Minimum essential region of CCG1/TAFII250 required for complementing the temperature-sensitive cell cycle mutants, tsBN462 and ts13 cells, of hamster BHK21 cells.

CCG1/TAFII250, the largest subunit of the TFIID complex, is mutated in ts cell cycle mutants of BHK21 cells, ts13 and tsBN462, which have a promoter-selective transcriptional defect. A series of deletion mutants of CCG1 cDNA were prepared and transfected into these mutants, in order to identify functional domains of CCG1 required for the complementation of ts 13/BN462 mutation. We determined the minimum size of CCG1:CCG1ME, essential for complementing the ts mutation, which possessed one proline cluster, an HMG1-like domain, and a nuclear localization signal, but which lacked the bromo domains and the acidic phosphorylation sites for casein kinase II common to transcriptional activators. It encodes a protein of 140 kDa. These characteristics of CCG1ME correspond to yeast TAFII145, the yeast homolog of human TAFII250. CCG1ME bound to TBP, creating its own TFIID complex different from that of the endogenous mutated CCG1 in ts+ transformants of tsBN462 cells.

The human CCG1 gene, essential for progression of the G1 phase, encodes a 210-kilodalton nuclear DNA-binding protein.

The human CCG1 gene complements tsBN462, a temperature-sensitive G1 mutant of the BHK21 cell line. The previously cloned cDNA turned out to be a truncated form of the actual CCG1 cDNA. The newly cloned CCG1 cDNA was 6.0 kb and encoded a protein with a molecular mass of 210 kDa. Using an antibody to a predicted peptide from the CCG1 protein, a protein with a molecular mass of over 200 kDa was identified in human, monkey, and hamster cell lines. In the newly defined C-terminal region, an acidic domain was found. It contained four consensus target sequences for casein kinase II and was phosphorylated by this enzyme in vitro. However, this C-terminal region was not required to complement tsBN462 mutation since the region encoding the C-terminal part was frequently missing in complemented clones derived by DNA-mediated gene transfer. CCG1 contains a sequence similar to the putative DNA-binding domain of HMG1 in addition to the previously detected amino acid sequences common in nuclear proteins, such as a proline cluster and a nuclear translocation signal. Consistent with these predictions, CCG1 was present in nuclei, possessed DNA-binding activity, and was eluted with similar concentrations of salt, 0.3 to 0.4 M NaCl either from isolated nuclei or from a DNA-cellulose column.