TRANSCRIPTION COFACTORS IN DIFFERENTIATION

Harvard University (Medical School), Boston MA

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Abstract

Transcriptional regulation is a key step in the cascade of events that control development and differentiation. The ability of transcription factors and cofactors to activate transcription in a repressive chromatin environment, in a correct temporal and spatial manner, is crucial for the proper execution of individual differentiation programs. The intent of this investigation is to study the molecular mechanisms by which CBP-1 regulates differentiation in a living organism, C. elegans. CBP-1 is a homolog of the mammalian p300 and CBP which are transcriptional cofactors that possess inherent and associated histone acetyltransferase (HAT) activity. Inactivation of p300 and CBP in mammalian cell is essential for viral oncogenic transformation and inhibition of differentiation, highlighting the importance of these proteins in cell growth and differentiation. We have recently shown that CBP-1 plays an indispensable role in C. elegans, along with the cell fate determining transcription factors such as SKN-1, in specifying multiple differentiation pathways. In this study, we will use both genetic and molecular approaches to delineate the mechanisms that underlie the differentiation-promoting activity of CBP-1 in C. elegans. Compared with the mammalian cell culture system, the unique advantage of C. elegans lies in the fact that we can study CBP-1 in a developmental context. In addition to the powerful "forward genetic" tools, unparalleled novel technologies such as RNAi and PCR-based mutant screening allow rapid reverse genetic analysis of gene function in vivo. We will undertake this goal by analyzing a cbp-1 deletion mutant that we have isolated recently, and by identifying domains of CBP-1 important for promoting differentiation and their interacting proteins in vivo. The functional relevance of the interacting proteins can be rapidly analyzed by RNAi assays. The systematic structure/function analysis will also determine the importance of the HAT activity of CBP-1 in differentiation. We will isolate conditional cbp-1 mutants and perform subsequent suppressor screening to identify novel genes involved in CBP-1 functions. Lastly, we will study regulation of potential physiologically relevant target genes by SKN-1, CBP-1 and histone deacetylases in differentiation. We will use biochemical and molecular assays to specifically test the model that CBP-1 functions as a coactivator for SKN-1 to activate zygotic programs important for C. elegans endoderm differentiation. Taken together, the proposed studies will provide significant novel insight into the biology and mechanism of action of cbp-1, in a living organism, C. elegans, and will shed light on how the homologous proteins p300 and CBP function in mammalian cells.