Probing the coordination of cell cycle progression and differentiation in hematopoietic stem cells

摘要 造血干细胞(HSCs)在成人中产生所有成熟的血细胞。他们保持着静止的状态 动态平衡，但在需要时，可以重新进入细胞周期，以自我更新或分化和补充 造血系统。虽然管理HSC自我更新的途径刚刚被发现，但什么 说明HSC的分化在很大程度上仍然难以捉摸。最近的研究表明，居住在离散的阶段 在不同来源的胚胎和成体干细胞中，细胞周期的不同促进分化。然而， 细胞周期滞留对HSC分化的影响尚未直接探讨。在这里，我们报告 由染色质修饰物Sin3B的遗传失活引起的造血表型。SIN3B充当 一种支架蛋白，通过与序列特异性的相互作用将抑制活动捆绑到不同的位置 转录因子和组蛋白修饰物。通过其调节细胞周期基因转录的能力 Sin3B通过细胞周期的早期阶段限制进展。Sin3B删除的HSC保持其自身 更新能力，但它们无法适当区分。对血统的恰当表达 规范转录因子表明染色质景观的变化可能决定了 SIN3B-/-HSCs分化缺陷。因此，Sin3B失活提供了一个独特的机会来研究 将细胞周期进程与转录环境的产生联系起来的过程 HSC分化。我们的中心假设是，造血干细胞分化的能力仅限于离散的 在细胞周期的早期阶段。我们将测试SIN3B丢失导致虚假的可能性 在细胞周期的早期阶段进行，这与正常的HSC分化不相容，原因是 染色质景观的变化。我们在这里建议：确定是否依赖于Sin3B的限制 细胞周期进程使HSC分化(目标1)。使用细胞周期报告器、谱系追踪工具和 单细胞转录分析，我们将利用Sin3B失活引起的表型来 确定HSCs细胞周期进程和分化之间的功能关系； 确定HSCs依赖Sin3B分化的分子基础(目标2).通过染色质 组蛋白标记的可及性分析和全基因组作图，我们将确定染色质特征 使HSCs在细胞周期进程中对促分化刺激做出反应。根据我们的初步调查 数据，我们将测试我们在Sin3B-/-HSCs中发现的特定信号通路改变的假设，包括 强效干扰素反应，调节造血干细胞的分化能力。我们提议的工作将定义一个 细胞周期进展和造血分化之间的分子联系研究不足，指出 为治疗目的调节HSC扩增和分化的潜在方法。

ABSTRACT Hematopoietic stem cells (HSCs) generate all mature blood cells in adults. They remain quiescent at homeostasis but when needed, can reenter the cell cycle to either self-renew or differentiate and replenish the hematopoietic system. While the pathways governing HSC self-renewal are just being uncovered, what dictates HSC differentiation remains largely elusive. Recent studies indicate that residence in discrete phases of the cell cycle potentiates differentiation in embryonic and adult stem cells of different origins. However, the impact of cell cycle residence on HSC differentiation has not been directly explored. Here, we report the hematopoietic phenotypes elicited upon genetic inactivation of the chromatin modifier Sin3B. Sin3B serves as a scaffold protein that tethers repressive activities to discrete loci by interacting with sequence-specific transcription factors and histone modifiers. Through its ability to modulate transcription of cell cycle genes Sin3B restricts progression through the early phases of the cell cycle. Sin3B-deleted HSCs retain their self- renewal capacities, but they are unable to properly differentiate. The appropriate expression of lineage specification transcription factors suggests that alterations of the chromatin landscape likely dictate the defective differentiation in Sin3B-/- HSCs. Thus, Sin3B inactivation provides a unique opportunity to study the processes linking cell cycle progression with the generation of a transcriptional environment permissive for HSC differentiation. Our central hypothesis is that the ability of HSCs to differentiate is restricted to a discrete window within the early phase of the cell cycle. We will test the possibility that loss of Sin3B induces spurious progression through early stages of the cell cycle, which is incompatible with proper HSC differentiation, due to alterations in the chromatin landscape. We propose here to: Establish whether Sin3B-dependent restriction of cell cycle progression enables HSC differentiation (aim 1). Using cell cycle reporters, lineage tracing tools and single cell transcriptomic analyses, we will leverage the phenotypes elicited upon Sin3B inactivation to determine the functional relationship between cell cycle progression and differentiation in HSCs; and Determine the molecular bases for Sin3B-dependent differentiation in HSCs (aim 2).Through chromatin accessibility assays and genome-wide mapping of histone marks, we will determine the chromatin features that enable HSCs to respond to pro-differentiation stimuli during cell cycle progression. Based on our preliminary data, we will test the hypothesis that specific signaling pathways we found altered in Sin3B-/- HSCs, including the tonic interferon response, modulate the ability of HSCs to differentiate. Our proposed work will define an understudied molecular link between cell cycle progression and hematopoietic differentiation, pointing to potential approaches to modulate HSC expansion and differentiation for therapeutic purposes.