Our goal is to understand the processes that control how human embryonic stem cells differentiate to become various distinct cell types. The control of several important genes that determine this process relies on a process known as DNA methylation. DNA methylation involves the reversible "decoration" of our DNA at specific gene locations, resulting in the silencing of that gene. This is a key step in the commitment of these cells to particular differentiated endpoints. Understanding what controls the DNA methylation and silencing of key genes in human embryonic stem cell differentiation is the goal of this proposal. RNA can regulate how proteins function in the cell, and based on many examples involving DNA methylation, we will test if RNA controls when and where DNA methylation occurs during the differentiation of human embryonic stem cells. We propose to use methods relying on the use of human embryonic stem cells and purified enzymes that carry out the DNA methylation itself. The identity of RNA molecules that bind to these enzymes will be determined by two distinct methods, and the biological importance of these RNA molecules to embryonic stem cell differentiation will be determined. The mechanism of how these RNA molecules alter the function of the the DNA methyltransferase enzymes will be determined. The realization of our goals is anticipated to provide the basis for controlling the function of the critical DNA methyltransferases, which has already been demonstrated to be important in influencing the cellular differentiation process.
The ability to use human embryonic stem cells depends on being able to control their differentiation. Thus, while these cells have the potential to become a different cells within different human tissues, our current ability to direct such changes is limited. This proposal will benefit the State of California and its citizens by providing improved ways of achieving this goal, of being able to direct these cells to particular differentiated cell types.