Project Summary/Abstract The long-term objectives of this proposal include the following: 1) to generate cell-type- specific chromatin profiles of the four cell types of the intestine, 2) to identify the effect crippled silencing in intestinal stem cells has on progenitor cells, and 3) to characterize the transcriptional changes associated with an age-related increase in the Polycomb Repressive Complex-2 mark, H3K27me2, in the intestine. Cell-type-specific chromatin profiles aid the specific gene expression patterns necessary for the function and identity of the cell. Complex tissues are comprised of both stem and differentiated cells with different phenotypes, transcriptional profiles, and roles within the tissue. The intestine contains a population of stem cells responsible for replenishing damaged cells from the multiple stimuli exerted on the intestine throughout the organism’s life span. However, the determining factors, especially at the chromatin level, that make each cell type unique are unknown. Investigation into the chromatin modifications of each cell type will provide insight into the unique roles each cell plays in the function of the tissue. To generate gut cell-specific chromatin profiles, I will use CUT&Tag, a robust and sensitive chromatin profiling technique that can be used with low cell numbers. I will generate landscapes for chromatin modifications for all cell types of the gut to comprehensively profile the epigenetic state of this tissue. Because of the importance of silencing modifications for cellular identity and previous observations of loss of chromatin silencing affecting the gut cells, I will investigate the effect of the loss of different chromatin silencing pathways in intestinal stem cells on progenitor cells. Since the intestinal stem cells are the only mitotically active cells in the intestine and the source of all other cells in the gut, their chromatin profiles likely affect the progenitor cells. Additionally, observations of age-related increases in H3K27me2 in the Drosophila melanogaster gut may indicate the protective role H3K27me2 has in preventing pervasive transcription as tissue ages. The insights gained from this project will help us understand the cell-type-specific chromatin profiles of cells of complex tissue and how chromatin modification changes in stem cells affect their progenitors. My proposed project fits the mission of NIGMS to fund research in fundamental biology that increases understanding of the principles that underlie biological processes.

Project Narrative Cells of complex tissues have specialized functions and identities to aid in the overall homeostasis of the tissue. However, the role chromatin modifications contribute to cell-type identity and function needs to be better understood. Using a sensitive chromatin profiling technique, I will characterize the genome-wide landscape of chromatin modifications and RNA Polymerase II in individual cell types based on a genetic marker or histone modification level to understand how chromatin varies from cell to cell and aid in cellular identity.