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

基本信息

批准号：
9042355
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
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-06-07 至 2018-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9042355
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 DNA replication fork 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 new therapeutic target novel overexpression oxidation prevent progenitor programs public health relevance self-renewal whole genome

项目摘要

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。在多能早期发育过程中，DNA 甲基化两次被全局消除。然后在整个体细胞分化过程中重新建立并维持它。直到最近，人们还认为正常体细胞中没有发生整体甲基化变化。我们发现红系分化与几乎所有基因组元件的 DNA 去甲基化相关，这一长期坚持的教条被推翻。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，对于红细胞成熟至关重要，但其机制尚不清楚。我们推测，整体红细胞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，最近在被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，其中去甲基化治疗取得了成功。我们的长期目标是阐明红细胞生成中整体 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) 使新生 DNA 甲基化，从而在细胞世代间维持 DNA 甲基化。红细胞整体去甲基化依赖于 DNA 复制，表明 Dnmt1 功能受损。去甲基化还需要加速 S 期内 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，成红细胞中的去甲基化与 Dnmt1 丢失无关，并且不能通过 Dnmt1 过度表达来预防。总之，这些观察结果表明了这样的假设：成红细胞含有限制 Dnmt1 甲基化能力的机制。我们建议通过以下三个目标来检验这一假设：目标 1：确定红细胞基因组 DNA 中的 5mC 是否会被 Tet2 酶氧化，从而导致复制依赖性、Dnmt1 抗性全局去甲基化目标 2：确定红细胞基因组 DNA 中的 5mC 的作用细胞周期蛋白依赖性激酶抑制剂 p57KIP2 在 S 期加速和红细胞整体 DNA 去甲基化中的作用。目标 3：确定红系分化开始时 S 期内 DNA 合成加速的根本机制：是由激发复制起点数量增加引起的，还是由复制叉进展速率增加引起的。询问该过程是否受到细胞周期蛋白依赖性激酶抑制剂 p57KIP2 的调节。这项工作的资助将阐明全球甲基化模式是如何调节的，并可能为治疗促红细胞生成素抵抗性贫血提供概念上的新途径。