were cultured in RPMI-1640, all with 10% decomplemented fetal bovine serum (FBS) and 100 U.ml21 penicillin-streptomycin at 37uC in a 5% CO2 incubator. MCF7 cells were treated with 200 nM of estradiol (E2) for 24 h, washed three times with 16 PBS and then incubated for 30 minutes in Locke’s solution (10 mM HEPES.HCl, pH 7.3, 150 mM NaCl, 5 mM KCl, 2 mM CaCl2, and 0.1% glucose) supplemented with 5 mM A23187 (C7522, Sigma). Jurkat cells were treated with phorbol myristate acetate (PMA) at 40 nM. Ionophores were used at 1 mM. PADI4 inhibitor, Cl-amidine (from Bertin Pharma) was dissolved in 16PBS as a 50 mM stock solution. MCF7 and Jurkat cells were treated with 200 mM Cl-amidine in complete cell culture medium for exactly 72 h. Generation of inducible PADI4-expressing HEK293 cells The cDNAs coding for either WT PADI4, hyperactive PADI4 (Mut+) [62], or hypoactive PADI4 (Mut2) [63] were inserted into the pi_tk_hygro vector for retroviral delivery [77]. Two days after transfection using FuGENE (Roche), the medium of packaging HEK293 cells was filtered on a 0.45 mm filter (Millipore) and supplemented with polybrene (AL-118, Sigma) at 100 mg.ml21. This medium was used to infect host HEK293 cells (3 consecutive infections) followed by selection with hygromycin (H3274, Sigma) at a final concentration of 100 mg.ml21. RNA interference, mRNA, and protein quantification HP1 small interfering RNAs (siRNAs) were described previously [35]. PADI4 (J-012471-05) and Glyceraldehyde-3-phosphate dehydrogenase control siRNAs were purchased from Dharmacon. Cells were harvested 72 h after transfection with DharmaFECT 1 (T 2001-03). Total RNA from PBMCs and MCF7 cells was extracted with RNeasy (Qiagen) and quantified with an ND-1000 (Nanodrop). After DNase treatment (Roche), reverse transcription was performed using SuperScript III (Invitrogen) and random hexanucleotides according to the manufacturer’s instructions. Complementary DNA was quantified by RT-qPCR as previously described [35]. PCR primers are listed in Table S2. Proteins were extracted as described previously [35] and detected by Western blotting. Acid extraction of histones was conducted as described by Shechter, et al., [78]. Immunofluorescence and image acquisition Immunofluorescent labeling was performed in MCF7 cells after sequential treatment with 200 nM of estradiol (E2) for 24 h in culture media followed by 5 mM A23187 for 30 min in Locke’s solution. Cells were permeabilized in ice cold CSK (20 mM PIPES pH 6.8, 200 mM NaCl, 600 mM sucrose, 6 mM MgCl2, and 2 mM EGTA) 0.5% Triton X-100 (v/v), 0.1 mM PMSF for 30 sec. Cells were fixed with CSK-3.7% paraformaldehyde at room temperature (RT) for 10 min, then blocked in PBS-0.05% Tween-20 (v/v), 10% (v/v) FBS for 30 min at RT. In peptide competition experiments, primary antibodies were pre-incubated with 1 mg of indicated histone H3 peptides or without peptide for 30 min. Cells were incubated with antibodies at 4uC overnight and then coverslips were washed three times in PBS-BSA 0.5% (w/v). Coverslips were incubated with secondary antibodies for 1 h at room temperature protected from light, before being washed three times in PBS-BSA 0.5% (w/v), and once in PBS, before final staining with DAPI. Imaging was conducted on an Axiovert 200 M microscope (Zeiss) coupled with an Apotome with Axiovision 4.7 (Zeiss). Supporting Information Figure S1 Depletion of HP1 proteins reactivates HERVs and TNFa. (A) Total RNA from MCF7 cells transfected with the PADI4 Regulates HP1 Binding to Histone H3 in MS indicated small interfering RNAs (siRNAs) was quantified with RT-qPCR. Changes in mRNA levels are shown relative to the siGAPDH transfection (set to 1). The data are presented as the means 6 SEM of triplicate experiments.(B and D) MCF7 cells were transfected with the indicated siRNA as mentioned in Figure 1B and Figure 3E–3F. Western blots were carried out with total extracts with the indicated antibodies. Blots are representa- tive of the experimental replicates. (PDF) Figure S2 PADI4 expression and activity is induced in MCF7 cells by a treatment with estradiol and an ionophore. (A) Extracts from MCF7 cells treated either estradiol (E2) or ionophore (A23187) and/or both as indicated were analyzed by Western-blot with anti-PADI4 & anti-Brg1 antibodies. Blots are representative of the experimental replicates. (B) Treatment of MCF7 cells with estradiol and an ionophore increases the expression of HERV-H/ env62 in 24 h. Total RNA from MCF7 cells uninduced (ethanol) or treated with E2 and/or A23187 for the indicated times, was quantified by RT-qPCR. Values were normalized to levels of RPLP0. Indicated values were averaged from three experimental replicates. (C) PADI4 activity does not reduce recruitment of H3cit8K9me3 on the promoter of RPLP0 in MCF7 cells. Relative enrichment of H3cit8K9me3 double mark on the RPLP0 promoter under the different condition as described in Figure 4. (PDF) Figure S3 PADI activity facilitates activation of HERVs in T cells. (A) ChIP with anti-HP1a antibodies was carried out with chromatin prepared from Jurkat cells either untreated or treated with PMA and ionophore (ionomycin). The relative enrichments of HP1a on the indicated LTRs were measured by qPCR. Data are presented relative to non-immune IgG. Changes in enrich- ment are presented relative to the un-induced control (set to 1). Values are means 6 SEM from two PCR measures of two independent ChIP experiments. (B) ChIP with anti-PADI4 antibodies was carried out as in A with indicated time points. Data are presented relative to non-immune IgG. Changes in enrichment are presented relative to the un-induced control (set to 1). Values are means 6 SEM from two PCR measures of two independent ChIP experiments. (C) ChIP with anti-H3cit8K9me3 antibodies was carried out as in A. Data are presented as a percentage of histone H3. Changes in enrichment are presented relative to the un-induced control (set to 1). Values are means 6 SEM from two PCR measures of two independent ChIP experiments. (D) Total RNA was isolated from Jurkat cells either un-stimulated or treated with ionomycin and PMA minus or plus PADI-inhibitor cl-amidine as indicated. Changes in mRNA levels for the indicated genes were quantified by RT-qPCR. The data are presented as the means 6 SEM of duplicate experiments. (PDF) Figure S4 HP1a in the repression of HERVs and cytokines. Correlation matrix representing the correlation between the presence of HP1a on the promoter regions of RPLP0, TNFa, and HERVs in study individuals analyzed in Figure 6C. Represented values are Spearman Rank correlation coefficients followed by p-values calculated using two sided student t test. p,0.05 was considered as significant. The colour bar at the bottom shows the colour scale according to Spearman’s Rank correlation coefficients. (PDF) Table S1 Clinical and demographic data for the lymphocyte samples. The data given for age and duration (years), and EDSS are: mean (range). Abbreviations are as follows: MS: MS patient; PLOS Genetics | www.plosgenetics.org 13 September 2012 | Volume 8 | Issue 9 | e1002934