Targeting the NuRD complex for fetal globin induction

摘要 血红蛋白病，例如镰状细胞病 (SCD) 和地中海贫血，是最严重的公共卫生问题之一 世界上的担忧。尽管目前正在测试新的治疗方式，例如基因疗法， 迫切需要治疗一般患者群体的药理学方法。我们的长期目标 是开发一种化合物，通过靶向转录因子来诱导胎儿型珠蛋白（HbF）的产生 复杂的调节珠蛋白转换。该应用的目的是确定分子机制 LRF-NuRD 介导的 γ-珠蛋白沉默的基础，并确定针对它们的方法。我们的中央 假设含有 LRF 的 NuRD 复合物是 HbF 再激活治疗的潜在靶点。的 拟议研究的基本原理是，了解 LRF/NuRD 介导的珠蛋白调节将有助于 更好地了解调节 γ-珠蛋白抑制的转录复合物并促进 开发新的 HbF 诱导治疗策略。在强有力的初步数据的指导下，我们 期望通过追求三个具体目标来实现我们的目标：1）确定γ-的分子基础 在缺乏 LRF/NuRD 的情况下珠蛋白重新激活； 2) 鉴定 γ- 必需的 CHD3/4 结构域 珠蛋白沉默； 3) 确定 LRF/CHD3 相互作用在控制 γ-珠蛋白中的功能意义 沉默。在 Aim1 中，我们将采用 ChIP-seq 和 ATAC-seq 足迹来确定 NuRD- 相关途径沉默成人红细胞中的 γ-珠蛋白表达以及 γ-珠蛋白是如何被诱导的 LRF 耗尽。在 Aim2 中，我们将确定负责 γ-珠蛋白沉默的 CHD3/4 最小结构域。至 为此，我们将使用 CRISPR-Cas9 基因诱变进行功能域映射。在Aim3中，我们将 确定 LRF/CHD3 相互作用在控制 γ-珠蛋白沉默中的功能意义。我们发表的 工作和初步数据强烈表明 NuRD 相关途径，其中 LRF 和 BCL11A 参与其中，代表了几乎整个“球蛋白开关”。我们期望综合方法 这里提出的将阐明 LRF 和 NuRD 复合物在 γ-珠蛋白沉默中的作用并促进 开发针对血红蛋白病的 HbF 再激活治疗新策略。

Abstract Hemoglobinopathies, such as sickle cell disease (SCD) and thalassemia, are among the greatest public health concerns in the world. Although new therapeutic modalities, such as gene therapy, are currently being tested, there is a pressing need for pharmacologic approaches to treat general patient populations. Our long-term goal is to develop a compound(s) that induces fetal-type globin (HbF) production by targeting the transcriptional complex regulating globin switching. The objective of this application is to determine molecular mechanisms underlying the LRF-NuRD-mediated γ-globin silencing and identify a mean(s) to target them. Our central hypothesis is that the LRF-containing NuRD complex is a potential target for HbF reactivation therapy. The rationale for the proposed research is that understanding the LRF/NuRD-mediated globin regulation will provide greater understanding of the transcriptional complex regulating γ-globin repression and facilitate development of novel therapeutic strategies for HbF induction therapy. Guided by strong preliminary data, we expect to achieve our objective by pursuing the three specific aims: 1) to determine the molecular basis for γ- globin reactivation in the absence of the LRF/NuRD; 2) to identify a domain(s) of CHD3/4 necessary for γ- globin silencing; and 3) to determine functional significance of LRF/CHD3 interaction in controlling γ-globin silencing. In Aim1, we will employ ChIP-seq and ATAC-seq foot-printing to determine how the NuRD- associated pathways silences γ-globin expression in adult erythroid cells and how γ-globin is induced upon LRF depletion. In Aim2, we will identify a minimal domain(s) of CHD3/4 responsible for γ-globin silencing. To do so, we will perform a functional domain mapping using CRISPR-Cas9 gene mutagenesis. In Aim3, we will determine functional significance of the LRF/CHD3 interaction in controlling γ-globin silencing. Our published work and preliminary data strongly suggest that the NuRD-associated pathways, in which LRF and BCL11A are involved, represent the near entirety of the “globin-switch”. We expect that the combined approaches proposed here will elucidate the role of LRF and the NuRD complex in γ-globin silencing and facilitate development of novel strategies for HbF reactivation therapy for hemoglobinopathies.