Targeting the NuRD complex for fetal globin induction

摘要 血红蛋白疾病，如镰状细胞病(SCD)和地中海贫血，是最大的公共健康之一。 世界关注的问题。尽管新的治疗方式，如基因疗法，目前正在测试中， 迫切需要药理学方法来治疗普通患者群体。我们的长期目标 是开发一种化合物(S)，通过靶向转录的 复杂的调节珠蛋白转换。这项应用的目的是确定分子机制 在LRF-NuRD介导的γ-珠蛋白沉默的基础上，确定一个平均(S)来靶向它们。我们的中央 假设含有LRF的NuRD复合体是HBF重新激活治疗的潜在靶点。这个 拟议研究的基本原理是，了解LRF/NuRD介导的珠蛋白调节将 提供对调节γ-珠蛋白抑制的转录复合体的更多了解，并促进 高铁诱导治疗的新治疗策略的发展。在强劲的初步数据指引下，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所必需的CHD3/4的一个结构域(S)- 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，我们将使用CHIP-SEQ和ATAC-SEQ足迹来确定NuRD- 相关通路沉默成人红系细胞中γ-珠蛋白的表达及γ-珠蛋白是如何诱导的 LRF耗尽。在AIM2中，我们将确定CHD3/4的一个最小结构域(S)，它负责γ-珠蛋白沉默。至 为此，我们将使用CRISPR-Cas9基因突变进行功能结构域的定位。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。我们出版的 工作和初步数据有力地表明，与NuRD相关的通路，其中LRF和BCL11A 几乎代表了“珠蛋白开关”的全部。我们预计，这些结合起来的方法 将阐明LRF和NuRD复合体在γ-珠蛋白沉默中的作用，并促进 血红蛋白疾病HBF复活治疗新策略的开发。

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.