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。在我们以前的工作中，我们表征了Skip(SKP-1)在线虫发育调控中的发育作用。Skip是一种转录辅因子，参与NHR、Notch和转化生长因子-β的调节，存在于所有已测序的真核生物中。我们研究了SKIP在线虫发育中的作用。我们证明了SKIP是线虫生存和发育所必需的。RT-PCR和GFP荧光法检测转基因株系中CeSKIP的表达始于胚胎，并持续到成年期。通过RNA介导的抑制(RNAi)导致CeSKIP活性的丧失会导致早期胚胎停滞，这与RNA聚合酶II的抑制类似。在CeSKIP RNAi处理的胚胎中，RNA聚合酶II的磷酸化在早期显示正常，尽管几个胚胎GFP报告基因的表达受到严重限制或缺失。SKIP是秀丽隐杆线虫发育过程中不可缺少的因子。我们已经发现，Skip是由一个操纵子和另一个基因组成，该基因是Survivin的同源基因，该基因的表达明显与癌症有关。由于排列在操纵子中的基因经常在功能上相连，我们问过BIR-1是否也在转录中发挥功能。BIR-1抑制导致多种发育缺陷，这些缺陷与CeSKIP功能丧失表型重叠：卵子滞留、矮胖、运动缺陷和致命性。BIR-1 RNAi降低了几个GFP转基因基因和内源基因dpy-7和hlh-1的表达。免疫印迹分析显示，Bir-1RNAi处理的蠕虫的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转录，并与SKIP具有相加作用。因此，我们表明BIR-1在转录和发育的调节中发挥作用。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α1(TRAlpha1)(N1RA1)的表达。RT-PCR检测到在所有被检测的癌症以及乳腺癌细胞系MCF-7和MDA-MB-231中都有trα基因的表达。用针对trα1的单抗进行的Western印迹分析显示，在大多数被检查的病例中以及在两种细胞系中都表达了多种N末端缺失的异构体。在约10%的癌症中，检测到全长的trα1是一条次要条带，并且缺失。在MCF-7细胞中，如果没有外源TRs的共表达，甲状腺激素受体调节的启动子不会被三碘甲腺原氨酸激活。从MCF-7细胞中扩增并克隆到pCDNA3表达载体中的组蛋白脱乙酰酶抑制剂trichostatin A.TrAlpha1不能恢复对T3的反应性。因此，起源于trα基因的N末端截短的异构体在浸润性乳腺癌中普遍表达，并可能作为甲状腺激素受体依赖性基因表达的抑制因子。

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.