In an effort to explore epigenetic factors controlling cell fate, our lab devised a cell fusion assay to dissect out contributions of cis and trans mechanisms in gene silencing. This assay entails fusing two cell types, with the goal of examining how gene expression in one cell is reset upon fusion with the other. In this way, we characterized a phenomenon called "occlusion," which is defined as stable transcriptional repression by cis-acting mechanisms in a manner that blocks the affected genes from responding to the trans-acting milieu of the cell. Based on the observation of occluded genes, we proposed that cell fate restriction in mammals is achieved via increasing occlusion of lineage-inappropriate genes as stem cells differentiate. Previous data have shown that embryonic stem cells (ESCs) can reprogram somatic cells to a pluripotent state via fusion. In contrast, our data show that upon fusion of two differentiated cells occluded genes fail to reprogram. These observations indicate that ESCs possess the ability to erase the occluded state. As this "deocclusion" ability is not present in terminally differentiated cells, a transition must take place during cellular differentiation whereby "deocclusion" ability is lost. To study this transition, I propose the following Specific Aims: 1. To distinguish the kinetics of deocclusion from transactivation during ESC reprogramming, 2. To identify the developmental stage at which the ability to deocclude genes is lost, and 3. To explore mechanisms of deocclusion through mRNA and chromatin analysis. In Aim 1 I will use ESC-fibroblasts fusions to investigate kinetics of gene transactivation and deocclusion during reprogramming. These studies will demonstrate that ESCs are capable of deocclusion and clarify the timing of ESC-mediated reprogramming. In Aim 2 I will fuse multiple murine stem cell types, representing a range of developmental stages, with differentiated fibroblasts to determine the stage at which deocclusion ability is lost. Deocclusion ability of stem cells will be monitored by activation of a GFP transgene that is normally occluded in fibroblasts, and by RT-PCR for activation of occluded fibroblast genes. In Aim 3, I plan to examine genome-wide changes in gene expression and chromatin marks that accompany the loss of deocclusion ability. The goal is to identify genes that are turned off and/or undergo dramatic chromatin modifications when differentiating stem cells lose their deocclusion ability. The occlusion of genes during lineage differentiation likely plays a central role in mammalian development. Similarly, aberrant gain or loss of occlusion may lead to disease states such as cancer. Thus, the study of gene occlusion may have broad relevance to many fields of biomedicine.