E2F6 negatively regulates BRCA1 in human cancer cells without methylation of histone H3 on lysine 9.
E2F6 contains a DNA binding domain that is very similar to that of the other members of the E2F family of transcriptional regulators. However, E2F6 cannot bind to all promoters that contain consensus E2F-binding sites. Therefore, we used a combination of chromatin immunoprecipitation and genomic microarrays to identify promoters bound by E2F6 in human cells. Although most of the identified promoters were bound by multiple E2F family members, one promoter was bound only by E2F6. To determine which of the newly identified promoters were regulated by E2F6, we reduced the level of E2F6 by using RNA interference technology. We found that mRNA transcribed from promoters bound by E2F6 was increased after reduction of the amount of E2F6 protein in the cell. Interestingly, many of the E2F6-regulated genes encoded functions involved in tumor suppression and the maintenance of chromatin structure. Specifically, our results suggest that E2F6 represses transcription of the brca1, ctip, art27, hp1alpha, and the rbap48 genes. E2F6 has been postulated to mediate transcriptional repression by recruiting a histone H3 methyltransferase to the DNA. However, we found that the E2F6-regulated promoters did not contain histone H3 methylated at lysine 9. To determine the mechanism by which E2F6 regulates transcription, we performed chromatin immunoprecipitation before and after the introduction of small inhibitory ribonucleic acids specific to E2F6. We found that depletion of E2F6 resulted in the recruitment of E2F1 to the target promoters. In summary, we have identified 48 endogenous target genes of E2F6 and have shown that E2F6 can repress target promoters in a manner that does not require histone H3 methylation at lysine 9.
Oberley
Matthew J
MJ
McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, 1400 University Avenue, Madison, WI 53706, USA.
Inman
David R
DR
Farnham
Peggy J
PJ
D019313:BRCA1 Protein / Q000096:biosynthesis*; D001665:Binding Sites; D018797:Cell Cycle Proteins / Q000378:metabolism / Q000502:physiology*; D045744:Cell Line, Tumor; D002843:Chromatin / Q000737:chemistry / Q000378:metabolism; D000090266:Chromobox Protein Homolog 5; D018899:CpG Islands; D050701:E2F6 Transcription Factor; D015972:Gene Expression Regulation, Neoplastic; D006657:Histones / Q000378:metabolism*; D006801:Humans; D008239:Lysine / Q000378:metabolism; D008745:Methylation; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000378:metabolism / Q000502:physiology; D014157:Transcription Factors
D019313:BRCA1 Protein; C000715594:CBX5 protein, human; D018797:Cell Cycle Proteins; D002843:Chromatin; D050701:E2F6 Transcription Factor; C491744:E2F6 protein, human; D006657:Histones; D012097:Repressor Proteins; D014157:Transcription Factors; D000090266:Chromobox Protein Homolog 5; D008239:Lysine