Mammalian somatic cells contain identical DNA content, yet gene expression varies widely from cell-type to cell-type. DNA within somatic cells is packaged into chromatin, a diverse and dynamic combination of DNA and proteins. The assembly of specialized chromatin provides a means for the establishment and maintenance of tissue-specific patterns of gene-expression. Therefore, specialized chromatin proteins represent candidate master regulators of tissue-specific patterns of gene expression. The specialized histone variant macroH2A1 has been implicated in developmentally-regulated gene silencing events, and is a member of the """"""""sternness"""""""" gene set. Much work on macroH2A1 has focused on its role in X chromosome inactivation, but macroH2A1 is widely expressed in both sexes, and is expressed at vastly different levels in specific adult organs. We will precisely map chromatin remodeling events involving macroH2A1 within the mouse genome. Preliminary data shows that the genomic distribution of macroH2A1 is altered in embryonic stem (ES) cells lacking Dnmt 1, the mouse maintenance cytosine methyltransferase gene. We will map the genomic distribution of macroH2A1 in wild-type and Dnmt 1-mutant ES cells to the level of DNA sequence. Use of these cells will allow us to develop and optimize much-needed technologies for mammalian chromatin research. We will then ascertain and map genomic redistributions of macroH2A1 (chromatin remodeling) in ES cells as they undergo directed differentiation. We will use female ES cells and male ES cells bearing Xist transgenes to provide internal controls involving known X chromosome inactivation events. This work will constitute the first comprehensive assessment of the assembly of a specialized chromatin component upon the mammalian genome in response to developmental cues. We will also complete needed developmental characterization of macroH2A1 expression during mouse embryogenesis, analyze cellular senescence, and compare the histological distribution of macroH2A1 in young versus aged organs. The differentiation of ES cells into therapeutic cells (stem cell therapy) is essentially an attempt to recapitulate development in vitro. Research proposed here will provide basic insights into differentiation biology and provide a framework for future attempts to achieve rationally-guided ES cell differentiation.
