Abstract
Histone deacetylases (HDACs) are involved in the deacetylation of core histones, which is an important event in transcription regulation in eukaryotes through alterations in the chromatin structure. We cloned cDNAs and genomic DNAs encoding two chicken HDACs (chHDAC-1 and -2), which are preferentially localized in nuclei. Treatment with trichostatin A reduced the HDAC activities in immunoprecipitates obtained with anti-chHDAC-1 and -2 antisera. Using gene targeting techniques, we generated homozygous DT40 mutants, DeltachHDAC-1 and -2, devoid of two alleles of the chHDAC-1 and -2 genes, respectively. The protein patterns on two-dimensional PAGE definitely changed for DeltachHDAC-2, and the amounts of the IgM H- and L-chains increased in it. Of the two IgM H-chain forms, the secreted form mu(s) increased in DeltachHDAC-2, but the membrane-bound form mu(m) decreased. The IgM H-chain gene was transcribed more in DeltachHDAC-2 than in DT40 cells. In the mutant, the alternative processing of IgM H-chain pre-mRNA preferentially occurred, resulting in an increase in the amount of mu(s) mRNA, whereas the stability of the two types of mRNA, mu(s) and mu(m), was unchanged. In DT40 cells, treatment with trichostatin A increased both the amounts of IgM H-chain mRNAs and the switch from mu(m) to mu(s) mRNAs. Based on these results, we propose a model for a role of chHDAC-2 in both the transcription and alternative processing steps, resulting in control of the amount of the mu(s) IgM H-chain in the DT40 cell line.
Highlights
Alterations in the chromatin structure have been thought to participate in the regulation of gene expression in eukaryotes
trichostatin A (TSA) Increases both Transcription of the IgM H-chain Gene and Alternative Processing of IgM H-chain Pre-mRNA in DT40 Cells—To confirm the participation of chHDAC-2 in the increases in both the transcription of the IgM H-chain gene and alternative processing from m to s mRNA, we studied the effect of TSA on these steps in DT40 cells
Recently have many studies revealed an attractive model for dynamic changes in the chromatin structure, based on both the acetylation and deacetylation of core histones, respectively, catalyzed by histone acetyltransferases and Histone deacetylases (HDACs) [1,2,3, 9, 10]
Summary
Cell Cultures—DT40 cells and all subclones were cultured essentially as described [24, 25]. Gene Constructs, Transfection, and Isolation of Transfectants—Two blunt-ended cassettes, carrying neo and hisD, respectively [41], transcribed by the chicken -actin promoter [42, 43], were obtained as described [26, 28] Using these cassettes, we generated targeting vectors for the disruption of chHDAC-1 and -2, as follows. Following the addition of 100 l of loading buffer, aliquots (4 l) of 1:50 dilutions of the resultant whole cell extracts, together with the conditioned media, were analyzed, essentially as mentioned above, except for the use of 7.5% SDS-PAGE and goat anti-chicken IgM -chain antibodies (Betyl Laboratories Inc.). 32P-Labeled antisense RNAs were synthesized with phage T7 RNA polymerase after cutting with BamHI and used as probe Cmu. regions of the IgM H-chain gene, was inserted into the EcoRV site of Bluescript II. IgM H-chain mRNAs were detected by hybridizing the 32P-labeled RNAs to DNA fragment VH, corresponding to the V, D, and J regions, spotted onto a nitrocellulose membrane
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