Systematic Genetic Dissection of Human Erythropoiesis

人类红细胞生成的系统遗传解剖

基本信息

批准号：
8797944
负责人：
Vijay Ganesh Sankaran
金额：
$ 39.56万
依托单位：
BOSTON CHILDREN'S HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2014
资助国家：
美国
起止时间：
2014-09-20 至 2019-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8797944
关键词：
Affect Algorithms Alleles Anemia Beryllium Binding Binding Sites Bioinformatics Biological Biological Assay Cells Child Chromatin Clustered Regularly Interspaced Short Palindromic Repeats Development Disease Dissection Enhancers Epigenetic Process Erythrocytes Erythroid Erythroid Cells Erythroid Progenitor Cells Erythropoiesis Etiology Functional RNA Future GATA1 gene GATA1 protein Gene Expression Genes Genetic Genetic Enhancer Element Genetic Variation Genome Genomic DNA Hematopoietic Human Investigation Lead Link Linkage Disequilibrium Measures Methodology Methods Modeling Morbidity - disease rate Mutagenesis Nucleotides Pattern Point Mutation Process Production Regulatory Pathway Reporter Reporter Genes Resources Role Signal Transduction Source Statistical Models Subfamily lentivirinae TAL1 gene Technology Transcription Factor 3 Transcriptional Regulation Variant Woman Work cell type cofactor cyclin D3 dosage follow-up genetic variant genome wide association study genome-wide high throughput screening human GATA1 protein human disease human tissue improved innovation interest mortality novel progenitor public health relevance success therapeutic target trait transcription factor

项目摘要

DESCRIPTION (provided by applicant): Anemia is a major source of morbidity and mortality worldwide, particularly among women and children. Much of this burden is attributable to defective red blood cell production (erythropoiesis). A greater understanding of erytkhropoiesis holds promise for the development of better therapies for various forms of anemia. Recent genome-wide association (GWA) studies have revealed over 75 loci associated with red blood cell traits. Furthermore, multiple bioinformatic analyses suggest that the majority of variation du to these common variants is intrinsic to red cell progenitors and precursors, allowing for directed studies on the effects of these genetic variants. Nevertheless, only a few studies of specific candidates have revealed new regulators of erythropoiesis. We and others have shown that many of the erythroid-trait associated loci (and variants in strong linkage disequilibrium (LD)) ar located in non-coding regions of the genome, enriched for empirically defined regions of open chromatin and erythroid enhancer elements marked by the erythroid transcription factors (TFs) GATA1, TAL1, KLF1, and NFE2. In select cases, we have shown that these variants are contained in and affect the activity of those enhancers and that they are dependent upon GATA1 activity. In this project, we propose to utilize a novel and innovate approach termed massively parallel reporter assay (MPRA) to systematically measure the activity of thousands of putative enhancer elements containing variants in strong LD with GWA study loci. Enhancers that show differential activity dependent upon the allele of the candidate variant contained will be prioritized for functional follow-up using cutting-edge CRISPR-Cas9 genome editing technology to confirm causality of that variant with respect to changes in proximal gene expression. Additionally, we will perform a second MPRA in GATA1-induced cells to examine whether each element is dependent upon the activity of the key hematopoietic TF GATA1. Increased differential enhancer activity will likely signal disruption of GATA1, co-factor, or nove TF binding sites, providing evidence of the underlying biological mechanism. Furthermore, each enhancer element of interest will be fully explicated per nucleotide using an innovative multiple mutagenesis approach in a similar MPRA, allowing for identification of TF binding site motifs linked to known or novel TFs involved in erythropoiesis. Successful execution of the proposed work will at a minimum identify multiple causal variants implicated in GWA studies of erythroid traits and create a public resource of erythroid-specific enhancer activity and important TF binding sites for future studies of erythropoiesis. Additionally, the proposed study establishes not just a new paradigm for high-throughput follow-up of GWA studies in troublesome non- coding regions of the genome, but creates a novel method for investigation of common transcriptional regulatory pathway disruption of cell-type or lineage-specific TFs and master regulators by common genetic variation which can be extended to many different cell types and human diseases.

描述（由申请人提供）：贫血是全球发病率和死亡率的主要来源，尤其是在妇女和儿童中。这种负担的大部分归因于有缺陷的红细胞产生（红细胞生成）。对Erytkhropoiesis的更多了解有望开发各种形式的贫血的更好疗法。最近的全基因组关联（GWA）研究揭示了与红细胞性状有关的75个基因座。此外，多次生物信息学分析表明，这些常见变体的大多数变异是红细胞祖细胞和前体的固有的，从而可以定向研究这些遗传变异的影响。然而，只有少数关于特定候选人的研究揭示了红细胞生成的新调节剂。我们和其他人已经表明，位于基因组非编码区域的强链不平衡（LD）AR的许多红斑性特性（以及具有牢固链接不平衡（LD）的变体，富含经验定义的开放染色质和红细胞增强剂元素的经验定义区域，由erythroid转录因子（TFS）gaa and1 galf kat katafe。在某些情况下，我们已经表明这些变体包含并影响这些增强剂的活性，并且它们取决于GATA1活性。在这个项目中，我们建议利用一种称为大规模平行的记者测定法（MPRA）的新颖而创新的方法，以系统地测量具有GWA研究基因座的强LD中包含变体的数千种推定增强子元素的活性。使用尖端的CRISPR-Cas9基因组编辑技术的功能随访，优先考虑显示差异活性的增强剂，以确认该变体在近端基因表达的变化方面的因果关系。此外，我们将在GATA1诱导的细胞中执行第二个MPRA，以检查每个元素是否取决于关键造血TF GATA1的活性。增加的增强剂活性增加可能会信号破坏GATA1，二型TF结合位点，从而提供潜在的生物学机制的证据。此外，使用创新的多重诱变方法在相似的MPRA中使用创新的多重诱变方法将每个感兴趣的增强子元素完全阐明，从而可以鉴定与涉及红细胞生成的已知或新型TF相关的TF结合位点基序。成功执行拟议的工作将最少确定与GWA有关红细胞性状研究的多种因果变异，并创造了红细胞特异性增强剂活性和重要的TF结合位点的公共资源，以供红细胞生成的未来研究。此外，拟议的研究不仅建立了在基因组的麻烦非编码区域进行高通量的GWA研究的新范式，而且还创建了一种新的方法，用于研究细胞类型或谱系特异性TFS和主体调节剂的常见转录途径破坏常见的遗传和人类的contenties contectors和许多不同的细胞类型。