Delineating the functions of EKLF during mammalian terminal erythroid differentiation

项目摘要 红系末端分化由3-4个快速的末端细胞分裂组成，也称为‘分化 分裂，伴随着形态的变化，如细胞和核的大小急剧减小。 红系细胞从自我更新到终末分裂的转换特殊地表现为短的G1 和S阶段，和快速的DNA复制。我们还不了解调节时间的过程， 完整性和这些快速终端部门的数量。当这些分歧出现问题时，就会导致严重的 由于DNA复制失败而引起的贫血，如先天性红细胞生成性贫血(CDA) 和/或胞质分裂，其特征是骨髓中的双核红细胞，在某些情况下 染色质是红细胞之间的桥梁。 EKLF/KLF1是导致CDA的一种基因，即CDA IV型。 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，但这还不是功能上的 调查过了。有趣的是，在终末分化过程中，EKLF-/-红系培养的比例增加了 指双核细胞和染色质桥；类似于在CDA疾病中观察到的。基于我们的 初步数据，我们假设EKLF在转录上上调了参与 维持DNA复制保真度(Aim1)和胞质分裂(AIM2)以适应快速的 终末红系细胞分裂；EKLF-/-红细胞中这一调节的损害导致复制 应激、胞质分裂失败和双核红细胞的形成。 我将通过量化DNA水平来研究EKLF在维持DNA复制保真度中的作用 损伤和复制压力、复制动态以及未解决的DNA损伤的程度 扰乱了细胞质分裂。我将通过研究EKLF的形成、结构和功能来研究EKLF在细胞质分裂中的作用 中体细胞器的功能，它形成在两个子细胞之间，对脱落是必不可少的。 最后，我还将检查EKLF受调控的候选基因对观察到的 EKLF-/-红细胞缺陷。这些研究将揭示红系中一种特殊的转录调控。 确保细胞周期机制能够适应细胞终末的快节奏 组织。他们还将为CDA等严重贫血的发病机制提供见解，其中一些 病因尚不清楚。 我在这里提出的学习和我在申请中描述的职业发展计划将使我能够 受益于詹姆斯·比克尔博士的指导，他发现了EKLF，并为 在红细胞生成领域，建立合作以扩大我的专业知识，并获得职业和科学方面的指导 从我的顾问委员会取得进展。总体而言，这将为我成功过渡到 独立。

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.