Computational and experimental insights into the structure and dynamics of heterochromatin

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Abstract Chemical, molecular and structural transformations of chromatin are intimately involved in critical cellular phenomena, including differentiation, signaling, and pathogenesis. A detailed knowledge of how molecular complexes involving multiple kilobases of DNA and hundreds of proteins respond to the finest changes in chemical structure is key to elucidating the role of chromatin transformations in life and disease. The overarching goal of this project is to develop and apply computational tools to investigate how the structure and dynamics of chromatin determine its functional states. Our central hypothesis is that physical properties and behavior of the chromatin fiber and associated proteins lend themselves to encoding into efficient and useful ultra-coarse- grained (UCG) representations. Our strategy to reach the goal is by bridging together several computational and experimental methodologies. We initiated the development of Molecular Biosystems (MB), a computational platform for UCG simulations specifically adapted to the chromatin biology. MB methodology represents a blend of physics-based mechanisms, such as dynamics of the chromatin fiber, with stochastic processes encompassing protein-protein interactions and enzymatic reactions. MB studies will be complemented by all-atom MD and CG simulations and experimentally tested using a unique chromatin in vivo assay (CiA) methodology. Specifically, we will investigate the chromatin-mediated repression of Oct4, a key gene regulating embryonic stem (ES) cell pluripotency at defined points in mammalian development. This is important because the ability to reverse the Oct4 repression would streamline production of induced pluripotent cells (iPSC) and advance regenerative medicine. The CiA technology at the Oct4 locus in mouse ES cells will be used for the exploration of changes to chromatin structure, as well as for testing the adequacy of MB simulations. Experimental endpoints that are directly comparable to computational hypotheses will be produced: (1) fraction of Oct4-repressed cells in cell culture; (2) H3K9 methylation patterns on Oct4 promoter; and (3) chromatin conformation capture. Three main components of our research are: (i) Extending and enhancing the UCG MB approach; (ii) Multi- scale simulations of chromatin processes to elucidate the structure and dynamics of heterochromatin of Oct4 regulatory elements; (iii) Experimental real-time monitoring of heterochromatin molecular signatures using Chromatin in vivo Assay (CiA) to study mechanisms and time course of Oct4 de-repression and provide feedback for the computational models. This work is important because of its focus on the physics of the gene repression, whose understanding will bring us one step forward toward the promise of regenerative medicine and new prospects for cancer therapy.