Therapeutic potential and broader applicability of forced chromatin looping

 描述(由申请人提供)：增强子和启动子之间的远程相互作用在发育过程中调节基因表达谱起着关键作用。基因座控制区(LCR)是一个强大的增强子，通过与胚胎、胎儿和成体珠蛋白基因启动子形成发育受限的长距离相互作用来激活B-珠蛋白基因。这些接触在一定程度上是由一种称为LDB1的环状因子调节的。这种核因子同时占据LCR和珠蛋白启动子，并被认为有利于自结合的环状相互作用。最近，我们的实验室发现，通过人工锌指(ZF)蛋白将LDB1连接到胎儿珠蛋白启动子上，可以触发启动子与LCR的接触，并在培养的成人红系细胞中重新激活胎儿基因，其珠蛋白合成总量高达85%。这一点特别令人感兴趣，因为高水平的胎儿珠蛋白水平减轻了诸如镰刀细胞病(SCD)等血红蛋白疾病的临床严重程度。虽然这种方法达到的水平对SCD患者是有治疗作用的，但目前还不清楚这种策略是否可以应用于临床环境。为了实现这一目标，我们将在专门表达人类镰状血红蛋白的转基因小鼠身上测试这一方法。这些小鼠含有自然配置的人类转基因基因，编码胎儿和成年镰状珠蛋白。我们将用ZF-LDB1融合载体或空载体转导这些小鼠的造血干细胞，并将转导的细胞移植到致死性照射的受者体内。我们将比较ZF-LDB1处理的小鼠和空载体处理的小鼠的完整血细胞计数、红细胞参数、器官病理和存活率。此外，尽管强制染色质环法似乎是控制珠蛋白基因的一种强有力的策略，但目前尚不清楚这种策略是否会更广泛地适用。CHIP-SEQ研究表明，LDB1在整个基因组中占据了大量的位置。基因敲除实验表明，LDB1是正确激活广泛的红系基因所必需的。然而，LDB1可能调节全基因组的特定长程相互作用仍不清楚。我们将用我们实验室建立的Capture-C方法，以一种公正的方式，在高分辨率下表征一组与LDB1结合的远端调控元件的远程相互作用。为了深入了解LDB1在全基因组范围内介导的环相互作用，我们将与加州大学欧文分校的Ali Mortazavi博士合作，使用染色质相互作用分析技术(CHIA-PET)。综上所述，这些实验旨在阐明染色质环的基本机制，探索LDB1介导的环治疗SCD的潜力，并拓宽该方法应用于其他基因和疾病的范围。

 DESCRIPTION (provided by applicant): Long-range interactions between enhancers and promoters play key roles in regulating gene expression profiles during development. The locus control region (LCR) is a powerful enhancer that activates ß-globin genes by forming developmentally restricted long-distance interactions with the embryonic, fetal, and adult globin gene promoters. These contacts are mediated in part by a looping factor termed Ldb1. This nuclear factor occupies both the LCR and globin promoters, and is thought to favor looping interactions by self- association. Recently, our laboratory has shown that tethering Ldb1 to the fetal globin promoter via artificial zinc finger (ZF) proteins triggers promoter contacts with the LCR, and re-activation of the fetal genes with up to 85% of total globin synthesis in cultured adult human erythroid cells. This is of particular interest as high fetal globin levels mitigate th clinical severity of hemoglobinopathies such as Sickle Cell Disease (SCD). While the levels achieved by this approach would be therapeutic for SCD patients, it is still unclear if this stratey can be applied in a clinical setting. Towards this goal, we will test this approach in transgenic mice that specifically express human sickle hemoglobin. These mice contain naturally configured human transgenes encoding the fetal and adult sickle ß-globin. We will transduce hematopoietic stem cells from these mice with either ZF-Ldb1 fusion constructs or empty vector and transplant the transduced cells into lethally irradiated recipients. We will compare complete blood counts, red blood cell parameters, organ pathologies, and survival rates of the ZF- Ldb1 treated mice to empty vector treated mice. Furthermore, while the forced chromatin looping approach appears to be a robust strategy to control globin genes, it is unclear if this strategy may be more broadly applicable. ChIP-seq studies have shown that Ldb1 occupies a substantial number of sites throughout the genome. Knockdown experiments have shown that Ldb1 is required for the proper activation of a wide range of erythroid genes. However, the specific long-range interactions Ldb1 may mediate genome-wide remain unclear. We will characterize long-range interactions in an unbiased manner for a select group of Ldb1-bound distal regulatory elements at high-resolution with the Capture-C method, which is well established in our laboratory. To gain insight into looping interactions mediated by Ldb1 genome-wide, we will use Chromatin Interaction Analysis using Paired-End Tag sequencing (ChIA-PET), in collaboration with Dr. Ali Mortazavi (UC Irvine). Together, the proposed experiments are designed to elucidate basic mechanisms of chromatin looping, to explore the therapeutic potential of Ldb1-mediated looping for the treatment of SCD, and broaden the scope of the approach to other genes and diseases.