Studies on the transcriptional regulatory factors controlling the expression of hCG and LH gene

hCGb亚基由6个基因（或假基因）组成的簇编码。与这个簇相连的是编码相关LHbeta亚基的单个基因。这些基因的总体结构非常相似。hCGbeta基因主要在妊娠早期胎盘中表达，LHbeta基因在垂体中表达。在这个项目中，我们试图阐明是什么因素控制hCGbeta基因和LHbeta基因的组织和阶段特异性表达。这些基因的启动子区域被分离并用于足迹分析和凝胶转移分析。虽然hCGbeta基因片段和LHbeta基因片段利用滋养层核提取物的足迹保护模式非常相似，但存在LHbeta受到保护而hCGbeta没有受到保护的区域。凝胶移位试验表明，结合该区域的因子在除垂体外的组织中均有表达。提示该因子抑制lhβ基因的表达。体外诱变制备LHbeta启动子突变体，取代hCGbeta核苷酸序列，分析其在滋养细胞中获得活性的关键序列。我们还使用不同妊娠阶段的滋养细胞核提取物进行了hCGbeta基因的足迹测定。他们没有表现出明显的差异。可能有许多核因子结合在同一个结构域上，有些因子的变化很难检测到。为了解活性hCGbeta基因与非活性hCGbeta基因的差异，利用hCGbeta基因5和hCGbeta基因7上游序列进行足迹分析。我们无法检测到保护模式的明显变化。这表明可能有一些因素是我们无法通过足迹分析检测到的。本研究结果为进一步研究与该区域结合的核因子调控hCGbeta基因和LHbeta基因提供了基础。

The hCGb subunit is encoded by a cluster of six genes (or pseudogenes). Linked to this cluster is the single gene encoding the related LHbeta subunit. The general structure of these genes is very similar. hCGbeta genes are expressed mainly in first trimester placenta and LHbeta gene is expressed in pituitary gland. In this project, we tried to elucidate what factors control the tissue and stage specific expression of hCGbeta gene and LHbeta gene. Promoter region of these genes were isolated and used for footprint assays and gel shift assays. Although the footprint protection pattern of the hCGbeta gene fragment and LHbeta gene fragment using trophoblastic nuclear extract was very similar, there was a region LHbeta was protected but hCGbeta was not protected. Gel shift assays showed the factor which binds to the region was expressed in tissues except pituitary. It suggests that the factor suppresses expression of LHbeta gene. In vitro mutagenesis to make LHbeta promoter mutants substituted with the hCGbeta nucleotide sequence was made to analyze key sequences to acquire activity in trophoblasts.We also performed footprint assays of hCGbeta gene using trophoblastic nuclear extracts from various stage of pregnancy. They did not show marked difference. Probably there are many nuclear factors binding to the same domain and it is hard to detect change of some factors.To know the difference of the active hCGbeta gene and inactive hCGbeta gene, upstream sequence of hCGbeta gene 5 and hCGbeta gene 7 were used for footprint assays. We could not detect visible change of the protection pattern. It suggests that there may be factors we cannot detect by footprint assay.The information obtained here provides a foundation for further studies on nuclear factors binding to this region regulating hCGbeta gene and LHbeta gene.