Targeted Disruption of H2B-V Encoding a Particular H2B Histone Variant Causes Changes in Protein Patterns on Two-dimensional Polyacrylamide Gel Electrophoresis in the DT40 Chicken B Cell Line

Abstract

The chicken H2B gene family comprises eight members (H2B-I to H2B-VIII), which are all located in two major histone gene clusters. All of them have been shown to encode four different protein variants (classes I to IV). In the DT40 chicken B cell line, the H2B-V gene, encoding the class III H2B variant, constituted about 10% of the total intracellular mRNA from all the H2B genes. To study the nature of this particular variant in vivo, we generated heterozygous (H2B-V, ؉/؊) and homozygous (H2B-V, ؊/؊) DT40 mutants by targeted integration. The remaining H2B genes were shown to be expressed more in these mutants than in the wild-type cell lines. The growth rate of DT40 cells was unchanged in the absence of the H2B-V gene. Two-dimensional polyacrylamide gel electrophoresis showed that the protein patterns were, on the whole, similar between the wildtype and homozygous cell lines. However, within this constant background, some cellular proteins disappeared or decreased quantitatively in the homozygous mutants, and several other proteins increased or newly appeared. These results suggest that the class III H2B variant participates negatively or positively in regulation of the expression of particular genes that encode the proteins that vary in DT40 cells. This type of regulation is possibly mediated through alterations in nucleosome structure over the restricted regions involving the putative genes of the DT40 genome.Chickens have fewer copies of the histone genes, ranging from six copies of the H1 gene to about ten copies of the core genes (H2A, H2B, H3, and H4) (1-3) than most higher eukaryotes, which possess large numbers of the genes of each histone subtype, ranging from several dozen to hundreds (4 -7). Recently, furthermore, compensation for disruption of particular histone genes has been shown in yeast and chickens (8 -15). Thus, in eukaryotes, this type of regulation, as well as the presence of multiple copies of the histone genes, should ensure that all the core histone subtypes remain in stoichiometric balance so that the chromatin structure is maintained precisely during cell proliferation.On the other hand, there are several protein variants for each histone subtype in many higher organisms (16 -20). Of the 44 chicken H1 and core genes, 30 have been sequenced, and available nucleotide sequence data indicate that the H1, H2A, H2B, and H3 families, respectively, comprise at least six, two, four, and three different protein variants (3,(21)(22)(23)(24)(25)(26)(27)(28)(29). All of the eight H2B genes belong to two major histone gene clusters with a total length of about 140 kb 1 (24). Five H2B genes (H2B-I, H2B-II, H2B-III, H2B-IV, and H2B-VI) encode the same amino acid sequence (class I), and that of H2B-VII differs from that of class I in three amino acid residues (Lys 31 3 Arg, Ser 32 3 Ala, Gly 60 3 Ser; class II) (24). H2B-V contains a single amino acid alteration (Lys 30 3 Arg; class III) (26), and the amino acid sequence of H2B-VIII is distinct from that of class I in two amino acid residues (Lys 30 3 A...

A single copy of linker H1 genes is enough for proliferation of the DT40 chicken B cell line, and linker H1 variants participate in regulation of gene expression

“…DT40 cells and all subclones were cultured essentially as described (Buerstedde & Takeda 1991;Takeda et al 1992;Takami et al 1995aTakami et al , 1995b) in Dulbecco's modified medium containing 10% (v/v) foetal calf serum, 1% (v/v) chicken serum, 2 mM L-glutamine, 0.01 mM 2-mercaptoetanol, and penicillin and streptomycin at 37 ЊC under 5% CO 2 in air in a humidified incubator. At the indicated times, the cells were counted to determine the growth rate.…”

“…Moreover, it has been reported that in Saccharomyces cerevisiae, the deletion of H4 N-terminal residues 4-23 or mutagenesis of the acetylatable lysines in this region decreases the activation of the GAL1 promoter, while the deletion of N-terminal residues 4-15 of H3 or the mutagenesis of the acetylatable lysines causes a hyperactivation of GAL1 (Fisher-Adams & Grunstein 1995). We recently established that the targeted disruption of H2B-V, encoding one of four H2B variants, causes changes in protein patterns in the DT40 chicken B cell line, indicating that the variant may participate in the regulation of expression of putative genes encoding the proteins that varied in the H2B-V deficient DT40 mutant (Takami et al 1995a). These results, together with other observations (van Daal & Elgin 1992), indicated that eukaryotic core histones affect the expression of specific genes both positively and negatively.…”

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

“…Transfection was carried out essentially as described (36,37). To obtain ⌬HIRA cells, transfectants with the p⌬HIRA/neo construct were first selected in medium containing 2 mg of neomycin (neo; G-418) per ml.…”

Chicken Histone Deacetylase-2 Controls the Amount of the IgM H-chain at the Steps of Both Transcription of Its Gene and Alternative Processing of Its Pre-mRNA in the DT40 Cell Line

“…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.…”

“…the growth rate and global chromatin structure, indicating that only one copy of the H1 genes is enough for cell proliferation (31). Third, the protein patterns are altered in the mutants, respectively, lacking particular H1 and H2B variants (26,28,30,31), and the variable proteins are specific for the corresponding mutants, suggesting that the H1 and core variants should be individually involved in regulation of the expression of specific genes. These results, together with those obtained for a number of different genes (33)(34)(35), suggest that the DT40 cell line should be most useful for an in vivo understanding of the roles of important genes in higher eukaryotes.…”