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

胎儿血红蛋白基因调控中β-珠蛋白位点下游非典型顺式调控元件的剖析

• 批准号: 10718028
• 负责人: Xiaotian Zhang
• 金额: $ 46.98万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-01 至 2028-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10718028
• 关键词: 3-Dimensional; Adult; Binding; Binding Sites; CRISPR/Cas technology; Categories; Cell Line; Cells; Chromatin; Classification; Code; Data; Development; Dimerization; Disease; Dissection; EEF1A2 gene; EP300 gene; Enhancers; Environment; Erythrocytes; Erythroid; Erythroid Cells; Erythropoiesis; Fetal Hemoglobin; GATA1 gene; Gene Cluster; Gene Expression; Gene Expression Regulation; Generations; Genes; Genetic; Genetic Diseases; Genome; Genomic Segment; Genomics; Globin; Hemoglobin; Hemoglobin F Disease; Hemoglobinopathies; Histones; Human; Human Genome; Locus Control Region; Mediating; Molecular Target; Pathogenicity; Play; Point Mutation; Population; Proteins; Publishing; Regulation; Regulatory Element; Research; Role; Sickle Cell Anemia; Site; Structure; System; Technology; Thalassemia; Transcriptional Regulation; Twin Multiple Birth; Untranslated RNA; Up-Regulation; Update; Work; beta Globin; beta Thalassemia; epigenome editing; epigenomic profiling; experimental study; fetal; gamma Globin; gene therapy; genome editing; genomic locus; mutant; novel; novel strategies; precise genome editing; prime editing; progenitor; promoter; reduce symptoms; targeted treatment; therapy development; tool; transcription factor

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.

项目摘要/摘要 在人类基因组中，只有10％的编码区域负责蛋白质编码。特别是，非 直接调节基因表达的编码区域 - 顺式调节元件（CRE） - 在正常发育和致病性发展中的重要性。 Cres通常受 转录因子（TFS）。 CRE在胎儿到成人红细胞生成期间的球蛋白转换中起着重要作用。 CRE充当增强子（LCR区域），副本（γ球蛋白启动子中的Bcl11a结合位点）或绝缘子（3'HS1 和HS5 CTCF结合位点（CBSS）在规范β-珠蛋白基因座边界上）。 CRE已被探索为新的 Globin基因表达的基因治疗靶标，这对于基因治疗的发展至关重要 血红蛋白病和丘脑贫血。传统上，调节β球蛋白基因座基因表达的CRE的研究 在5'Hs和3'HS1 CTCF结合位点的边界之间受到限制。最近，我们与其他人一起 揭示了围绕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 结构扩展了β-珠蛋白基因簇的调节景观，下游延伸145kb （形成一个称为D-TAD的3D基因组结构）和110KB tad上游（形成3D基因组结构 称为U-Tad）。利用与β-珠蛋白基因接壤的多个CBS（缺失和反转）的基因组编辑 群集，我们发现CBS在3'HS1处的删除经常下游边界上的β-珠蛋白基因座 受HPFH缺失破坏 - 足以诱导成年红细胞中的γ-球蛋白。重要的是，我们找到了 长期以来一直猜测并置激活HPFH中的胎儿血红蛋白的HPFH增强剂是负责 用于γ-球蛋白的重新激活。通过删除48KB区域以使HPFH增强器更靠近β-珠蛋白 基因座，我们还观察到胎儿血红蛋白的上调，这表明HPFH增强剂是一种新颖 条件增强子用于γ球蛋白表达。我们已发表的作品与他人一起强烈暗示 嵌套在β-珠蛋白基因簇的下游亚tad中的CRE确实在 调节球蛋白基因表达。这些数据使我们假设D-TAD中的CRE正在调节β球蛋白 通过远程3D基因组相互作用的基因表达。借助最新的技术，我们建议 两个研究目的。在AIM1中，我们将使用基于CRISPR/CAS9的Prime编辑来介绍TF绑定突变体 精确地询问位于D-TAD中的TF结合枢纽中的TF结合配位。表观基因组编辑 还将应用于HUDEP-2细胞中D-TAD的TF结合集线器，以检查组蛋白PTM在调节中的作用 基因表达。我们还将应用基因组和表观基因组编辑，以使CRE在促进γ-方面更加活跃 球蛋白表达。在AIM2中，我们建议研究由D-TAD和 规范β-珠蛋白簇。我们将使用Hichip剖析主动TF（例如GATA1）介导的相互作用 和BCL11A等反射性TF。总体而言，我们提议的研究将扩大中毒的机械观点 γ-球蛋白的表达，并为镰状细胞疾病或β-丘脑贫血提供新的基因治疗靶标。