The determination of the hSNF5 target genes and the clarification of the transcriptional mechanism of hSNF5

恶性横纹肌样瘤（MRT）是一种针对幼儿的高度侵袭性癌症，在原发性肿瘤和细胞系中表现出hSNF 5基因的失活。我们以前曾报道，hSNF 5在MRT细胞系中的再表达通过p21 WAF 1/CIP 1（p21）mRNA诱导引起G1期阻滞。由于p21是p53抑癌基因的转录靶点，我们确定了p53在hSNF 5诱导p21激活中的作用。我们在A204和TTC 642 MRT细胞系中重新表达hSNF 5，使用表达hSNF 5和GFP或GFP的腺病毒载体，有或没有p53表达的稳定敲除。虽然p53表达的丧失在24小时显著抑制了A204细胞中由hSNF 5诱导的p21转录活性，但在24小时后在TTC 642细胞中没有改变hSNF 5诱导的p21转录活性。这些结果提示hSNF 5通过p53依赖性和p53介导上调p21基因转录。独立机构 ...更多信息 sms分别在A204和TTC 642细胞系中。接下来，我们使用p21启动子的染色质免疫沉淀（ChIP）分析来确定hSNF 5出现在p21启动子的位置，以及它的募集是否改变了其他转录因子或染色质景观的结合。我们的研究结果表明，重新表达hSNF 5结合在1 kb的转录起始位点（TSS），最大的富集在TSS在两个细胞系。此外，通过在A204细胞中再表达hSNF 5，将组蛋白乙酰转移酶（HAT）、RNA聚合酶II（RNAPII）和BRG-1与p53和CDK 8（p53转录程序的共激活因子）组装。相比之下，虽然p53和CDK 8的占有率没有改变后，hSNF 5在TTC 642细胞中的再表达，HAT，RNAPII，和BRG-1水平增加在p21启动子。这些发现与p53敲低实验的结果相关。此外，组蛋白H3 K36三甲基化在两种细胞系的TSS下游增加。这些结果表明，hSNF 5通过自身或p53募集来调节p21启动子的起始活性，从而导致mRNA的延伸。我们的研究结果表明，虽然hSNF 5在A204细胞中诱导p21转录依赖于p53，但在TTC 642细胞中，它不依赖于p53。缺乏对p53的依赖似乎延伸到我们实验室测试的其他MRT细胞系。此外，我们的研究结果表明，hSNF 5可能会改变p21转录启动本身或通过招募其他未知的转录因子。少

Malignant rhabdoid tumor(MRT), a highly aggressive cancer of young children, displays inactivation of the hSNF5 gene in primary tumors and cell lines. We have previously reported that reexpression of hSNF5 in MRT cell lines causes a G1 arrest via p21WAF1/CIP1(p21) mRNA induction. However, the mechanisms of p21 promoter activation by hSNF5 remain unclear.Because p21 is a transcriptional target of the p53 tumor suppressor gene, we determined the role of p53 in hSNF5-induced p21 activation. We reexpressed hSNF5 in the A204 and TTC642 MRT cell lines, with or without stable knockdown of p53 expression, using adenoviral vectors expressing either hSNF5 and GFP or GFP. While loss of p53 expression significantly inhibited p21 transcriptional activity induced by hSNF5 in A204 cells at 24 hours, it did not alter its activity by hSNF5 after 24 hours in the TTC642 cells. These results suggested that the up-regulation p21 transcription by hSNF5 operated through p53-dependent and. independent mechani … More sms in the A204 and TTC642 cell lines, respectively. Next, we used chromatin immunoprecipitation(ChIP) analysis of the p21 promoter to determine where hSNF5 appeared at the p21 promoter and whether its recruitment altered binding of other transcription factors or the chromatin landscape. Our results indicated that reexpressed hSNF5 binds within 1 kb of transcript start site(TSS), with maximal enrichment at the TSS in both cell lines. Furthermore, histone acetyltransferases(HATs), RNA polymerase II(RNAPII), and BRG-1 are assembled with p53 and CDK8(co-activator of p53 transcriptional program) by reexpression of hSNF5 in A204 cells. In contrast, while p53 and CDK8 occupancy did not change after hSNF5 reexpression in the TTC642 cells, HATs, RNAPII, and BRG-1 levels increased at the p21 promoter. These findings correlated with the results of p53 knock-down experiments. Furthermore, histone H3K36 tri-methylation increased downstream of the TSS in both cell lines. These results suggested hSNF5 regulates the initiation activity of the p21 promoter by either itself or p53 recruitment, resulting in the mRNA elongation.Our findings demonstrate that while induction of p21 transcription by hSNF5 in A204 cells depends on p53, it occurs independently of p53 in TTC642 cells. The lack of dependence upon p53 appears to extend to other MRT cell lines tested in our laboratory. Furthermore, our results suggest that hSNF5 may alter p21 transcriptional initiation by itself or through the recruitment of other uncharacterized transcription factors. Less