The Polycomb complex is a major chromatin repressor that controls stem cell identity, tissue development, and tumorigenesis. Its activity is divided between two multi-protein complexes called Polycomb repressive complex (PRC) 1 and 2. The classical model of Polycomb-mediated repression postulates a strict hierarchical recruitment of the PRC complexes, where PRC2 recruits PRC1 to establish chromatin compaction and gene repression. Our recent biochemical and genetic data, however, questioned this model. First, in contrast to the hierarchical model, we found that the recruitment of PRC1 occurs independently of PRC2. Second, we have generated mice that lack the PRC1/2 complexes in epidermal progenitors by ablating the essential subunits of each complex - EED (PRC2) and Ring1b (PRC1). While mice lacking solely EED or Ring1b had no obvious epidermal alterations, double knockout EED/Ring1b animals had striking changes in epidermal formation characterized by the altered architecture of the basal layer, a loss of basal cell attachment to the basement membrane, and defective basal/suprabasal cell-cell adhesion. This is again in contrast to the model, which states that concomitant loss of PRC1 and PRC2 should be identical to the loss of PRC1 or PRC2 alone. These findings provide us with the unique opportunity to demonstrate that the classical model of Polycomb repression is too simplistic, at least for the epidermis. The main goal of this grant is to understand how the PRC1 and PRC2 complexes regulate epidermal development. As the Polycomb complexes function to control gene expression, we hypothesize that PRC1 and PRC2 regulate genes that are critical for epidermal development. To test this hypothesis, first, we will analyze the EED/Ring1b-null epidermis to pinpoint when the first phenotypic abnormalities are observed. Second, by performing transcriptional and biochemical studies, we will identify PRC1/PRC2 target genes that are activated in the EED/Ring1b-null epidermal cells. These analyses will be followed by functional studies to determine the genes responsible for the EED/Ring1b-null epidermal phenotypes. Third, recent studies in ES cells have identified a non-canonical PRC1 complex called RYBP- PRC1 that is recruited to chromatin without the pre-existent binding of the PRC2 complex. Our preliminary data have led us to hypothesize that the RYBP-PRC1 complex functions with PRC2 to regulate genes that are critical for epidermal development. Here, we will perform biochemical, transcriptional, and loss-of-function studies to determine the role of the RYBP-PRC1 complex and how it cooperates with PRC2 to control epidermal development. In summary, the outlined studies will provide novel insights into the mechanisms of Polycomb-mediated gene control and uncover regulators controlling the development of the epidermis, a tissue that is essential for our survival.