Elucidation of the role of Setd8 and H4K20me1 in Erythropoiesis

阐明 Setd8 和 H4K20me1 在红细胞生成中的作用

• 批准号: 9204831
• 负责人: LAURIE A. STEINER
• 金额: $ 34.59万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-01-15 至 2020-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9204831
• 关键词: Abnormal Cell; Affect; Anemia; Apoptosis; CD34 gene; Cell Cycle Progression; Cell division; Cell physiology; Cells; Cessation of life; Chromatin; Complex; Coupled; DNA Damage; Data; Defect; Deposition; Disease; Embryo; Enzymes; Epigenetic Process; Erythroblasts; Erythrocytes; Erythroid; Erythroid Cells; Erythropoiesis; Gene Expression; Genes; Genetic Transcription; Goals; Health; Hematological Disease; Hematopoietic stem cells; Higher Order Chromatin Structure; Histone H4; Human; Impairment; Inherited; Knock-out; Knowledge; Laboratories; Link; Lysine; Mammals; Mediating; Methyltransferase; Molecular; Mono-S; Mus; Nuclear; Nucleic Acid Regulatory Sequences; Nucleosomes; Pathway interactions; Phenotype; Physical condensation; Population; Process; Production; Proteins; Recruitment Activity; Regulation; Role; Severities; Site; Stem cells; TFRC gene; Time; Tissues; Trans-Activators; Transcription Process; Transcription Repressor/Corepressor; Transcriptional Regulation; cell type; condensin; erythroid differentiation; gene repression; genetic manipulation; genome-wide analysis; histone methyltransferase; histone modification; human disease; in vivo; insight; knock-down; member; novel; overexpression; progenitor; transcriptome

More than 1/3 of the world’s population is anemic, making the process of erythropoiesis central to human health and disease. The maturation of a committed erythroid progenitor to a functional erythrocyte is characterized by a global decline in transcription and progressive chromatin condensation that ultimately culminates in enucleation. Although these processes occur in parallel, the molecular mechanisms that coordinate them are unknown. We hypothesize that Setd8 is a critical regulator of mammalian erythropoiesis that functions to coordinate the fundamental processes of transcriptional repression and chromatin condensation. Setd8 is the sole enzyme in mammals capable of mono-methylating H4K20 (H4K20me1), and is expressed at significantly higher levels in CD71+ erythroid cells than in any other cell- or tissue-type. In addition, Setd8 has functions that are independent of its methyltransferase activity. The overarching goal of this proposal is to elucidate the role of Setd8 in the regulation of mammalian erythropoiesis. Data from our laboratory demonstrates that conditional erythroid Setd8 deletion results in severe defects in primitive erythropoiesis, with visible anemia at E9.5 and death from anemia by ~E12.5. Erythroblasts from these embryos fail to undergo the typical semi-synchronous maturation observed in erythroblasts from control embryos and have a profound defect in nuclear condensation. The goal of Aim 1 is to delineate the function of Setd8 during mammalian erythropoiesis. In Aim 1, in vivo analyses of conditional Setd8 disruption will be coupled with knockdown of Setd8 in human CD34+ hematopoietic stem and progenitor cells to gain a comprehensive understanding of the function of Setd8 in mammalian erythropoiesis. Preliminary data from our laboratory further demonstrates that Setd8 functions as a transcriptional repressor in erythroid cells, and that the Gata2 locus is a direct Setd8 target. Knockdown of Setd8 leads to increased Gata2 expression, with loss of H4K20me1 and increased H4Acetylation in the Gata2 locus. Although Gata2 is a key regulator of erythroid differentiation, Gata2 overexpression alone does not account for the severity of the Setd8 phenotype, especially the profound impairment in nuclear condensation. The Condensin II complex recognizes H4K20me1 and mediates both transcriptional repression and higher order chromatin condensation. Interestingly, Setd8 and a member of the Condensin II complex co-occupy key regulatory regions of the Gata2 locus. The goal of Aim 2 is to delineate the molecular mechanism(s) underlying the role of Setd8 in erythropoiesis. In Aim 2 we will determine the mechanisms by which Setd8 regulates key erythroid loci, and the degree to which the Setd8-H4K20me1-Condensin II pathway mediates transcriptional repression and chromatin condensation in erythroid cells. Taken together, the studies in this proposal will delineate the function of a novel regulator of erythropoiesis and provide critical insights into pathways involved in both inherited and acquired human disease.

世界上超过三分之一的人口患有贫血，这使得红细胞生成过程对人类来说至关重要 健康和疾病。一个确定的红系祖细胞成熟为一个有功能的红细胞是 以转录的全球下降和染色质的渐进性凝聚为特征，最终 最终导致眼球摘除。尽管这些过程是并行进行的，但分子机制 它们的坐标是未知的。我们推测，Setd8是哺乳动物的一个重要调节因子 协调转录抑制的基本过程的红细胞生成 和染色质凝聚。Setd8是哺乳动物体内唯一能够单甲基化H4K20的酶 (H4K20me1)，并且在CD71+红系细胞中的表达水平显著高于任何其他细胞-或 组织类型。此外，Setd8具有不依赖于其甲基转移酶活性的功能。这个 这项建议的首要目标是阐明SET 8在哺乳动物的调节中的作用 红血球生成。我们实验室的数据表明，条件性红系Setd8缺失会导致 原始红细胞生成严重缺陷，E9.5可见贫血，~E12.5死于贫血。 这些胚胎的红细胞没有经历典型的半同步成熟 来自对照胚胎的红细胞，在核凝聚方面存在严重缺陷。目标1的目标是 目的：阐明Setd8在哺乳动物红细胞生成中的作用。在目标1中，条件条件的活体分析 人CD34+造血干/祖细胞中Set8基因的破坏伴随着Setd8基因的敲除 目的是全面了解Setd8在哺乳动物红细胞生成中的作用。初步 我们实验室的数据进一步证明了Setd8在红系中作为转录抑制因子发挥作用 细胞，并且Gata2基因是一个直接的Setd8靶基因。敲除Set8导致Gata2增加 在Gata2基因座，H4K20me1缺失，H4乙酰化增加。尽管Gata2是一把钥匙 红系分化的调节因子，Gata2的过度表达本身并不能解释Setd8的严重程度 表型，尤其是核凝聚的严重损害。凝聚素II复合体识别 H4K20me1，介导转录抑制和高阶染色质缩合。 有趣的是，Setd8和凝聚素II复合体的一个成员共同占据了Gata2的关键调控区域 轨迹。目标2的目标是描述Setd8在糖尿病中的作用的分子机制(S)。 红血球生成。在目标2中，我们将确定Setd8调节关键红系基因座的机制，以及 Setd8-H4K20me1-conducin II途径在多大程度上介导转录抑制和 红系细胞内染色质凝集。综上所述，这项建议中的研究将勾勒出 一种新的红细胞生成调节因子的功能，并提供了对两者所涉及的途径的关键见解 遗传性和获得性人类疾病。