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Functional proteomics in differentiating erythroid cells

分化红细胞的功能蛋白质组学

基本信息

批准号：
8460913
负责人：
JORG BUNGERT
金额：
$ 24.83万
依托单位：
UNIVERSITY OF FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-05-15 至 2016-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8460913
关键词：
Adult Affect Anemia Binding Biochemical Biological Assay Biotin Biotinylation Cells Chromatin Chromatin Loop Chromosomes Code Complex DNA DNA Sequence Data Development Disease Elements Enhancers Erythroid Erythroid Cells Fetal Liver Future Gene Expression Gene Expression Regulation Gene Targeting Genes Genetic Transcription Globin Goals Hemoglobinopathies Human Knowledge Life Locus Control Region MEL Gene Mass Spectrum Analysis Mediating Methodology Modeling Molecular Molecular Conformation Mus Mutation Population Precipitation Proteins Proteomics Recruitment Activity Regulation Regulatory Element Role Sampling Sickle Cell Anemia Site Staging Streptavidin Technology Thalassemia Transcription factor genes Transcriptional Activation Transgenic Mice USF2 gene Undifferentiated Yolk Sac chromatin immunoprecipitation chromatin protein cofactor crosslink embryonic stem cell gain of function human GATA1 protein insight loss of function magnetic beads novel promoter protein complex protein crosslink protein function public health relevance research study restriction enzyme transcription factor

项目摘要

DESCRIPTION (provided by applicant): The human ?-globin gene locus consists of five genes that are expressed in a developmental stage- and erythroid-specific manner and regulated by a locus control region (LCR) located far upstream of the genes. Mutations in the ?-globin gene locus are quite frequent in the human population and associated with mild to severe anemias. Most of these mutations are located within the coding region of the adult ?-globin gene or within regulatory sequences affecting expression of the adult gene. Among the most prominent of ?-globin associated diseases are sickle cell anemia (SCA) and ?- thalassemias. Current treatments for these diseases are unsatisfactory and much effort is being invested in developing novel forms of therapies. We anticipate that further knowledge of how the globin genes are activated during development will create new opportunities for the treatment of hemoglobinopathies. For example, recent evidence suggests that the regulatory elements of the globin locus, including the LCR and the gene promoters, come in close proximity during activation of gene expression. If it is understood how genes are brought into close proximity to the LCR it may be possible in the future to modulate these interactions in such a way that associations of the ?-globin genes are favored over that of the adult ?-globin gene in definitive erythroid cells. We propose to perform a comprehensive analysis of the composition and function of protein complexes containing transcription factors involved in globin gene regulation. In these studies we will utilize novel methodology, including expression of biotin tagged proteins in differentiating erythroid cells and in transgenic mice, to examine the role of transcription factors and associated co-factors in the regulation of chromatin accessibility, globin locus conformational changes, and recruitment of transcription complexes. Biotinylated transcription factors will be isolated from cells using streptavidin coated magnetic beads and subjected to mass-spectrometry for identifying associated proteins, to chromatin immunoprecipitation (ChIP) for identifying associated DNA sequences, and to a combined ChIP and chromosome conformation capture (3C) assay for identifying proteins that function in the context of a globin locus configuration in which the LCR and promoters are in close proximity.

描述(申请人提供)：人类？-珠蛋白基因座由五个基因组成，这些基因以发育阶段和红系特有的方式表达，并受位于基因上游的一个基因座控制区(LCR)调控。-珠蛋白基因突变在人类群体中相当频繁，并与轻度到重度贫血有关。这些突变大多位于成体-珠蛋白基因的编码区或影响成体基因表达的调控序列内。与β-珠蛋白相关的最突出的疾病是镰状细胞性贫血(SCA)和β-地中海贫血。目前对这些疾病的治疗方法并不令人满意，正在投入大量精力开发新的治疗形式。我们预计，进一步了解珠蛋白基因在发育过程中是如何被激活的，将为治疗血红蛋白疾病创造新的机会。例如，最近的证据表明，珠蛋白基因座的调控元件，包括LCR和基因启动子，在基因表达激活过程中非常接近。如果了解了基因是如何接近LCR的，未来就有可能以这样一种方式来调节这些相互作用，即在确定的红系细胞中，？-珠蛋白基因的关联比成年？-珠蛋白基因更受青睐。我们建议对含有参与珠蛋白基因调控的转录因子的蛋白质复合体的组成和功能进行全面的分析。在这些研究中，我们将利用新的方法，包括在红系细胞分化和转基因小鼠中表达生物素标记的蛋白，来研究转录因子和相关辅助因子在调节染色质可及性、珠蛋白基因座构象变化和转录复合体招募中的作用。生物素化的转录因子将使用链霉亲和素包裹的磁珠从细胞中分离出来，并经过质谱仪鉴定相关蛋白质，染色质免疫沉淀(CHIP)鉴定相关DNA序列，以及结合芯片和染色体构象捕捉(3C)分析鉴定在珠蛋白基因座构型(其中LCR和启动子非常接近)中起作用的蛋白质。

项目成果

期刊论文数量（5）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Distinct and overlapping DNMT1 interactions with multiple transcription factors in erythroid cells: Evidence for co-repressor functions.

DOI：
10.1016/j.bbagrm.2016.09.007
发表时间：
2016-12
期刊：
Biochimica et biophysica acta
影响因子：
0
作者：
Dimitris N. Papageorgiou;Elena Karkoulia;Alexandra Amaral-Psarris;P. Burda;Katarzyna E. Kolodziej;J. Demmers;J. Bungert;T. Stopka;J. Strouboulis
通讯作者：
Dimitris N. Papageorgiou;Elena Karkoulia;Alexandra Amaral-Psarris;P. Burda;Katarzyna E. Kolodziej;J. Demmers;J. Bungert;T. Stopka;J. Strouboulis

GATA-1 genome-wide occupancy associates with distinct epigenetic profiles in mouse fetal liver erythropoiesis.

DOI：
10.1093/nar/gkt167
发表时间：
2013-05
期刊：
Nucleic acids research
影响因子：
14.9
作者：
Papadopoulos GL;Karkoulia E;Tsamardinos I;Porcher C;Ragoussis J;Bungert J;Strouboulis J
通讯作者：
Strouboulis J

GATA1 and PU.1 Bind to Ribosomal Protein Genes in Erythroid Cells: Implications for Ribosomopathies.