The dissection of non-canonical cis-regulatory elements downstream of beta-globin locus in the fetal hemoglobin gene regulation

项目摘要/摘要 在人类基因组中，只有10%是负责蛋白质编码的编码区。尤其是非- 直接调节基因表达的编码区--顺式调节元件(CRE)--非常重要 在正常发育和致病发展中的重要性。Cre通常受以下约束 转录因子(TF)。Cres在胎儿向成人红细胞生成过程中的珠蛋白转换中起着重要作用。 CRE可作为增强子(LCR区)、抑制子(γ珠蛋白启动子中的BCL11A结合部位)或绝缘体(3‘HS1 和规范的β-珠蛋白基因座边界上的HS5CTCF结合位点(CBS)。Cre作为新的探索已被探索 基因治疗的靶点是珠蛋白基因的表达，这对基因治疗的发展至关重要。 血红蛋白病和地中海贫血。传统上，Cres调控β珠蛋白基因表达的研究 限制在5‘HS和3’HS1 CTCF结合位点的边界之间。最近，我们和其他人一起， 揭示了β-珠蛋白基因簇周围的嵌套多环3D基因组结构。多环3D基因组 结构延伸了β-珠蛋白基因簇的调控格局，下游延伸了145kb (形成称为d-TAD的3D基因组结构)和110kb的亚TAD上游(形成3D基因组结构 称为u-tad)。利用与β-珠蛋白基因相邻的多个CBS(缺失和倒置)的基因组编辑 我们发现，3‘HS1-β-珠蛋白基因座下游边界CBS的缺失频率较高 被hpfh缺失打乱--足以在成人红细胞中诱导γ-珠蛋白。重要的是，我们找到了一个 HPFH增强子是HPFH中激活胎儿血红蛋白的原因之一，长期以来人们一直推测HPFH增强子并置激活胎儿血红蛋白 用于γ-珠蛋白的重新激活。通过删除48kb区域使HPFH增强子更接近β-珠蛋白 我们还观察到胎儿血红蛋白的上调，这表明HPFH增强子是一种新的 γ珠蛋白表达的条件增强子。我们发表的研究，与其他人一起，强烈地表明 嵌套在β-珠蛋白基因簇下游亚TAD的Cres确实在 调控珠蛋白基因的表达。这些数据使我们假设d-TAD中的Cres正在调节β珠蛋白 通过远程3D基因组相互作用的基因表达。凭借最新的技术，我们建议 有两个研究目的。在Aim1中，我们将使用基于CRISPR/Cas9的Prime编辑来引入Tf结合突变体 准确地询问位于d-TAD的Tf结合中枢中的Tf结合协同性。表观基因组编辑 也将应用于HUDEP-2细胞中d-TAD处的TF结合中心，以检验组蛋白PTM在调节 基因表达。我们还将应用基因组和表观基因组编辑，使CRES在促进γ- 珠蛋白表达。在AIM2中，我们建议研究由d-TAD和TAD之间的CRE形成的3D CRE中枢 典型的β-珠蛋白星系团。我们将使用HiChIP来剖析由GATA1等活跃的TF介导的相互作用 以及像BCL11A这样的压抑的TF。总体而言，我们提出的研究将扩展镇定者的机械论观点 γ-珠蛋白的表达，为镰状细胞病或β-地中海贫血提供新的基因治疗靶点。

Project Summary/Abstract In the human genome, only 10% are coding regions responsible for protein coding. Particularly, the non- coding regions that directly regulate the gene expression – cis-regulatory elements (CREs) – are of great importance in normal development and pathogenic disease development. CREs are generally bound by transcriptional factors (TFs). CREs play an important role in the globin switch during fetal to adult erythropoiesis. CREs act as enhancers (LCR region), repressors (BCL11A binding site in γ globin promoter), or insulators (3'HS1 and HS5 CTCF binding sites (CBSs) on canonical β-globin locus border). CREs have been explored as new gene therapy targets for the globin gene expression, which is critical for gene therapy development of hemoglobinopathies and thalassemia. Traditionally, studies of CREs regulating β globin locus gene expression are limited between the border of 5'HS and 3'HS1 CTCF binding sites. Recently, together with others, we revealed a nested multi-loop 3D genomic structure around the β-globin gene cluster. The multi-loop 3D genome structure extends the regulatory landscape of the β-globin gene cluster with an extension of 145kb downstream (forming a 3D genomic structure called d-TAD) and a 110kb sub-TAD upstream (forming a 3D genomic structure called u-TAD). Utilizing genome editing of multiple CBSs (deletions and inversions) bordering the β-globin gene cluster, we found that the deletion of the CBS at 3'HS1 – the β-globin locus downstream boundary frequently disrupted by HPFH deletions – is sufficient to induce γ-globin in adult red blood cells. Importantly, we located an HPFH enhancer that's long speculated for the juxtaposition to activate fetal hemoglobin in HPFHs is responsible for the reactivation of γ-globin. By deleting the 48kb region to bring the HPFH enhancer closer to the beta-globin locus, we also observed an upregulation of fetal hemoglobin, suggesting the HPFH enhancer is a novel conditional enhancer for γ globin expression. Our published work, together with others, strongly suggests the CREs nested in the downstream sub-TAD of the β-globin gene cluster indeed played an important role in regulating globin gene expression. These data lead us to hypothesize that CREs in d-TAD are regulating β globin gene expression through long-range 3D genomic interactions. With the most updated technologies, we propose two research aims. In Aim1, we will use CRISPR/Cas9-based prime editing to introduce TF binding mutant precisely to interrogate the TF binding cooperativity in the TF binding hubs located in d-TAD. Epigenomic editing will also be applied to TF binding hubs at d-TAD in HUDEP-2 cells to examine the histone PTM's role in regulating gene expression. We will also apply genomic and epigenome editing to make CREs more active in promoting γ- globin expression. In Aim2, we propose to study the 3D CRE hubs formed by CREs between d-TAD and canonical β-globin cluster. We will use HiChIP to dissect the interactions mediated by active TFs like GATA1 and repressive TFs like BCL11A. Overall, our proposed research will expand the mechanistic view of the poised expression of γ-globin and provide new gene therapy targets for sickle cell disease or β-thalassemia.