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.

项目概要/摘要 在人类基因组中，只有10%是负责蛋白质编码的编码区，特别是非编码区。 直接调控基因表达的编码区——顺式调控元件（CRE）——非常重要 CRE 在正常发育和致病性疾病发展中的重要性通常受到限制。 转录因子 (CRE) 在胎儿到成人红细胞生成过程中的珠蛋白转换中发挥重要作用。 CRE 充当增强子（LCR 区域）、阻遏子（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 (CBS) 位于规范 β-珠蛋白位点 CRE 上）已被探索为新的。 基因治疗的目标是珠蛋白基因表达，这对于基因治疗的发展至关重要 血红蛋白病和地中海贫血的传统研究是调节 β 珠蛋白基因座基因表达。 最近，我们与其他人一起，限制在 5'HS 和 3'HS1 CTCF 结合位点之间。 揭示了β-珠蛋白基因簇周围的嵌套多环3D基因组结构。 结构扩展了 β-珠蛋白基因簇的调控景观，向下游延伸了 145kb （形成称为 d-TAD 的 3D 基因组结构）和上游 110kb 子 TAD（形成 3D 基因组结构 利用与 β-珠蛋白基因接壤的多个 CBS（删除和倒位）的基因组编辑。 簇中，我们发现 3'HS1（β-珠蛋白基因座下游边界）处的 CBS 经常被删除 HPFH 缺失破坏 – 足以在成人红细胞中诱导 γ-珠蛋白。 长期以来，人们推测 HPFH 增强剂与激活 HPFH 中胎儿血红蛋白的并置有关 通过删除 48kb 区域，使 HPFH 增强子更接近 β 珠蛋白，从而重新激活 γ 珠蛋白。 位点，我们还观察到胎儿血红蛋白的上调，表明 HPFH 增强剂是一种新型的 我们与其他人一起发表的工作强烈表明了γ珠蛋白表达的条件增强子。 嵌套在 β-珠蛋白基因簇下游 sub-TAD 中的 CRE 确实在 这些数据使我们认为 d-TAD 中的 CRE 正在调节 β 珠蛋白。 我们建议利用最新的技术通过远程 3D 基因组相互作用进行基因表达。 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。 精确地询问位于 d-TAD 的 TF 结合中心的 TF 结合协同性。 还将应用于 HUDEP-2 细胞中 d-TAD 处的 TF 结合中心，以检查组蛋白 PTM 在调节中的作用 我们还将应用基因组和表观基因组编辑，使 CRE 在促进 γ- 方面更加活跃。 在 Aim2 中，我们建议研究 d-TAD 和 d-TAD 之间的 CRE 形成的 3D CRE 中心。 我们将使用 HiChIP 来剖析由 GATA1 等活性 TF 介导的相互作用。 总体而言，我们提出的研究将扩展平衡的机制观点。 γ-珠蛋白的表达并为镰状细胞病或β-地中海贫血提供新的基因治疗靶点。