PADI4 Regulates HP1 Binding to Histone H3 in MS Figure 1. HP1a involved in silencing of HERVs and cytokines. (A) HP1a is present on the promoter of HERVs and cytokines in MCF7 cells. Chromatin prepared from MCF7 cells were subjected to ChIP using anti-HP1a antibodies. The relative enrichment was measured by qPCR on Satellite 2 sequences, the LTR regions of shown HERVs and on the promoter regions of TNFa, pS2 and GAPDH. Data are normalized to the values obtained with non-immune IgGs and expressed relatively to GAPDH (set to 1). Shown data are means +/2 SEM from four experiments. (B and C) Total RNA from MCF7 cells transfected with the indicated small interfering RNAs (siRNAs) was quantified with RT-qPCR. Changes in mRNA levels are shown relative to the siGAPDH transfection (set to 1), which was not affecting the mRNA levels of the genes of interest (Figure. S1B). The data are presented as the means 6 SEM of triplicate experiments. doi:10.1371/journal.pgen.1002934.g001 double modification when the cells were expressing WT or hyperactive versions of PADI4. Under these conditions, we also observed a general increase in histone H3 citrullination, but no change in the levels of H3K9me3, indicating that H3R8 citrullination and H3K9 tri-methylation are not antagonistic. Taken together, these experiments demonstrate that the H3cit8K9me3 double modification exists in vivo and that its formation is favored by increased PADI4 activity. Citrullination of H3R8 inhibits binding of HP1a to the histone H3 tail We next investigate the impact of the H3R8 citrullination on the binding of HP1a to histone H3 tails tri-methylated on K9. When histone H3 peptides were spotted on membrane, HP1a bound a peptide carrying the single K9me3 modification, but not a peptide with a double cit8K9me3 modification (Figure 3A). We also tested the ability of these peptides to interfere with the binding of HP1a to its endogenous target sites. For this, we took advantage of the fact that recombinant GST-HP1a protein incubated on fixed permeabilized cells distributes in a pattern indistinguishable from that of the endogenous protein [64]. In this assay, while H3R8K9me3 peptide competed with the cellular sites for GST- HP1a binding, H3cit8K9me3 peptide did not (Figure 3B). As expected, H3cit8K9 and H3R8K9 peptides also failed to compete for GST-HP1a binding. Surface plasmon resonance allowed us to quantify the effect of H3R8 citrullination and indicated a more than 200-fold decrease in the affinity of HP1a for H3cit8K9me3 compared to H3R8K9me3 (Kd 313628 mM and 1.3960.06 mM, respectively; Figure 3C–3D). We noted also that citrullination of H3R8 exerted a 10-fold higher effect on HP1a binding than did its methylation (Kd 29.661.3 mM). To document that citrullination compromises transcriptional repression of HERVs and cytokines, we finally used siRNAs against PADI4 in the MCF7 cells known to express relatively high levels of PADI4 [65]. Depletion of this protein had an effect inverse to that of HP1a depletion and resulted in decreased levels of HERV transcripts (Figure 3E, Figure S1A and S1C). Levels of Satellite 2 transcripts (but not a-Satellite transcripts) were also decreased, suggesting a broad yet selective effect of citrullination on the silencing of repeats. PADI4 depletion also decreased expression of the immune genes TNFa, IL16, and IL8, as well as IL23 and IL1A, but not TGFß1 (Figure 3F). The control pS2 gene known to be negatively regulated by PADI4 [44,45], was, as expected, moderately stimulated. Endogenous PADI activity can be artificially regulated and directly controls HP1a-mediated repression MCF7 cells are estrogen-responsive and an estradiol (E2) treatment combined with an ionophore increases total levels of both PADI4 (Figure S2A and [65]) and H3cit8K9me3 modifica- tion (Figure 4A, compare lanes 1 and 2). In contrast, reduced PADI activity can be obtained with the specific inhibitor Cl- amidine [66]. This drug affects the nuclear PADI4 as illustrated by the decreased levels of H3Cit8K9me3 in MCF7 cells (Figure 4A; compare lanes 1 and 3). Thus, treatment with either E2/A23187 or Cl-amidine allowed us to control endogenous nuclear PADI activity at will. As in the PADI4-depletion experiments, treatment of the MCF7 cells with Cl-amidine reduced expression of HER V-H/ env62, ERVWE1, and the selected cytokine genes, but not a- Satellite and TGFb1 (Figure 4B, black bars). Inversely, augment- ing PADI4 activity by treating the MCF7 cells with E2 and ionophore resulted in a Cl-amidine-sensitive increase in expres- sion of the same HERVs and cytokines (Figure 4B, grey and white bars & S2B). Finally, we performed ChIP to follow the impact of endoge- nous nuclear PADI activity on the citrullination of histone H3 and the recruitment of HP1a to the LTRs of HERV-H and ERVWE1 (HERV-W/LTR) and the promoter of TNFa. These assays confirmed that stimulation of PADI4 activity with E2 and ionophore locally increases levels of citrullinated histone H3, as detected with either anti-H3cit (2, 8, 17) or anti-H3cit8K9me3 antibody (Figure 4C–4D and Figure S2C), while the levels of HP1a recruitment were markedly decreased (Figure 4E, black and grey bars). These levels of HP1a occupancy were partially restored by further treating the cells with Cl-amidine (Figure 4E, white bars), illustrating that this inhibitor can overcome the detrimental effect of excessive PADI activity. The level of HP1a recruitment at the pS2 control promoter region was low and was not substantially affected by changes in citrullination levels (Figure 4C–4E). Taken together, these results demonstrate that HP1a and citrullination antagonistically regulate several immune genes and HERVs, and that this regulation is druggable. PADI activity is required for normal activation of T cells An inflammatory response can be induced in Jurkat T cells stimulated with an ionophore and the phorbol ester PMA. The stimulation of the Jurkat cells correlates with an eviction of HP1a from the promoter region of TNFa and IL8 (Figure 5A), and also from HERV-H/LTR62 and HERV-W/LTR (Figure S3A). The treatment also results in an approx. 6-fold increase of PADI4 accumulation (Figure 5B) and is expected to increase PADI activity as a consequence of the ionophore-induced calcium influx. We therefore used this system to determine whether increased PADI activity is associated with normal transcriptional activation of immune genes. Stimulation of the Jurkat cells resulted in a rapid and very transient recruitment of PADI4 to the promoters of TNFa and IL8, and at HERV-H/LTR62 and HERV-W/LTR (Figure S3B). This recruitment correlated with increase levels of H3Cit8K9me3 at these positions (Figure 5C and Figure S3C). Finally, inhibition of PADI activity with Cl-amidine reduced the kinetic and the abundance of TNFa and IL8 mRNA accumulation in Jurkat cells stimulated by ionomycin and PMA (Figure 5D and Figure S3D). Together, these observations suggest that PADI activity participates in the modification of the epigenetic landscape at the promoter of immune genes upon stimulation of T cells, and thereby facilitate the transcriptional activation of these genes. PLOS Genetics | www.plosgenetics.org 4 September 2012 | Volume 8 | Issue 9 | e1002934