Interaction between Transactivation Domain of p53 and Middle Part of TBP-Like Protein (TLP) Is Involved in TLP-Stimulated and p53-Activated Transcription from the p21 Upstream Promoter

Abstract

TBP-like protein (TLP) is involved in transcriptional activation of an upstream promoter of the human p21 gene. TLP binds to p53 and facilitates p53-activated transcription from the upstream promoter. In this study, we clarified that in vitro affinity between TLP and p53 is about one-third of that between TBP and p53. Extensive mutation analyses revealed that the TLP-stimulated function resides in transcription activating domain 1 (TAD1) in the N-terminus of p53. Among the mutants, #22.23, which has two amino acid substitutions in TAD1, exhibited a typical mutant phenotype. Moreover, #22.23 exhibited the strongest mutant phenotype for TLP-binding ability. It is thus thought that TLP-stimulated and p53-dependent transcriptional activation is involved in TAD1 binding of TLP. #22.23 had a decreased transcriptional activation function, especially for the upstream promoter of the endogenous p21 gene, compared with wild-type p53. This mutant did not facilitate p53-dependent growth repression and etoposide-mediated cell-death as wild-type p53 does. Moreover, mutation analysis revealed that middle part of TLP, which is requited for p53 binding, is involved in TLP-stimulated and p53-dependent promoter activation and cell growth repression. These results suggest that activation of the p21 upstream promoter is mediated by interaction between specific regions of TLP and p53.

TBP-like Protein (TLP) Disrupts the p53-MDM2 Interaction and Induces Long-lasting p53 Activation

“…This mutant is found in cervical cancer that expresses wild-type p53, and the mutation is located in the p53 binding region (Fig. 6B) (27). As expected, the p53 binding ability of D99H was considerably lower than that of wild-type TLP (Fig.…”

“…Expression Plasmids, siRNAs, and shRNAs-pCI-neo-FH-mTLP and pCI-neo-HA-mTLP were prepared as described previously (27). The siRNA-resistant TLP mutant that has nucleotide substitutions but retains the native amino acid sequence was constructed by PCR mutagenesis strategy.…”

“…4D). TLP has been shown to bind to the p53 TAD (27); hence, we hypothesized that TLP prevents p53 binding of MDM2.…”

“…Upon genotoxic stress, TLP represses cell growth via regulation of cell cycle-associated genes such as wee1, Cdkn1a (p21), and Trp63 (23,24). We have also found that TLP binds to the TAD of p53, as does TBP, and enhances p21 expression in a p53-dependent manner (25)(26)(27). However, little is known about the most fundamental question of how TLP regulates p53 target genes or p53 itself.…”

Nucleosome distortion as a possible mechanism of transcription activation domain function

“…There are significant distinctions in the interaction of ADs (with interaction partners) in the direct-recruitment model and in the nucleosome-distortion model. For the recruitment model, the requirement is an establishment of a functional link by attraction of a specific enzymatic activity(s) to the promoter; thus, the more functionally productive interactions have to have higher affinity to specific targets interacting in a specific structural region of the target as it was demonstrated for TBP, Gal11, Tra1, and others [ 15 , 87 , 104 , 105 ]. In contrast, for the nucleosome-distortion model, in order to avoid nucleosome stabilization at the promoter (as this outcome impedes transcription initiation), the interactions must be of low or very low affinity, occurring likely in multiple structural points of the nucleosome with these points competing with each other and preventing creation of a stable link and thus maximally destabilizing the nucleosome.…”