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

描述(申请人提供)：X染色体失活(XCI)，雌性哺乳动物(XX)与雄性哺乳动物(XY)相比平衡X连锁基因表达的过程，由非编码RNA Xist的表达启动，并包含一系列染色质变化，建立并维持非活性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新编程形式逆转，这种形式被称为X-再激活，发生在体内的早期胚胎和生殖系中，或在体外诱导的多能干细胞(IPS)形成期间。虽然XCI已经被深入研究了很多年，并对基因调控的一般机制有了深入的了解，但对X再激活的了解要少得多。我们认为，在小鼠细胞中识别靶向X-重新激活的新分子也将被证明对iPS细胞的重新编程有用。与小鼠iPS细胞相比，大多数人iPS细胞株在X-再激活方面都失败了，对于这个拟议的项目之后的未来计划，我们期待着在人iPS细胞上测试新的小分子探针。IPS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，具有巨大的治疗潜力。按照目前的做法，这一过程效率低下，并不总是能够产生真正等同于多能胚胎干细胞的细胞。一些小分子，包括DNA甲基转移酶抑制剂5-氮杂-2‘-脱氧胞苷(5-aza-2’-deoxcytidine，5-aza-DC)，已被发现部分解除了体细胞中非活性X基因的抑制，并有助于iPS细胞的形成，进一步强调了XCI与iPS细胞重编程之间的密切关系。携带X连锁绿色荧光蛋白报告基因的雌性小鼠成纤维细胞系将被用于研究X-再激活。应用阳性对照5-aza-DC可引起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。二次筛查将测试四个内源X连锁基因的非活性等位基因的重新激活。从这些屏幕上出现的探针将是剖析X-重新激活的有用工具，或许还可以进一步阐明XCI。在最后的测试中，将测试X-再激活探针是否有能力增强成纤维细胞重新编程为iPS细胞的能力。虽然大多数高通量筛选针对的是特定的酶或途径，但这里提出的初步筛选是基于生物学结果，这是许多表观遗传机制的结果，这些机制迄今尚不完全清楚。这种方法的优点是，我们希望找到在维持X染色体的非活动状态方面起关键作用的新蛋白质的探针，这些蛋白质是X染色体重新激活所必需的，也与重新编程有关。

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.