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Genome-Wide uH2A Localization Analysis Highlights Bmi1-Dependent Deposition of the Mark at Repressed Genes

Figure 4

uH2A deposition is linked to DNA methylation.

(A) Bmi1-dependent per base pair uH2A tag density was determined for genes defined as high CpG dinucleotide promoter content (HCP, blue) (n = 10,310), intermediate CpG dinucleotide promoter content (ICP, red) (n = 2889), low CpG dinucleotide promoter content (LCP, green) (n = 2668). (B) HCP class genes with available DNA methylation data were grouped based on the presence (+) (n = 266) or absence (−) (n = 8230) of a promoter bound peak of Bmi1-dependent uH2A and the distribution of DNA methylation values for each group was visualized using a standard box plot. Red lines indicate median values. P value derived from Wilcoxon signed-rank test. (C) Western blotting of uH2A was carried out using cell lysate prepared from wild-type and Dnmt1 null MEFs. H3 was used as a loading control. (D) Cytosine extension analysis was carried out using wild-type and Bmi1 null MEFs as well as Dnmt1 null and control cells. Biotin labeled restriction digests were spotted in triplicate onto a Nylon membrane and visualized using alkaline phosphatase. The procedure was repeated three independent times and data for one representative experiment is shown. (E) Data presented in panel D were quantified and presented as the ratio between methylation sensitive HpaII incorporation versus methylation insensitive MspI incorporation. Error bars represent s.e.m. (n = 3).

Figure 4