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. 2011 Jan 13;6(1):e16244. doi: 10.1371/journal.pone.0016244

Figure 3. Candidate approach by Western blot analysis does not reveal the methyltransferase and demethylase responsible for H2B lysine 37 methylation.

Figure 3

(A) Following validation of correct deletion of the ORF of interest and replacement with kanMX by genomic PCR (data not shown), histones were acid-extracted from candidates from the Yeast Knockout Collection (Open Biosystems), and putative histone methyltransferase activity was tested by Western blot analysis using the purified α-H2BK37me2 antibody. A Coomassie-stained gel illustrating a representative purification of histones is shown in upper panel, and representative Western blots results from the candidate screen are shown below. The blots were first probed with the α-H2BK37me2 antibody (upper) and then striped and reprobed with an α-H2B antibody (lower) to demonstrate equal loading. Histones derived from strains harboring wild-type Flag-H2B (YKG001) and Flag-H2B K37R (YKG006) or K37A (YKG007) were loaded on all gels to demonstrate loss-of-signal upon mutation of lysine 37, thereby serving as a control for antibody specificity. The presence of a Flag-tag on histone H2B results in the slight shift in electrophoretic mobility observed in the control strains, as compared to untagged H2B species in the candidate deletion strains. Deletion of candidate genes did not reveal a putative H2BK37me2 histone methyltransferase by Western blot analysis. (B) Histones were acid-extracted from the five JmjC-domain-containing protein deletions in Saccharomyces cerevisiae, and putative histone demethylase activity was analyzed by Western blot analysis using the purified α-H2BK37me2 antibody. Shown are Western blot results from the candidate screen, in which the blots were first probed with the α-H2BK37me2 antibody (upper) and then striped and reprobed with an α-H2B antibody (lower) to demonstrate equal loading. Again, histones derived from strains harboring wild-type Flag-H2B (YKG001) and Flag-H2B K37A (YKG007) were used as a control for antibody specificity, and the presence of a Flag-tag on histone H2B results in the slight shift in electrophoretic mobility observed in the control strains, as compared to untagged H2B species in the candidate deletion strains. Deletion of each individual candidate did not result in an enhanced signal, suggesting that none of these candidates function as the histone demethylase for H2BK37me2.