N-terminal region, C-terminal region, nuclear export signal, and deacetylation activity of histone deacetylase-3 are essential for the viability of the DT40 chicken B cell line.
Histone deacetylases (HDACs) are involved in the deacetylation of core histones, which is related to transcription regulation in eukaryotes through alterations in the chromatin structure. We cloned cDNA and genomic DNA encoding a chicken HDAC, chHDAC-3, which was localized in both the nuclei and cytoplasm in DT40 cells. Although one of the two chHDAC-3 alleles could be disrupted with high efficiency, no homozygous mutants were obtained after a second round of the gene-targeting technique due to its necessity for DT40 cells. We introduced a chHDAC-3 transgene under the control of a tetracycline-responsive promoter into the heterozygous mutant and subsequently disrupted the remaining endogenous chHDAC-3 allele to generate the homozygous chHDAC-3-deficient mutant, DeltachHDAC-3/FHDAC3. Inhibition of the expression of the regulatable chHDAC-3 transgene caused apoptotic cell death of the mutant. Complementation experiments involving truncated and missense chHDAC-3 mutant proteins revealed that the 1-23 N-terminal sequence, the 389-417 C-terminal sequence, the nuclear export signal, and the deacetylation activity of chHDAC-3 were essential for the cell viability. Taken together, these results indicate that chHDAC-3 plays an essential role, probably as a scavenger in the cytoplasm, in the proliferation of DT40 cells.
Takami
Y
Y
Department of Biochemistry, Miyazaki Medical College, Kihara, Kiyotake, Miyazaki 889-1692, Japan.
Nakayama
T
T
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000235:genetics; D001402:B-Lymphocytes / Q000201:enzymology* / Q000378:metabolism; D002455:Cell Division / Q000235:genetics; D002460:Cell Line; D002467:Cell Nucleus / Q000201:enzymology; D002470:Cell Survival; D002645:Chickens; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000201:enzymology; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation; D005816:Genetic Complementation Test; D006655:Histone Deacetylases / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D016415:Sequence Alignment; D014162:Transfection; D019076:Transgenes
D006655:Histone Deacetylases