Regulation of Hemoglobin Switching

摘要 发育基因表达中最古老和最深入研究的问题之一是从胎儿 红血球前体中的成人型血红蛋白。这个问题的相关性激起了人们对这个问题的兴趣 遗传性血液疾病，如镰状细胞病和地中海贫血，以及有关调控的知识 这一过程正在转化为基因疗法和其他治疗方法。BCL11A是一个关键 但令人惊讶的是，它是如何在发育过程中受到调控的，在很大程度上仍然不清楚。我们的 初步数据表明，BCL11A主要在转录水平上受到控制。通过CRISPR-CAS9 基因筛查，我们发现转录抑制因子HIC2是一种新的血红蛋白转换调节因子。 HIC2在胎儿红系细胞中高表达，在成人红系细胞中表达减弱。我们的初步数据 进一步提示HIC2通过直接抑制BCL11A在胎儿型细胞中的转录 停用胎儿期特异的BCL11A增强子元件。总而言之，这些观察结果定义了 这一应用的基本假设：HIC2在成人红细胞中的表达消失，允许 激活BCL11A增强子促进成体细胞表达BCL11A并触发胎儿细胞沉默 确定珠蛋白基因的类型。这将HIC2置于BCL11A上游控制血红蛋白的调节电路中 正在切换。更多的初步数据表明，HIC2促进了更广泛的胎儿转录程序。在……里面 具体目标1我们将在体内功能获得和丧失实验中检查HIC2的生物学。 细胞培养模型和整体动物研究相结合。具体目标2侧重于机械学 实验，定义HIC2调节染色质特征和转录因子结合的方式 靶基因，包括BCL11A，以及哪些HIC2辅助因子参与其中。《特定目标3》致力于 明确HIC2表达的发育调控。这将通过将 胎儿和成人红细胞中HIC2基因位点的调控格局与发育 阶段性摄动实验。 总之，拟议的研究旨在了解HIC2在血红蛋白转换和 在生物和分子水平上建立胎儿红系状态。这项提案预计将 为更广泛地理解发育中的造血以及 治疗镰状细胞病和地中海贫血。

Abstract One of the oldest and most deeply studied problems in developmental gene expression is the switch from fetal to adult type hemoglobin in red blood cell precursors. Interest in this question has been fueled by its relevance to genetic blood disorders such as sickle cell disease and thalassemia, and knowledge about regulatory processes is being translated into gene therapies and other therapeutic approaches. BCL11A is a critical player in the globin switch, but how it is regulated developmentally is surprisingly still largely unclear. Our preliminary data show that BCL11A is controlled predominantly at the transcriptional level. Via a CRISPR-Cas9 genetic screen we identified the transcriptional repressor HIC2 as a novel regulator of hemoglobin switching. HIC2 is expressed highly in fetal erythroid cells and extinguished in adult erythroid cells. Our preliminary data further suggest that HIC2 represses BCL11A transcription specifically in fetal type cells by directly decommissioning a fetal stage-specific BCL11A enhancer element. Together, these observations define the foundational hypothesis of this application: HIC2 expression is extinguished in adult red cells, allowing for the activation of a BCL11A enhancer to boost BCL11A expression in adult cells and trigger the silencing of fetal type globin genes. This places HIC2 upstream of BCL11A in the regulatory circuitry that controls hemoglobin switching. Additional preliminary data suggest that HIC2 promotes a broader fetal transcriptional program. In Specific Aim 1 we will examine the biology of HIC2 in gain- and loss-of-function experiments in vivo using a combination of cell culture models and whole animal studies. Specific Aim 2 is focused on mechanistic experiments, defining the way by which HIC2 regulates chromatin features and transcription factor binding at target genes, including BCL11A, and what HIC2 co-factors are involved. Specific Aim 3 is dedicated to defining the developmental control of HIC2 expression. This will be accomplished by characterizing the regulatory landscape of the HIC2 locus in fetal and adult erythroblasts in combination with developmental stage specific perturbative experiments. In sum, the proposed studies aim to understand the role of HIC2 in hemoglobin switching and in the establishment of a fetal erythroid state at an organismal and molecular level. This proposal is expected to produce results with ramifications for a broader understanding of developmental hematopoiesis as well as the treatment of sickle cell disease and thalassemia.