Epigenetic and Cell Cycle Functions of Glucocorticoids in Erythropoietic Stress

描述（由申请人提供）：糖皮质激素（GCs）是应激激素调节剂。它们加速了红细胞的生成速度，这一效应已被小鼠遗传学、体外红细胞生成系统和GCs失调的人类疾病综合征所证实。然而，由于其严重的副作用，GCs在贫血治疗中的使用是复杂的，因此仅限于红细胞生成素（Epo）治疗难治性或禁忌症，包括Diamond Blackfan贫血和其他骨髓衰竭综合征。从目前正在开发的用于体外生成输血用红细胞的系统中，GCs的转化重要性是显而易见的。了解GCs在红细胞祖细胞中的分子作用，有助于开发出比GCs副作用更小的新型促红细胞生成药物，并提高体外红细胞生成效率。在功能上，GCs通过延缓红细胞祖细胞从自我更新到分化的转变来提高红细胞生成率。这种作用背后的分子机制在很大程度上是未知的。基于我们最近发表的工作和初步数据，我们提出了一个新的GC作用假设，暗示细胞周期S期和DNA甲基化是新的调控靶点。我们最近发现，胎儿和成人的红细胞生成都需要全基因组DNA去甲基化，这是体细胞中一种独特的全局表观遗传修饰（Shearstone etal ., Science 2011）。全局去甲基化与红系基因启动子的去甲基化密切相关，是其转录激活的限速因素。此外，全局去甲基化蛋白依赖于细胞周期S期的显著变化，随着细胞从自我更新到分化的转变，S期变短，速度加快50%。周期蛋白依赖性激酶抑制剂（CDKI） p57KIP2是这一开关的关键负调控因子。p57KIP2也是gc的直接转录靶点。我们的初步数据显示，在GCs存在的情况下，红系祖细胞不能下调p57KIP2，不能加速S期，不能进行DNA去甲基化，从而延迟红系基因的转录。在本研究中，我们研究了红细胞生成应激过程中高水平的GCs通过诱导p57KIP2抑制从自我更新到分化的转换，从而抑制S期加速、整体DNA去甲基化和红细胞基因诱导的假设。我们将在体内使用红细胞生成应激小鼠模型和p57KIP2、GC受体或DNA甲基转移酶1 （Dnmt1）缺失或突变的小鼠模型来验证这一假设，目的有以下三个：1)确定p57KIP2在GC介导的红细胞生成应激反应中的作用；2)确定GCs是否在应激时延长红系祖细胞的S期；3)确定GCs是否在应激时延迟全局DNA去甲基化的发生。这项工作的重点是一种独特的表观遗传修饰，并有可能确定概念上新颖的调控机制，对epoo抵抗性贫血的治疗具有转化意义。

DESCRIPTION (provided by applicant): Glucocorticoids (GCs) are hormonal regulators of stress. They accelerate red blood cell production rate, an effect that is well established by mouse genetics, by in vitro erythropoiesis systems, and by human disease syndromes in which GCs are dysregulated. The use of GCs in the treatment of anemia is complicated, however, by their severe side effects, and is therefore limited to conditions where Erythropoietin (Epo) treatment is refractory or contraindicated, including Diamond Blackfan Anemia and other bone-marrow failure syndromes. The translational importance of GCs is apparent from their use in systems currently under development for the in-vitro generation of red blood cells for transfusion. Understanding the molecular action of GCs in erythroid progenitors could facilitate the development of novel erythropoiesis-stimulating agents that have fewer side-effects than GCs, and that improve the efficiency of generating red blood cells in vitro. Functionally, GCs increase erythropoietic rate by delaying the switch from self-renewal to differentiation in erythroid progenitors. The molecular mechanisms underlying this action are largely unknown. Based on our recently published work and on preliminary data, we propose a novel hypothesis of GC action that implicates the cell cycle S phase and DNA methylation as novel regulatory targets. We recently showed that both fetal and adult erythropoiesis entail genome-wide DNA demethylation, a unique global epigenetic modification in somatic cells (Shearstone et al., Science 2011). Global demethylation is tightly correlated with demethylation at erythroid gene promoters, and is a rate-limiting for their transcriptional activation. Further, global demethylatin is dependent on a marked change in S phase of the cell cycle, which becomes shorter and 50% faster with the switch from self-renewal to differentiation. The cyclin-dependent kinase inhibitor (CDKI) p57KIP2 is a key negative regulator of this switch. p57KIP2 is also a direct transcriptional target of GCs. Our preliminary data show that, in the presence of GCs, erythroid progenitors fail to downregulate p57KIP2, fail to accelerate S phase, and fail to undergo DNA demethylation, thereby delaying erythroid gene transcription. In this proposal, we investigate the hypothesis that high levels of GCs during erythropoietic stress inhibit the switch from self-renewal to differentiation by inducing p57KIP2, thereby inhibiting S phase acceleration, global DNA demethylation and erythroid gene induction. We will test this hypothesis in vivo using mouse models of erythropoietic stress and mice deleted or mutated for either p57KIP2, the GC receptor, or DNA methyl transferase 1 (Dnmt1), with the following three aims: 1) Determine the role of p57KIP2 in the GC-mediated erythropoietic stress response 2) Determine whether GCs prolong S phase in erythroid progenitors during stress 3) Determine whether GCs delay the onset of global DNA demethylation during stress. This work focuses on a unique epigenetic modification and has the potential to identify conceptually novel regulatory mechanisms, with translational implications for therapy of Epo-resistant anemia.