Metazoans possess two TATA-binding protein homologs: the general transcription factor TBP and a related factor called TLF. In C. elegans, TLF is required to express a subset of RNA Polymerase II (Pol II) genes, including
pes-10 in pregastrula embryos (1). Two models could explain the non-redundant role of TLF during embryogenesis. First, TLF could function as a component of the basal transcription machinery and replace TBP at certain promoters. This model predicts that individual promoters should depend on either TBP or TLF, but not both. Alternatively, TLF could have co-opted the TBP DNA binding domain for a new purpose, such as functioning as an enhancer-binding activator that recruits co-activators or basal machinery. Our current goal is to distinguish between these models by using a putative TLF target (the
pes-10promoter) to identify TLF binding sites and determine the role of TLF binding in vivo. RNAi against TLF or a TBP-specific TAF (TAF250) both abolish or severely reduce
pes-10::GFP expression at the earliest detectable stage, suggesting both proteins are required for
pes-10 transcription. These data support the second model, suggesting TLF has co-opted the TBP-like DNA binding domain for some other purpose. We made site-specific mutations in the
pes-10 promoter from 300bp upstream of the ATG through the trans-splice site and analyzed the effects on GFP expression at the earliest detectable stage, the 28-cell stage. We have identified specific sequences within the
pes-10 promoter that affect
pes-10::GFP expression at the 28-cell stage. We hypothesize that one of these sequences is a TLF binding site. We have previously shown TLF associates with the
pes-10 promoter in vivo using a nuclear spot assay (1). We are using wild-type and mutated versions of these sequences in the nuclear spot assay to determine if TLF or TBP bind these sites in vivo.