Functional analysis of thyroid hormone receptor interacting protein

为了了解甲状腺激素作用的分子方面，我们利用酵母双杂交系统克隆了甲状腺激素受体相互作用蛋白。用人甲状腺激素受体β(TRbeta)在Gal4 DNA结合域载体(PGBT9)中筛选构建在Gal4激活结构域载体(PGAD-GH)中的HeLa细胞cDNA文库。通过核苷酸序列测定，分离到一个与TRbeta相互作用的Done(命名为B1)，并被认为是人核受体共抑制因子(Hn-COR)的部分片段。通过菌落杂交和5‘-RACE技术，克隆了hN-CORS的野生型和其他三个变异体。其中一个变异体(hn-CoRv1)由56个氨基酸组成(A.A.)插入到A.A.1018个野生型N-CoR。第二个基因(hn-CoRv2)是在A.A.1857年和1977年。第三个是一个羧基末端变体，在120A.A.为了研究hN-COR的染色体定位，用hN-Co…进行了荧光原位杂交更多的R基因作为探针。将Hn-cor基因定位于染色体17p11.2。由于Hn-COR通过其羧基末端与受体和维甲酸受体(RAR)相互作用，N-COR变异体(克隆H3)的羧基末端的改变可能影响受体相互作用的特异性。N-COR异构体的存在将支持N-COR异构体的不同表达可能调节甲状腺激素的转录调控。提取的RNA被逆转录，然后用PCR扩增N-COR变异体。在所有研究的细胞系中，与野生型相比，两个N-COR变异体(hn-CoRv2和v3)的mRNA表达最低。然而，Hn-CoRv1在不同细胞系中的表达有所不同。在三种细胞系(NB-1神经母细胞瘤细胞系、Hela宫颈癌细胞系和HOS骨肉瘤细胞系)中，Hn-CoRv1的表达强于野生型。Hn-COR变异体的不同表达可能通过共抑制因子改变抑制水平，调节激素信号。较少

To understand the molecular aspect of thyroid hormone action, we performed to clone TR interacting protein by yeast two hybrid system. Hela cell cDNA library constructed in Gal4 activation domain vector (pGAD-GH) was screened by human thyroid hormone receptor beta (TRbeta) in Gal4 DNA binding domain vector (pGBT9). One done (named B1) to interact with TRbeta was isolated and considered to be a partial fragment of human nuclear receptor co-repressor (hN-CoR) by nucleotide sequencing. By colony hybridization technique and 5'-RACE, we cloned wild type and other three variants of hN-CoRs. One variant (hN-CoRv1) is 56 amino acids (a.a.) insertion in to a.a. 1018 of wild type N-CoR.The second one (hN-CoRv2) is a deletion of 120 a.a between a.a. 1857 and 1977. The third one is a carboxyterminal variant, in that 120 a.a. of wild type sequence is replaced with distinct 50 a.a..To study the chromosomal localization of hN-CoR, fluorescence in situ hybridization technique was performed using hN-Co … More R cDNA as a probe. Then the locus of hN-CoR was assigned to chromosome 17p11.2. Since hN-CoR localizes at one locus, the variants are considered to be generated by alternative splicings.Since mN-CoR interacts with TR and retinoic acid receptor (RAR) through its carboxy-terminal region, the alteration of carboxy-terminus in N-CoR variant (clone H3) might affect specificity of receptor interactions. The existence of N-CoR isoforms will support the possibility that the different expression of these isoforms may modulate transcriptional regulation by thyroid hormone.Expression profile of N-CoR variants was analyzed in 8 human cell lines by RT-PCR method. The extracted RNA was reverse-transcribed and followed by the amplification of N-CoR variants with PCR.In all cell lines studied, mRNA expression of two N-CoR variants (hN-CoRv2 and v3) were detected minimally in comparison with Wild type. However, the expression of hN-CoRv1 was different in the cell lines. In three cell lines (NB-1 neuroblastoma cell line, Hela cervival carcinoma cell line and HOS osteosarcoma cell line), hN-CoRv1 was expressed predominant than wild type. The diffrent expression of hN-CoR variants might modify repression level by co-repressor and modulate hormonal signalings. Less