A Novel Citrullinated Modification of Histone 3 and Its Regulatory Mechanisms Related to IPO-38 Antibody-Labeled Protein

IPO-38 is a potential biomarker for early diagnosis of gastric cancer that we recently identified. Although we characterized its chemical nature as a nucleosome histone, we suspected the existence of histone modification for the IPO-38 antibody-labeled protein. Here, we used a commercially available modified histone peptide array to identify the type and site of histone modification labeled by the IPO-38 monoclonal antibody. In protein array analysis, the citrulline modification of histone 3 on arginine 26 (H3R26Cit) yielded the strongest signal. Although peptidyl arginine deiminase-2 and -4 (PADI2 and PADI4, respectively) can catalyze the conversion of arginine to citrulline, we observed that only PADI4 expression correlated with the citrulline histone modification of H3R26Cit. Overexpression of PADI4, via transfection of a eukaryotic expression vector, and knockdown of PADI4 gene expression, by a PADI4 CRISPR/Cas9 vector, confirmed the crucial function of PADI4 on the increased level of H3R26Cit in gastric cancer cell lines. By immunoprecipitation and immunoblotting, we found an interaction between H3R26Cit and H3K27me3. Our study established the first link between the IPO-38 antigen and citrullinated histone 3, and clarified the upstream regulatory enzyme PADI4. The new findings suggest an important role for the citrullination modification of histone in gastric cancer biology, and should help us optimize the development of a sensitive and specific diagnostic reagent.