Citrullination of Histone H3 Interferes with HP1- Mediated Transcriptional Repression Priyanka Sharma1, Saliha Azebi1, Patrick England2, Tove Christensen3, Anne´ Møller-Larsen3, Thor Petersen4, Eric Batsche´ 1, Christian Muchardt1* 1 Institut Pasteur, De´partement de Biologie du De´veloppement, CNRS URA2578, Unite´ de Re´gulation Epige´ne´tique, Paris, France, 2 Institut Pasteur, De´partement de Biologie Structurale et Chimie, CNRS UMR3528, Plate-Forme de Biophysique des Macromole´cules et de Leurs Interactions, Paris, France, 3 Department of Biomedicine, Aarhus University, Aarhus, Denmark, 4 Department of Neurology, Aarhus University Hospital, Aarhus, Denmark Abstract 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 TNFa 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 HP1a 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 HP1a 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 TNFa, and several HERVs share a deficit in HP1a 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 HP1a 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. Citation: Sharma P, Azebi S, England P, Christensen T, Møller-Larsen A, et al. (2012) Citrullination of Histone H3 Interferes with HP1-Mediated Transcriptional Repression. PLoS Genet 8(9): e1002934. doi:10.1371/journal.pgen.1002934 Editor: Beth A. Sullivan, Duke University, United States of America Received February 22, 2012; Accepted July 16, 2012; Published September 13, 2012 Copyright: 2012 SHARMA et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported in part by the Agence National pour la Recherche, by the Fondation pour la Recherche Medicale, and by the ARSEP foundation. PS received support from the Sandwich Fellowship Program of the French Embassy in India and from the Institut National de la Sante´ et de la Recherche Me´dicale. TC, AM-L, and TP were supported by the Aase and Ejnar Danielsen Foundation and the Danish MS Society. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: muchardt@pasteur.fr Introduction Multiple Sclerosis (MS) is a progressive inflammatory disease of the central nervous system in which leukocytes and antibodies attack myelin sheaths, resulting in demyelination and ultimately destruction of the axons [1]. Many lines of evidence point at inappropriate activation of T cells as an initiating event of the pathological process, although the mechanism at the root of this T cell activation is still poorly defined (for a recent review see [2]). In MS patients, activation of the T cell population is associated with increased expression of a series of cytokines [3,4]. The abnormally abundant expression of the genes encoding these regulators of the immune system may be a consequence, but also possibly a cause of the activation of the T cells. It is therefore essential to explore the mechanisms that keep these genes in check in normal cells and that may be defective in MS patients. Interestingly, in MS and other autoimmune diseases including Rheumatoid Arthritis and Systemic Lupus Erythematosus, tran- scription of Human Endogenous RetroViruses (HERVs) is also increased in T cells [5,6,7]. HERVs are abundant vestigial retroviral sequences that in healthy cells are largely silenced by the epigenetic mechanisms repressing most repeated DNA sequences. These mechanisms include DNA methylation and histone H3 lysine 9 (H3K9) methylation. DNA methylation favors chromatin compaction by promoting recruitment of histone deacetylases (HDACs) or alternatively by directly reducing the affinity of transcription factors to their cognate DNA binding sites [8]. Consistent with this, deletion of the de novo DNA methyltransferase Dnmt1 results in massive re-expression of ERVs in the mouse embryo [9]. DNA methylation at ERV promoters is particularly high in differentiated mouse cells [10], while it may be partially dispensable in mouse embryonic stem (ES) cells [11]. In these cells, the major source of silencing appears to be H3K9 methylation [12,13,14]. This histone modification is recognized by a number of proteins containing either chromo- [15,16,17, 18,19,20], MBT- [21], PHD- [22], or Tudor- [21,23] domains. Mainly HP1 proteins have been detected on mouse ERV promoter sequences [24,25], although their role in the repression of these sequences in mouse ES cells is still at debate [26]. HP1 proteins are particularly interesting in the context of MS because in addition to their possible function in the silencing of repeated DNA [27,28], they are present on the promoters of a number of genes involved in immune defense, including the immunomodulatory cytokine TNFa [29], the interleukins IL1b [30,31], IL6 [32], and IL8 [33], and several interferon-inducible genes [34]. They also participate in the regulation of the HIV1 PLOS Genetics | www.plosgenetics.org 1 September 2012 | Volume 8 | Issue 9 | e1002934