In mammalian cells, the SWI–SNF chromatin‐remodeling complex is a regulator of cell proliferation, and overexpression of the catalytic subunit Brm interferes with cell cycle progression. Here, we show that treatment with histone deacetylase (HDAC) inhibitors reduces the inhibitory effect of Brm on the growth of mouse fibroblasts. This observation led to the identification of two carboxy‐terminal acetylation sites in the Brm protein. Mutation of these sites into non‐acetylatable sequences increased both the growth‐inhibitory and the transcriptional activities of Brm. We also show that culture in the presence of HDAC inhibitors facilitates the isolation of clones overexpressing Brm. Removal of the HDAC inhibitors from the growth medium of these clones leads to downregulation of cyclin D1. This downregulation is absent in cell transformed by oncogenic ras .