Embryonic stem cells hold great promise for the treatment of numerous degenerative diseases. Understanding the mechanisms involved in altering the embryonic stem cell transcriptional program during differentiation will be crucial if these cells are to be used for therapeutic purposes. Key developmental regulator genes are repressed by polycomb proteins in embryonic stem cells. As an embryonic stem cell commits to specific cell lineages these genes are selectively expressed, however the underlying mechanisms of activation remain unclear. Evidence from Drosophila indicates that polycomb-repressed genes are activated by trithorax proteins and this is coincidental with the expression of upstream noncoding RNAs. I propose to use microarray technology to determine if noncoding transcripts alter the mouse embryonic stem cell transcriptional program by recruiting trithorax proteins to promote gene activation. I have designed a microarray that covers a significant portion upstream of polycomb-bound developmental regulator genes in mouse embryonic stem cells to detect noncoding transcripts and trithorax protein binding. Using the microarray, I will first test whether upstream noncoding transcription at polycomb-bound developmental regulator genes results in activation of gene expression in differentiated cell types (Aim 1). Then I will determine by chromatin immunoprecipitation and microarray analysis if upstream noncoding transcripts activate expression by recruiting trithorax proteins (Aim 2). Taken together, these results will determine the contribution of noncoding transcription and trithorax proteins to the regulation of gene expression and differentiation of mouse embryonic stem cells. ? ? Relevance to Public Health: Embryonic stem cells have the potential to be used for treating various diseases since they retain the ability to form virtually any cell in the body, and could be used to replace damaged or nonfunctioning cells. The mechanisms that are involved in causing an embryonic stem cell to become a differentiated cell (neurons, muscle cells, skin cells, etc) need to be elucidated if this is to become a reality. The goal of this research is to identify and gain insight into these mechanisms. ? ? ?
