Early zygotic transcription activates differentiation-specific genes necessary for blastomere specification and gene products needed for core biological functions.
ama-1 (RNA polymerase II) RNAi embryos fail to initiate zygotic transcription, arresting at about 100 undifferentiated cells. We are evaluating the requirements for general transcription regulators in early transcriptional events. Biochemical and genetic analysis in yeast have identified many regulatory cofactors which function between the polymerase and gene specific activators. Some, such as the TAFIIs, have been predicted to coordinately regulate cell cycle genes or to interact with activators. However, their role in metazoan gene regulation is unclear. Our approach has been to (1) identify C. elegans homologues of regulatory cofactors and obtain corresponding clones (from Y. Kohara or the Sanger Centre), (2) evaluate the RNAi phenotype and (3) analyze the RNAi effect on reporter genes in vivo. RNAi of orthologs for human TAFII250, TAFII130, TAFII100, and TAFII31/32 produced an early embryonic arrest, similar to the
ama-1 RNAi. We have also analyzed CeTAFII250 and CeTAFII100 RNAi in the
end-1 ::gfp strain (obtained from J. Rothman).
end-1 is expressed in the E2-E8 cells and is regulated by the maternal factor SKN-1. Ectopically expressed
end-1 induces exogenous gut and adoption of the long Ea/Ep-like cell cycle (Zhu (98) G&D 12:3809). RNAi of
skn-1 and
ama-1 in
end-1 ::gfp worms blocks gfp expression. RNAi of CeTAFII250 or CeTAFII100 does not affect gfp expression, even while the embryos show the early
ama-1 like arrest. Thus, CeTAFII250 and CeTAFII100 are required for core biological functions, but not to express a differentiation specific gene. Lineage analysis of the CeTAFII250 RNAi embryos shows that the E daughters divide early, with the MS daughters. Our results show that RNAi of CeTAFII250 uncouples
end-1 expression from the
end-1 dependent cell cycle lengthening in the E daughters. We will continue to evaluate requirements for specific TAFIIs and other regulatory cofactors on early reporter genes in vivo , and use this as a model system to investigate how these biochemically defined factors are required for specific gene regulation in vivo .