Genes Ancestral To The Thyroid/steroid Receptor Family

我们正在研究核受体对转录和发育的调控。在我们之前的工作中，我们描述了SKIP （skp-1）在秀丽隐杆线虫发育调控中的发育作用。SKIP是一种参与nhr、Notch和tgf - β调控的转录辅助因子，存在于迄今已测序的所有真核生物基因组中。我们研究了SKIP对秀丽隐杆线虫的发育作用。我们发现SKIP是秀丽隐杆线虫生存和发育所必需的。通过RT-PCR和GFP荧光检测转基因系中CeSKIP的表达始于胚胎并持续到成年。通过RNA介导的抑制（RNAi）导致CeSKIP活性的丧失导致早期胚胎停滞，类似于RNA聚合酶II的抑制。RNA聚合酶II磷酸化在CeSKIP RNAi处理的胚胎中早期表现正常，尽管一些胚胎GFP报告基因的表达严重受限或缺失。SKIP是秀丽隐杆线虫发育过程中不可缺少的因子。我们发现SKIP在一个操纵子中与另一个与Survivin同源的基因组织在一起，而Survivin的表达显然与癌症有关。由于排列在操纵子中的基因经常在功能上连接，我们想知道BIR-1是否也在转录中起作用。bir1抑制导致多种发育缺陷，这些缺陷与CeSKIP重叠，功能表型丧失：卵子保留、矮墩墩、运动缺陷和致命。bir1 RNAi降低了几种gfp转基因基因和内源基因dpy-7和hhl -1的表达。免疫印迹分析显示，在bir1 RNAi处理的蠕虫中，磷酸化组蛋白减少。在异源转染系统中，bir1增强甲状腺激素调节转录，并与SKIP具有加性效应。因此，我们证明了BIR-1在转录和发育调控中起作用。在我们的第三个项目中，我们研究了40例浸润性乳腺癌中甲状腺激素受体α 1 (TR α 1) （N1RA1）的表达。RT-PCR检测到TR α基因座在所有肿瘤和乳腺癌细胞系MCF-7和MDA-MB-231中的表达。使用针对TR alpha1的单克隆抗体进行Western blot分析，结果显示在大多数情况下，在两种细胞系中都有多个n端缺失亚型的表达。全尺寸TR α 1在约10%的癌症中被检测到为小条带，缺失。在MCF-7细胞中，如果没有外源性TRs的共同表达，甲状腺激素受体调节的启动子不会被三碘甲状腺原氨酸激活。组蛋白去乙酰化酶抑制剂Trichostatin A. TR alpha1从MCF-7细胞中扩增并克隆到pCDNA3表达载体中，抑制了甲状腺激素应答启动子的表达。因此，源于TR α基因的n端截短异构体通常在浸润性乳腺癌中表达，并可能作为甲状腺激素受体依赖基因表达的抑制剂。

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.