Article

Methods for Analyzing Histone Citrullination in Chromatin Structure and Gene Regulation

Center for Eukaryotic Gene Regulation, University Park, PA, USA.
Methods in molecular biology (Clifton, N.J.) (Impact Factor: 1.29). 01/2012; 809:473-88. DOI: 10.1007/978-1-61779-376-9_31
Source: PubMed

ABSTRACT

Histone posttranslational modifications play significant roles in regulating chromatin structure and gene expression. One of the histone modifications, histone citrullination, is catalyzed by an enzyme called peptidylarginine deiminase 4 (PAD4, also called PADI4), which converts both histone arginine (Arg) and mono-methyl arginine residues to citrulline. Recent studies have found that histone citrullination counteracts the effect of histone arginine methylation and functions as a repressive marker to turn off gene expression. Here, we describe assays to study histone citrullination by PAD4 in vitro and in vivo. We also describe approaches to measure histone citrullination levels at gene promoters using chromatin immunoprecipitation assay and analyze the effects of PAD4 inhibitor on cell cycle and apoptosis by flow cytometry. These methods would be useful techniques to study this unique histone modification.

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    • "Results showed that embryos arrested either at the 2-4 cell stage (83%, n = 100) or at the 1-cell stage (17%, n = 100) in the Cl-amidine group, while 86.1% of embryos (n = 94) in the H-amidine group and 72.3% of embryos (n = 36) in KSOM medium alone developed to the morula stage (Figure 2F and Table 1). We note here that (1) the concentration of Cl-amidine used in our study is within the range of that used to functionally block PADI activity in somatic cells [14,23] and that (2) lower concentrations of Cl-amidine did not affect embryonic development (Additional file 5A and - 5B). Our finding that Cl-amidine suppressed histone citrullination in cleavage-stage embryos suggested that the observed effects of Cl-amidine on development were due to specific inhibition of PADI activity. "
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