Transcriptional Control of Hemoglobin Synthesis

血红蛋白合成的转录控制

• 批准号: 9302889
• 负责人: Emery H Bresnick
• 金额: $ 38.47万
• 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/9302889
• 关键词: Address; Adopted; Anabolism; Anemia; Award; Beryllium; Binding; Binding Sites; Biological Assay; Biology; Boxing; 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; Gene Targeting; Generations; Genes; Globin; Hematopoietic; 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; Stem cells; System; Technology; Testing; Thalassemia; Therapeutic; Transcriptional Activation; Transcriptional Regulation; Work; base; chromatin modification; chromosome conformation capture; cohort; design; heme biosynthesis; in vivo; insight; loss of function; small hairpin RNA; stem; 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.

此申请是为了申请延长MERIT奖。我们的进展证实了我们的假设， 基因表达和血红素生物合成通过加塔-1依赖性机制相互关联。目标1.到 区分血红素如何放大加塔-1活性以协调血红蛋白的模型 生物合成和红系细胞发育/功能。我们将测试血红素是否允许或增强 将靶基因座从核周边排出的加塔-1依赖性亚核转换（模型1），或 增强了转变后的加塔-1活性（模型2）。如果血红素增强了依赖于加塔-1的 Bach 1敏感基因的亚核转换，我们将分析Bach 1与其他基因的关系。 驱动基因座重定位的因子/辅助调节因子。由于亚核跃迁和 染色质环仍然难以捉摸，我们将确定血红素和Bach 1是否调节加塔-1介导的 循环如果血红素增强激活后的过渡，我们将剖析后期的机制步骤。 目标二。组装激活和抑制矩阵，并使用这些独特的资源来阐明 加塔-1/血红素回路如何建立红系细胞转录组的关键部分。我们 假设加塔-1靶向基因群需要独特因子集合（包括血红素）， 协同调节因子有共同的机制/途径。我们组装了第一代矩阵， 靶基因表达与因子/辅助调节因子需求之间的关系。大大扩展 将制定第二代矩阵。解开机制/途径将产生重要的见解， 血红蛋白合成和红系细胞发育/功能。目标3.利用合成生物学 一种涉及顺式元件工程以确定加塔-1结合顺式元件如何控制 血红素生物合成和红系生物学。使用CRISPR/Cas9，我们将重新连接顺式元件电路， 控制血红素的生物合成，以确定为什么Alas 2内含子1加塔-1结合顺式元件 比内含子8加塔-1结合顺式元件更重要。我们将检验这个假设， 反映了元件之间的内在差异，或者不同的侧翼序列使元件 在内源基因座上有差异活性。我们将产生G1 E-ER-加塔-1细胞，其中顺式元件 以确定它们是否在异位染色体位点保留或采用新的属性。概念 将在原代成红细胞和体内进行验证。这些研究将建立顺式元件的规则 在红系细胞中起作用，这将告知加塔因子依赖性机制、生物学和病理学。 相关性（参见说明）： 拟议的研究将提供基本的洞察机制的疾病， 血红蛋白合成，包括地中海贫血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，该工作应为设计提供概念框架 以及针对这些疾病的转化和治疗策略的实施。