Genetic Modifiers of Beta-like Globin Gene Switching

 描述(申请人提供)：贫血影响全球约16亿人，对医疗资源造成巨大负担。在遗传性贫血中，血红蛋白疾病，特别是镰状细胞病和β-地中海贫血，由于其患病率和严重性而脱颖而出。在SCD和β-地中海贫血中，通过共同遗传产生Hbf的阳性遗传修饰物或羟基脲(HU)治疗，出生后提高胎儿Hbf水平的能力可以显著减轻疾病严重程度。然而，HU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表达的新疗法。在人类中，包括BCL11A在内的三个主要基因座改变HBF的表达。然而，它们加在一起只占HbF水平变化的50%左右。因此，关于HBF生产的遗传控制方面的知识仍然存在重大差距。我们将利用两个强大的小鼠资源来寻找β样珠蛋白开关的新调节者，小鼠突变株NAN(新生儿贫血)和新开发的高分辨率外交多样性(DO)定位资源。在NAN中，KLF1第二个锌指(EKLF)的单个氨基酸改变会导致与其靶基因子集的序列选择性结合中断，导致严重的贫血和显著的血红蛋白转换失败。与野生型相比，胚胎βH1珠蛋白在成年南脾中的表达上调了100倍，这是通过BCL11A独立的机制实现的，该机制不受应激红细胞生成的影响。在高度遗传多样性的DO小鼠中，βH1珠蛋白在成人中的表达在个体之间有很大的差异。因此，NaN和DO小鼠是检测β样珠蛋白开关的新调节子的理想工具。 强大的、无偏见的遗传QTL(数量性状基因座/基因座)定位与全球基因组和蛋白质组策略相结合。这项提议的总体目标是识别调控β样珠蛋白开关的新基因。为了实现这一目标，其具体目的是(1)定位Nan F2杂交和DO小鼠胚胎珠蛋白表达的修饰基因，以分别识别KLF1依赖和独立的基因座；(2)在红系人群中进行芯片序列分析，以比较野生型(WT)和突变型(Nan)KLF1差异结合的dna靶标；(3)获得红系整体转录组(rna-seq，miR-seq)和磷酸化蛋白质组图谱，以确定基因表达和转录后差异；以及(4)分析和整合所有数据，以识别、优先排序和启动最引人注目的功能分析 候选基因。识别调节β样珠蛋白转换的遗传位点、NAN-KLF1DNA靶标与WT-KLF1DNA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集中于识别β样珠蛋白转换的新调控因子，从而为血红蛋白疾病提供重要的新的治疗靶点。

 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.