Different roles of N-terminal and C-terminal halves of HIRA in transcription regulation of cell cycle-related genes that contribute to control of vertebrate cell growth.

We reported previously that chicken HIRA, a homolog of Saccharomyces cerevisiae transcriptional co-repressors Hir1p and Hir2p, possesses seven WD dipeptide motifs and an LXXLL motif in its N-terminal and C-terminal halves, respectively, required for transcription regulations. Here, by using the gene targeting technique, we generated the homozygous HIRA-deficient DT40 mutant DeltaHIRA. The HIRA deficiency caused slightly delayed cell growth and affected the opposite transcriptions of cell cycle-related genes, i.e. repressions for P18, CDC25B, and BCL-2, activations for P19 and cyclin A, and histones H2A, H2B, H3, and H4. These altered expressions were completely revived by the artificial stable expression of hemagglutinin-tagged HIRA in DeltaHIRA. The ability to rescue the delayed growth rate was preferentially aided by the N-terminal half instead of the C-terminal half. We cloned the chicken P18 genomic DNA, and we established that its promoter was located surrounding the sequence GCGGGCGC at positions -1157 to -1150. Chromatin immunoprecipitation assay revealed that the N-terminal half interacted directly or indirectly with the putative promoter region of the p18 gene, resulting in up-regulation of the gene. These results indicated that the N-terminal half of HIRA should contribute positively to the growth rate via up-regulation of a set of cell cycle-related genes, whereas the C-terminal half down-regulated another set of them without exhibiting any effect on the cell growth.