The goal of this research proposal is to utilize synthetically generated chromatin templates to facilitate the biochemical and biophysical characterization of the Oct4, Sox2, Klf4, and c-Myc (OSKM) pluripotency regulators. When ectopically expressed in somatic cells, OSKM act synergistically to reprogram cell fate to pluripotency, resulting in self-renewing induced pluripotent stem cells (iPSCs) that can differentiate into cell types of the three germ layers. iPSCs are an invaluable tool in the fields f regenerative medicine and custom cell therapies because they are functionally indistinguishable from embryonic stem cells (ESCs) and circumvent all ESC-related ethical concerns. Furthermore, iPSCs that are generated from patients with complex genetic syndromes can be differentiated into the afflicted cell type to afford unparalleled insight into the pathogenesis ofa given disease and to provide a model that is compatible with therapeutic screening. Currently, less than 1% of the starting cell population will reach pluripotency in a typical reprogramming experiment, and the process needs to be substantially optimized to fully realize the clinical applications of iPSCs. OSKM bind to the genome, where they influence epigenetic modifications and control gene expression by recruiting a variety of transcriptional regulators to chromatin. Not surprisingly, OSKM localization patterns in iPSCs and ESCs are highly similar and mislocalization is observed in cells that fail to achieve pluripotency. Interestingly, certain epigenetic marks are able to act as barriers to the reprogramming process by disrupting OSKM activity. Despite the fact that genome-wide studies continue to emphasize the importance of epigenetic signatures in OSKM localization and function, there is a lack of mechanistic information describing the crosstalk between histone modifications and OSKM. I propose to recapitulate reprogramming factor-nucleosome interactions in vitro by combining techniques in peptide synthesis, protein engineering, site-specific protein modification, and fluorescence spectroscopy. These studies will elucidate the effects that histone marks have on OSKM binding and function in the context of mononucleosomes and nucleosome arrays. Additionally, I will engineer multivalent Oct4, Sox2 and Klf4 proteins that target epigenetic barriers, which will be used to generate iPSCs.
The specific aims of this research are: 1) characterize the effect of OSKM binding on nucleosome stability and chromatin structure in vitro, 2.) determine the role of key epigenetic modifications in OSKM binding and function, and 3.) design chimeric factors that can overcome epigenetic barriers to reprogramming. This proposed research is designed to delineate valuable mechanistic information underlying OSKM binding and function, which is crucial for the development of reprogramming strategies that can overcome epigenetic barriers and generate high quality iPSCs on a more consistent basis.
Induced pluripotent stem cells (iPSCs) are an invaluable tool in the fields of regenerative medicine and custom cell therapies because they are functionally indistinguishable from embryonic stem cells (ESCs) and circumvent all ESC-related ethical concerns. Generation of iPSCs is a highly inefficient process due to the fact that the proteins responsible for inducing cellular reprogramming, Oct4, Sox2, Klf4, and c-Myc (OSKM), must overcome specific epigenetic hurdles to interact with the genome and convert somatic cells to a pluripotent state. The goal of this proposal is to delineate mechanisms of OSKM binding and function in the presence of various histone modifications, which may lead to more efficient reprogramming strategies that will allow the clinical potential of iPSC technology to be fully realized.
|MÃ¼ller, Manuel M; Fierz, Beat; Bittova, Lenka et al. (2016) A two-state activation mechanism controls the histone methyltransferase Suv39h1. Nat Chem Biol 12:188-93|