Identification of sequences and factors required for HbF repression

 描述(由申请人提供)：胎儿血红蛋白水平是反映β-血红蛋白紊乱、镰状细胞病和β-地中海贫血临床严重程度的关键指标。这个 这项建议的目标是确定调节HbF水平的机制，并可作为重新诱导β-Hb疾病的HbF的合理靶点。全基因组关联研究(GWAS)已确定BCL11A是一个关键的HBF相关基因座。功能研究表明，BCL11A是一种真正的γ-珠蛋白转录抑制因子。自然发生的常见遗传变异调节BCL11A的红系特异性增强子。我们先前的研究发现BCL11A标记的红系增强子是与HBF水平相关的常见遗传变异的底物。基因组编辑揭示了这种增强子内的关键序列，这是成年阶段抑制HBF所必需的。我假设这些序列招募了关键的反式作用因子，而这些因子本身是抑制HBF水平所必需的。此外，我推测BCL11A和HBS1L-MYB基因座上的其他关键序列也需要在成体阶段抑制HBF。为了验证这些假设，我将：(1)利用高分辨率、高通量的基因组编辑方法来确定与HBF水平相关的BCL11A和HBS1L-MYB的非编码序列；以及(2)定义与BCL11A和HBS1L-MYB的关键编码序列在物理和功能上相互作用的反式作用因子。我期望这些目标的实现不仅将加深我们目前对珠蛋白基因表达发育调控的顺式和反式作用决定因素的理解，而且还将产生新的见解来指导重新诱导HBF的治疗努力。

 DESCRIPTION (provided by applicant): The level of fetal hemoglobin (HbF) is a key modifier of the clinical severity of the β-hemoglobin disorders sickle cell disease and β-thalassemia. The goal of this proposal is to identify mechanisms that regulate HbF level and could serve as rational targets for efforts to re-induce HbF for the β-hemoglobin disorders. Genome- wide association studies (GWASs) have identified BCL11A as a critical HbF-associated locus. Functional studies have demonstrated that BCL11A is a bona fide repressor of γ-globin transcription. Naturally occurring common genetic variation modulates an erythroid-specific enhancer of BCL11A. Our prior studies identified the GWAS-marked erythroid enhancer of BCL11A as the substrate for common genetic variation associated with HbF level. Genome editing has revealed critical sequences within this enhancer required for HbF repression in the adult stage. I hypothesize that these sequences recruit critical trans-acting factors, which themselves are required for suppressing HbF levels. In addition, I hypothesize that other critical sequences at the BCL11A and HBS1L-MYB loci are required to repress HbF in the adult-stage. To test these hypotheses, I will: (1) utilize a high-resolution, high-throughput genome editing approach to identify non-coding sequences at BCL11A and HBS1L-MYB associated with HbF level; and (2) define trans-acting factors that physically and functionally interact with critical on-coding sequences at BCL11A and HBS1L-MYB. I expect that accomplishment of these aims will not only deepen our current understanding of cis- and trans-acting determinants of developmental regulation of globin gene expression but also yield novel insights to guide therapeutic efforts for HbF re-induction.