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: 7885399
• 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/7885399
• 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; HERVs; Hematopoietic; Human; Immunoprecipitation; In Vitro; K562 Cells; Lentivirus Vector; Locus Control Region; Maps; Messenger RNA; Methylation; Molecular; Molecular Conformation; Mus; 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 Organisms; base; bisulfite; cellular transduction; hydroxyurea; in vivo; promoter; public health relevance; 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（由申请人提供）：ERV-9人内源性逆转录病毒的孤立ltr与3000-4000个人类基因位点相关，包括？-珠蛋白基因位点，其中ERV-9 LTR位于基因座控制区（LCR）上游40-70 kb的5'端附近。-然后呢？球蛋白推动者。ERV-9 LTR增强子含有循环的CCAAT和GATA基序。CCAAT基序结合无处不在的转录因子NF-Y，在活性LTR增强子复合物NF-Y/GATA-2的组装中，将红系因子GATA-2募集到邻近的同源位点。在转基因（Tg）小鼠中携带跨越整个人类的100 kb BAC ？-珠蛋白基因位点，ERV-9 LTR的缺失抑制了2-珠蛋白基因的转录并重新激活了？-珠蛋白基因在Tg小鼠红细胞发育中的作用。我们随后发现，在LTR Tg小鼠中，LTR缺失降低了？-珠蛋白启动子却增加了这些因子在？-珠蛋白启动子，与珠蛋白基因转录的相互转换有关。本应用的目的是测试与ERV-9 LTR相互作用以及与LTR相关的NF-Y、GATA -2和CBP/p300结合的竞争假设。-然后呢？-球蛋白启动子在BAC Tg小鼠和进行体外红细胞生成的原代人红细胞祖细胞中调节球蛋白基因的转录和开关。在目的1中，我们将使用电泳迁移率转移法（EMSA）和染色质免疫沉淀法（ChIP）来确定NF-Y、GATA-2和CBP/p300的水平是否与？——还有？-球蛋白启动子在BAC Tg小鼠和人红细胞祖细胞红细胞发育过程中与球蛋白基因转录活性相关的变化，以及这些因子的水平是否与？-然后呢？-珠蛋白启动子复合物在LTR Tg小鼠各自的发育阶段发生改变。我们将使用gst -pull - down、共免疫沉淀（co-IP）和转染技术，在体外和体内红细胞中鉴定NF-Y、GATA-2和CBP/p300中参与NF-Y/GATA-2/CBP/p300转录复合物组装的相互作用亚域。在Aim 2中，我们将使用ChIP和染色体构象捕获（3C）来确定ERV-9 LTR是否与LCR和珠蛋白启动子物理相互作用，根据BAC Tg小鼠和人红细胞祖细胞中各自的珠蛋白启动子活性。我们将确定这些远程相互作用对wt BAC Tg小鼠中LCR和珠蛋白启动子表观遗传标记的影响，以及LTR缺失对LTR Tg小鼠中珠蛋白位点表观遗传标记的影响。在Aim 3中，我们将使用慢病毒载体在人红细胞祖细胞中过表达或敲低NF-Y和GATA-2，其中？-珠蛋白基因沉默，2-珠蛋白基因高水平转录，以确定改变NF-Y和GATA-2水平是否可以重编程？-然后呢？-珠蛋白基因转录。公共卫生相关性：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，可能为发现能够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的小化合物铺平道路。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（如羟基脲）的不良副作用。