Interconversion between active and inactive TATA-binding protein transcription complexes in the mouse genome.

ABSTRACT: The conserved core domain of the TATA binding protein (TBP) interacts with multiple partners forming the complexes required for transcription by RNA Polymerases I, II and III. We use genetically modified mouse embryonic fibroblasts to show that many TBP core domain mutants complement loss of endogenous TBP, but this often results in a slow growth phenotype. Two TBP mutations, R188E and K243E, disrupt the TBP-BTAF1 interaction and B-TFIID complex formation. Transcriptome and ChIP-seq analyses show that loss of B-TFIID does not affect global genomic distribution of TBP, but positively or negatively affects TBP and/or RNA Polymerase II (Pol II) recruitment to a selected set of promoters. We identify a set of promoters where wild-type TBP assembles a partial inactive preinitiation complex lacking Pol II and TAF1. Our results suggest that an exchange of the B-TFIID complex in wild-type cells for TFIID in R188E and K243E mutant cells at these primed promoters recruits Pol II to activate their expression. We also observe that both Wt and mutant TBP can occupy promoters without concurrent Pol II recruitment and active transcription. Our data reveal a novel regulatory mechanism involving the formation of a partial preinitiation complex that primes the promoter for productive preinitiation complex formation in mammalian cells. Using homologous recombination, we generated ES cells and mice harbouring a null allele in the Tbp gene by insertion of a hygromycin resistance cassette in exon III and a floxed allele, in which exon III is surrounded by LoxP sites. From these mice, we generated an embryonic fibroblast Tbplox/- cell line in which TBP expression can be inactivated by expression of the Cre recombinase leading to cell death. We adopted a two-step strategy to generate Tbp-/- cell lines expressing human (h)TBP. Cells were first infected with pBABE retrovirus vectors expressing Wt hTBP or a series of mutants in the TBP core region, all of which carry a Flag-HA tag on their N-terminus. Cells expressing endogenous mTBP and exogenous hTBP were then infected with a second retrovirus expressing the 4 hydroxy-tamoxifen (OHT) inducible Cre-ERT2. Subsequently, multiple clonal populations from the OHT treated cells were isolated and genotyped by PCR to identify the Tbp-/-clones. At least two independent clones where expression of the endogenous mTBP was lost and replaced by the exogenous hTBP were isolated.

ORGANISM(S): Mus musculus

SUBMITTER: Celine Keime 

PROVIDER: E-GEOD-27908 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress