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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

基本信息

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
项目类别：
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

项目摘要

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/文摘