Hypoxia-Inducible Factor 3 Is an Oxygen-Dependent Transcription Activator and Regulates a Distinct Transcriptional Response to Hypoxia

1. Download : Download high-res image (216KB)
2. Download : Download full-size image

     

PGC-1 Coactivators Regulate MITF and the Tanning Response

1. Download : Download high-res image (175KB)
2. Download : Download full-size image

Highlights? PGC-1 induces pigment formation in melanocytes ? PGC-1s activate expression of MITF ? α-MSH induces PGC-1s, which are required for induction of melanogenic genes ? eQTLs in human PGC-1β are associated with tanning ability and melanoma protection     

利用染色质免疫共沉淀技术确定转录因子RIN调控的靶基因

以粉红期番茄果实为材料,用含不同浓度甲醛的缓冲液交联DNA和蛋白质,利用超声波将其染色质随机断裂成大小为200-1 000 bp的片段,用RIN蛋白的特异性抗体免疫沉淀与RIN蛋白结合的DNA片段,然后解交联和纯化DNA片段,最终用普通PCR试验和测序验证与转录因子RIN结合的DNA序列.结果表明,适用于番茄果实的最佳ChIP试验条件为:用1％甲醛溶液交联DNA和蛋白质的复合物;用20％功率,工作6s,间隔10s,脉冲3次超声破碎该复合物,可以得到适当大小的片段,用于后续的试验.普通PCR和测序验证结果证明转录因子RIN与LeACS2和LeACS4启动子区域的CArG box序列结合.     

BRG1/BRM and prohibitin are required for growth suppression by estrogen antagonists

Estrogen antagonists are universally employed in the breast cancer therapy, although antagonist therapy is limited by the inevitable development of cellular resistance. The molecular mechanisms by which these agents inhibit cellular proliferation in breast cancer cells are not fully defined. Recent studies have shown the involvement of the E2F pathway in tamoxifen-induced growth arrest. We show that an E2F repressor, prohibitin, and the chromatin modifiers Brg1/Brm are required for estrogen antagonist-mediated growth suppression through the estrogen receptor, and that their recruitment to native promoter-bound E2F is induced via a JNK1 pathway. In addition, we demonstrate major mechanistic differences among the signaling pathways initiated by estrogen, estrogen deprivation, and estrogen antagonists. Collectively, these findings suggest that the prohibitin/Brg1/Brm node is a major cellular target for estrogen antagonists, and thereby also implicate prohibitin/Brg1/Brm as potentially important targets for breast cancer therapy.     

运用染色质免疫沉淀技术筛选AP-2α的靶基因

在后基因组时代,DNA-蛋白质的相互作用是研究基因表达调控的一个重要领域.染色质免疫沉淀技术(chromatin immunoprecipitation assay,简称CHIP)是目前唯一研究体内DNA与蛋白质相互作用的方法.对与ChIP有关的实验条件进行了优化,获得了较优的实验条件,并运用ChIP实验筛选出了转录因子activator protein-2 alpha (AP-2a)的未知靶基因,对于进一步研究AP-2a的功能和调控网络打下了基础.     

KLF7通过抑制缺氧诱导因子1α转录影响肉鸡脂肪组织发育

Krüppel样转录因子7(Krüppel-like factor 7, KLF7)是脂肪形成的负调控因子,而缺氧诱导因子1(hypoxia-inducible factor 1, HIF1)促进缺氧诱导的哺乳动物脂肪组织发育。本实验室前期利用ChIP-seq技术,发现在鸡的缺氧诱导因子1α(hypoxia-inducible factor 1 alpha, HIF1α)基因上游存在1个KLF7的结合峰,提示KLF7可能调控HIF1α基因转录。为此,本研究首先利用ChIP-PCR技术验证ChIP-seq结果,发现KLF7能够与HIF1α基因5′侧翼区结合。双荧光素酶报告基因与qRT-PCR结果显示,过表达KLF7能够显著下调HIF1α(-4 432/-4 182)的荧光素酶报告基因活性（P<0.01）,抑制HIF1α基因表达。与野生型质粒相比,将生物信息学预测的KLF7结合基序“TGCGCAGCAA”(-4 300/-4 290)缺失突变后, HIF1α(-4 432/-4 182)报告基因活性显著增强（P<0.01）。此外,选取第19代1～7周龄东北农业大学高、低脂双向选择品系肉鸡(Northeast Agricultural University broiler lines divergently selected for abdominal fat content, NEAUHLF)作为实验材料,利用qRT-PCR检测HIF1α基因在肉鸡腹部脂肪组织中的表达规律。结果显示,HIF1α在1～7周龄高脂系肉鸡中的相对表达量均高于低脂系,提示HIF1α对鸡脂肪组织发育具有促进作用。在永生化鸡前体脂肪细胞系(immortalized chicken preadipocyte cell line, ICP1)诱导分化过程中,HIF1α相对表达量逐渐升高。综上所述,HIF1α是转录因子KLF7的一个靶基因,KLF7可能通过抑制HIF1α基因转录参与鸡脂肪组织发育过程。     

Residual Complexes Containing SMARCA2 (BRM) Underlie the Oncogenic Drive of SMARCA4 (BRG1) Mutation

Collectively, genes encoding subunits of the SWI/SNF (BAF) chromatin remodeling complex are mutated in 20% of all human cancers, with the SMARCA4 (BRG1) subunit being one of the most frequently mutated. The SWI/SNF complex modulates chromatin remodeling through the activity of two mutually exclusive catalytic subunits, SMARCA4 and SMARCA2 (BRM). Here, we show that a SMARCA2-containing residual SWI/SNF complex underlies the oncogenic activity of SMARCA4 mutant cancers. We demonstrate that a residual SWI/SNF complex exists in SMARCA4 mutant cell lines and plays essential roles in cellular proliferation. Further, using data from loss-of-function screening of 165 cancer cell lines, we identify SMARCA2 as an essential gene in SMARCA4 mutant cancer cell lines. Mechanistically, we reveal that Smarca4 inactivation leads to greater incorporation of the nonessential SMARCA2 subunit into the SWI/SNF complex. Collectively, these results reveal a role for SMARCA2 in oncogenesis caused by SMARCA4 loss and identify the ATPase and bromodomain-containing SMARCA2 as a potential therapeutic target in these cancers.