Epigenetic and Cell Cycle Functions of Glucocorticoids in Erythropoietic Stress

描述（由申请人提供）：糖皮质激素（GC）是应激的激素调节剂。它们加速红细胞生成速率，这是由小鼠遗传学、体外红细胞生成系统和GC失调的人类疾病综合征充分确立的效果。然而，GC在治疗贫血中的使用是复杂的，由于其严重的副作用，因此仅限于促红细胞生成素（Epo）治疗难治或禁忌的情况，包括Diamond Blackfan贫血和其他骨髓衰竭综合征。GC的转化重要性从其在目前正在开发的用于输血用红细胞体外生成的系统中的使用中显而易见。了解GCs在红系祖细胞中的分子作用可以促进新的红细胞生成刺激剂的开发，其具有比GCs更少的副作用，并且提高体外生成红细胞的效率。在功能上，GC通过延迟红系祖细胞从自我更新到分化的转换来增加红细胞生成速率。这种作用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的数据，我们提出了一个新的假设GC的行动，牵连细胞周期S期和DNA甲基化作为新的调控目标。我们最近表明，胎儿和成人红细胞生成都需要全基因组DNA去甲基化，这是体细胞中独特的整体表观遗传修饰（Shearstone et al.，Science 2011）。整体去甲基化与红细胞基因启动子的去甲基化密切相关，并且是其转录激活的限速因子。此外，全局去甲基化依赖于细胞周期S期的显著变化，随着从自我更新到分化的转变，S期变得更短并且快50%。细胞周期蛋白依赖性激酶抑制剂（CDKI）p57 KIP2是该开关的关键负调节因子。p57KIP2也是GC的直接转录靶点。我们的初步数据显示，在GC存在下，红系祖细胞不能下调p57 KIP 2，不能加速S期，不能进行DNA去甲基化，从而延迟红系基因转录。在这个建议中，我们调查的假设，高水平的GC在红细胞生成应激抑制开关从自我更新到分化诱导p57 KIP 2，从而抑制S期加速，全球DNA去甲基化和红细胞基因诱导。我们将使用红细胞生成应激的小鼠模型和p57 KIP 2、GC受体或DNA甲基转移酶1（Dnmt 1）缺失或突变的小鼠在体内检验这一假设，目的如下：1）确定p57 KIP 2在GC介导的红细胞生成应激反应中的作用2）确定GC是否在应激期间延长红系祖细胞中的S期3）确定GC是否延迟了应激过程中整体DNA去甲基化的发生。这项工作的重点是一个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，与翻译的影响，治疗环氧乙烷耐药性贫血。

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.