Genes Ancestral To The Thyroid/steroid Receptor Family

甲状腺/类固醇受体家族的祖先基因

• 批准号: 6821132
• 负责人: JOSEPH EDWARD RALL
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6821132
• 关键词: Caenorhabditis elegans; Cnidaria; DNA directed RNA polymerase; biochemical evolution; developmental genetics; gene mutation; genetic mapping; genetic regulation; genetic transcription; hormone receptor; invertebrate embryology; invertebrate hormone; lens; phosphorylation; polymerase chain reaction; protein structure function; reporter genes; steroid hormone receptor; thyroid hormones; tissue /cell culture; transforming growth factors

We are working on regulation of transcription and development by nuclear receptors. In our previous work, we characterized the development role of SKIP (skp-1) on regulation of development in C. elegans. SKIP is a transcription cofactor participating in regulation by NHRs, Notch and TGF-beta and is present in all eukaryotes which genome was sequenced to date. We have studied the developmental role of SKIP on C. elegans. We showed that SKIP is required for C. elegans viablity and development. Expression of CeSKIP assayed by RT-PCR and by GFP fluoresence in transgenic lines starts in embryos and continues to adulthood. Loss of CeSKIP activity by RNA-mediated inhibition (RNAi) results in early embryonic arrest similar to that seen following inhibition of RNA Polymerase II. RNA Polymerase II phosphorylation appears normal early in CeSKIP RNAi treated embryos although the expression of several embryonic GFP reporter genes is severely restricted or absent. SKIP is an indispensable factor for Caenorhabditis elegans development. We have found the SKIP is organized in an operon with another gene that is an orthologue of Survivin, a gene which expression is clearly linked to cancer. Since genes arranged in operons are frequently linked functionally, we have asked if BIR-1 also functions in transcription. bir-1 inhibition resulted in multiple developmental defects that overlapped with CeSKIP loss of function phenotypes: retention of eggs, dumpy, movement defects and lethality. bir-1 RNAi decresed expression of several gfp transgenes and endogenous genes dpy-7 and hlh-1. Immunoblot analysis revealed decreased phosphacetylated histones in bir-1 RNAi treated worms. In a heterologous transfection system, BIR-1 augments thyroid hormone regulated transcription and has an additive effect with SKIP. Thus we show that BIR-1 functions in the regulation of transcription and development. In our third project, we studied the expression of thyroid hormone receptor alpha 1 (TR alpha1) (N1RA1) in 40 cases of infiltrating breast carcinoma. RT-PCR detected expression from the TR alpha locus in all cancers examined and in breast cancer cell lines MCF-7 and MDA-MB-231. Western blot analysis using a monoclonal antibody directed against TR alpha1 revealed expression of multiple N-terminally deleted isoforms in most cases examined and in both cell lines. Full size TR alpha1 was detected as a minor band and was missing in ~10% of cancers. Thyroid hormone receptor regulated promoters are not activated by triodothyronine without co-expression of exogenous TRs in MCF-7 cells. Responsiveness to T3 is not restored by histone deacetylase inhibitor, Trichostatin A. TR alpha1 amplified from MCF-7 cells and cloned in the pCDNA3 expression vector repressed expression from thyroid hormone responsive promoters. Thus, N-terminally truncated isoforms originating from TR alpha gene are commonly expressed in infiltrative breast cancers and are likely to function as inhibitors of thyroid hormone receptor dependent gene expression.

我们正在研究核受体对转录和发育的调控。在我们以前的工作中，我们描述了SKIP（skp-1）在C.优美的SKIP是参与NHR、Notch和TGF-β调节的转录辅因子，并且存在于迄今为止基因组测序的所有真核生物中。我们研究了SKIP在C.优雅的我们证明了C需要SKIP。elegans活力和发展。通过RT-PCR和GFP荧光测定的CeSKIP在转基因系中的表达开始于胚胎并持续至成年。RNA介导的抑制（RNAi）导致CeSKIP活性丧失，导致早期胚胎停滞，类似于RNA聚合酶II抑制后所见。RNA聚合酶II磷酸化在CeSKIP RNAi处理的胚胎早期表现正常，尽管几种胚胎GFP报告基因的表达受到严重限制或不存在。SKIP是秀丽隐杆线虫发育不可缺少的因子。我们已经发现SKIP与另一个基因一起组织在一个操纵子中，该基因是Survivin的直系同源基因，Survivin的表达与癌症明显相关。由于操纵子中排列的基因经常在功能上相连，我们已经问过BIR-1是否也在转录中起作用。bir-1抑制导致与CeSKIP功能表型丧失重叠的多种发育缺陷：卵滞留、矮胖、运动缺陷和致死。bir-1 RNAi降低了几种gfp转基因和内源基因dpy-7和hlh-1的表达。免疫印迹分析显示bir-1 RNAi处理的蠕虫中磷酸乙酰化组蛋白减少。在异源转染系统中，BIR-1增强甲状腺激素调节的转录，并与SKIP具有累加效应。因此，我们表明，BIR-1的功能在转录和发育的调节。 在我们的第三个项目中，我们研究了甲状腺激素受体α 1（TR α 1）（N1 RA 1）在40例浸润性乳腺癌中的表达。RT-PCR检测到TR α基因座在所有检查的癌症和乳腺癌细胞系MCF-7和MDA-MB-231中的表达。使用针对TR α 1的单克隆抗体的Western印迹分析显示，在大多数情况下，在两种细胞系中，多个N-末端缺失的亚型表达。完整大小的TR α 1被检测为次要条带，并且在约10%的癌症中缺失。在MCF-7细胞中，甲状腺激素受体调节的启动子在没有外源TR共表达的情况下不被三碘甲状腺原氨酸激活。组蛋白去乙酰化酶抑制剂曲古抑菌素A不能恢复对T3的反应性。从MCF-7细胞扩增并克隆到pCDNA 3表达载体中的TR α 1抑制了甲状腺激素应答启动子的表达。因此，源自TR α基因的N-末端截短的同种型通常在浸润性乳腺癌中表达，并且可能作为甲状腺激素受体依赖性基因表达的抑制剂起作用。