Author Summary 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 chro- matin factors. In this study, we have searched for regulatory mechanisms shared between the cytokines and the retroviral sequences. We find that the repressor protein HP1a 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 HP1a 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. long terminal repeat (LTR) that shares several regulatory mechanisms with immune genes [35,36,37]. Consistent with their role in the transcriptional control of inducible genes, the binding of HP1 proteins to chromatin is subject to regulation. In particular, the methylation mark on histone H3 can be removed by histone demethylases [38]. A more transient regulation of HP1 binding may occur by modification of residues neighboring H3K9, including phosphorylation of serine 10 and acetylation of lysine 14 [39,40,41]. The histone H3 arginine 8 (H3R8) located immediately upstream of H3K9, is also subject to modifications [42] that theoretically could interfere with HP1 binding, although this has never been investigated. This arginine can be either methylated [43] or converted into the non- coding amino acid citrulline [44,45,46,47,48]. Citrullination of H3R8 is catalyzed by the calcium-dependent peptidylarginine deiminase PADI4. This enzyme, that is the only member of its family to enter the nucleus, also citrullinates histone H3 on arginines 2, 17, and 26, as well as histones H2A and H4 on their respective arginine 3 [44,45,46,49]. Many reports describe PADI4 as a regulator of transcription. On p53 targets [50,51], and estrogen-regulated genes, including pS2 [44,45,52], it functions as a repressor either by interfering with activating arginine-methyl- ation events [44], or by favoring recruitment of HDACs [52]. Inversely, PADI4 also associates with a number of transcriptionally active promoters and functions as an activator of c-Fos via a mechanism that involves facilitated phosphorylation of the ETS- domain protein Elk-1 [53]. In Asians, hyperactivity of PADI4 has been associated with Rheumatoid Arthritis [54,55,56], and contributes to the generation of antibodies directed against citrullinated proteins during the development of this disease [57]. An earlier study has also reported increased nuclear localization of this enzyme in white brain matter of MS patients [58] The transcriptional deregulation of both cytokines and HERVs in MS patient T cells prompted us to investigate a possibly co- regulation of these two types of transcription units by HP1 proteins. At cytokine genes and HERVs we examined in tissue culture cells, transcriptional repression required HP1a, while PADI4 functioned as an activator by destroying the HP1a binding site on the tail of histone H3. Consistent with this, we observed PADI4 Regulates HP1 Binding to Histone H3 in MS that in circulating blood cells from MS patients, recruitment of HP1a to the promoter of the master cytokine TNFa and to HERV sequences is significantly reduced, while citrullination of H3R8 at these positions is increased. Taken together, our data strongly suggest that increased citrullination of histone H3 can antagonize gene-specific chromatin-mediated silencing in T cells and thereby participate in increased cytokine expression during the normal inflammatory response and in MS patients. Results The transcriptional regulator HP1a is shared by HERVs and cytokines While several reports describe an implication of HP1 proteins in the regulation of genes involved in immune defense [29,30,31,32,33,34], the role of these proteins in the silencing of HERVs in human cells needed to be clarified. We carried out these experiments in MCF7 cells, a breast tumor-derived cell line frequently used to examine expression of HERVs (see for example [59]). Chromatin Immunoprecipitation (ChIP) assays demonstrated that in these cells, HP1a accumulates on HERV- K, HERV-W, and HERV-H promoters at levels similar to those observed on Satellite-2 sequences (Figure 1A). As expected, HP1a was also detected on the promoters of cytokines TNFa and IL8. Consistent with this, depletion of HP1a with small interfering RNAs (siRNAs) resulted in increased expression of the HERVH/ env62, HERVH/env59, HERVH/env60, ERVWE1, HERVK/ env102, TNFa, IL8, and IL16, a cytokine also relevant for MS [60] (Figure 1B, Figure S1A and S1B). In these experiments, expression of IL23 [61] was unaffected, while the control estrogen-responsive pS2 gene was repressed rather than activated. Reactivation of HERVs and TNFa was also observed upon depletion of HP1b and HP1c, two other members of the HP1 family (Figure 1C). PADI4 citrullinates the binding site of HP1a on histone H3 in vivo HP1a binds tri-methylated histone H3 lysine 9 (H3K9me3). The neighboring H3R8 residue is one of the 3 arginines recognized by the anti-H3cit (2, 8, 17) antibody used to show increased histone H3 citrullination in MS patients [58]. This raised a possibility of interference between citrullination of H3R8 and HP1a binding to histone H3. We therefore explored whether citrullination of histone H3R8 occurs in vivo on histone H3 tails already tri-methylated on K9. To this end, we generated an antibody recognizing the double citrullination-methylation mod- ification H3cit8K9me3 (Figure 2A). The specificity of this antibody was verified by dot blots using synthetic peptides mimicking modified histone tails (Figure 2B) and by testing the ability of the same peptides to compete with the binding of the antibody to cellular targets in fixed breast cancer- derived MCF7 cells (Figure 2C). In the later assay, the anti- H3cit8K9me3 antibody yielded an immunofluorescent staining very similar to that obtained with anti-H3cit(2,8,17) antibody (Figure 2D). We next focused our attention on PADI4, the partially nuclear peptidylarginine deiminase responsible for the citrullination of histone H3. To determine whether this enzyme can citrullinate H3R8 when H3K9 is methylated, we generated HEK293-derived cell lines expressing either wild-type (WT), hyperactive [62], or hypoactive [63] versions of PADI4 under the control of a ponasterone-inducible promoter (Figure 2E). Induction of PADI4 synthesis and activity with ponasterone and the ionophore A23187, respectively, allowed detection of the H3cit8K9me3 PLOS Genetics | www.plosgenetics.org 2 September 2012 | Volume 8 | Issue 9 | e1002934