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.
Ahmad
Ahyar
A
Department of Life Science, Frontier Science Research Center, Section of Biochemistry and Molecular Biology, Department of Medical Sciences, Miyazaki Medical College, University of Miyazaki, Japan.
Kikuchi
Hidehiko
H
Takami
Yasunari
Y
Nakayama
Tatsuo
T
D020816:Amino Acid Motifs; D000818:Animals; D017209:Apoptosis; D001483:Base Sequence; D015139:Blotting, Southern; D015153:Blotting, Western; D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000378:metabolism; D002460:Cell Line; D049109:Cell Proliferation; D002645:Chickens; D047369:Chromatin Immunoprecipitation; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D015536:Down-Regulation; D005091:Exons; D006367:HeLa Cells; D056488:Histone Chaperones; D006657:Histones / Q000737:chemistry; D006720:Homozygote; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D008954:Models, Biological; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000737:chemistry / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection; D015854:Up-Regulation
D018797:Cell Cycle Proteins; D018076:DNA, Complementary; C086057:HIRA protein, human; D056488:Histone Chaperones; D006657:Histones; D009687:Nuclear Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D008156:Luciferases