The goal of this proposal is to reveal molecular mechanisms by which cell fates are controlled in mammalian development and to use the information to enhance the reprogramming of differentiated cells, ultimately for modeling disease and cellular therapeutics. Work on this grant led to the discovery of pioneer transcription factors; they initiate cellular programming by targeting unmarked nucleosomal sequences in silent chromatin. Indeed, recent Epigenome studies show that 40-60% of the chromatin in a cell is in a low signal nucleosomal state, unmarked by activating or inhibitory marks. Yet the molecular features that permit or preclude pioneer factors' targeting of nucleosomal DNA and the chromatin perturbations that immediately succeed binding, to promote cooperative interactions with other factors, remain to be understood. We recently found that the FoxA pioneer factor makes specific contacts with core histones that initiate chromatin opening. With mice engineered to perturb FoxA-mediated chromatin opening, we are poised to understand the developmental role. We also discovered that large genomic domains spanned by histone H3 lysine 9 trimethylation (H3K9me3) prevent nucleosome targeting by FoxA and other pioneer factors. We are poised to test whether this role of H3K9me3-chromatin helps explain the stability of cell fate and developmental restriction, whereby certain fates are excluded from progenitor cells. With our Aims to reveal how pioneer factors target unmarked chromatin, initiate chromatin opening, and can be restricted from H3K9me3 domains, we will unveil mechanisms by which cells stabilize their fate and how fates can be destabilized for cell type conversions.
Aim 1. To test whether shared and unique features endow different pioneer factors with the ability to target nucleosomes, and to use such features to improve cell reprogramming.
Aim 2. To understand how pioneer factors initiate chromatin opening in embryonic development and the impact on cell fate specification.
Aim 3. To reveal the interplay between H3K9me3-based heterochromatin and pioneer factor binding at differentiation genes during development. This proposal distinctively combines in vitro chromatin binding studies, using purified components, with natural chromatin from different stages of embryonic development, using purified cell populations. Fulfilling our Aims will reveal mechanisms by which a subset of transcription factors can engage nucleosomal DNA and open the local chromatin, and the interplay between such activities and restrictive heterochromatin, thereby illuminating ways to enhance our ability to create new cells for research and, eventually, autologous cell transplantation.

Public Health Relevance

The long term public health goal of this work is to enhance the ability to reprogram cell fates at will. The efficiency of induced cell conversions is now poor and the reprogrammed cells do not fully express the differentiated transcriptional repertoire that is necessary for proper tissue function. By seeking to understand how cell reprogramming factors scan the genome, bind target sites, and make local regions of the chromosome accessible to other factors, and by revealing how such events are impeded by repressive regions of the genome, the proposed work will bring us closer to being able to reprogram cells efficiently and fully, to create new cells for research and, eventually, autologous cell transplantation.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Molecular Genetics B Study Section (MGB)
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Gibbs, Kenneth D
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University of Pennsylvania
Anatomy/Cell Biology
Schools of Medicine
United States
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Zaret, Kenneth S (2018) Pioneering the chromatin landscape. Nat Genet 50:167-169
Palozola, Katherine C; Donahue, Greg; Liu, Hong et al. (2017) Mitotic transcription and waves of gene reactivation during mitotic exit. Science 358:119-122
Moreno, Jonathan; Gearhart, John; Zoloth, Laurie et al. (2017) Managing cell and human identity. Science 356:139-140
Kim, Jungsun; Bamlet, William R; Oberg, Ann L et al. (2017) Detection of early pancreatic ductal adenocarcinoma with thrombospondin-2 and CA19-9 blood markers. Sci Transl Med 9:
Iwafuchi-Doi, Makiko; Zaret, Kenneth S (2016) Cell fate control by pioneer transcription factors. Development 143:1833-7
Bhat, Neha; Park, Jeehye; Zoghbi, Huda Y et al. (2016) The Chromatin Modifier MSK1/2 Suppresses Endocrine Cell Fates during Mouse Pancreatic Development. PLoS One 11:e0166703
Zaret, Kenneth S; Lerner, Jonathan; Iwafuchi-Doi, Makiko (2016) Chromatin Scanning by Dynamic Binding of Pioneer Factors. Mol Cell 62:665-7
Iwafuchi-Doi, Makiko; Donahue, Greg; Kakumanu, Akshay et al. (2016) The Pioneer Transcription Factor FoxA Maintains an Accessible Nucleosome Configuration at Enhancers for Tissue-Specific Gene Activation. Mol Cell 62:79-91
Zaret, Kenneth S; Mango, Susan E (2016) Pioneer transcription factors, chromatin dynamics, and cell fate control. Curr Opin Genet Dev 37:76-81
Becker, Justin S; Nicetto, Dario; Zaret, Kenneth S (2016) H3K9me3-Dependent Heterochromatin: Barrier to Cell Fate Changes. Trends Genet 32:29-41

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