PADI4 Regulates HP1 Binding to Histone H3 in MS Figure 3. The H3cit8K9me3 double histone modification reduces the affinity of HP1a for the H3K9me3 single modification. (A) Dot blot showing that HP1a does not bind to histone H3 peptides carrying a H3Cit8K9me3 double modification. Indicated histone H3 peptides of 1, 0.2, and 0.04 mg were spotted on the membranes. Then binding of recombinant GST-HP1a was tested by labeling of the retained protein with anti-GST antibodies. Blots are representative of the experimental replicates. (B) Purified recombinant GST-HP1a (red) was bound to fixed MCF7 cells in ‘‘overlay’’ assays, and then challenged by competition with 1 mg of the indicated peptides. DNA is labeled with DAPI (blue), scale bar: 5 mm. (C) Real- time association and dissociation surface plasmon resonance (SPR) profiles corresponding to the injection of the indicated H3 peptides at 12.5 mM over immobilized GST-HP1a. (D) Percentage of bound GST-HP1a sites as a function of the peptide concentration. (E–F) Total RNA from MCF7 cells transfected with the PADI4 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 S1C). The data are presented as the means 6 SEM of triplicate experiments. doi:10.1371/journal.pgen.1002934.g003 Methylation and citrullination may therefore allow for a gradual activation of HP1a target genes in response to external stimuli. The fact that PADI4 is a regulator of cytokines that can be either positively regulated by cellular stimuli or negatively regulated by specific inhibitors provides yet unexplored avenues to the control of inflammation, and in the case of MS, molecules such as Cl-amidine may potentially allow restoring chromatin- mediated repression of over-activated cytokine genes. While HP1 proteins are best described as heterochromatic silencers and suppressors of variegation, our observations confirm that these proteins are also highly relevant for the transcriptional control of inducible genes that require a transient phase of silencing. The sharing of regulatory mechanisms between euchro- matic cytokine genes and repeated sequences such as HERVs suggests that many bridges may exist between active and inactive chromatin, and that there is a continuum and not a clear-cut boarder between euchromatin and heterochromatin. Therefore, probing the status of heterochromatic silencing as well as its defects may provide much new insight on the transcriptional programs in which cells are engaged. Materials and Methods Study population: Ethics statement Blood samples were collected from each participant after informed consent as approved by the local Danish ethical committee. Study population: Description and methods The study population consisted of 36 subjects, encompassing 18 MS patients clinically diagnosed for MS and fulfilling the diagnostic criteria of Poser et al., 1993 [75] and 18 unaffected (healthy) first or second degree relatives, one for each of the MS patients. The participants were from a homogenous population (Caucasian, Northern European descent). Venous blood was drawn and processed on the same day in our laboratory. The clinical and demographic data of each participant are summarized in Table S1. The mean age of both the MS patients and their unaffected relatives was 52 years. The gender ratios for MS patients (11 female/7 male) and unaffected relatives (9 female/9 male) were also comparable. Peripheral blood mononuclear cells (PBMCs) were prepared by standard Isopaque-Ficoll centrifuga- tion. The separated cells were cryo-preserved in RPMI with addition of 20% inactivated human serum (HS) and 10% DMSO, at 2135uC until use. For the assays, PBMCs were thawed and cultured for 24 h in RPMI-1640 with 10% inactivated human serum, and 100 Uml-1 penicillin-streptomycin at 37uC in a 5% CO2 incubator prior to use. For each family, PBMCs from the patient and the control individual were analyzed at the same time.The data were analyzed by using the software XLSTAT (version 2010.5.06, www.xlstat.com). When indicated in the text, Wilcoxon signed rank test [76] was used to determine whether a significant (p,0.05) difference. Antibodies, peptides, and chemicals Anti-H3cit8K9me3 antibody was produced in rabbits using a peptide coupled to KLH with the following sequence: ARTKQTA (cit)(Kme3)STGGKAPRC. Anti-PADI4 (ChIP: P4749; immuno- blots: ab50332), anti-H3cit(2,8,17) (ab5103), anti-H3(ab1791), and anti-H3K9me3 (ab8898) antibodies were purchased from Abcam. Anti-HP1a (ChIP: 1H5; immunoblots: 2G9) and anti-Brg1 (2E12) were from Euromedex. Calcium ionophore A23187 (used on HEK293 and MCF7 cells) and ionomycin (only ionophore tolerated by Jurkat cells), and 17-ß-estradiol (E2758) were purchased from Sigma. DNA was labeled with 49,6-diamidino-2- phenylindole (DAPI; Invitrogen) at a concentration of 150 ng.ml21. The peptide ARTKQTARKSTGGKAPRC was used for competition, overlay, and surface plasmon experiments, either unmodified or with K9me3, R8meK9me3, cit8K9me3, or cit8 modifications. Peptides were carefully quantified by amino acid analysis, and the presence of the modifications was confirmed by mass spectrometry. Chromatin immunoprecipitation (ChIP) ChIP was carried out essentially as previously described [74], with minor alterations. MCF7 or Jurkat cells, or PBMCs were cross-linked in phosphate-buffered saline (PBS) containing 1% formaldehyde (Sigma) for 10 min at room temperature. The crosslinking reaction was quenched with PBS containing 125 mM glycine, followed by three washes with ice-cold PBS. The chromatin was fragmented by sonication to produce average DNA lengths of 0.5 kb. After ChIP, the eluted DNAs were detected by quantitative PCR using the primers listed in Table S2. Levels of histone modifications are expressed as % of H3, and levels of HP1a are expressed relatively to the signal obtained for ChIP using non-immune IgGs. Values are averaged from 3 independent experiments. In vitro binding studies by surface plasmon resonance (SPR) Real-time SPR assays were performed at 25uC in PBS. GST- HP1a was covalently coupled to a CM5 sensor chip, using a Biacore 2000 instrument and an Amine Coupling Kit (GE Healthcare), achieving three different immobilization densities (Rimmo) of 3500, 6500, and 15000 resonance units (RU; 1RU <1 pg.mm22). On the remaining flow cell, 5700 RU of GST were immobilized to prepare a reference surface. A series of 10 concentrations of peptides (50 nM–25 mM for H3R8K9me3, 200 nM–100 mM for the H3cit8K9me3, H3cit8K9, and unmod- ified peptides) were injected for 2 min over the GST-HP1a and GST surfaces at a flow rate of 50 mL.min21. After following the dissociation for 5 min, the surfaces were regenerated with a 3-min wash of 2 M NaCl, and two 15-sec washes with 10 mM glycine- HCl (pH 1.5) and 0.05% SDS. The association and dissociation profiles were double-refer- enced using the Scrubber 2.0 software (BioLogic Software) (i.e. PLOS Genetics | www.plosgenetics.org 8 September 2012 | Volume 8 | Issue 9 | e1002934