Delineating the functions of EKLF during mammalian terminal erythroid differentiation

描述 EKLF 在哺乳动物终末红细胞分化过程中的功能

• 批准号: 10440030
• 负责人: Merlin Nithya Gnanapragasam
• 金额: $ 14.38万
• 依托单位: CLEVELAND STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-03-01 至 2024-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10440030
• 关键词: ASF1B gene; Advisory Committees; Affect; Anemia; Architecture; Bone Marrow; Candidate Disease Gene; Cell Cycle; Cell division; Cells; ChIP-seq; Chromatin; Collaborations; Comb animal structure; Congenital dyserythropoietic anemia; Coupled; Cytokinesis; Cytoskeleton; DNA; DNA Damage; DNA Maintenance; DNA Repair; DNA biosynthesis; DNA replication fork; Data; Defect; Development Plans; Disease; Ectopic Expression; Electron Microscopy; Ensure; Equilibrium; Erythroblasts; Erythrocytes; Erythroid; Erythroid Cells; Erythropoiesis; Etiology; Event; Excision; Failure; Frequencies; G1 Phase; Genes; Genetic Transcription; Image; Impairment; Investigation; Light; Measures; Mentorship; Morphology; Mutate; Mutation; Nuclear; Organelles; Pathogenesis; Process; Regulation; Replication Origin; Reporting; Role; Speed; Stains; Structure; Time; Transcriptional Regulation; Ultrafine; alpha Tubulin; base; career; career development; daughter cell; erythroid Kruppel-like factor; erythroid differentiation; genome-wide analysis; insight; programs; protein distribution; repaired; replication stress; self-renewal; transcription factor; transcriptome sequencing

Project Summary Erythroid terminal differentiation is comprised of 3-4 rapid terminal cell divisions also known as ‘differentiation divisions’, which are coupled with morphological changes such as a dramatic decrease in cell and nuclear size. The switch from self-renewal to terminal divisions in erythroid cells are peculiarly characterized by short G1 and S phases, and fast DNA replication. We do not yet understand the processes that regulate the timing, integrity and the numbers of these rapid terminal divisions. When these divisions go awry, it leads to severe anemias such as Congenital Dyserythropoietic Anemia (CDA), which arise due to failures in DNA replication and/or cytokinesis, and are characterized by binucleate erythroblasts in the bone marrow and in some cases chromatin bridges between erythroblasts. EKLF/KLF1 is one the genes when mutated causes a type of CDA, CDA type IV. Although previous genome wide studies have alluded to its roles in DNA replication/repair and cytokinesis, this has not been functionally investigated. Interestingly, EKLF-/- erythroid cultures during terminal differentiation have increased proportions of binucleate cells and chromatin bridges; similar to what has been observed in CDA disorders. Based on our preliminary data, we hypothesize that EKLF transcriptionally upregulates the genes involved in the maintenance of DNA replication fidelity (Aim1) and cytokinesis (Aim2) to accommodate the rapid pace of terminal erythroid cell divisions; impairment of this regulation in EKLF-/- erythroblasts results in replication stress, cytokinesis failure and the formation of binucleate erythroblasts. I will study the role of EKLF in the maintenance of DNA replication fidelity by quantifying the levels of DNA damage and replication stress, replication dynamics, and the extent to which unresolved DNA damage perturbs cytokinesis. I will investigate the role of EKLF in cytokinesis by studying the formation, structure, and the function of the midbody organelle, which forms between two daughter cells and is essential for abscission. Finally, I will also examine the extent to which EKLF regulated candidate genes contribute to the observed defects in EKLF-/- erythroblasts. These studies will reveal a specialized transcriptional regulation in erythroid cells to ensure that the cell cycle machinery is able to accommodate the rapid pace of the terminal cell divisions. They will also provide insights on the pathogenesis of severe anemias such as CDA, some of whose etiology is unknown. The studies proposed here along with career development plan described in my application, will enable me to benefit from the mentorship of Dr. James Bieker, who discovered EKLF and has contributed immensely to the field of erythropoiesis, forge collaborations to expand my expertise, and gain guidance on career and scientific progression from my advisory committee. Overall, this will pave the way for my successful transition to independence.

项目摘要 红系终末分化由3-4次快速终末细胞分裂组成，也称为“分化 分裂“，这与形态学变化如细胞和核大小的急剧减少相结合。 红系细胞从自我更新到终末分裂的转变以短G1期为特征 和S期，以及快速DNA复制。我们还不了解控制时间的过程， 完整性和这些快速终端部门的数量。当这些分歧出了差错，就会导致严重的 贫血，如先天性红细胞生成不良性贫血（CDA），由于DNA复制失败而引起 和/或胞质分裂，其特征是骨髓中的双核成红细胞，在某些情况下， 成红细胞之间的染色质桥。 EKLF/KLF 1是一个基因突变时，导致一种类型的CDA，CDA IV型。虽然以前的基因组 广泛的研究已经暗示了它在DNA复制/修复和胞质分裂中的作用，这在功能上还没有被证实。 研究了有趣的是，在终末分化过程中，EKLF-/-红系细胞培养物中 双核细胞和染色质桥;类似于在CDA疾病中观察到的。基于我们 根据初步数据，我们假设EKLF在转录水平上上调了参与细胞凋亡的基因。 维持DNA复制保真度（Aim 1）和胞质分裂（Aim 2），以适应快速的 终末红系细胞分裂; EKLF-/-成红细胞中这种调节的受损导致复制 应激、胞质分裂失败和双核成红细胞的形成。 我将研究EKLF在维持DNA复制保真度的作用，通过定量DNA水平， 损伤和复制压力，复制动力学，以及未解决的DNA损伤 扰乱胞质分裂。我将研究EKLF在胞质分裂中的作用，通过研究EKLF的形成、结构和功能， 中间体细胞器的功能，它形成于两个子细胞之间，对细胞分裂至关重要。 最后，我还将研究EKLF调节的候选基因在多大程度上有助于观察到的 EKLF-/-成红细胞缺陷。这些研究将揭示红系细胞中一种特殊的转录调控机制 细胞，以确保细胞周期机制能够适应终端细胞的快速节奏 分裂他们还将提供关于严重贫血如CDA的发病机制的见解，其中一些 病因不明。 沿着在此提出的学习计划以及我申请中所描述的职业发展计划，将使我能够 受益于James Bieker博士的指导，他发现了EKLF，并为EKLF的发展做出了巨大贡献。 红细胞生成领域，建立合作，以扩大我的专业知识，并获得职业和科学指导 我的顾问委员会的进展总的来说，这将为我成功过渡到 独立

项目成果

EKLF/KLF1 expression defines a unique macrophage subset during mouse erythropoiesis.

• DOI: 10.7554/elife.61070
• 发表时间: 2021-02-11
• 期刊: eLife
• 影响因子: 7.7
• 作者: Mukherjee K;Xue L;Planutis A;Gnanapragasam MN;Chess A;Bieker JJ
• 通讯作者: Bieker JJ