DNA Replication and Genome-Wide Demethylation in Erythropoiesis

红细胞生成过程中的 DNA 复制和全基因组去甲基化

• 批准号: 8824527
• 负责人: Merav Socolovsky
• 金额: $ 36.43万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-06-07 至 2016-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8824527
• 关键词: Aberrant DNA Methylation; Acceleration; Accounting; Anemia; Behavior; Cell Cycle; Cell Differentiation process; Cells; Characteristics; Comb animal structure; Cyclin-Dependent Kinase Inhibitor; Cytosine; DNA; DNA Methylation; DNA Modification Methylases; DNA biosynthesis; Daughter; Development; Dinucleoside Phosphates; Down-Regulation; Dysmyelopoietic Syndromes; Elements; Embryonic Development; Enzymes; Epigenetic Process; Erythroblasts; Erythrocytes; Erythroid; Erythropoiesis; Erythropoietin; Fetal Liver; Funding; Gene Expression; Generations; Genes; Genetic Transcription; Genome; Genomic DNA; Genomics; Goals; Hematopoietic; Hematopoietic stem cells; Knockout Mice; Maintenance; Mediating; Methylation; Modeling; Modification; Process; Proteins; Refractory anemias; Replication Origin; Research; Resistance; Role; S Phase; Science; Somatic Cell; Syndrome; Testing; Tetanus Helper Peptide; Time; Tissues; Wild Type Mouse; Work; base; demethylation; epigenetic regulation; erythroid differentiation; gene induction; genome wide methylation; genome-wide; knock-down; mammalian genome; methyl group; methylation pattern; neuronal cell body; novel; overexpression; oxidation; prevent; progenitor; programs; public health relevance; self-renewal; targeted treatment

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.

描述（由申请人提供）：在哺乳动物基因组中，5‘-CpG-3’二核苷酸内约70%的胞嘧啶被甲基化，这是一种与转录沉默相关的表观遗传修饰。在多能性早期发育过程中，DNA甲基化在全球范围内被抹去两次。然后在整个体细胞分化过程中重新建立和维持它。直到最近，人们才认为正常体细胞中不会发生全局甲基化变化。这个长期持有的教条被我们的发现所推翻，即红系分化与几乎所有基因组元素的DNA去甲基化有关。快速红系基因诱导需要全局去甲基化。它代表了一种新的全基因组表观遗传转化，对红系成熟至关重要，其机制尚不清楚。我们假设红细胞整体去甲基化的破坏可能是促红细胞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DNA甲基化，其中去甲基化治疗是成功的。我们的长期目标是阐明红细胞生成过程中整体DNA去甲基化的机制和功能及其失调的后果。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。去甲基化进一步需要加速s期DNA合成，这是最近发现的红细胞分化开始时的细胞周期行为，其机制尚不清楚。然而，红母细胞的去甲基化与Dnmt1缺失无关，并且不能通过Dnmt1过表达来阻止。总之，这些观察结果提出了红母细胞含有限制Dnmt1甲基化能力的机制的假设。我们提出通过以下三个目的来验证这一假设：目的1：确定红细胞基因组DNA中的5mC是否受到Tet2酶的氧化，从而导致复制依赖性，dnmt1抗性的全局去甲基化；目的2：确定周期蛋白依赖性激酶抑制剂p57KIP2在S期加速和红细胞全局DNA去甲基化中的作用。目的3：确定红细胞分化开始时s期内DNA合成加速的潜在机制：它是由激活复制起点数量增加引起的，还是由复制叉进展速度增加引起的？询问这一过程是否受周期蛋白依赖性激酶抑制剂p57KIP2的调控。这项工作的资助将阐明全球甲基化模式是如何被调节的，并可能为红细胞生成素抵抗性贫血的治疗提供概念上的新途径。