TATA-Binding protein-interacting protein 120, TIP120, stimulates three classes of eukaryotic transcription via a unique mechanism.

We previously identified a novel TATA-binding protein (TBP)-interacting protein (TIP120) from the rat liver. Here, in an RNA polymerase II (RNAP II)-reconstituted transcription system, we demonstrate that recombinant TIP120 activates the basal level of transcription from various kinds of promoters regardless of the template DNA topology and the presence of TFIIE/TFIIH and TBP-associated factors. Deletion analysis demonstrated that a 412-residue N-terminal domain, which includes an acidic region and the TBP-binding domain, is required for TIP120 function. Kinetic studies suggest that TIP120 functions during preinitiation complex (PIC) formation at the step of RNAP II/TFIIF recruitment to the promoter but not after the completion of PIC formation. Electrophoretic mobility shift assays showed that TIP120 enhanced PIC formation, and TIP120 also stimulated the nonspecific transcription and DNA-binding activity of RNAP II. These lines of evidence suggest that TIP120 is able to activate basal transcription by overcoming a kinetic impediment to RNAP II/TFIIF integration into the TBP (TFIID)-TFIIB-DNA-complex. Interestingly, TIP120 also stimulates RNAP I- and III-driven transcription and binds to RPB5, one of the common subunits of the eukaryotic RNA polymerases, in vitro. Furthermore, in mouse cells, ectopically expressed TIP120 enhances transcription from all three classes (I, II, and III) of promoters. We propose that TIP120 globally regulates transcription through interaction with basal transcription mechanisms common to all three transcription systems.

A serine residue in the N-terminal acidic region of rat RPB6, one of the common subunits of RNA polymerases, is exclusively phosphorylated by casein kinase II in vitro.

RPB6 is one of the common subunits of all eukaryotic RNA polymerases and is indispensable for the enzyme function. Here, we isolated a rat cDNA encoding RPB6. It contained 127 amino acid (a.a.) residues. From alignment of RPB6 homologues of various eukaryotes, we defined two conserved regions, i.e. an N-terminal acidic region and a C-terminal core. In this study, we investigated in vitro phosphorylation of rat RPB6 by casein kinase II (CKII), a pleiotropic regulator of numerous cellular proteins. Three putative CKII-phosphorylated a.a. within rat RPB6 were assigned. We found that serines were phosphorylated by CKII in vitro. Mutagenesis studies provided evidence that a serine at a.a. position 2 was exclusively phosphorylated. Finally, an RPB6-engaged in-gel kinase assay clarified that CKII was a prominent protein kinase in rat liver nuclear extract that phosphorylates RPB6. Therefore, RPB6 was implied to be phosphorylated by CKII in the nucleus. We postulate that the N-terminal acidic region of the RPB6 subunit has some phosphorylation-coupled regulatory functions.

TIP120B: a novel TIP120-family protein that is expressed specifically in muscle tissues.

TATA-binding protein (TBP) forms complexes with various nuclear proteins and plays roles in all eukaryotic transcription. We previously identified TBP-interacting protein 120 (TIP120) from rat liver. TIP120 stimulates in vitro transcription generally. Homologs of TIP120 exist in various higher eukaryotes including D. melanogaster, C. elegans, and A. thaliana. Here, we isolated cDNA of a novel rat TIP120-like protein, named TIP120B. Rat TIP120B was composed of 1,235 amino acids and was 60% identical to the original TIP120 (re-named TIP120A). However, TIP120B gene was expressed specifically in the muscle tissues, which was contrary to the ubiquitous expression of TIP120A. Moreover, TIP120B protein was observed exclusively in the muscle tissues. TIP120B is therefore suggested to be a muscle-specific protein. Northern blot analysis of the mouse embryo revealed that the expression of TIP120B was temporarily increased during the embryogenesis, whereas TIP120A maintained a constant expression level. Pull-down assay using GST-fused TBP demonstrated that TBP specifically associated with TIP120B in the nuclear extract. These results indicate that TIP120B is a muscle-specific TIP120 family protein and can also interact with TBP. TIP120B is supposed to have a specific role in muscle tissues, which may be diffrerent from that of TIP120A.

Molecular cloning of a novel 120-kDa TBP-interacting protein.

TATA-binding protein (TBP) is a central component for transcriptional regulation and is a target for various transcription regulators. Using histidine-tagged TBP as a ligand for affinity-purification of proteins bound to TBP, we purified a 120-kD protein, termed TBP-interacting protein 120 (TIP120), from rat liver nuclear extracts. The entire cDNA sequence of TIP120 contained an open reading frame encoding a novel polypeptide of 1230 amino acids. The recombinant TIP120 interacted directly with TBP under a physiological condition in vitro. Immunoprecipitation analysis indicated that TIP120 was associated with TBP in nuclear extracts. Interestingly, the N-terminal region of TIP120 exhibited sequence similarity to that of Drosophila TAF80, which was shown to bind directly to TBP. This novel TBP-binding protein is considered to participate in transcription regulation through the interaction with TBP.

Multiple mammalian proteasomal ATPases, but not proteasome itself, are associated with TATA-binding protein and a novel transcriptional activator, TIP120.

BACKGROUND: SUG1 belongs to proteasomal ATPase. Previous studies have demonstrated that SUG1 is associated with TBP. It is assumed to be involved in transcriptional regulation in addition to proteolysis. In this study, we investigated the association of mammalian SUG1 with TBP in more detail. RESULTS: Pull-down experiments with TBP revealed multiple TBP-interacting proteins (TIPs) that were recovered dependent upon the presence of C-terminal conserved domain of TBP. By 2-D electrophoresis, we identified SUG1 in TIPs. By using far-Western analysis, we identified two proteins that could directly bind to TBP: SUG1 and another proteasomal ATPase (S4). Protein microsequencing and Western blotting identified all the remaining proteasomal ATPases (MSS1, TBP1, TBP7, and SUG2) in the TIP preparations. We present evidence that TBP and at least SUG1, MSS1, and S4 form a complex in the cell. However, no evidence of association of TBP with the 26S proteasome or its 19S regulatory unit was obtained. The molecular mass of the TBP/ATPases-complex, which also included a novel transcription regulatory factor, TIP120, was estimated to be approximately 800 kDa. CONCLUSION: These results suggest that there is a novel multisubunit complex containing TBP and proteasomal ATPases. Based on our findings, we hypothesize that proteasomal ATPases are involved in transcriptional regulation in addition to proteolysis.

Induced expression, localization, and chromosome mapping of a gene for the TBP-interacting protein 120A.

TBP-interacting protein 120A (TIP120A) is a novel eukaryotic transcriptional regulator and has been suggested to be involved in the general regulation of transcription because of its ability to potentiate transcription of all classes of genes and to interact with common transcriptional machineries. In the present study, we investigated the expression of the tip120a gene. TIP120A transcripts were expressed abundantly in the heart and liver, moderately in the brain and skeletal muscle, and only slightly in the spleen and lung. This ubiquitous expression pattern was similar to that of TBP. Gene expression of TIP120A in the rat liver was not stimulated by hepatocarcinogenesis or liver regeneration. TIP120A was thus suggested not to be a growth-related protein. On the other hand, in P19 mouse embryonal carcinoma cells, TIP120A expression was elevated upon retinoic acid treatment, which induces differentiation. Notably, the foci-like nuclear localization pattern of TIP120A was transformed into a speckle-like pattern. The level of TIP120A was also elevated in such stem-like cells as F9 and HL60 after each differentiation procedure, retinoic acid and DMSO, respectively. In HEp-2 cells, TIP120A was observed as a limited number of nuclear foci, and the localization coincided with that of the PML oncogenic domain. FISH detection revealed that the human tip120a gene was located at 12q14, the position to which a myopathic type scapuloperoneal syndrome locus also mapped. Our study suggests that, contrary to an early assumption, TIP120A is involved in tissue-specific and/or differentiation-related gene expression.

Structures of the rat proteasomal ATPases: determination of highly conserved structural motifs and rules for their spacing.

We have isolated rat cDNAs for all of the five known proteasomal ATPases. The protein sequences of rat TBP1, TBP7, MSS1, S4, and SUG1 predicted from the open reading frames consist of 439, 418, 433, 440, and 406 amino acid residues, respectively, and exhibit striking similarities to each human counterpart with only several amino acid substitutions. These five rat ATPases are also highly homologous with each other. The N-terminal region in rat TBP1, TBP7, and SUG1 contains a heptad repeat of hydrophobic amino acids reminiscent of a leucine zipper. Also, in the central region of each rat ATPase, we found four conserved motifs, Gx4GKT, DEID, SAT, and H/QRxGRx2R, that are characteristic of a large family of ATP-dependent RNA/DNA helicases. The spacing between individual motifs was strictly conserved in the rat ATPases. These findings suggest a common function of the rat proteasomal ATPases in ATP-dependent RNA/DNA unwinding.