DNA Replication and Genome-Wide Demethylation in Erythropoiesis

描述(申请人提供)：在哺乳动物基因组中，5‘-CpG-3’二核苷酸中约70%的胞嘧啶发生甲基化，这是一种与转录沉默相关的表观遗传修饰。在多能性的早期发育中，DNA甲基化两次被全球消除。然后它被重新建立并在整个体细胞分化过程中保持。直到最近，人们还认为在正常的体细胞中没有发生全球甲基化变化。我们发现，红系分化与几乎所有基因组元素的DNA去甲基化有关，这一长期持有的教条被推翻了。红系基因的快速诱导需要整体去甲基化。它代表了一种新的全基因组表观遗传转化，对红系成熟是必要的，其机制尚不清楚。我们推测，红系整体去甲基化的中断可能是红血球生成素抵抗性贫血综合征的基础。事实上，最近在归类为骨髓增生异常综合征(MDS)的难治性贫血中发现了全基因组异常的DNA甲基化，在MDS中，去甲基化治疗是成功的。我们的长期目标是阐明全球DNA去甲基化在红细胞生成中的机制和功能，以及其失调的后果。在复制过程中，DNA甲基转移酶1(DNMT1)甲基化新生DNA，从而在细胞代间维持DNA甲基化。红系整体去甲基化依赖于DNA复制，提示Dnmt1功能受损。去甲基化还需要加速的S相DNA合成，这是最近发现的一种在红系分化开始时的细胞周期行为，其机制尚不清楚。然而，红细胞中去甲基化与Dnmt1的丢失无关，也不能通过Dnmt1的过度表达来阻止。总之，这些观察结果表明，红细胞含有限制Dnmt1甲基化能力的机制的假设。我们建议通过以下三个目的来验证这一假说：目的1：确定红系基因组DNA中的5mC是否受到TET2酶的氧化，从而导致复制依赖的、DNMT1抵抗的全局去甲基化。目的2：确定细胞周期蛋白依赖的激酶抑制物p57Kip2在S期加速和红系DNA全局去甲基化中的作用。目的3：确定红系分化开始时S相内DNA合成加速的潜在机制：是由更多的激发复制起始点引起的，还是由复制分叉进展速度增加引起的。询问这一过程是否受细胞周期蛋白依赖性激酶抑制因子p57Kip2的调控。这项工作的资金将阐明全球甲基化模式是如何受到调节的，并可能从概念上为红细胞生成素耐药贫血的治疗提供新的途径。

DESCRIPTION (provided by applicant): In the mammalian genome, ~70% of cytosines within 5'-CpG-3' dinucleotides are methylated, an epigenetic modification correlating with transcriptional silencing. Twice in pluripotent early development, DNA methylation is globally erased. It is then re-established and maintained throughout somatic cell differentiation. Until recently, no global methylation changes were thought to occur in normal somatic cells. This long-held dogma was overturned by our discovery that erythroid differentiation is associated with DNA demethylation at nearly all genomic elements. Global demethylation is required for rapid erythroid gene induction. It represents a novel genome-wide epigenetic transformation essential for erythroid maturation, whose mechanism is as yet unknown. We hypothesize that disruption of global erythroid demethylation may underlie syndromes of erythropoietin-resistant anemias. Indeed, genome-wide aberrant DNA methylation was recently found in the refractory anemias classified as myelodysplastic syndromes (MDS), where demethylation therapy is successful. Our long-term goal is to elucidate the mechanism and function of global DNA demethylation in erythropoiesis and the consequences of its dysregulation. During replication, DNA methyltransferase 1 (Dnmt1) methylates nascent DNA, thereby maintaining DNA methylation across cell generations. Erythroid global demethylation is dependent on DNA replication, suggesting impaired Dnmt1 function. Demethylation further requires an accelerated intra-S phase DNA synthesis, a recently-discovered cell cycle behavior at the onset of erythroid differentiation whose mechanism is unknown. Demethylation in erythroblasts is not, however, associated with Dnmt1 loss, and cannot be prevented by Dnmt1 overexpression. Together, these observations suggest the hypothesis that erythroblasts contain mechanisms limiting the methylation capacity of Dnmt1. We propose to test this hypothesis with the following three aims: Aim 1: Determine whether 5mC in erythroid genomic DNA is subject to oxidation by the enzyme Tet2, resulting in replication-dependent, Dnmt1-resistant global demethylation Aim 2: Determine the role of the Cyclin-dependent kinase inhibitor p57KIP2 in S phase acceleration and in erythroid global DNA demethylation. Aim 3: Determine the underlying mechanism of the accelerated intra-S phase DNA synthesis at the onset of erythroid differentiation: is it caused by an increased number of firing replication origins, or by increased rate of replication fork progression. Ask whether this process is regulated by the cyclin-dependent kinase inhibitor p57KIP2. Funding of this work will elucidate how global methylation patterns are regulated, and may provide conceptually new avenues for therapy of erythropoietin-resistant anemias.