Genome-wide analysis using ChIP to identify isoform-specific gene targets

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• dc.contributor.author Beshiri, Michael L.ca
• dc.contributor.author Islam, Abul, 1978-ca
• dc.contributor.author DeWaal, Dannielle C.ca
• dc.contributor.author Richter, William F.ca
• dc.contributor.author Love, Jenniferca
• dc.contributor.author López Bigas, Núriaca
• dc.contributor.author Benevolenskaya, Elizaveta V.ca
• dc.date.accessioned 2016-01-18T16:14:30Z
• dc.date.available 2016-01-18T16:14:30Z
• dc.date.issued 2010
• dc.description.abstract Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the protein domains that bind to specific histone modifications. One such domain is the plant homeodomain (PHD), found in several chromatin-binding proteins. The epigenetic factor RBP2 has multiple PHD domains, however, they have different functions (Figure 4). In particular, the C-terminal PHD domain, found in a RBP2 oncogenic fusion in human leukemia, binds to trimethylated lysine 4 in histone H3 (H3K4me3). The transcript corresponding to the RBP2 isoform containing the C-terminal PHD accumulates during differentiation of promonocytic, lymphoma-derived, U937 cells into monocytes. Consistent with both sets of data, genome-wide analysis showed that in differentiated U937 cells, the RBP2 protein gets localized to genomic regions highly enriched for H3K4me3. Localization of RBP2 to its targets correlates with a decrease in H3K4me3 due to RBP2 histone demethylase activity and a decrease in transcriptional activity. In contrast, two other PHDs of RBP2 are unable to bind H3K4me3. Notably, the C-terminal domain PHD of RBP2 is absent in the smaller RBP2 isoform. It is conceivable that the small isoform of RBP2, which lacks interaction with H3K4me3, differs from the larger isoform in genomic location. The difference in genomic location of RBP2 isoforms may account for the observed diversity in RBP2 function. Specifically, RBP2 is a critical player in cellular differentiation mediated by the retinoblastoma protein (pRB). Consistent with these data, previous genome-wide analysis, without distinction between isoforms, identified two distinct groups of RBP2 target genes: 1) genes bound by RBP2 in a manner that is independent of differentiation; 2) genes bound by RBP2 in a differentiation-dependent manner. To identify differences in localization between the isoforms we performed genome-wide location analysis by ChIP-Seq. Using antibodies that detect both RBP2 isoforms we have located all RBP2 targets. Additionally we have antibodies that only bind large, and not small RBP2 isoform (Figure 4). After identifying the large isoform targets, one can then subtract them from all RBP2 targets to reveal the targets of small isoform. These data show the contribution of chromatin-interacting domain in protein recruitment to its binding sites in the genome.ca
• dc.description.sponsorship This work was supported by 115347-RSG-08-271-01-GMC from ACS, and by CA138631 grant from NIH.
• dc.format.mimetype application/pdfca
• dc.identifier.citation Beshiri ML, Islam A, DeWaal DC, Richter WF, Love J, Lopez-Bigas N et al. Genome-wide analysis using ChIP to identify isoform-specific gene targets. Journal of visualized experiments. 2010;(41) pii:2101. DOI: 10.3791/2101ca
• dc.identifier.doi http://dx.doi.org/10.3791/2101
• dc.identifier.issn 1940-087X
• dc.identifier.uri http://hdl.handle.net/10230/25594
• dc.language.iso engca
• dc.publisher MYJoVE Corporationca
• dc.relation.ispartof Journal of visualized experiments. 2010;(41) pii:2101
• dc.rights Copyright © 2010 Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported Licenseca
• dc.rights.accessRights info:eu-repo/semantics/openAccessca
• dc.rights.uri https://creativecommons.org/licenses/by-nc-nd/3.0/ca
• dc.subject.keyword Biochemistry
• dc.subject.keyword Issue 41
• dc.subject.keyword Chromatin immunoprecipitation
• dc.subject.keyword ChIP-Seq
• dc.subject.keyword RBP2
• dc.subject.keyword JARID1A
• dc.subject.keyword KDM5A
• dc.subject.keyword Isoform-specific recruitment
• dc.subject.other Cromatinaca
• dc.subject.other Genoma humàca
• dc.title Genome-wide analysis using ChIP to identify isoform-specific gene targetsca
• dc.type info:eu-repo/semantics/articleca
• dc.type.version info:eu-repo/semantics/publishedVersionca