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期基因表达的转换， 成人型血红蛋白的前体红细胞。这个问题的相关性激发了人们对它的兴趣 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， 基因治疗和其他治疗方法正在被转化为基因治疗和其他治疗方法。BCL11A是一种关键的 在球蛋白开关的球员，但它是如何调节发展令人惊讶的是，仍然在很大程度上不清楚。我们 初步数据显示BCL11A主要在转录水平上受到控制。通过CRISPR-Cas9 通过遗传筛选，我们鉴定出转录抑制因子EST2作为血红蛋白转换的新型调节因子。 EST2在胎儿红系细胞中高度表达，在成人红系细胞中消失。我们的初步数据 进一步表明，BCL11A在胎儿型细胞中特异性抑制BCL 11A转录， 使胎儿阶段特异性BCL11A增强子元件退役。总之，这些观察定义了 本申请的基本假设：在成体红细胞中，CD12表达消失， BCL11A增强子的激活以促进BCL11A在成体细胞中的表达并触发胎儿细胞的沉默 型珠蛋白基因。这将BCL11A的上游BCL2置于控制血红蛋白的调节回路中 切换额外的初步数据表明，BMP2促进了更广泛的胎儿转录程序。在 具体目的1我们将使用免疫组织化学方法在体内功能获得和丧失实验中检查BMP2的生物学。 细胞培养模型和整个动物研究的组合。具体目标2侧重于机械 实验，定义了BMP2调节染色质特征和转录因子结合的方式， 靶基因，包括BCL11A，以及参与的BCL12辅助因子。具体目标3致力于 明确了BMP2表达的发育调控。这将通过表征 在胎儿和成人成红细胞中， 阶段特定微扰实验。 总之，所提出的研究旨在了解BMP2在血红蛋白转换中的作用，以及BMP2在血红蛋白转换中的作用。 在生物体和分子水平上建立胎儿红系状态。预计该提案将 产生的结果，为更广泛地了解发育造血以及 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.