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


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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    • "Other than DOT1L (KMT4), all lysine methyltransferases contain the conserved SET (Suppressor of variegation, Enhancer of zeste, and Trithorax) domain. Recently, the notion that demethylation occurs only on synthesis of new histones was over-turned with the discovery of enzymes that convert arginine to citrulline, to remove arginine methylation, and lysine demethylases, including LSD1 (KDM1) and the Jumonji C family (Johansson et al., 2014;Li et al., 2012;Wang et al., 2009). HMTi are at their very early phases of development and include chaetocin, 3- Deazaneplanocin A (DZNep) and BIX- 01294. "
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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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    ABSTRACT: The peptidylarginine deiminases (PADIs) convert positively charged arginine residues to neutrally charged citrulline on protein substrates in a process that is known as citrullination or deimination. Previous reports have documented roles for histone citrullination in chromatin remodeling and gene regulation in several tissue types, however, a potential role for histone citrullination in chromatin-based activities during early embryogenesis has not been investigated. In the present study, we tested by laser scanning confocal indirect immunofluorescence microscopy whether specific arginine residues on the histone H3 and H4 N-terminal tails (H4R3, H3R2 + 8 + 17, and H3R26) were citrullinated in mouse oocytes and preimplantation embryos. Results showed that all of the tested residues were deiminated with each site showing a unique localization pattern during early development. Given these findings, we next tested whether inhibition of PADI activity using the PADI-specific inhibitor, Cl-amidine, may affect embryonic development. We found that treatment of pronuclear stage zygotes with Cl-amidine reduces both histone H3 and H4 tail citrullination and also potently blocks early cleavage divisions in vitro. Additionally, we found that the Cl-amidine treatment reduces acetylation at histone H3K9, H3K18, and H4K5 while having no apparent effect on the repressive histone H3K9 dimethylation modification. Lastly, we found that treatment of zygotes with trichostatin A (TSA) to induce hyperacetylation also resulted in an increase in histone citrullination at H3R2 + 8 + 17. Given the observed effects of Cl-amidine on embryonic development and the well documented correlation between histone acetylation and transcriptional activation, our findings suggest that histone citrullination may play an important role in facilitating gene expression in early embryos by creating a chromatin environment that is permissive for histone acetylation.
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