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.

项目总结/摘要 ERV-9人内源性逆转录病毒的孤立LTR与3000-4000个人基因位点相关 包括ε-珠蛋白基因座，其中ERV-9 LTR位于基因座控制区（LCR）的5'端附近， 在珠蛋白启动子和珠蛋白启动子上游40-70 kb。ERV-9 LTR增强子含有复发性CCAAT和 加塔图案。CCAAT基序结合普遍存在的转录因子NF-Y，其募集红系因子加塔 -2与活性LTR增强子复合物NF-Y/加塔-2组装中的相邻同源位点。转基因 (Tg)携带100 kb BAC的小鼠跨越整个人类珠蛋白基因座，ERV-9 LTR缺失 在红系发育过程中抑制了<$-珠蛋白基因的转录并重新激活了<$-珠蛋白基因， Tg小鼠的红系细胞。我们随后发现，在LTR Tg小鼠中，LTR缺失降低了 NF-Y、加塔-2和CBP在<$-珠蛋白启动子上的水平，但在<$-珠蛋白启动子上这些因子的水平增加 在成人红细胞启动子，与珠蛋白基因转录的相互转换。的 本申请的目的是检验与ERV-9 LTR相互作用的竞争和 结合LTR相关的NF-γ，加塔-2和CBP/p300之间的<$-和<$-珠蛋白启动子调节 BAC Tg小鼠和原代人红系祖细胞中珠蛋白基因的转录和转换 进行体外红细胞生成。在目标1中，我们将使用电泳迁移率变动分析（EMSA）， 染色质免疫沉淀（ChIP）以确定NF-Y、加塔-2和CBP/p300的水平是否与 珠蛋白启动子和珠蛋白启动子的变化与珠蛋白基因的转录活性相关， BAC Tg小鼠和人红系祖细胞中的红系发育以及这些因子的水平 与g-和b-珠蛋白启动子复合物相关的基因在相应的发育阶段发生改变， LTR Tg小鼠。我们将使用GST-下拉，免疫共沉淀（co-IP）和转染试验来鉴定 参与NF-Y/加塔-2/CBP/p300组装的NF-Y、加塔-2和CBP/p300中的相互作用亚结构域 体外转录复合物和体内红系细胞中。在目标2中，我们将使用ChIP和染色体 构象捕获（3C），以确定ERV-9 LTR是否与LCR和球蛋白发生物理相互作用 启动子与BAC Tg小鼠红系细胞中相应的球蛋白启动子活性一致， 人红系祖细胞。我们将确定这些长程相互作用对表观遗传的影响， 野生型BAC Tg小鼠中LCR和珠蛋白启动子的标记以及LTR缺失对表观遗传学的影响 LTR Tg小鼠中珠蛋白位点的标记。在目标3中，我们将使用慢病毒载体来过度表达或敲低 NF-Y和加塔-2在人红系祖细胞中的表达，其中<$-珠蛋白基因沉默，<$-珠蛋白基因沉默。 以高水平转录，以确定改变NF-Y和加塔-2的水平是否可以重新编程<$-和<$-球蛋白 基因转录项目叙述/相关性 了解NF-Y/加塔-2/CBP/p300复合物在ERV-9 LTR上的功能和组装模式 增强子和球蛋白启动子可能为发现小的化学化合物铺平道路， 特异性地调节该复合物以优先激活β-珠蛋白基因而不影响其他基因。 这种珠蛋白基因特异性药物可以避免产生目前处方的不良副作用。 细胞毒性药物如羟基脲。