A high throughput screen for X-reactivation and reprogramming by small molecules

描述（由申请人提供）：通过小分子X染色体失活（XCI）对X染色体再激活和重编程进行高通量筛选，雌性哺乳动物（XX）与雄性（XY）相比平衡X连锁基因表达的过程是由非编码RNA Xist的表达启动的，并包含一系列染色质变化，这些变化建立并维持了失活X的沉默状态。这种抑制环境被一种称为X再激活的表观遗传重编程形式逆转。它发生在体内早期胚胎和生殖系，或在体外诱导多能干细胞（iPS）形成期间。尽管人们对XCI进行了多年的深入研究，并对基因调控的一般机制有了深入的了解，但对x再激活的了解却少得多。我们提出，在小鼠细胞中鉴定靶向x -再激活的新分子也将证明对iPS细胞重编程是有用的。与小鼠iPS细胞相比，大多数人类iPS细胞系在x -再激活中失败，在本项目提出后的未来计划中，我们期待在人类iPS细胞上测试新的小分子探针。诱导多能干细胞的产生改变了我们对遗传学和表观遗传学之间相互作用的理解，并具有巨大的治疗潜力。根据目前的实践，这种方法效率低下，而且并不总是能够产生真正等同于多能胚胎干细胞（ES）的细胞。一些小分子，包括dna甲基转移酶抑制剂5-Aza-2'-脱氧胞苷（5-aza-dC），已经被发现可以部分地抑制体细胞中失活X上的基因，这也有助于iPS细胞的形成，进一步强调XCI与iPS细胞重编程之间的密切关系。携带X连锁GFP报告基因的雌性小鼠成纤维细胞系将用于研究X-再激活。应用阳性对照5-aza-dC引起X-GFP的剂量依赖性再激活。对于高通量筛选（HTS），应用测试化合物产生的GFP荧光将通过自动显微镜进行分析。二次筛选将测试四种内源性x连锁基因的特异性失活等位基因的再激活。从这些屏幕中出现的探针将是解剖x -再激活的有用工具，也可能进一步阐明XCI。在最后的实验中，将测试x -再激活探针增强成纤维细胞重编程成iPS细胞的能力。虽然大多数高通量筛选针对特定的酶或途径，但这里提出的初级筛选是基于许多尚未完全理解的表观遗传机制的生物学结果。这种方法的优点是，我们期望鉴定出在维持X染色体的非活性状态中起关键作用的新蛋白质探针，这是X-再激活所必需的，也与重编程有关。

DESCRIPTION (provided by applicant): A high throughput screen for X-reactivation and reprogramming by small molecules X chromosome inactivation (XCI), the process by which female mammals (XX) balance X-linked gene expression compared to males (XY), is initiated by the expression of the non-coding RNA Xist and encompasses a series of chromatin changes that establishes and maintains the silent state of the inactive X. This repressive environment is reversed by a form of epigenetic reprogramming known as X-reactivation, which occurs in vivo in the early embryo and the germ line, or in vitro during the formation of induced pluripotent stem (iPS) cells. While XCI has been intensively studied for many years and has yielded insight into the general mechanisms of gene regulation, much less is known about X-reactivation. We propose that identifying new molecules that target X-reactivation in mouse cells will also prove useful for reprogramming iPS cells. In contrast to mouse iPS cells, most human iPS cell lines fail in X-reactivation, and for future plans after this proposed project, we look forward to testin new small molecule probes on human iPS cells. The generation of iPS cells has transformed our understanding of the interplay between genetics and epigenetics and has tremendous therapeutic potential. As currently practiced, this process is inefficient and not always capable o generating cells that are truly equivalent to pluripotent embryonic stem (ES) cells. A few small molecules, including the DNA-methyltransferase inhibitor 5-Aza-2'-deoxycytidine (5-aza-dC), have been identified that partially de-repress genes on the inactive X in somatic cells and that also aid the formation of iPS cells, further underscoring the tight relationship between XCI and iPS cell reprogramming. A female mouse fibroblast cell line carrying an X-linked GFP reporter gene will be used to study X- reactivation. Application of the positive control 5-aza-dC causes dose-dependent reactivation of X-GFP. For the high-throughput screen (HTS), GFP fluorescence resulting from the application of test compounds will be assayed by automated microscopy. A secondary screen will test the reactivation of specifically the inactive alleles of four endogenous X-linked genes. Probes emerging from these screens will be useful tools to dissect X-reactivation and perhaps shed further light on XCI as well. In a final assay, X-reactivation probes will be tested for their ability to enhance the reprogramming of fibroblasts into iPS cells. While most high throughput screens target a specific enzyme or pathway, the primary screen proposed here is based on a biological outcome that is the result of many epigenetic mechanisms that are as yet incompletely understood. The advantage of this approach is that we expect to identify probes of novel proteins that play crucial roles in maintaining the inactive state of the X-chromosome, that are required for X- reactivation, and that are also relevant to reprogramming.