Transcription Factor Induced Reprogramming

 描述（由申请人提供）：将体细胞重编程为诱导多能干细胞（iPSC）的效率极低。这被认为是由于在成体组织中保留细胞身份并抵抗细胞命运改变的表观遗传修饰。因此，剖析限制体细胞重编程为iPSC的机制有望为正常建立和维持细胞身份及其失调可能导致疾病的机制提供见解。为了发现细胞身份的新调节因子，我们在iPSC形成期间进行了染色质聚焦的RNAi筛选。这导致鉴定染色质组装因子-1（CAF-1）复合物的所有亚基作为诱导多能性的主要障碍。CAF-1是一种组蛋白伴侣，负责在DNA复制过程中将组蛋白H3和H4沉积到染色质上。虽然CAF-1以前被证明对异染色质维持，DNA修复和早期发育至关重要，但在细胞重编程和出生后生活中的作用尚未报道。在这里，我们概述了三个互补的目的，剖析CAF-1在细胞重编程，组织稳态和癌症过程中发挥的分子机制和功能作用。在第一个目标中，我们将探索CAF-1丢失对细胞命运改变的细胞后果。简而言之，我们将确定CAF-1耗竭是否在多种情况下增强细胞命运变化，包括将不同细胞类型重编程为iPSC以及两种转分化范例。我们将进一步研究CAF-1沉默如何通过分析iPSC形成的定义中间阶段影响诱导多能性的轨迹。鉴于CAF-1作为复制依赖性组蛋白伴侣的作用，我们将测试以下假设：它的耗尽减少了获得多能性所需的最小细胞分裂次数，并且它使iPSC形成成为确定性过程。在第二个目标中，我们将阐明CAF-1抵抗细胞命运改变的分子机制。具体来说，我们将测试的假设，CAF-1的损失，促进转录因子的目标基因开放的染色质调控元件。此外，我们将使用有针对性的和公正的方法来定义那些组蛋白标记和组蛋白变体负责观察到的表型。在第三个目标中，我们将探索CAF-1在体内调节细胞命运变化中的作用。为了支持体细胞祖细胞的调节功能，我们发现CAF-1在未成熟骨髓细胞系中的敲低触发分化。因此，我们将使用转基因RNAi系统表征CAF-1减少对造血的影响。然后，我们将这些研究扩展到AML和CML的小鼠模型，以询问CAF-1在稳定癌症细胞身份中的作用。总的来说，R 01的应用将有助于我们理解细胞命运如何通过使用CAF-1作为工具在不同的细胞环境中稳定。我们预计， 这些研究将改进产生用于再生目的的所需细胞类型的策略，并可能为癌症治疗提供新的途径。

 DESCRIPTION (provided by applicant): The reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) is extremely inefficient. This is thought to be due to epigenetic modifications that preserve cell identity and resist cell fate change in adult tissues. Dissecting the mechanisms that limit the reprogramming of somatic cells into iPSCs is thus expected to provide insights into the mechanisms that normally establish and maintain cellular identity and whose dysregulation may contribute to disease. In order to uncover new regulators of cell identity, we performed a chromatin-focused RNAi screen during iPSC formation. This led to the identification of all subunits of the chromatin assembly factor-1 (CAF-1) complex as major roadblocks of induced pluripotency. CAF-1 is a histone chaperone responsible for depositing histones H3 and H4 onto chromatin during DNA replication. While CAF-1 was previously shown to be critical for heterochromatin maintenance, DNA repair and early development, a role in cellular reprogramming and postnatal life has not yet been reported. Here, we outline three complementary aims to dissect the molecular mechanisms and functional role CAF-1 plays during cellular reprogramming, tissue homeostasis and cancer. In the first aim, we will explore the cellular consequences of CAF-1 loss on cell fate change. Briefly, we will determine whether CAF-1 depletion enhances cell fate change in multiple contexts including reprogramming of distinct cell types into iPSCs as well as two trans differentiation paradigms. We will further investigate how CAF-1 silencing influences the trajectory of induced pluripotency by analyzing defined intermediate stages of iPSC formation. Given CAF-1's role as a replication-dependent histone chaperone, we will then test the hypotheses that its depletion reduces the minimal number of cell divisions required to attain pluripotency and that it renders iPSC formation a deterministic process. In the second aim, we will elucidate the molecular mechanisms by which CAF-1 resists cell fate change. Specifically, we will test the hypothesis that CAF-1 loss facilitates transcription factor access to target genes by opening up chromatin at regulatory elements. Moreover, we will use targeted and unbiased approaches to define those histone marks and histone variants responsible for the observed phenotype. In the third aim, we will explore CAF-1's role in regulating cell fate changes in vivo. In support of a regulatory function i somatic progenitors, we find that CAF-1 knockdown in immature myeloid cell lines triggers differentiation. We will therefore characterize the consequence of CAF-1 reduction on hematopoiesis using a transgenic RNAi system. We will then extend these studies to mouse models of AML and CML in order to interrogate the role of CAF-1 in stabilizing cell identity in cancer. Collectively, this R01 application will contribute to our understanding of how cell fates are stabilized in different cellular contexts by using CAF-1 as a tool. We expect that results from these studies will improve strategies for the generation of desired cell types for regenerative purposes, and may provide new avenues for cancer therapy.