Vertebrate TBP-like protein (TLP/TRF2/TLF) stimulates TATA-less terminal deoxynucleotidyl transferase promoters in a transient reporter assay, and TFIIA-binding capacity of TLP is required for this function.
The TBP-like protein (TLP/TRF2/TLF), which belongs to the TBP family of proteins, is present in all metazoan organisms. Although the human TLP has been reported to interfere with transcription from TATA-containing promoters, the transcription activation potential of TLP in higher animals is obscure. We previously demonstrated that artificially promoter-recruited TLP behaves like an unconventional transcriptional activator. In this study, we investigated the effects of TLP on TATA-less promoters of mouse and human terminal deoxynucleotidyl transferase (TdT) genes by transient reporter assays. As expected, TLP repressed both basal and activator-augmented transcription from the TATA-containing adenovirus major late promoter (MLP) and E1B promoter. On the other hand, however, TLP significantly stimulated both basal and activated transcription from TdT promoters. We investigated the strength of the promoters in chicken DT40 cells that lack the TLP gene. The MLP showed higher activity but the TdT promoter showed lower activity in TLP-null cells than in the wild-type cells. Moreover, ectopic expression of mouse TLP in the TLP-null cells considerably stimulated the TdT promoter. Insertion of a TATA element upstream from the TdT core promoter resulted in a loss of TLP-mediated activation. The mouse TLP was demonstrated to bind specifically to TFIIA with greater strength than TBP. We constructed mutated TLPs having amino acid substitutions that impair TFIIA binding. A representative TLP mutant lacking TFIIA-binding ability could not stimulate transcription from the TdT promoter, whereas that mutation suppressed TLP-mediated transcription repression of TATA promoters. The results of the present study suggest that the vertebrate TLP potentiates exogenous TATA-less promoters and that TFIIA plays an important role in the TLP function.
Ohbayashi
T
T
Department of Biology, Faculty of Science, Chiba University, 1-33 Yayoicho, Inage-ku, Chiba 263-8522, Japan.
Shimada
M
M
Nakadai
T
T
Wada
T
T
Handa
H
H
Tamura
T
T
D000818:Animals; D001667:Binding, Competitive; D002460:Cell Line; D002645:Chickens; D004253:DNA Nucleotidylexotransferase / Q000235:genetics*; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016385:TATA Box / Q000235:genetics; D035182:TATA Box Binding Protein-Like Proteins / Q000235:genetics / Q000378:metabolism*; D035561:Transcription Factor TFIIA / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000235:genetics
D011993:Recombinant Fusion Proteins; D035182:TATA Box Binding Protein-Like Proteins; C441409:TBPL1 protein, human; D035561:Transcription Factor TFIIA; D008156:Luciferases; D004253:DNA Nucleotidylexotransferase