Oberley Matthew J, Inman David R, Farnham Peggy J
McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, 1400 University Avenue, Madison, WI 53706, USA.
J Biol Chem. 2003 Oct 24;278(43):42466-76. doi: 10.1074/jbc.M307733200. Epub 2003 Aug 8.
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.
E2F6含有一个DNA结合结构域，该结构域与转录调节因子E2F家族的其他成员非常相似。然而，E2F6不能结合所有含有共有E2F结合位点的启动子。因此，我们结合使用染色质免疫沉淀和基因组微阵列来鉴定人类细胞中与E2F6结合的启动子。尽管大多数鉴定出的启动子与多个E2F家族成员结合，但有一个启动子仅与E2F6结合。为了确定新鉴定出的启动子中哪些受E2F6调控，我们使用RNA干扰技术降低了E2F6的水平。我们发现，在细胞中E2F6蛋白量减少后，从与E2F6结合的启动子转录的mRNA增加。有趣的是，许多受E2F6调控的基因编码参与肿瘤抑制和染色质结构维持的功能。具体而言，我们的结果表明E2F6抑制brca1、ctip、art27、hp1alpha和rbap48基因的转录。据推测，E2F6通过招募组蛋白H3甲基转移酶到DNA上来介导转录抑制。然而，我们发现受E2F6调控的启动子不包含赖氨酸9处甲基化的组蛋白H3。为了确定E2F6调节转录的机制，我们在引入针对E2F6的小干扰核糖核酸之前和之后进行了染色质免疫沉淀。我们发现E2F6的缺失导致E2F1募集到靶启动子上。总之，我们已经鉴定出E2F6的48个内源性靶基因，并表明E2F6可以以不依赖赖氨酸9处组蛋白H3甲基化的方式抑制靶启动子。
