(from the application): The histone acetyl transferase proteins p300/CBP and ATP-dependent chromatin remodeling complexes of the SWI/SNF family are implicated in the regulation of cellular proliferation and differentiation. BRG1, a protein member of the SWI/SNF complex, associates with the retinoblastoma protein (RB) and histone deacetylases (HDACI), to form complexes with repressor activity. However, BRG1 activity is counteracted by cyclin E, a cell cycle regulatory protein. Cyclin E/CDK2 can also inactivate RB function whereas association with p300/CBP results in p300/CBP phosphorylation and increased activity. Therefore cyclin E has the ability to regulate both, p300/CBP and SWI/SNF complexes. Our preliminary data suggests that cyclin E, p300/CBP and RB are involved in melanocyte senescence and transformation since: a) senescent melanocytes do not express the proteins p300, CBP and cyclin E; b) expression of the catalytic subunit of telomerase (hTERT) results in an extended melanocyte lifespan characterized by high levels of cyclin E and p300/CBP; c) cyclin E and p300 are highly expressed in invasive primary melanoma tumors, d) RB is rapidly dephosphorylated in melanocytes undergoing senescence. The experiments proposed here aim to elucidate the role of members of the chromatin remodeling complexes of the SWI/SNF and p300 family in melanocyte senescence and transformation. Specifically, we will: 1)Determine whether loss of cyclin E results in increased RB/BRG1/HDAC association and activity and replicative senescence. Determine whether forced cyclin E expression or expression of a dominant negative BRG1 mutant in pre-senescent melanocytes extends the lifespan of these cells. 2) Determine whether inactivation of the histone acetyl-transferase domain of p300/CBP results in senescence and whether that is followed by loss of major cell cycle regulatory genes such as cyclin E, E2F and p21. We will test these aims by over expressing wild-type and mutant proteins with dominant negative activity, using retroviral and adenoviral vectors in primary cultures of human normal melanocytes. Induction of senescence will be monitored by testing the expression of senescent markers (SA-B-gal, osteonectin), loss of BrdU incorporation, and appearance of a flat phenotype. The studies proposed have the potential to unravel master pathways that could limit proliferation in human cells and selectively induce patterns of gene expression characteristic of senescent melanocytes.
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