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Structure and function of the human beta-globin locus control region

人β-珠蛋白基因座控制区的结构和功能

基本信息

批准号：
9341939
负责人：
JORG BUNGERT
金额：
$ 32.87万
依托单位：
UNIVERSITY OF FLORIDA
依托单位国家：
美国
项目类别：
财政年份：
1997
资助国家：
美国
起止时间：
1997-06-01 至 2019-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9341939
关键词：
Adult Affinity Antibodies Binding Binding Proteins Binding Sites Biotin CD34 gene Cell Culture Techniques Cell Differentiation process Cells Chromatin Chromatin Structure Chromatin Structure Alteration Complex DNA DNA Binding DNA Binding Domain DNA Polymerase II DNA Sequence DNA-Binding Proteins Data Deoxyribonuclease I Development Dimerization Distant Elements Enhancers Erythroid Cells Funding Gene Expression Gene Expression Profile Gene Expression Regulation Generations Genes Genetic Transcription Genome Globin Goals Hemoglobinopathies Human Hypersensitivity In Vitro K562 Cells Knowledge Lead Link Locus Control Region Mediating Methodology Methods Modeling Molecular Conformation Mutation Pattern Play Population Procedures Proteins Protocols documentation RNA Recombinant DNA Recruitment Activity Regulatory Element Role Sickle Cell Anemia Site Specificity Streptavidin Structure Testing Thalassemia Transcript Transcription Elongation Transcription Initiation Site Transcription Process Transgenic Mice Transgenic Organisms Umbilical Cord Blood Untranslated RNA Zinc Fingers beta Globin crosslink dimer disease-causing mutation erythroid differentiation experimental study fetal globin follow-up genome-wide improved in vivo novel nuclear factor-erythroid 2 promoter protein complex public health relevance reconstitution synthetic construct

项目摘要

DESCRIPTION (provided by applicant):The human -type globin genes are expressed exclusively in erythroid cells and regulated by a locus control region (LCR) that is located far upstream of the genes. Mutations in the -globin locus are among the most common disease-causing mutations in the human population. These mutations lead to thalassemias, characterized by reduced or absent adult -globin gene expression, or sickle cell anemia. A possible therapy for these hemoglobinopathies is the reactivation of the normally repressed -globin genes in adult erythroid cells. We propose to continue our efforts to understand how the LCR regulates globin gene expression during development and differentiation and will develop new methodology for changing expression patterns in the - globin gene locus. We established a novel procedure utilizing small synthetic DNA binding domains to analyze and neutralize specific cis-regulatory DNA elements in the -globin gene locus. We will target these synthetic DNA binding domains to known repressor binding sites in the -globin gene promoters and to activator binding sites in the gene encoding for the -globin repressor Bcl11A. Targeting these sites with synthetic DNA binding proteins is expected to increase -globin expression in adult erythroid cells. We and others have shown previously that the LCR recruits transcription complexes which produce enhancer RNAs (eRNAs). Transcription factors NF-E2 and USF have been implicated in transcription complex recruitment to the LCR. We will utilize the synthetic DNA binding domains to analyze the contribution of cis-regulatory elements in the LCR that interact with transcription factors NF-E2 and USF. Furthermore, we will examine if LCR associated transcripts or the process of transcription contributes to LCR mediated activation of globin gene expression. The four aims of this proposal will focus on optimizing the DNA binding specificity and delivery methods for synthetic DNA binding proteins (aim 1), to use synthetic DNA binding proteins to analyze cis-regulatory elements in the -globin locus and to reactivate -globin expression in adult erythroid cells (aim 2), to identify all proteins associated with the human LCR in erythroid cells of transgenic mice (aim 3), and to analyze the mechanism(s) by which the LCR and LCR associated non-coding transcription activates the globin genes (aim 4).

描述（由申请人提供）：人型珠蛋白基因仅在红系细胞中表达，并受到位于基因上游远处的基因座控制区（LCR）的调节。 β珠蛋白基因座的突变是人类中最常见的致病突变之一。这些突变导致地中海贫血，其特征是成人珠蛋白基因表达减少或缺失，或镰状细胞性贫血。这些血红蛋白病的一种可能的治疗方法是重新激活成人红细胞中通常受抑制的β珠蛋白基因。我们建议继续努力了解LCR如何在发育和分化过程中调节珠蛋白基因表达，并将开发新的方法来改变珠蛋白基因座的表达模式。我们建立了一种新的程序，利用小的合成 DNA 结合域来分析和中和 β 珠蛋白基因座中的特定顺式调节 DNA 元件。我们将这些合成的 DNA 结合域靶向 β 珠蛋白基因启动子中已知的阻遏物结合位点，以及编码 β 珠蛋白阻遏物 Bcl11A 的基因中的激活物结合位点。用合成的 DNA 结合蛋白靶向这些位点预计会增加成体红细胞中的β珠蛋白表达。我们和其他人之前已经证明，LCR 招募产生增强子 RNA (eRNA) 的转录复合物。转录因子 NF-E2 和 USF 参与了转录复合物向 LCR 的募集。我们将利用合成的 DNA 结合域来分析 LCR 中与转录因子 NF-E2 和 USF 相互作用的顺式调控元件的贡献。此外，我们将检查 LCR 相关转录物或转录过程是否有助于 LCR 介导的珠蛋白基因表达的激活。该提案的四个目标将重点是优化合成 DNA 结合蛋白的 DNA 结合特异性和递送方法（目标 1），使用合成 DNA 结合蛋白分析 β 珠蛋白基因座中的顺式调控元件并重新激活成体红细胞中的 β 珠蛋白表达（目标 2），识别与人类红细胞中的人类 LCR 相关的所有蛋白质。转基因小鼠（目标 3），并分析 LCR 和 LCR 相关非编码转录激活珠蛋白基因的机制（目标 4）。

项目成果

期刊论文数量（48）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Engineered Zinc Finger DNA-Binding Domains: Synthesis, Assessment of DNA-Binding Affinity, and Direct Protein Delivery to Mammalian Cells.

工程化锌指 DNA 结合域：合成、DNA 结合亲和力评估以及将蛋白质直接递送至哺乳动物细胞。

DOI：
10.1007/978-1-4939-7231-9_27
发表时间：
2017
期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
0
作者：
Hossain,MirA;Knudson,IsaacJ;Thakur,Shaleen;Shen,Yong;Stees,JaredR;Barrow,JoevaJ;Bungert,Jörg
通讯作者：
Bungert,Jörg

Identification of a Novel Enhancer/Chromatin Opening Element Associated with High-Level γ-Globin Gene Expression.

DOI：
10.1128/mcb.00197-18
发表时间：
2018-10-01
期刊：
Molecular and cellular biology
影响因子：
5.3
作者：
Shen Y;Bassett MA;Gurumurthy A;Nar R;Knudson IJ;Guy CR;Perez A;Mellen RW;Ikeda M;Hossain MA;Huang S;Igarashi K;Bungert J
通讯作者：
Bungert J

Context-dependent EKLF responsiveness defines the developmental specificity of the human epsilon-globin gene in erythroid cells of YAC transgenic mice.

背景依赖性 EKLF 反应性定义了 YAC 转基因小鼠红细胞中人ε珠蛋白基因的发育特异性。

DOI：
10.1101/gad.822500
发表时间：
2000
期刊：
Genes & development
影响因子：
10.5
作者：
Tanimoto,K;Liu,Q;Grosveld,F;Bungert,J;Engel,JD
通讯作者：
Engel,JD

Combining chromatin immunoprecipitation and DNA footprinting: a novel method to analyze protein-DNA interactions in vivo.

结合染色质免疫沉淀和 DNA 足迹：一种分析体内蛋白质-DNA 相互作用的新方法。

DOI：
10.1093/nar/30.10.e44
发表时间：
2002
期刊：
Nucleic acids research
影响因子：
14.9
作者：
Kang,Sung-HaeLee;Vieira,Karen;Bungert,Jörg
通讯作者：
Bungert,Jörg

High Fractional Occupancy of a Tandem Maf Recognition Element and Its Role in Long-Range β-Globin Gene Regulation.

串联 Maf 识别元件的高分数占用及其在长程 β-珠蛋白基因调控中的作用。

DOI：
10.1128/mcb.00723-15
发表时间：
2016
期刊：
Molecular and cellular biology
影响因子：
5.3
作者：
Stees,JaredR;Hossain,MirA;Sunose,Tomoki;Kudo,Yasushi;Pardo,CarolinaE;Nabilsi,NancyH;Darst,RussellP;Poudyal,Rosha;Igarashi,Kazuhiko;Huang,Suming;Kladde,MichaelP;Bungert,Jörg
通讯作者：
Bungert,Jörg