MULTIPLE MECHANISMS OF SKN-1 FUNCTION IN VIVO

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DESCRIPTION (provided by applicant): The C. elegans transcription factor SKN-1 specifies early embryonic cells which form the digestive system, but also has postembryonic functions that have not been elucidated. SKN-1 binds DNA by an unusual mechanism, but is related to the two vertebrate NRF bZIP proteins, especially within a unique transactivation element called DIDLID. Vertebrate NRF proteins are required for resistance to oxidative stresses, and directly activate antioxidant enzyme genes. SKN-1 is similarly required for oxidative stress resistance, and directly activates intestinal expression of a critical antioxidant enzyme. This suggests that SKN-1 and NRF proteins have maintained parallel functions, and mechanisms of action, and that insights obtained into SKN-1 functions in C. elegans will be applicable to vertebrate antioxidant defenses. Our preliminary findings suggest three related hypotheses that we will now test in vivo: that SKN-1 has multiple functions which parallel those of NRF proteins, that SKN-1 acts at different target genes through mechanisms that are dependent upon or independent of cbp-i, the C. elegans p300/CBP histone acetyl transferase (HAT), and that the conserved DIDLID element is critical for its cbp-1-independent functions. To test these models, we will investigate the involvement of SKN-1 in antioxidant defenses, and in direct detoxification gene activation. We wifi also use site-directed mutagenesis to test in vivo whether SKN-1 elements that include DIDLID and its CBP-1--binding region are required to fulfil its antioxidant and embryonic functions, and to activate the respective target genes involved. Finally, we will investigate the mechanisms through which SKN-1 functions by determining how it binds to CBP-1 and whether its functions involve other HATs, and by identifying physiological protein targets of DIDLID and other CBP-1-independent SKN-1 elements. This work will elucidate conserved transcriptional regulatory interactions that have been conserved in transcriptional responses to oxidative stress, an ancestral digestive system function, and that in C. elegans are fundamental to initial specification of the digestive system. It will also apply the unique advantages of the C. elegans system to elucidate in vivo how a metazoan transcription factor regulates cell-type-specific genes through distinct mechanisms in different regulatory contexts.