Lei Li, Lu Lu, Xiang Lv, Xue-song Wu, De-pei Liu, Chih-chuan Liang
Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences, Beijing 100005, China.
Chin Med Sci J. 2010 Dec;25(4):199-205. doi: 10.1016/s1001-9294(11)60002-3.
To study the regulatory mechanism of SATB1 repression in cells other than T cells or erythroid cells, which have high expression level of SATB1.
HeLa epithelial cells were treated with either histone deacetylase inhibitor (HDACi) trichostatin A (TSA) or DNA methylation inhibitor 5-Aza-C before detecting SATB1 expression. Luciferase reporter system was applied to measure effects of EZH2 on SATB1 promoter activity. Over-expression or knockdown of EZH2 and subsequent quantitative reverse transcription-polymerase chain reaction were performed to determine the effect of this Polycomb group protein on SATB1 transcription. Chromatin immunoprecipitation (ChIP) assay was applied to measure enrichment of EZH2 and trimethylated H3K27 (H3K27me3) at SATB1 promoter in HeLa cells. K562 cells and Jurkat cells, both having high-level expression of SATB1, were used in the ChIP experiment as controls.
Both TSA and 5-Aza-C increased SATB1 expression in HeLa cells. Over-expression of EZH2 reduced promoter activity as well as the mRNA level of SATB1, while knockdown of EZH2 apparently enhanced SATB1 expression in HeLa cells but not in K562 cells and Jurkat cells. ChIP assay Results suggested that epigenetic silencing of SATB1 by EZH2 in HeLa cells was mediated by trimethylation modification of H3K27. In contrast, enrichment of EZH2 and H3K27me3 was not detected within proximal promoter region of SATB1 in either K562 or Jurkat cells.
SATB1 is a bona fide EZH2 target gene in HeLa cells and the repression of SATB1 by EZH2 may be mediated by trimethylation modification on H3K27.
研究SATB1在除T细胞或红细胞外高表达SATB1的细胞中的抑制调控机制。
在检测SATB1表达之前，用组蛋白去乙酰化酶抑制剂（HDACi）曲古抑菌素A（TSA）或DNA甲基化抑制剂5-氮杂胞苷（5-Aza-C）处理HeLa上皮细胞。应用荧光素酶报告系统来检测EZH2对SATB1启动子活性的影响。进行EZH2的过表达或敲低以及随后的定量逆转录-聚合酶链反应，以确定这种多梳蛋白家族蛋白对SATB1转录的影响。应用染色质免疫沉淀（ChIP）分析来检测HeLa细胞中SATB1启动子处EZH2和三甲基化H3K27（H3K27me3）的富集情况。ChIP实验中使用SATB1高表达的K562细胞和Jurkat细胞作为对照。
TSA和5-Aza-C均增加了HeLa细胞中SATB1的表达。EZH2的过表达降低了启动子活性以及SATB1的mRNA水平，而EZH2的敲低则明显增强了HeLa细胞中SATB1的表达，但在K562细胞和Jurkat细胞中未增强。ChIP分析结果表明，HeLa细胞中EZH2对SATB1的表观遗传沉默是由H3K27的三甲基化修饰介导的。相反，在K562细胞或Jurkat细胞的SATB1近端启动子区域内未检测到EZH2和H3K27me3的富集。
SATB1是HeLa细胞中真正的EZH2靶基因，EZH2对SATB1的抑制可能由H3K27的三甲基化修饰介导。
