Use of chromatin immunoprecipitation to clone novel E2F target promoters

Weinmann, A. S., Bartley, S. M., Zhang, T., Zhang, M. Q., Farnham, P. J. (October 2001) Use of chromatin immunoprecipitation to clone novel E2F target promoters. Molecular and Cellular Biology, 21 (20). pp. 6820-6832. ISSN 0270-7306

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URL: http://www.ncbi.nlm.nih.gov/pubmed/11564866
DOI: 10.1128/MCB.21.20.6820-6832.2001

Abstract

We have taken a new approach to the identification of E2F-regulated promoters. After modification of a chromatin immunoprecipitation assay, we cloned nine chromatin fragments which represent both strong and weak in vivo E2F binding sites. Further characterization of three of the cloned fragments revealed that they are bound in vivo not only by E2Fs but also by members of the retinoblastoma tumor suppressor protein family and by RNA polymerase II, suggesting that these fragments represent promoters regulated by E2F transcription complexes. In fact, database analysis indicates that all three fragments correspond to genomic DNA located just upstream of start sites for previously identified mRNAs. One clone, ChET 4, corresponds to the promoter region for beclin 1, a candidate tumor suppressor protein. We demonstrate that another of the clones, ChET 8, is strongly bound by E2F family members in vivo but does not contain a consensus E2F binding site. However, this fragment functions as a promoter whose activity can be repressed by E2F1. Finally, we demonstrate that the ChET 9 promoter contains a consensus E2F binding site, can be activated by E2F1, and drives expression of an mRNA that is upregulated in colon and liver tumors. Interestingly, the characterized ChET promoters do not display regulation patterns typical of known E2F target genes in a U937 cell differentiation system. In summary, we have provided evidence that chromatin immunoprecipitation can be used to identify E2F-regulated promoters which contain both consensus and nonconsensus binding sites and have shown that not all E2F-regulated promoters show identical expression profiles.

Item Type: Paper
Uncontrolled Keywords: TUMOR-SUPPRESSOR GENE S-PHASE ENTRY TRANSCRIPTION FACTOR CELL-CYCLE IN-VIVO GROWTH-REGULATION FIBROBLASTS LEADS BINDING-SITES LIVING CELLS MOUSE E2F1
Subjects: bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification
bioinformatics > genomics and proteomics > genetics & nucleic acid processing
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > Chromatin dynamics
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > DNA expression > promoter
CSHL Authors:
Communities: CSHL labs > Zhang lab
Depositing User: Matt Covey
Date: October 2001
Date Deposited: 15 Jan 2014 17:18
Last Modified: 15 Jan 2014 17:18
PMCID: PMC99859
Related URLs:
URI: http://repository.cshl.edu/id/eprint/29327

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