Genetic Modifiers of Beta-like Globin Gene Switching

类β球蛋白基因转换的遗传修饰剂

• 批准号: 8882861
• 负责人: LUANNE L PETERS
• 金额: $ 39.38万
• 依托单位: JACKSON LABORATORY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-16 至 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8882861
• 关键词: Accounting; Adult; Adverse effects; Affect; African; African American; Amino Acids; Anemia; Animal Model; Binding; Birth; Blood Circulation; Bone Marrow; Candidate Disease Gene; ChIP-seq; DNA; Data; Development; Disease; Embryo; Employee Strikes; Erythrocytes; Erythroid; Erythroid Cells; Erythropoiesis; Etiology; European; Failure; Fetal Development; Fetal Hemoglobin; Fetal Liver; Gene Expression; Gene Expression Profile; Gene Targeting; Genes; Genetic; Genomics; Globin; Goals; Health; Hemoglobin; Hemoglobin F Disease; Hemoglobinopathies; Human; Human Development; Inbreeding; Individual; Inherited; Knowledge; Lead; Life; Maps; Medical; Missense Mutation; Modeling; Mus; Mutant Strains Mice; Mutation; Natural Increases; Neonatal Anemia; Newborn Infant; Pathway interactions; Patients; Population; Prevalence; Production; Proteome; Proteomics; Quantitative Trait Loci; Research; Resolution; Resources; Secondary to; Severities; Severity of illness; Sickle Cell Anemia; Sickle Hemoglobin; Spleen; Stress; Switch Genes; Symptoms; United States; Variant; Work; Zinc Fingers; beta Globin; beta Thalassemia; differential expression; erythroid Kruppel-like factor; fetal globin; genetic variant; hydroxyurea; in utero; man; mutant; new therapeutic target; novel; postnatal; protein expression; tool; transcription factor; transcriptome sequencing

DESCRIPTION (provided by applicant): Anemia affects approximately 1.6 billion people worldwide, imposing an enormous burden on medical resources. Of the inherited anemias, the hemoglobinopathies, particularly sickle cell disease (SCD) and β-thalassemia, stand out due to their prevalence and severity. The ability to postnatally elevate fetal hemoglobin (HbF) levels in SCD and β-thalassemia via co-inheritance of positive genetic modifiers of HbF production or hydroxyurea (HU) treatment can significantly alleviate disease severity. HU, however, has serious side effects and may even be carcinogenic. Novel therapies aimed at elevating HbF expression in adults are, therefore, desperately needed. Three major loci, including BCL11A, modify HbF expression in humans. Together, however, they account for only ~50% of the variation in HbF levels. Hence, significant gaps in knowledge remain regarding the genetic control of HbF production. We will take advantage of two powerful mouse resources to identify novel regulators of β-like globin switching, the mouse mutant Nan (neonatal anemia) and the newly developed high resolution Diversity Outbred (DO) mapping resource. In Nan a single amino acid change in the second zinc finger of KLF1 (erythroid Krüppel-like factor, EKLF) causes sequence selective disruption of binding to a subset of its target genes resulting in severe anemia and a striking failure of hemoglobin switching. Expression of embryonic βh1 globin is upregulated 100-fold in adult Nan spleen vs. wild type via a BCL11A independent mechanism that is not secondary to stress erythropoiesis. In highly genetically diverse DO mice, expression of βh1 globin in adults varies substantially from individual to individual. Thus, Nan and DO mice are ideal tools with which to detect novel regulators of β-like globin switching using powerful, unbiased genetic QTL (quantitative trait locus/loci) mapping integrated with global genomic and proteomic strategies. The overall goal of this proposal is to identify novel genes regulating β-like globin switching. To accomplish this goal, the specific aims are to (1) map modifiers of embryonic globin expression in Nan F2 intercrosses and in DO mice to identify KLF1-dependent and independent loci, respectively; (2) perform ChIP-seq in erythroid populations to compare DNA targets differentially bound by wild type (WT) and mutant (Nan) KLF1; (3) obtain erythroid global transcriptome (RNA-seq, miR-seq) and phospho-proteome profiles to identify gene expression and post-transcriptional differences; and (4) analyze and integrate all data to identify, prioritize, and initiate functional analysis of the most compelling candidate genes. Identification of genetic loci regulating β-like globin switching, differences i Nan- vs. WT-KLF1 DNA targets, and differences in the transcriptome and proteome of Nan and WT erythroid cells will converge to identify novel regulators of β-like globin switching, thereby providing important new therapeutic targets for hemoglobinopathies.

描述（申请人提供）：贫血影响全球约16亿人，给医疗资源带来巨大负担。在遗传性贫血中，血红蛋白病，特别是镰状细胞病（SCD）和β-地中海贫血，由于其患病率和严重性而突出。SCD和β-地中海贫血中通过HbF产生的阳性遗传修饰物的共遗传或羟基脲（HU）治疗而在出生后升高胎儿血红蛋白（HbF）水平的能力可显著减轻疾病严重程度。然而，HU具有严重的副作用，甚至可能致癌。因此，迫切需要旨在提高成人HbF表达的新型疗法。包括BCL 11 A在内的三个主要基因座修饰人类中的HbF表达。然而，它们一起仅占HbF水平变化的约50%。因此，关于HbF产生的遗传控制的知识仍然存在重大差距。我们将利用两种强大的小鼠资源来鉴定β样珠蛋白转换的新型调节因子，即小鼠突变体Nan（新生儿贫血）和新开发的高分辨率多样性远交（DO）作图资源。在Nan中，KLF 1（红细胞Krüppel样因子，EKLF）第二锌指中的单个氨基酸变化导致与其靶基因子集结合的序列选择性破坏，导致严重贫血和血红蛋白转换的显著失败。与野生型相比，成人Nan脾中胚胎βh1珠蛋白的表达上调100倍，这是通过不继发于应激红细胞生成的BCL 11 A独立机制实现的。在高度遗传多样性的DO小鼠中，βh1珠蛋白在成年小鼠中的表达因个体而异。因此，Nan和DO小鼠是使用免疫荧光检测β样珠蛋白转换的新型调节剂的理想工具。 强大的，无偏见的遗传QTL（数量性状基因座/基因座）作图与全球基因组和蛋白质组学策略相结合。该提案的总体目标是鉴定调节β样珠蛋白转换的新基因。为了实现这一目标，具体的目标是（1）在Nan F2杂交小鼠和DO小鼠中绘制胚胎珠蛋白表达的修饰物，以分别鉴定KLF 1依赖性和独立性基因座;（2）在红细胞群体中进行ChIP-seq，以比较野生型（WT）和突变型（Nan）KLF 1差异结合的DNA靶标;（3）获得红系整体转录组（RNA-seq，miR-seq）和磷酸化蛋白质组谱，以鉴定基因表达和转录后差异;以及（4）分析和整合所有数据，以鉴定、优先排序和启动最引人注目的功能分析。 候选基因 调节β样珠蛋白转换的遗传基因座的鉴定、Nan-与WT-KLF 1 DNA靶标的差异以及Nan和WT红系细胞的转录组和蛋白质组的差异将汇聚于鉴定β样珠蛋白转换的新型调节剂，从而为血红蛋白病提供重要的新治疗靶标。