Name: genome-wide analysis using chip to identify isoform-specific gene targets
Uploaded: 2010-07-07T17:00:00
Duration: 11 min 19 s
Description: Watch this Scientific Journal Video about Genome-wide Analysis using ChIP to Identify Isoform-specific Gene Targets at JoVE.com

इस काम के लिए 115,347-RSG-08-271 01-जीएमसी ACS से, और CA138631 द्वारा NIH से अनुदान द्वारा समर्थित किया गया.

<ol>
	<li>Wang, G. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Haematopoietic+malignancies+caused+by+dysregulation+of+a+chromatin-binding+PHD+finger.">Haematopoietic malignancies caused by dysregulation of a chromatin-binding PHD finger.</a> Nature. 459 (7248), (2009).</li><li>Benevolenskaya, E. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Binding+of+pRB+to+the+PHD+protein+RBP2+promotes+cellular+differentiation.">Binding of pRB to the PHD protein RBP2 promotes cellular differentiation.</a> Mol Cell. 18 (6), 623-623 (2005).</li><li>Lopez-Bigas, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+analysis+of+the+H3K4+histone+demethylase+RBP2+reveals+a+transcriptional+program+controlling+differentiation.">Genome-wide analysis of the H3K4 histone demethylase RBP2 reveals a transcriptional program controlling differentiation.</a> Mol Cell. 31 (4), 520-520 (2008).</li><li>Benevolenskaya, E. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+H3K4+demethylases+are+essential+in+development+and+differentiation.">Histone H3K4 demethylases are essential in development and differentiation.</a> Biochem Cell Biol. 85 (4), 435-435 (2007).</li><li>Odom, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+pancreas+and+liver+gene+expression+by+HNF+transcription+factors.">Control of pancreas and liver gene expression by HNF transcription factors.</a> Science. 303 (5662), 1378-1378 (2004).</li><li>Guenther, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aberrant+chromatin+at+genes+encoding+stem+cell+regulators+in+human+mixed-lineage+leukemia.">Aberrant chromatin at genes encoding stem cell regulators in human mixed-lineage leukemia.</a> Genes Dev. 22 (24), 3403-3403 (2008).</li></ol>

ब्याज की कोई संघर्ष की घोषणा की.

RBP2 isoforms के बीच कार्यात्मक मतभेद नहीं निर्धारित किया गया है. हम एक व्यापक दृष्टिकोण का इस्तेमाल किया है जीनोमिक RBP2 isoforms द्वारा बाध्य क्षेत्रों की पहचान और परिभाषित कार्यात्मक श्रेणियों कि इन क्षेत्रों का प्रतिनिधित्व करते हैं. इस चिप Seq प्राप्त अनुक्रम के जैव सूचना विज्ञान विश्लेषण पढ़ता द्वारा पीछा विश्लेषण के द्वारा पूरा किया गया था. RBP2 histone पूंछ पर methylated लाइसिन अवशेषों को संशोधित. हमने पाया है कि RBP2 बड़े isoform methylated histone lysine और RBP2 छोटे मानव जीनोम (चित्रा 5A) में विभिन्न क्षेत्रों के लिए इस मॉड्यूल बाँध कमी isoform के लिए मान्यता मॉड्यूल से युक्त है. महत्वपूर्ण बात, isoform विशिष्ट क्षेत्रों और अतिव्यापी क्षेत्रों अलग आणविक कार्यों (चित्रा 5B) के साथ जीन के हैं. उदाहरण के लिए, &quot;chromatin बाध्यकारी&quot; और &quot;प्रतिलेखन कारक बाध्यकारी&quot; कार्यों, RBP2 छोटे isoform के जीन लक्ष्य को जिम्मेदार माना जा सकता है लेकिन RBP2 बड़े नहीं isoform (चित्रा 5B). वास्तविक जीन की तुलना करके उत्पन्न सेट सभी isoforms और RBP2 बड़े isoform (नहीं दिखाया डेटा है) के लिए, हम भी परिभाषित कर सकते हैं अगर बड़े isoform विशेष रूप से कुछ जीनों के लिए भर्ती है.

Oligonucleotide दृश्यों 2006 Illumina, इंक सभी अधिकार सुरक्षित. <ul><li> Sol_PCR_1 अनुक्रम 5&#39;-AAT GAT ACG GCG एसीसी एसीसी भूमिकाः एटीसी टीएसी अधिनियम सीटीटी टीसीसी CTA सीएसी GAC GCT सीटीटी CCG एटीसी T-3 &#39; </li><li> Sol_PCR_2 अनुक्रम 5&#39;-CAA GAA GAC GGC एटीए CGA GCT सीटीटी CCG एटीसी GCA T-3 &#39; </li></ul>

genome-wide analysis using chip to identify isoform-specific gene targets

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)1. The transcript corresponding to the RBP2 isoform containing the C-terminal PHD accumulates during differentiation of promonocytic, lymphoma-derived, U937 cells into monocytes2. 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 H3K4me33. 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 isoform4. 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.

Watch this Scientific Journal Video about Genome-wide Analysis using ChIP to Identify Isoform-specific Gene Targets at JoVE.com

Genome-wide Analysis using ChIP to Identify Isoform-specific Gene Targets

यहाँ हम जीनोम चौड़ा प्रोटीन isoforms है कि एक histone बाध्यकारी डोमेन में अलग स्थान विश्लेषण के लिए एक chromatin immunoprecipitation प्रक्रिया (चिप) प्रस्तुत कर रहे हैं. हम यह चिप Seq विश्लेषण के लिए आवेदन कर रहे हैं KDM5A/JARID1A/RBP2 histone demethylase के लक्ष्यों की पहचान.

जीनोम चौड़ा चिप का उपयोग isoform विशेष जीन लक्ष्य पहचानें विश्लेषण

Recruitment of transcriptional and epigenetic factors to their targets is a key step in their regulation. Prominently featured in recruitment are the ...

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