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: 7528008
• 负责人: DOROTHY Y TUAN
• 金额: $ 36.75万
• 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 2013-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7528008
• 关键词: Acetylation; 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; Green Fluorescent Proteins; HERVs; Hematopoietic; Human; Immunoprecipitation; In Vitro; K562 Cells; Lentivirus Vector; Locus Control Region; Maps; Messenger RNA; Methylation; Molecular; Molecular Conformation; Mus; Pharmaceutical Preparations; Plasmids; Public Health; Range; Recombinants; Recruitment Activity; Recurrence; Reverse Transcriptase Polymerase Chain Reaction; Site; Small Interfering RNA; Spleen; Staging; Subfamily lentivirinae; Switch Genes; Techniques; Testing; Time; Transfection; Transgenic Organisms; base; bisulfite; cellular transduction; day; hydroxyurea; in vivo; promoter; transcription factor

DESCRIPTION (provided by applicant): 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 2-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 ?- and ?-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 2-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. PUBLIC HEALTH 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 3-globin promoter may pave the way for discovery of small chemical compounds that can specifically modulate this complex to preferentially activate the 3-globin gene without affecting other genes. Such 3-globin gene-specific drugs may avoid producing the undesirable side effects of currently prescribed cytotoxic drugs such as hydroxyurea.

描述（申请人提供）：ERV-9人内源性逆转录病毒的单独LTR与包括α-珠蛋白基因座在内的3000-4000个人类基因座相关，其中ERV-9 LTR位于靠近α-和β-珠蛋白启动子上游40-70kb的基因座控制区（LCR）的5'端。 ERV-9 LTR 增强子包含重复出现的 CCAAT 和 GATA 基序。 CCAAT 基序结合普遍存在的转录因子 NF-Y，将红细胞因子 GATA -2 招募到活性 LTR 增强子复合物 NF-Y/GATA-2 组装过程中的邻近同源位点。在携带跨越整个人类 β-珠蛋白基因座的 100 kb BAC 的转基因 (Tg) 小鼠中，ERV-9 LTR 的缺失抑制了 2-珠蛋白基因的转录，并在 Tg 小鼠红系细胞的红系发育过程中重新激活了 β-珠蛋白基因。我们随后发现，在LTR Tg小鼠中，LTR缺失降低了α-珠蛋白启动子上的NF-Y、GATA-2和CBP的水平，但增加了成年红细胞中α-珠蛋白启动子上这些因子的水平，这与珠蛋白基因转录的相互转换相关。本申请的目的是测试以下假设：α-和β-珠蛋白启动子之间与 ERV-9 LTR 相互作用以及与 LTR 相关 NF-Y、GATA -2 和 CBP/p300 结合的竞争调节 BAC Tg 小鼠和经历体外红细胞生成的原代人红系祖细胞中珠蛋白基因的转录和转换。在目标 1 中，我们将使用电泳迁移率变动分析 (EMSA) 和染色质免疫沉淀 (ChIP) 来确定与 α- 和 β- 珠蛋白启动子相关的 NF-Y、GATA-2 和 CBP/p300 的水平是否与 BAC Tg 小鼠和人红系祖细胞红系发育过程中珠蛋白基因的转录活性相关。 这些与α-和β-珠蛋白启动子复合物相关的因子在LTR Tg小鼠的各自发育阶段发生改变。我们将使用 GST 下拉、免疫共沉淀 (co-IP) 和转染测定来鉴定 NF-Y、GATA-2 和 CBP/p300 中参与 NF-Y/GATA-2/CBP/p300 转录复合物体外组装和体内红细胞细胞组装的相互作用子结构域。在目标 2 中，我们将根据 BAC Tg 小鼠红系细胞和人红系祖细胞中各自的珠蛋白启动子活性，使用 ChIP 和染色体构象捕获 (3C) 来确定 ERV-9 LTR 是否与 LCR 和珠蛋白启动子发生物理相互作用。我们将确定这些远程相互作用对 wt BAC Tg 小鼠中 LCR 和珠蛋白启动子的表观遗传标记的影响，以及 LTR 缺失对 LTR Tg 小鼠中珠蛋白基因座表观遗传标记的影响。在目标3中，我们将使用慢病毒载体在人红系祖细胞中过表达或敲低NF-Y和GATA-2，其中α-珠蛋白基因被沉默，2-珠蛋白基因在高水平转录，以确定改变NF-Y和GATA-2的水平是否可以重新编程α-和β-珠蛋白基因转录。公共健康相关性：了解 NF-Y/GATA-2/CBP/p300 复合物在 ERV-9 LTR 增强子和 3-珠蛋白启动子上的功能和组装模式可能为发现小化合物铺平道路，这些化合物可以特异性调节该复合物以优先激活 3-珠蛋白基因而不影响其他基因。这种3-珠蛋白基因特异性药物可以避免产生目前处方的细胞毒性药物例如羟基脲的不期望的副作用。