Background: Chromatin structure regulates fundamental DNA-related processes and is thought to play an important role in pluripotency. While chromatin structure and pluripotency have been linked, how nucleosomes are positioned across key genetic loci in pluripotent cells and how this contributes to the self-renewal and differentiation of these cells is unclear. Objective/hypothesis: The goal of this proposal is to determine the position and density of nucleosomes at key pluripotency and differentiation-specifying regions in mouse embryonic stem (ES) cells, induced-pluripotent stem (iPS) cells, and various somatic cell types. We hypothesize that nucleosome positioning is a key regulator of gene expression that is established in pluripotent cells by key chromatin modifying factors, and that this regulation plays a role in the self renewal and differentiation of pluripotent cells.
Specific Aims : (1) Perform structural analysis of chromatin in ES cells, differentiated cells, iPS cells and their parental somatic fibroblasts at key pluripotent and differentiation-related gene loci. We will further correlate nucleosome positioning and transcriptional status at gene loci to covalent histone modifications located there. (2) Interrogate effects of modulating key chromatin regulators on nucleosome positioning in pluripotent cells and the subsequent ability of these cells to self renew and differentiate. Study Design: By comparing nucleosome positioning across hundreds of genes associated with pluripotency and differentiation in multiple pluripotent and lineage-specified cell types via high-resolution tiling microarrays, this study will generate a detailed description of the chromatin structure associated with pluripotency and how this structure changes in lineage-specified cells and potentially influences developmentally-timed transcriptional programs. Furthermore, the extent to which somatic-cell reprogramming to iPS cells erases the somatic epigenetic memory and establishes a pluripotent signature with regard to nucleosome positioning can be determined. Lastly, we will use these data to determine the effects of gene knockdown of several chromatin modifying factors on nucleosome positioning in ES cells, linking the functional regulation of chromatin structure to gene expression and pluripotency. These studies will provide the framework for understanding programs influencing self-renewal and differentiation of pluripotent cells. As aberrant self-renewal or differentiation can lead to disease, these findings can be used for new lines of research into chromatin structure and how it relates to stem-cell self renewal, lineage commitment, and developmental malignancies, including cancer.
Organization of chromatin is of great interest due to its potential to illuminate the mechanisms of activation and repression of genomic loci which plays a role not only during development but also in disease, including cancer. By profiling nucleosome patterns broadly across hundreds of genes involved in pluripotency and lineage specification, including multiple oncogenic factors, this study will determine how chromatin structure correlates to pluripotency in embryonic stem cells and induced- pluripotent stem cells. Together, these studies will illuminate the functional consequences of chromatin structure on not only during development, but also on how chromatin structure may contribute to tumorigenesis.
|West, Jason A; Cook, April; Alver, Burak H et al. (2014) Nucleosomal occupancy changes locally over key regulatory regions during cell differentiation and reprogramming. Nat Commun 5:4719|
|West, Jason A; Davis, Christopher P; Sunwoo, Hongjae et al. (2014) The long noncoding RNAs NEAT1 and MALAT1 bind active chromatin sites. Mol Cell 55:791-802|
|Riedel, Christian G; Dowen, Robert H; Lourenco, Guinevere F et al. (2013) DAF-16 employs the chromatin remodeller SWI/SNF to promote stress resistance and longevity. Nat Cell Biol 15:491-501|