H3 citrullination cross-talk within the histone code

Post translational modifications (PTMs) alter protein structure and function, thus impacting cellular dynamics. As an understudied protein post translational modification (PTM), positively charged arginine is converted to neutral citrulline (Rcit) by peptidylarginine deiminase (PAD) enzymes, with no known means of reversal. The PTM is particularly extensive in NETosis, a form of neutrophil programmed cell death with an extensive citrullinome characterized by Neutrophil Extracellular Traps (NETs). Here the Citrullinome is extensive and includes the citrullination of histone tails neutralizes arginine charge and is linked to chromatin de-condensation during extracellular release. Given its assumed irreversibility, any role for histone citrullination outside of terminal NETosis is controversial. However, any investigation of site-specific histone Rcit(s) in combinatorial chromatin regulation is severely hindered by the lack of reliable detection reagents. To address, we used nucleosome-based screening to develop recombinant monoclonal antibodies that individually recognize histone H3R2cit, H3R8cit or H3R17cit independent of the cis PTM context. These show PAD4 citrullinates nucleosomal H3R17 and H3R8 more efficiently than H3R2; is inhibited at each position by pre-existing arginine methylation; but strikingly unaffected by neighboring lysine methylations or acetylations. As expected, each citrullination occurs in mouse primary neutrophils upon calcium ionophore induced NETosis. However, H3R8cit and H3R17cit also show broad genomic enrichment patterns in multiple cell lines, suggesting potential roles in epigenetic regulation.