PADI4 Regulates HP1 Binding to Histone H3 in MS Figure 4. PADI4 activity controls HP1a occupancy and histone H3 citrullination at TNFa and HERV promoters. (A) Total protein extracts from MCF7 cells treated either estradiol (E2) or ionophore (A23187) and/or Cl-amidine were immunoblotted with the indicated antibodies. (B) Total RNA from MCF7 cells either untreated or treated with estradiol followed by ionophore (E2+A23187) and/or Cl-amidine (E2+A23187+Cl-amidine) treatment was quantified by RT-qPCR. Data are shown relative to the un-induced condition (set to 1). Values are mean 6 SEM from four experimental replicates. (C–E) ChIP with the listed antibodies was carried out with chromatin from MCF7 cells either untreated or treated with estradiol and ionophore (E2+A23187) in the absence or presence of Cl-amidine. The relative enrichments of the indicated antibodies on the shown LTRs or promoters were measured by qPCR. Data are presented as a percentage of histone H3 or relative to non-immune IgG as indicated. Enrichments are presented relative to indicated controls (set to 1). Values are means 6 SEM from four PCR measures of representative ChIP experiments. doi:10.1371/journal.pgen.1002934.g004 Figure 5. PADI activity facilitates activation of TNFa and IL8 transcription in T cells. (A) ChIP with anti-HP1a antibodies was carried out with chromatin from Jurkat cells either untreated or treated with PMA and ionophore (ionomycin). The relative enrichments of HP1a on the indicated promoters were measured by qPCR. 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. (B) Total RNA was isolated from Jurkat cells either un-stimulated or treated with ionomycin and PMA for 2 hour. Changes in mRNA levels for the indicated genes were quantified by RT-qPCR. The data are presented as the means 6 SEM of triplicate 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 treated as in B 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. doi:10.1371/journal.pgen.1002934.g005 PLOS Genetics | www.plosgenetics.org 10 September 2012 | Volume 8 | Issue 9 | e1002934