Systematic Genetic Dissection of Human Erythropoiesis

描述（由申请人提供）：贫血是世界范围内发病率和死亡率的主要来源，特别是在妇女和儿童中。这种负担的大部分是由于有缺陷的红细胞产生（红细胞生成）。对红细胞生成的更深入了解有望为各种形式的贫血提供更好的治疗方法。最近的全基因组关联（GWA）研究已经揭示了超过75个与红细胞性状相关的基因座。此外，多种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细胞固有的，从而允许定向的细胞分化。 研究这些基因变异的影响。然而，只有少数研究特定的候选人揭示了新的调节红细胞生成。我们和其他人已经表明，许多红系性状相关基因座（和强连锁不平衡（LD）中的变体）位于基因组的非编码区，富含经验定义的开放染色质区域和红系转录因子（TF）GATA 1、TAL 1、KLF 1和NFE 2标记的红系增强子元件。在选择的情况下，我们已经表明，这些变体包含在这些增强子中，并影响这些增强子的活性，并且它们依赖于GATA 1活性。在这个项目中，我们建议利用一种新的和创新的方法，称为大规模平行报告分析（MPRA），系统地测量数千个推定的增强子元件的活性，包含强LD与GWA研究位点的变体。根据所含候选变体的等位基因显示差异活性的增强子将优先用于使用尖端CRISPR-Cas9基因组编辑技术进行功能随访，以确认该变体与近端基因表达变化的因果关系。此外，我们将在GATA 1诱导的细胞中进行第二次MPRA，以检查每个元素是否依赖于关键造血TF GATA 1的活性。差异增强子活性的增加将可能是GATA 1、辅因子或新TF结合位点的信号破坏，为潜在的生物学机制提供证据。此外，每个感兴趣的增强子元件将在类似的MPRA中使用创新的多重诱变方法完全解释每个核苷酸，允许鉴定与红细胞生成中涉及的已知或新TF连接的TF结合位点基序。成功执行拟议的工作将在最低限度上确定涉及GWA研究红细胞性状的多种因果变异，并创建红细胞特异性增强子活性的公共资源和重要的TF结合位点，用于未来的红细胞生成研究。此外，所提出的研究不仅建立了用于在基因组的麻烦的非编码区中高通量跟踪GWA研究的新范例，而且创建了用于研究通过常见遗传变异对细胞类型或谱系特异性TF和主调节因子的常见转录调节途径破坏的新方法，其可以扩展到许多不同的细胞类型和人类疾病。

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.