Genetic Modifiers of Beta-like Globin Gene Switching

项目摘要/摘要 贫血影响着全球约16亿人，给医疗带来巨大负担 资源。在遗传性贫血中，血红蛋白病，特别是镰状细胞病和... 地中海贫血，由于其患病率和严重性而脱颖而出。出生后提高胎儿血红蛋白的能力 SCD和地中海贫血的HbF水平通过HbF产生或产生的正遗传修饰物的共同遗传 羟基脲(HU)治疗可明显减轻病情严重程度。然而，胡舒立也不是没有立场 影响，甚至可能是致癌的。旨在提高成人HBF表达的新疗法是 因此迫切需要。 已知包括BCL11A在内的三个主要基因座可以改变人类HBF的表达。然而，在一起，他们 仅占HbF水平变化的约50%。因此，知识上的重大差距依然存在。 关于HBF生产的遗传控制。我们将利用两个强大的鼠标资源 为了确定类珠蛋白开关的新调节因子，小鼠突变株Nan(新生儿贫血)和新的 开发了高分辨率的DO图谱资源。在Nan中，单个氨基酸的变化 红系Kr？ppel样因子(EKLF)的第二个锌指导致序列选择性干扰 与其目标基因的一个子集结合。结果是严重的贫血伴随着一种令人震惊的失败 血红蛋白转换。胚胎H1珠蛋白在成人南脾中的表达是野生脾的100倍 通过BCL11A独立机制输入。在高度遗传多样性的DO小鼠中，H1珠蛋白的表达 成人的发病率因个人而异。因此，Nan和DO MICE这两个资源， 具备检测类珠蛋白开关的新型调节剂所需的先决条件， 无偏见的遗传(QTL定位)和基因组(芯片序列、RNA序列)策略。 这项提议的目的是识别调控类珠蛋白开关的新基因。为了实现这一目标， 我们将：(A)在南F2杂交和DO小鼠中对H1表达的MAP修饰物；(B)在 比较野生型(WT)EKLF和突变型(NAN)差异结合的DNA靶点的红系群体 EKLF和RNA-seq以识别基因表达差异；以及(C)分析和整合所有数据以识别， 确定候选基因的优先顺序，并开始分析。好了！ 确定调控H1表达的遗传位点，NAN-EKLF和WT-EKLF DNA靶标的差异，以及 NAN和WT红系细胞基因表达的差异将汇聚在一起，以确定新的调控因子。 如珠蛋白转换，从而为血红蛋白疾病提供了重要的新治疗靶点。好了！

PROJECT SUMMARY/ABSTRACT 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 ¿- thalassemia, stand out due to their prevalence and severity. The ability to postnatally elevate fetal hemoglobin (HbF) levels in SCD and ¿-thalassemia via co-inheritance of positive genetic modifiers of HbF production or hydroxyurea (HU) treatment can significantly alleviate disease severity. However, HU is not without 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, are known to 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 ¿-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 the erythroid Kr¿ppel-like factor (EKLF) causes sequence selective disruption of binding to a subset of its target genes. The result is severe anemia accompanied by a striking failure of hemoglobin switching. Expression of embryonic ¿h1 globin is upregulated 100-fold in adult Nan spleen vs. wild type via a BCL11A independent mechanism. In highly genetically diverse DO mice, expression of ¿h1 globin in adults varies substantially from individual to individual. Thus, these two resources, Nan and DO mice, possess the prerequisites required to detect novel regulators of ¿-like globin switching using powerful, unbiased genetic (QTL mapping) and genomic (ChIP-seq, RNA-seq) strategies. The aim of this proposal is to identify novel genes regulating ¿-like globin switching. To accomplish this aim, we will: (a) map modifiers of ¿h1 expression in Nan F2 intercrosses and in DO mice; (b) perform ChIP-seq in erythroid populations to compare DNA targets differentially bound by wild type (WT) EKLF and mutant (Nan) EKLF, and RNA-seq to identify gene expression differences; and (c) analyze and integrate all data to identify, prioritize, and initiate analysis of candidate genes. ! Identifying genetic loci regulating ¿h1 expression, differences in Nan- vs. WT-EKLF DNA targets, and differences in gene expression in Nan vs. WT erythroid cells will converge to identify novel regulators of ¿- like globin switching, thereby providing important new therapeutic targets for the hemoglobinopathies. !