MECHANISMS OF CHROMATIN STRUCTURE AND TRANSCRIPTION REGULATION BY THE NUCLEAR PORE COMPLEX

ABSTRACT Nuclear structure-chromatin interactions underlie spatial genome organization and gene regulation during development and disease. However, the mechanisms by which distinct nuclear structures control these processes remain poorly defined. Understanding these mechanisms is critical, especially given that the changes in the nuclear structure can propagate to alterations in cell signaling, cell division and genome stability. The nuclear structure is in part composed of the nuclear pore complex (NPC) and lamina. Proteins that are the building blocks of the NPCs, called Nucleoporins (Nups), have been implicated in transcription and chromatin regulation by directly binding to chromatin. The NPC provides a nuclear compartment that accommodates subnuclear organization of genes, transcription factors and chromatin regulatory proteins. However, we still have very limited understanding on the molecular determinants and mechanisms of Nup- mediated transcription and chromatin structure in mammalian cells, and how these processes are governed during early development. Work to date suggests functional roles for Nups in cell type-specific gene regulation. Findings of this proposal will fill remaining gaps in knowledge regarding the exact mechanisms of how the NPCs influence binding of chromatin regulatory proteins at different genes, and how this mechanism influences transcription, peripheral chromatin organization and spatial positioning of genes. We recently provided new evidence that a particular Nup, NUP153, influences transcription and chromatin structure of developmentally regulated genes by mediating POL II pausing and binding of chromatin architectural proteins, CTCF and cohesin, at cis-regulatory elements. Towards dissecting the molecular basis of NUP153- mediated CTCF binding, we identified the catalytic subunit of the SWI/SNF chromatin remodeling complex, BRG1, as an NUP153 interacting protein. We hypothesize that the NPC-chromatin interactions through NUP153 mediate transcription, chromatin structure and peripheral chromatin organization by controlling BRG1 and CTCF binding. We propose that this mechanism in coordination with POL II pausing is necessary for cell type-specific gene regulation during early development. We will test this hypothesis by utilizing human HCT116 cells and mouse embryonic stem (ES) cells and performing genome-wide and genic assays. In Aim 1, we will determine the functional significance of NUP153-BRG1 interactions in NPC-mediated chromatin structure and transcription. In Aim 2, we will define regulatory function of NPC in spatial chromatin organization across the lamina. In Aim 3, we will elucidate the functional relevance of NPC-chromatin interactions in early development. Collectively, findings of this study will provide critical insights into the functional role for the NPC in integrating transcriptional regulation with chromatin remodeling, and spatial organization of chromatin, and how POL II pausing and activity participate in these processes during cell type-specific gene regulation in early development.