Long-range function of the ERV-9 LTR in regulating globin gene switching

ERV-9 LTR 在调节珠蛋白基因转换中的长程功能

• 批准号: 8321551
• 负责人: DOROTHY Y TUAN
• 金额: $ 36.38万
• 依托单位: AUGUSTA UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-15 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8321551
• 关键词: Acute Erythroblastic Leukemia; Adult; Adverse effects; Affect; Binding; Biological Assay; Cell Nucleus; Chemicals; Chromosomes; Co-Immunoprecipitations; Complex; CpG Islands; Cytotoxic agent; DNA Methylation; Development; Dissection; EP300 gene; Electrophoretic Mobility Shift Assay; Endogenous Retroviruses; Enhancers; Epigenetic Process; Erythroblasts; Erythroid; Erythroid Cells; Erythroid Progenitor Cells; Erythropoiesis; Fetal Liver; Fluorescence; Genes; Genetic Transcription; Globin; HERVs; Hematopoietic; Histone Acetylation; Human; Immunoprecipitation; In Vitro; K562 Cells; Lentivirus Vector; Locus Control Region; Maps; Messenger RNA; Methylation; Molecular; Molecular Conformation; Pharmaceutical Preparations; Plasmids; Recombinants; Recruitment Activity; Recurrence; Reverse Transcriptase Polymerase Chain Reaction; Site; Small Interfering RNA; Spleen; Staging; Subfamily lentivirinae; Switch Genes; Techniques; Testing; Time; Transfection; Transgenic Mice; abstracting; base; bisulfite; cellular transduction; hydroxyurea; in vivo; promoter; transcription factor

Project Summary/Abstract The solitary LTRs of the ERV-9 human endogenous retrovirus are associated with 3000-4000 human gene loci including the ¿-globin gene locus, where the ERV-9 LTR is located near the 5' end of the locus control region (LCR) 40-70 kb upstream of the ¿- and ¿-globin promoters. The ERV-9 LTR enhancer contains recurrent CCAAT and GATA motifs. The CCAAT motifs bind the ubiquitous transcription factor NF-Y, which recruits erythroid factor GATA -2 to the neighboring cognate site in the assembly of an active LTR enhancer complex NF-Y/GATA-2. In transgenic (Tg) mice carrying a 100 kb BAC spanning the entire human ¿globin gene locus, deletion of the ERV-9 LTR suppressed transcription of the ¿-globin gene and re-activated the ¿-globin gene during erythroid development in the erythroid cells of the Tg mice. We subsequently found that in the ¿LTR Tg mice, LTR deletion lowered the levels of NF-Y, GATA-2 and CBP on the ¿-globin promoter but increased the levels of these factors on the ¿-globin promoter in adult erythroid cells, in correlation with the reciprocal switching of globin gene transcription. The objective of this application is to test the hypothesis that the competition for interaction with the ERV-9 LTR and binding to LTR-associated NF-Y, GATA -2 and CBP/p300 between the ¿- and ¿-globin promoters regulates transcription and switching of the globin genes in the BAC Tg mice and in primary human erythroid progenitor cells undergoing in vitro erythropoiesis. In aim 1, we will use electrophoretic mobility shift assay (EMSA) and chromotin immunoprecipitation (ChIP) to determine if the levels of NF-Y, GATA-2 and CBP/p300 associated with the ¿- and the ¿-globin promoters change in correlation with the transcriptional activities of the globin genes during erythroid development in BAC Tg mice and human erythroid progenitor cells and if the levels of these factors associated with the g- and b-globin promoter complexes are altered at the respective developmental stages in ¿LTR Tg mice. We will use GST-pulldown, co-immunoprecipitation (co-IP) and transfection assays to identify the interacting sub-domains in NF-Y, GATA-2 and CBP/p300 involved in the assembly of the NF-Y/GATA-2/CBP/p300 transcriptional complex in vitro and in erythroid cells in vivo. In Aim 2, we will use ChIP and chromosome conformation capture (3C) to determine if the ERV-9 LTR physically interacts with the LCR and the globin promoters in accordance with the respective globin promoter activities in erythroid cells of the BAC Tg mice and human erythroid progenitor cells. We will determine the effects of these long-range interactions on the epigenetic marks of the LCR and the globin promoters in the wt BAC Tg mice and the effects of LTR deletion on the epigenetic marks of the globin locus in ¿LTR Tg mice. In Aim 3, we will use lentiviral vectors to over-express or knockdown NF-Y and GATA-2 in human erythroid progenitor cells, in which ¿-globin gene is silenced and ¿-globin gene is transcribed at a high level, to determine if changing the levels of NF-Y and GATA-2 can reprogram ¿- and ¿-globin gene transcription. Project Narrative/Relevance Understanding the function and mode of assembly of the NF-Y/GATA-2/CBP/p300 complex on the ERV-9 LTR enhancer and the ¿-globin promoter may pave the way for discovery of small chemical compounds that can specifically modulate this complex to preferentially activate the ¿-globin gene without affecting other genes. Such ¿-globin gene-specific drugs may avoid producing the undesirable side effects of currently prescribed cytotoxic drugs such as hydroxyurea.

项目摘要/摘要 ERV-9人内源性逆转录病毒的单个LTRS与3000-4000个人类基因座相关 包括？-珠蛋白基因座位，其中ERV-9 LTR位于座位控制区(LCR)的5‘端附近 珠蛋白启动子上游40-70kb。ERV-9 LTR增强子包含复发性CCAAT和 Gata图案。CCAAT基序结合了普遍存在的转录因子NF-Y，它招募红系因子GATA 到活性LTR增强子复合体-2\f25 NF-Y/GATA-2-2组装中的相邻同源位置。在转基因中 (TG)携带跨越整个人类珠蛋白基因座的100kb BAC的小鼠，ERV-9 LTR的缺失 在红系发育过程中抑制-珠蛋白基因转录并重新激活-珠蛋白基因 TG小鼠的红系细胞。我们随后发现，在LTRTG小鼠中，LTR缺失降低了 核因子-Y、GATA-2和CBP在珠蛋白启动子上的水平但这些因子在珠蛋白启动子上的水平增加 成人红系细胞中的启动子，与珠蛋白基因转录的相互切换相关。这个 本申请的目的是检验这样一个假设，即与ERV-9 LTR和ERV-9相互作用的竞争 珠蛋白启动子和珠蛋白启动子之间与LTR相关的NF-Y、GATA-2和CBP/p300的结合调节 BACTG小鼠和原代人红系祖细胞中珠蛋白基因的转录和转换 正在进行体外红细胞生成。在目标1中，我们将使用凝胶迁移率改变分析(EMSA)和 染色质免疫沉淀法(CHIP)检测核因子-Y、GATA-2和CBP/p300水平是否与 珠蛋白启动子和珠蛋白启动子与珠蛋白基因转录活性的变化 BAC-TG小鼠和人红系祖细胞的红系发育及这些因子的水平 与g-和b-珠蛋白启动子复合体相关的基因在不同发育阶段都会发生变化 ？LTRTg小鼠。我们将使用GST-Pull-Down，共免疫沉淀(co-IP)和转染法来鉴定 参与NF-Y/GATA-2/CBP/p300组装的NF-Y、GATA-2和CBP/p300中相互作用的亚结构域 体外和体内红系细胞的转录复合体。在目标2中，我们将使用芯片和染色体 构象捕获(3C)以确定ERV-9 LTR是否与LCR和珠蛋白物理上相互作用 根据BAC-TG小鼠和BAC-TG小鼠红系细胞中各自珠蛋白启动子活性的启动子 人类红系祖细胞。我们将确定这些远程相互作用对表观遗传学的影响 Wt BAC转基因小鼠LCR和珠蛋白启动子的标记及其缺失对表观遗传学的影响 LTRTg小鼠的珠蛋白基因座标记。在目标3中，我们将使用慢病毒载体来过度表达或敲除 人红系祖细胞中的核因子-Y和GATA-2，其中珠蛋白基因沉默，珠蛋白基因沉默。 在高水平转录，以确定改变核因子-Y和GATA-2的水平是否可以重新编程？和？珠蛋白 基因转录。项目说明/相关性 了解NF-Y/GATA-2/CBP/p300复合体在ERV-9 LTR上的功能和组装方式 增强子和珠蛋白启动子可能为发现能够 特异性地调节这个复合体，优先激活珠蛋白基因，而不影响其他基因。 这种针对珠蛋白基因的药物可能会避免产生目前处方的不良副作用 羟基脲等细胞毒性药物。