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Figure 1. KDM2B binds to non-methylated CpG DNA and localises to CGIs genome-wide.(A) Electrophoretic mobility shift assay (EMSA) demonstrating that the recombinant KDM2B ZF-CxxC domain binds to a DNA probe containing non-methylated CpGs (left panel) in a concentration-dependent manner. Binding is abrogated by CpG methylation (right panel). (B) An analogous EMSA to part (A) using the known CGI binding factor KDM2A for comparison. (C) Input, KDM2A, and KDM2B ChIP-seq profiles over a region of the genome containing CGI and non-CGI associated genes. Bio-CAP-seq profiles are shown to indicate the location of non-methylated DNA (Blackledge et al., 2012). Above the sequencing traces individual genes are shown with the arrow indicating the transcription start site and vertical black lines corresponding to exons. The location of CGIs are indicated by green bars. Both KDM2A and KDM2B associate specifically with CGIs containing non-methylated DNA. (D) KDM2A and KDM2B ChIP-seq signal segregates specifically with CGI associated gene promoters (left panel) and is excluded from gene promoters not associated with CGIs (right panel). (E) KDM2A and KDM2B ChIP-seq signal is centred over CGIs, in agreement with their capacity to recognize non-methylated DNA at these sites.DOI: http://dx.doi.org/10.7554/eLife.00205.003Figure 1—figure supplement 1. KDM2B is found throughout the nucleus and not concentrated in the nucleolus.(A) Endogenous KDM2B immunofluorescence in five different mammalian cell lines. KDM2B signal is in red (left) and DAPI signal in blue (right). In all cell types KDM2B is a broadly distributed throughout the nucleus and not concentrated in the nucleolus as previously reported (Frescas et al., 2007). (B) Immunofluorescence in HeLa cells for Flag-tagged KDM2B and endogenous fibrillarin, a nucleolus marker. KDM2B is largely excluded from nucleoli. (C) The KDM2A and KDM2B antibodies specifically recognize their respective antigens. Empty expression vector, Flag-KDM2A, and Flag-KDM2B were transiently transfected into 293T cells. Whole cell extracts were made and probed with anti-KDM2A (left panel), anti-KDM2B (centre panel), or anti-Flag (right panel).DOI: http://dx.doi.org/10.7554/eLife.00205.004 Image published in: Farcas AM et al. (2012) Copyright © 2012, Farcas et al. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution license Larger Image Printer Friendly View

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