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3000-4000人类基因位点有关，包括？-Globin基因基因座，ERV-9 LTR位于基因座控制区域（LCR）40-70 kb的5'末端附近的ERV-9 LTR位于？？ ERV-9 LTR增强子包含经常性的CCAAT和GATA图案。 CCAAT基序结合了无处不在的转录因子NF-Y，该转录因子NF-Y募集了红细胞因子GATA -2与邻近的同源位点，在活性LTR增强剂复合物NF-Y/GATA-2的组装中。在横跨整个人类的100 kb BAC的转基因（TG）小鼠中，Globin基因基因座的缺失，ERV-9 LTR的缺失抑制了2-蛋白基因的转录，并重新激活了 - 粉刺基因在TG小鼠的肾上腺细胞中毛状腺发育过程中。随后，我们发现，在LTR TG小鼠中，LTR缺失降低了？ - 珠蛋白启动子上的NF-Y，GATA-2和CBP的水平，但与Globin Gene Gene转录的相关性相关的成人红细胞启动子在成人红细胞启动子上的这些因素水平增加。该应用的目的是检验以下假设：与ERV-9 LTR相互作用并与？ - globin启动子之间在BAC TG小鼠中的全球基因的转录和转换型在eRyth erythroid erythroid reconity-eryththeriD-necromeniton中，与？ - globin启动子之间的竞争与与？ - globin启动子之间的竞争结合。 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在LTR TG小鼠的各个发育阶段，这些因素与？ - 和？ - 珠蛋白启动子络合物相关。我们将使用GST-Pulldown，共免疫沉淀（CO-IP）和转染测定法确定NF-Y，GATA-2和CBP/P300在NF-Y/GATA-Y/GATA-2/CBP/CBP/CBP/P300转录复合物中的NF-Y，GATA-2和CBP/p300中的相互作用亚构域。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是否根据BAC TG小鼠和人类红细胞祖细胞的细菌细胞中的各个球蛋白启动子在相应的球蛋白启动子中与LCR和球蛋白启动子进行物理相互作用。我们将确定这些远程相互作用对WT BAC TG小鼠中LCR和球蛋白启动子的表观遗传标记的影响，以及LTR缺失对LTR TG小鼠球蛋白基因座表观遗传标记的影响。在AIM 3中，我们将使用喉病毒载体在人红骨祖细胞中过度表达或敲除NF-Y和GATA-2，其中？ - 格珠蛋白基因是沉默的，并且2-蛋白基因在高水平上转录，以确定是否会更改NF-Y-Y和GATA-2可以更改nf-y和gata-2可以重新分析？公共卫生相关性：了解ERV-9 LTR增强剂上NF-Y/GATA-2/CBP/P300复合物的组装功能和模式，3-珠蛋白启动子可能为发现的小型化学化合物铺平了道路，这些化合物可以发现可以特异性地调节这种复合物以优惠地激活3-蛋白基因而不会影响其他基因而不会影响其他基因。这种3-珠蛋白基因特异性药物可能避免产生当前处方的细胞毒性药物（如羟基脲）的不良副作用。