Systematic Genetic Dissection of Human Erythropoiesis

说明(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。这种负担在很大程度上可归因于红细胞产生缺陷(红细胞生成)。对红细胞生成的更多了解有望开发出更好的治疗各种形式的贫血的方法。最近的全基因组关联(GWA)研究揭示了超过75个与红细胞特征相关的基因座。此外，多种生物信息学分析表明，这些常见变异体的大多数Du变异是红细胞祖细胞和前体固有的，允许定向 对这些遗传变异的影响的研究。然而，只有少数针对特定候选者的研究揭示了红细胞生成的新调节因素。我们和其他人已经证明，许多与红系性状相关的基因座(和强连锁不平衡(LD)的变体)位于基因组的非编码区，富含由红系转录因子(TF)GATA1、TAL1、KLF1和NFE2标记的经验区的开放染色质和红系增强子元件。在某些情况下，我们已经证明这些变体包含在这些增强子中并影响它们的活性，并且它们依赖于GATA1的活性。在这个项目中，我们建议利用一种名为大规模平行报告实验(MPRA)的新颖和创新的方法，系统地利用GWA研究基因座来系统地测量数千个含有强LD变异体的假定增强子元件的活性。使用尖端的CRISPR-Cas9基因组编辑技术，对表现出依赖于所包含的候选变体的等位基因的不同活性的增强子进行优先功能跟踪，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GATA1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的造血因子TFGATA1的活性。差异增强子活性的增加可能会发出GATA1、辅因子或新的TF结合位点的破坏信号，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中使用创新的多重突变方法在每个核苷酸中得到充分解释，从而能够识别与参与红细胞生成的已知或新的TF相关联的TF结合位点基序。这项拟议工作的成功实施将至少识别GWA红系特征研究中涉及的多种因果变异，并为未来的红系生成研究创建红系特异性增强子活性和重要的转铁蛋白结合位点的公共资源。此外，这项拟议的研究不仅为在麻烦的基因组非编码区进行GWA研究的高通量后续研究建立了一个新的范式，而且为研究常见的细胞类型或谱系特定的TF的共同转录调控途径的破坏和通过共同的基因变异掌握调控因子创造了一种新的方法，这些共同的基因变异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.