PADI4 Regulates HP1 Binding to Histone H3 in MS Figure 2. An antibody specific for histone H3cit8K9me3 reveals increased levels of this double modification catalyzed by PADI4. (A) Epitope used to generate the anti-H3cit8K9me3 antibody. The antibody was raised against a peptide mimicking histone H3 citrullinated at position 8 and tri-methylated on K9. (B) Dot blot highlighting the specificity of the anti-H3cit8K9me3, performed with 1, 0.2, and 0.04 mg of H3 peptides bearing the indicated post-translational modifications. (C) Specificity of the anti-H3cit8K9me3 antibody test by competition with H3 peptides. MCF7 cells were extracted, fixed and incubated with the indicated peptides along with anti-H3cit8K9me3 antibodies and DAPI. Scale bar 5 mm. (D) Similar staining with anti-H3cit8K9me3 and anti-H3cit(2,8,17) antibodies. Indirect immunofluorescent staining of MCF7 cells with DAPI to visualize the DNA and either anti-H3cit8K9me3 (b1–b2) or anti-H3cit(2,8,17) (b3–b4). (E) HEK293 cells that stably integrate a ponasterone A-inducible construct with either no insert (NI) or an insert coding for wild-type (WT), hyperactive (Mut+), or hypoactive (Mut2) PADI4 were cultured without or with ionophore (A23187). Total protein extracts were immunoblotted with the indicated antibodies. Signals were quantified with the Image J software (NI without treatment set to 1). doi:10.1371/journal.pgen.1002934.g002 Reduced recruitment of HP1a on the promoter of the TNFa gene and on several HERVs in MS patients To investigate whether levels of HP1a recruitment and H3cit8K9me3 double modification at HERV and cytokine promoters were affected in MS, we collected PBMCs from 18 families, each family consisting of one MS patient and a genetically related healthy control (Table S1). The patients suffered from either relapsing-remitting (n = 10), or secondary progressive (n = 8) MS. As PBMCs yield only minute amounts of chromatin, our analysis was restricted to the LTRs of the unique HERV-H locus LTR59 [67] and the unique HERV-W locus ERVWE1 [68], and to the promoter of the cytokine TNFa. Consistent with previous observations [4,5,6,7], transcription of these loci was significantly augmented in the MS patients (Figure 6A–6B). ChIP assays revealed that recruitment of HP1a to the TNFa and the examined HERV promoters was significantly reduced in the MS patients compared to their genetically related healthy controls, while recruitment to a control promoter (RPLP0) was unchanged (Figure 6C). We also observed a significant correlation between HP1a levels on the TNFa promoter and HP1a levels on the LTRs of the HERVs, further suggesting that a single pathway regulates HP1a binding to both types of transcription units (Figure S4). In ChIP assays, the anti-H3cit8K9me3 antibody was functional, but with a relatively poor sensitivity. Therefore, our analysis was restricted to the 9 families (9 patients and their respective healthy relatives) from whom we had the most abundant material. In these samples, levels of H3cit8K9me3 at the promoters of HERV-W/ ERVWE1 and TNFa were significantly increased in the patients when compared to the genetically related healthy controls (Figure 6D–6E). On HERV-H/LTR59, levels of H3cit8K9me3 also appeared increased in the patients, but the p value associated with this data (0.06) is above the significance level of 0.05. We finally questioned whether PADI4 could be linked to the increased levels of H3cit8K9me3. To this end, we examined PBMCs collected from the 18 families described above. Analysis of these samples by RT-PCR showed that PADI4 mRNA levels were significantly elevated in MS patients compared to the genetically related healthy controls (approx. 1.5-fold, Figure 6F). Altogether, these experiments showed that in the patients, increased expression HERV-W/ERVWE1 and TNFa transcripts and decreased recruitment of HP1a at their promoter region is accompanied by a local increase in H3R8 citrullination and a moderate up-regulation of PADI4 expression. Discussion In this report, we show that dependence on HP1a-mediated silencing is a common denominator between cytokines and HERVs, both expressed at abnormally high levels in T cells from MS patients, and we suggest that a decreased efficiency of the HP1-mediated silencing may participate in the pathological deregulation of these transcription units. In this context, we find that one source of defective HP1- mediated silencing is citrullination of H3R8. This histone modification reduces the affinity of the chromo domain of the HP1 proteins to the methylated histone H3K9 residue and thereby defines a novel mechanism regulating HP1-binding to chromatin. Using an antibody specifically recognizing H3cit8K9me3, we show that this double modification is induced in the presence of elevated levels of PADI4, the only known nuclear peptidylarginine deiminase. Interestingly, when an inflammatory response is induced in Jurkat T cells, expression of PADI4 is increased and levels of H3cit8K9me3 rise at the promoters of the immune genes IL8 and TNFa. Under these conditions, inhibiting PADI activity with the chemical inhibitor Cl-amidine results in reduced kinetic and amplitude in the activation of the two immune genes. These observations show that PADI activity and citrullination of histone H3 are required for normal activation of immune genes and define PADI4 as a novel regulator of cytokine expression. We speculate that H3cit8, together with other histone modifications such as H3S10 and H3S28 phosphorylation participate in creating an epigenetic landscape favorable for the transcriptional activation of a subset of immune genes. PADI4 activity could also be artificially increased in MCF7 cells treated with estradiol and an ionophore. This allowed us to show that abnormally elevated levels of PADI activity result in transcriptional stimulation of several immune genes. Consistent with this, PBMCs collected from MS patients (and compared to healthy relatives) showed in average increased expression of TNFa, increased levels of H3cit8K9me3 at the promoter of this gene, and increased expression of PADI4. These observations strongly suggest that inappropriate activity of PADI4 can participate in the deregulation of immune genes relevant for MS (see model Figure 7). We here note that the estrogen/ionophore treatment inducing PADI4 expression in MCF7 cells also stimulated production of this enzyme in Jurkat T cells (data not shown). We therefore speculate that PADI4 could be involved in the activating effect of estrogen on TNFa expression observed in T cells under some conditions ([69] and references therein) and could thereby play a role in the higher incidence of MS in females [70]. Other enzymes affecting the affinity of HP1 proteins for chromatin may also be good candidates for an implication in MS. For example, Jak2 that is expressed at increased levels in MS Th17 cells [71] also cause exclusion of HP1a from chromatin by phosphorylating H3Y41, a residue contacted by the C-terminal region of the HP1 proteins [72]. Along the same lines, we note that levels of arginine methylation of myelin basic protein MBP is increased in MS patients [73], while we find that methylation of H3R8 reduces affinity of HP1a for the neighboring methylated H3K9 approximately 10-fold. Possibly, the same arginine meth- ylases may be involved in the modification of both MBP and histones. In addition, PADI4 has earlier been described as involved in arginine demethylation, although methylated arginines are rather poor substrates for this enzyme in vitro [44,45,47]. PLOS Genetics | www.plosgenetics.org 6 September 2012 | Volume 8 | Issue 9 | e1002934