Citrullination of histone occurs on arginine residues. Catalyzed by the peptidyl arginine deiminase family (Padis), this modification alters the charge of the histone tail and modulates its interaction with DNA and chromatin interacting proteins. Although a growing number of studies demonstrated the gene regulatory role of histone citrullination in developmental processes and cancer progression, and revealed its cross-talk with various histone modifications, its characteristics at the genome-wide level are unclear. This study employs proliferating and differentiated mouse keratinocytes as models, aiming to examine the genome-wide enrichment, dynamics, and function of histone citrullination in the context of cell differentiation.

To start, ChIP-seq targeting to H3R2+8+17Cit, and H3R8Cit have been conducted. However, the enrichment of these citrullination marks is highly similar to total histone H3, and no differential enrichment was observed between cell states. These results suggest the existence of technical hurdles in profiling H3Cit or that these marks are persistent and unchanged during differentiation. Currently, cells depleted for multiple Padis are being generated by CRISPR to test for H3Cit ChIP antibodies specificity, and to examine whether Padis depletion impact keratinocyte differentiation.

Besides H3Cit ChIP-seq, Padi4 binding on the chromatin will be profiled to indirectly predict a subset of citrullinated loci. In parallel, ATAC-seq, RNA-seq, and ChIP-seq targeting the reported H3Cit-associated histone marks will be conducted for integrative analysis.

Moreover, our previous finding suggests that Padi4 regulates translation, restricting proliferation and lineage commitment of hair follicle progenitors. Diving deeper into the mechanisms, immunoprecipitation-mass spectrometry (IP-MS) will be performed to identify Padi4 interactors. Also, Padi4-associated chromatin and transcription factors will be revealed to understand its epigenetic regulations.