Transcription Factor Induced Reprogramming

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 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.