Methylation of chromatin and noncoding RNA featured in Cold Spring Harbor Protocols|
COLD SPRING HARBOR, N.Y. (Fri., Oct. 1, 2010) – Post-translational modifications of histones play an important role in regulating chromatin dynamics and function. One such modification, methylation, is involved in the regulation of the epigenetic program of a cell, determining chromatin structure, and regulating transcription. Methylation of histones occurs on both lysine and arginine residues, and until recently, was thought to be an irreversible process. The recent discovery of histone demethylases revealed that histone methylation is more dynamic than previously recognized. The October issue of Cold Spring Harbor Protocols (http://cshprotocols.cshlp.org/TOCs/toc10_10.dtl) features a set of methods from Keiichi Nakayama and colleagues from Kyushu University (http://hyoka.ofc.kyushu-u.ac.jp/search/details/K001794/english.html) for investigating demethylase activity. The protocol, In Vitro Histone Demethylase Assay, describes two different in vitro histone demethylase enzyme reactions and three different methods for measuring histone demethylase activity. These methods can be applied to measuring histone demethylase activity in tissues and cell lysates, identification of novel histone demethylases, and screening for inhibitors of histone demethylases. This featured article is freely available on the journal’s website (http://cshprotocols.cshlp.org/cgi/content/full/2010/10/pdb.prot5512).
While post-transcriptional modifications are a characteristic feature of noncoding RNAs, the biological function of these modifications is unknown. Cytosine-5 methylation has been detected in abundant RNA molecules including ribosomal RNAs and transfer RNAs, but the methylation status of cytosines in other noncoding RNAs is not known. To further investigate these modifications, Matthias Schaefer and colleagues from the German Cancer Research Center (http://www.dkfz.de/en/index.html) have developed a protocol for Detection of Cytosine Methylation in RNA Using Bisulfite Sequencing. The method, featured in the October issue of Cold Spring Harbor Protocols (http://cshprotocols.cshlp.org/TOCs/toc10_10.dtl), uses a bisulfite treatment of RNA to chemically deaminate nonmethylated cytosines to uracil, leaving methylated cytosines unaffected. Subsequent cDNA synthesis and PCR amplification offers researchers material for high-throughput sequencing analysis of the methylation patterns in any RNA molecule, including noncoding RNAs and low-abundance RNAs. This featured article is freely available on the journal’s website (http://cshprotocols.cshlp.org/cgi/content/full/2010/10/pdb.prot5505).
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