ERV-9 IN THE BETA-GLOBIN LOCUS CONTROL REGION

人内源性逆转录病毒的ERV-9 LTR是在U3增强子区域中含有不寻常序列特征的中等重复DNA。 广泛的长期目标是确定分散在人类基因组中的ERV-9 LTR在个体发育和造血谱系分化期间转录激活并因此标记早期祖细胞中造血基因和基因家族的顺式连锁位点中的功能意义。 具体目的是检验以下假设：位于人β-珠蛋白基因座控制区（LCR）5'边界中的HS 5位点附近的单个ERV-9 LTR在个体发育的早期阶段启动LCR的转录，并且LCR的这种转录过程在红细胞生成期间调节更下游的β-样珠蛋白基因的转录激活。 将对这一假设的具体方面进行检验。 1）。5 'HS 5 LTR的U3增强子重复序列可以结合在红系和胎盘滋养层细胞中大量表达的有限的同源转录因子。 这些转录因子将通过电泳迁移率变动分析来鉴定。 将克隆编码新转录因子的基因。 将通过含有绿色荧光蛋白报告基因的试验质粒中U3重复序列的定点诱变和转染试验，检查U3增强子复合物的分子结构和功能。 2）。用来自不同的人内源性逆转录病毒家族的LTR替换5 'HS 5 LTR破坏了β-珠蛋白LCR的转录和跨越整个人β-珠蛋白基因座的YAC克隆中的其它下游人β-样珠蛋白基因的转录激活。 本研究将创建携带突变YAC克隆的转基因小鼠。 3）。在小鼠β-珠蛋白LCR的5 ′边界区域中可能存在鼠内源性逆转录病毒的功能等同的单独LTR. 将测序克隆在BAC载体中的β-珠蛋白LCR的鼠5'边界区，以试图鉴定具有与人5' HS 5 LTR相似的序列特征和体外功能特征的鼠LTR。 这项工作可能提供人类基因组功能组织的信息，并有助于人类遗传性疾病的基因治疗。

The ERV-9 LTRs of human endogenous retroviruses are middle repetitive DNAs that contain unusual sequence features in the U3 enhancer region. The broad long-term objective is to ascertain the functional significance of the ERV-9 LTRs dispersed in the human genome in transcriptionally activating and thus marking the cis-linked loci of hematopoietic genes and gene families in early progenitor cells during ontogeny and hematopoietic lineage differentiation. The specific aim is to test the hypothesis that the solo ERV-9 LTR located near the HS5 site in the 5' border of the human beta-globin locus control region (LCR) initiates transcription of the LCR during early stages of ontogeny and that this transcription process of the LCR regulates the transcriptional activation of the further downstream beta-like globin genes during erythropoiesis. Specific aspects of this hypothesis will be tested. 1). The U3 enhancer repeats of the 5'HS5 LTR may bind to a limited set of cognate transcription factors abundantly expressed in erythroid and placental trophoblast cells. These transcription factors will be identified by electrophoretic mobility shift assays. Genes encoding new transcription factors will be cloned. The molecular architecture and function of the U3 enhancer complex will be examined by site-directed mutagenesis of the U3 repeats in test plasmids containing the Green Fluorescent Protein reporter gene and by transfection assays. 2). Replacement of the 5'HS5 LTR with an LTR from a different family of human endogenous retroviruses disrupts the transcription of the beta-globin LCR and the transcriptional activation of the further downstream human beta-like globin genes in a YAC clone spanning the entire human beta-globin gene locus. Transgenic mice harboring the mutant YAC clones will be created for this study. 3). A functionally equivalent solo LTR of a murine endogenous retrovirus may exist in the 5' boundary area of the mouse beta- globin LCR. The murine 5' boundary area of the beta-globin LCR cloned in a BAC vector will be sequenced in an attempt to identify a murine LTR with similar sequence features and in vitro functional characteristics as the human 5'HS5 LTR. The proposed work may provide information on the functional organization of the human genome and aid in the effort on gene therapy of hereditary human diseases.