| The molecular mechanisms of transcription initiation are pivotal for the control of gene regulation. TATA binding protein (TBP) is a key initiation factor which is known to nucleate the preinitiation complex containing RNA polymerase II and other basal factors. In vertebrates two TBP-related factors exist that are not found in yeast: TBP-like factor (TLF / TRF2) and TBP2 (also known as TRF3). In a variety of model systems, the three factors are each required for distinct subsets of transcripts. How TBP and TBP2, which are co-expressed in early development, mediate initiation of transcription in a gene-selective fashion is unknown. Our data indicate that promoters can selectively require either TBP or TBP2 for high levels of transcription initiation even if both TBP and TBP2 are recruited. The objective for this application is to understand the molecular basis of non-universal, gene-selective initiation of transcription by TBP and TBP2 in Xenopus embryos. To obtain such an understanding, the relation between TBP and TBP2 recruitment, other factors at the promoter, and productive transcription initiation need to be uncovered. Here it is proposed (1) to determine the genome-wide recruitment of TBP and TBP2 in Xenopus embryos using chromatin immunoprecipitation and deep sequencing (ChIP-seq); (2) to determine the relationships between TBP and TBP2-dependent transcription initiation, core promoter elements, promoter-enriched histone modifications and other factors recruited to the promoter. The mechanisms involved will be analyzed using ChIP, antisense knockdown and analysis of promoter elements. The experiments will provide global maps of TBP and TBP2 recruitment and establish the molecular mechanisms that link this recruitment to the core promoter, epigenetic marks, other proteins recruited to the promoter, and transcription initiation. This is important because the basal transcription machinery, which is far less universal then previously thought, provides a paradigm for how interactions of general factors contribute to gene-selective transcriptional regulation. |
