Mapping Gamma Globin Regulatory Elements

绘制伽马珠蛋白调控元件

基本信息

批准号：
9751639
负责人：
Phillip A Doerfler
金额：
$ 6.37万
依托单位：
ST. JUDE CHILDREN'S RESEARCH HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-07-05 至 2021-07-04
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9751639
关键词：
ATAC-seq Adult Affect Age-Months Alleles American Benign Binding Binding Sites Birth CD34 gene CRISPR/Cas technology Cell Line Cells Cellular Assay Chromatin Chromatin Structure Clinical DNA DNA Double Strand Break DNA Sequence Development Differentiated Gene Dimensions Disease Distal Elements Epigenetic Process Erythroblasts Erythrocytes Erythroid Erythroid Cells Exhibits Fetal Hemoglobin Frequencies Functional disorder Gene Expression Genes Genetic Genetic Diseases Genetic Transcription Globin Hematopoietic stem cells Hemoglobin Hemoglobin F Disease Hemoglobinopathies Human Impairment In Vitro Intercistronic Region Introns Life Locus Control Region Maps Mediating Modification Molecular Morbidity - disease rate Mutation Mutation Analysis Nucleotides Pattern Pharmaceutical Preparations Production Promoter Regions Regulatory Element Research Series Severities Sickle Cell Anemia Switch Genes Testing Therapeutic Trans-Activators Transcription Initiation Site Transcription Repressor/Corepressor Upstream Enhancer base beta Globin beta Thalassemia chromatin immunoprecipitation falls fetal reactivity functional genomics gamma Globin gene therapy genome editing infancy insight interest mortality mutant novel novel therapeutic intervention postnatal precision medicine promoter repaired synergism tool transcription factor

项目摘要

PROJECT SUMMARY/ABSTRACT I am studying developmental globin gene expression in order to better understand and treat sickle cell disease (SCD) and β-thalassemia, common disorders that cause substantial morbidity and early mortality around the world. Both diseases become symptomatic between birth and 6 months age, as the γ-globin (HBG1/2) genes switch to β-globin (HBB) causing fetal hemoglobin (HbF, α2γ2) to be replaced by adult hemoglobin (HbA, α2β2). Elevated postnatal levels of HbF, including a benign condition termed "hereditary persistence of fetal hemoglobin (HPFH)", alleviates SCD and β-thalassemia. We are seeking to reverse the γ-to-β-globin gene switch in order to induce HbF therapeutically. Our preliminary studies show that CRISPR/Cas9-mediated genome editing of primary human hematopoietic stem and progenitor cells (HPSCs) can recreate an HPFH-associated 13- nucleotide deletion in the HBG1 promoter by removing nucleotides -102 to -114 upstream of the transcriptional start site. In vitro differentiation of the gene-edited HSPCs generates erythroid progeny with HbF elevated to potentially therapeutic levels. Now, I will fine-map the -102 to -114 region using genome editing-mediated homology directed repair and cellular assays for Hb switching in order to better define the relevant nucleotide motif(s) and their interacting transcription factors (Aim 1). Because HBG1 and HBG2 are nearly identical, CRISPR/Cas9 gene editing of the -102 promoter regions introduces double-stranded DNA breaks in both genes simultaneously, which could cause deletion of the 5 kb intervening sequence, including HBG2. Preliminary studies indicate that approximately 20% of alleles harbor the 5 kb deletion after editing of human CD34+ HSPCs. Surprisingly, however, erythroid clones with this deletion exhibit increased γ-globin production, despite loss of HBG2. Epigenetic analysis and functional testing of targeted deletions within the deleted HBG1-HBG2 intergenic region identified a potential DNA regulatory element that represses HBG1 transcription. I will better define this element by mutational analysis using cellular assays for globin switching and epigenetic studies to map associated transcription factors and effects on three-dimensional chromatin structure (Aim 2). By characterizing the cis elements and the corresponding trans-acting factors that regulate γ-to-β globin switching, I hope to develop new ways to induce HbF for treating SCD and β-thalassemia.

项目概要/摘要我正在研究发育珠蛋白基因表达，以便更好地了解和治疗镰状细胞病（SCD）和β-地中海贫血是导致大量发病率和早期死亡率的常见疾病由于 γ-珠蛋白 (HBG1/2) 基因的影响，这两种疾病都会在出生至 6 个月期间出现症状。转换为 β-珠蛋白 (HBB)，导致胎儿血红蛋白 (HbF、α2γ2) 被成人血红蛋白 (HbA、α2β2) 取代。产后 HbF 水平升高，包括一种称为“胎儿血红蛋白遗传性持续存在”的良性病症 (HPFH)”，缓解 SCD 和 β-地中海贫血。我们正在寻求逆转 γ-至-β-珠蛋白基因开关，以便我们的初步研究表明 CRISPR/Cas9 介导的基因组编辑。原代人类造血干细胞和祖细胞 (HPSC) 可以重建 HPFH 相关的 13- 通过去除转录上游的核苷酸 -102 至 -114 来删除 HBG1 启动子中的核苷酸基因编辑的 HSPC 的体外分化产生 HbF 升高至的红系后代。现在，我将使用基因组编辑介导的精细图谱-102 至-114 区域。用于 Hb 转换的同源定向修复和细胞测定，以便更好地定义相关核苷酸因为 HBG1 和 HBG2 几乎相同， -102 启动子区域的 CRISPR/Cas9 基因编辑在两个基因中引入双链 DNA 断裂同时，这可能会导致 5 kb 插入序列（包括 HBG2）的删除。研究表明，在编辑人类 CD34+ HSPC 后，大约 20% 的等位基因存在 5 kb 缺失。然而，令人惊讶的是，具有这种缺失的红系克隆表现出增加的 γ-珠蛋白产量，尽管 HBG2。删除的 HBG1-HBG2 基因间靶向删除的表观遗传学分析和功能测试。区域确定了一个抑制 HBG1 转录的潜在 DNA 调控元件，我将更好地定义这一点。使用珠蛋白转换的细胞测定和表观遗传学研究进行突变分析来绘制元素相关转录因子及其对三维染色质结构的影响（目标 2）。调节γ-β珠蛋白转换的顺式元件和相应的反式作用因子，我希望开发诱导 HbF 治疗 SCD 和 β-地中海贫血的新方法。