PADI4 Regulates HP1 Binding to Histone H3 in MS Figure 6. Modified accumulation of HP1a and H3cit8K9me3 at HERVs and TNFa in MS patients. (A–B) Expression of HERVs and TNFa in PBMCs from 18 families (n = 36 individuals) each consisting of one MS patient and a genetically related unaffected relative. Total RNA was prepared from the PBMCs and indicated transcripts were quantified by RT-qPCR. Values are means 6SE from at least 3 qPCR reactions for each individual. For each family, the basal level used to calculate the fold-change was the average of at least 3 qPCR values obtained from the healthy control. (C) ChIP with anti-HP1a on chromatin extracted at same time from PBMCs from above mentioned 18 families. The relative enrichment of HP1a on the LTR or promoter region of HERVs and TNFa was measured by qPCR and normalized to the signal obtained with irrelevant non-immune IgGs. The reported values are means 6 SEM from at least three qPCRs for each individual (n = 36) from a representative ChIP experiment (* p,0.05, significant and up.0.05 not significant, Wilcoxon signed rank test). (D–E) ChIP with anti-H3cit8K9me3 on chromatin extracted from PBMCs from 9 families. The relative enrichment of H3cit8K9me3 on the LTR or promoter region was measured by qPCR and data are presented as a percentage of histone H3. The reported values are means 6 SEM from at least three qPCRs for each individual (n = 18) from a representative ChIP experiment. The enrichment of H3cit8K9me3 on the HERV/LTR59 is also higher in MS patients as compared to healthy individuals, but due to the small sample size, the p value (*u p = 0.06) is not significant and could not be interpreted (* p,0.05, significant and up.0.05, not significant, Wilcoxon signed rank test). (F) PADI4 mRNA levels were quantified by RT-qPCR in total RNA prepared from PBMCs of 18 families each including one MS patients and one genetically related control individual. Values are means +/2SE from at least 3 qPCR reactions for each individual. The p value (p,0.0007) was calculated by Wilcoxon signed rank test. In each panel, the boxes represent the interquartile range. The whiskers extend the box to the highest and lowest value. The line across the box indicates the median value. doi:10.1371/journal.pgen.1002934.g006 both the signals from the reference GST surface and from blank experiments using PBS instead of peptide were subtracted). The steady-state SPR responses (Req) were plotted against the peptide concentration (C) and fitted according to the following equation: Req~ðRmax CÞ=ðKdzCÞ 1 ð Þ The percentage of bound HP1a sites was determined as follows: % bound HP1a sites~ Req Rimmo MWðGST{HP1aÞ MWðpeptiedÞ 100 2 ð Þ where Kd is the equilibrium dissociation constant of the peptide/ GST-HP1a interaction and Rmax the maximal binding capacity of GST-HP1a. Cell culture MCF-7 and HEK293 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco BRL), and Jurkat cells Figure 7. Model. HP1a silences HERVs and represses several cytokines until they are stimulated. It binds the tri-methylated lysine 9 of histone H3 on these promoters.This binding is in part regulated by PADI4 conversion of histone H3 arginine 8 into a citrulline, thereby reducing the affinity of HP1a for the neighboring methylated lysine 9. In MS patients, PADI4 induces an enrichment of H3cit8K9me3 double histone modification resulting in reduced accumulation of HP1a on HERVs, on the promoter of TNFa and possibly on other cytokines genes. This in turn may be responsible for excessive expression of HERVs and cytokines that inappropriately activate T lymphocytes and ultimately damages the central nervous system. It may be possible to interrupt this sequence of event with the PADI4 specific inhibitor Cl-amidine. doi:10.1371/journal.pgen.1002934.g007 PLOS Genetics | www.plosgenetics.org 12 September 2012 | Volume 8 | Issue 9 | e1002934