Transcriptional Control of Hemoglobin Synthesis

血红蛋白合成的转录控制

• 批准号: 9752268
• 负责人: Emery H Bresnick
• 金额: $ 35.97万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-15 至 2021-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9752268
• 关键词: Address; Adopted; Anabolism; Anemia; Award; BACH1 gene; Binding; Binding Sites; Biological Assay; Biology; CD34 gene; CRISPR/Cas technology; Cell Line; Cells; Chromatin Loop; Development; Disease; Down-Regulation; Elements; Engineering; Enhancers; Erythroblasts; Erythroid; Erythroid Cells; Etiology; Foundations; GATA1 gene; Gene Expression; Generations; Genes; Globin; Hematopoietic stem cells; Heme; Hemoglobin; Human; Instruction; Introns; Link; Maintenance; Mediating; Modeling; Mus; Mutant Strains Mice; Nuclear; Nucleotides; Pathology; Pathway interactions; Porphyrias; Principal Investigator; Reagent; Regulation; Repression; Research; Resources; Site; Statistical Data Interpretation; System; Technology; Testing; Thalassemia; Therapeutic; Transcriptional Activation; Transcriptional Regulation; Work; base; chromatin modification; chromosome conformation capture; cohort; design; heme 1; heme biosynthesis; in vivo; insight; loss of function; small hairpin RNA; synthetic biology; transcriptome

This application is to request a MERIT Award extension. Our progress validated our hypothesis that globin gene expression and heme biosynthesis are interlinked through GATA-1-dependent mechanisms. Aim 1. To distinguish between models for how heme amplifies GATA-1 activity to coordinate hemoglobin biosynthesis and erythroid cell development/function. We will test whether heme permits or enhances the GATA-1-dependent subnuclear transition that expels target loci from the nuclear periphery (Model 1) or enhances GATA-1 activity subsequent to the transition (Model 2). If heme enhances the GATA-1-dependent subnuclear transition of Bach1-sensitive genes, we will analyze the relationship between Bach1 and other factors/coregulators that drive locus relocalization. As the relationship between subnuclear transitions and chromatin looping remains elusive, we will determine whether heme and Bach1 regulate GATA-1-mediated looping. If heme enhances activation subsequent to the transition, we will dissect late mechanistic steps. Aim 2. To assemble activation and repression matrices and use these unique resources to elucidate how the GATA-1/heme circuit establishes a critical sector of the erythroid cell transcriptome. We hypothesize that GATA-1 target gene cohorts requiring unique ensembles of factors (including heme) and coregulators share common mechanisms/pathways. We assembled first-gen. matrices illustrating relationships between target gene expression and factor/coregulator requirements. Considerably expanded second-gen. matrices will be developed. Unraveling the mechanisms/pathways will yield vital insights into hemoglobin synthesis and erythroid cell development/function. Aim 3. To use a synthetic biology approach involving cis-element engineering to ascertain how GATA-1-binding cis-elements control heme biosynthesis and erythroid biology. Using CRISPR/Cas9, we will rewire the cis-element circuitry controlling heme biosynthesis to determine why the Alas2 intron1 GATA-1-binding cis-element is much more important than the intron8 GATA-1-binding cis-element. We will test the hypothesis that the difference reflects intrinsic differences between the elements, or distinct flanking sequences render the elements differentially active at endogenous loci. We will generate G1E-ER-GATA-1 cells in which the cis-elements are swapped to determine if they retain or adopt new attributes at the ectopic chromosomal site. Concepts will be validated in primary erythroblasts and in vivo. These studies will establish rules governing cis-element function in erythroid cells, which will inform GATA factor-dependent mechanisms, biology, and pathologies. RELEVANCE (See instructions): The proposed studies shall provide fundamental insights into mechanisms underlying disorders of hemoglobin synthesis, including thalassemias and anemias and diseases associated with aberrant heme biosynthesis, including porphyrias. Moreover, the work shall provide a conceptual framework for the design and implementation of translational and therapeutic strategies for these disorders.

本申请是要求征收奖励扩展。我们的进步证实了我们的假设 基因表达和血红素生物合成通过GATA-1依赖性机制相互联系。目标1 区分血红素如何放大GATA-1活性以坐标为血红蛋白 生物合成和红细胞发育/功能。我们将测试血红素允许还是增强 依赖GATA-1依赖性亚核跃迁，从核外围排出目标基因座（模型1）或 增强过渡之后的GATA-1活性（模型2）。如果血红素增强了GATA-1依赖性 Bach1敏感基因的核下临界过渡，我们将分析Bach1与其他的关系 驱动基因座重新定位的因素/核心节。作为亚核过渡与 染色质循环仍然难以捉摸，我们将确定血红素和Bach1是否调节GATA-1介导 循环。如果血红素增强过渡后的激活，我们将剖析较晚的机械步骤。 目标2。组装激活和抑制矩阵并使用这些独特的资源来阐明 GATA-1/血红素电路如何建立红细胞转录组的关键部门。我们 假设GATA-1靶基因组需要独特的因素（包括血红素）和 核心节共享共同的机制/途径。我们组装了第一代。矩阵说明 靶基因表达与因子/核心节的需求之间的关系。大大扩展 第二代。矩阵将开发。解开机制/途径将产生重要的见解 血红蛋白合成和红细胞的发育/功能。目标3。使用合成生物学 涉及顺式元素工程的方法，以确定gata-1结合符号元素如何控制 血红素生物合成和红斑生物学。使用CRISPR/CAS9，我们将重新连接顺式元素电路 控制血红素生物合成以确定为什么ALAS2 Intron1 GATA-1结合顺式元素更大得多 重要的是Intron8 GATA-1结合顺式元素。我们将检验以下假设 反映元素之间的内在差异或不同的侧翼序列使元素 内源性位点有差异性。我们将生成g1e-er-gata-1细胞，其中顺式元素 交换以确定它们在异位染色体部位保留还是采用新属性。概念 将在主要的红细胞和体内进行验证。这些研究将制定有关顺式元素的规则 在红细胞细胞中的功能，这将为GATA因子依赖性机制，生物学和病理学提供信息。 相关性（请参阅说明）： 拟议的研究应提供对疾病潜在机制的基本见解 血红蛋白的合成，包括丘脑d和贫血以及与异常血红素相关的疾病 生物合成，包括卟啉症。此外，工作应为设计提供概念框架 以及针对这些疾病的翻译和治疗策略的实施。