The highly conserved amino-terminal region of the protein encoded by the v-myb oncogene functions as a DNA-binding domain.

Klempnauer, Karl‐Heinz; Sippel, Albrecht E.

doi:10.1002/j.1460-2075.1987.tb02565.x

Cited by 159 publications

(113 citation statements)

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“…The motif of WTTEEEFNV is in a single copy starting at amino acid 319. This is similar to that of yeast REB1 (26), a DNA-binding protein that binds rDNA and the promoters of many RNA polymerase II-transcribed genes (27). Therefore, we cannot exclude the possibility that HOS3 binds DNA directly.…”

Section: Discussionmentioning

confidence: 74%

Yeast HOS3 forms a novel trichostatin A-insensitive homodimer with intrinsic histone deacetylase activity

Carmen

Griffin

Calaycay

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

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Histone deacetylases such as human HDAC1 and yeast RPD3 are trichostatin A (TSA)-sensitive enzymes that are members of large, multiprotein complexes. These contain specialized subunits that help target the catalytic protein to histones at the appropriate DNA regulatory element, where the enzyme represses transcription. To date, no deacetylase catalytic subunits have been shown to have intrinsic activity, suggesting that noncatalytic subunits of the deacetylase complex are required for their enzymatic function. In this paper we describe a novel yeast histone deacetylase HOS3 that is relatively insensitive to the histone deacetylase inhibitor TSA, forms a homodimer when expressed ectopically both in yeast and Escherichia coli, and has intrinsic activity when produced in the bacterium. Most HOS3 protein can be found associated with a larger complex in partially purified yeast nuclear extracts, arguing that the HOS3 homodimer may be dissociated from a very large nuclear structure during purification. We also demonstrate, using a combination of mass spectrometry, tandem mass spectrometry, and proteolytic digestion, that recombinant HOS3 has a distinct specificity in vitro for histone H4 sites K5 and K8, H3 sites K14 and K23, H2A site K7, and H2B site K11. We propose that while factors that interact with HOS3 may sequester the catalytic subunit at specific cellular sites, they are not required for HOS3 histone deacetylase activity.acetylation ͉ mass spectrometry W e previously have identified a yeast (Saccharomyces cerevisiae) histone deacetylase HDA1 (1) that was found to have similarity to four other histone deacetylases, RPD3, HOS1, HOS2, and HOS3, inferred from the yeast genome database (2). Of these enzymes, RPD3 is homologous to a mammalian histone deacetylase subunit, HDAC1 (3), that is recruited in a multiprotein complex (4) to various regulatory DNA sequences, such as Mad-Max binding sites, to repress adjacent genes (5, 6). This involves proteins such as mSIN3 and p48, which interact with repressor proteins and histones, respectively (5-7). Similar mechanisms are involved in repression by the unliganded thyroid hormone receptor (8), retinoblastoma protein (9-11), notch protein (12), and other proteins (13-15). In certain HDAC complexes (e.g., Mi2), ATPases are also implicated in histone deacetylase function (16)(17)(18). Surprisingly, although HDAC1 binds small-molecule deacetylase inhibitors such as trichostatin A (19), apicidin (20), and trapoxin (3), it has not yet been shown that HDAC-like proteins have intrinsic deacetylase activity. This suggests that other proteins in the HDAC complex are required for activity of the catalytic subunit.In yeast, the RPD3 deacetylase is also found in a large, multiprotein complex even after extensive enzyme purification (2,21,22). This trichostatin A (TSA)-sensitive enzyme complex is targeted by SIN3 to UME6-regulated genes in a manner similar to that involving Mad-Max (23). Other yeast histone deacetylases that include HDA1, HOS1, and HOS2 are also found in large comple...

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Section: Discussionmentioning

confidence: 74%

Yeast HOS3 forms a novel trichostatin A-insensitive homodimer with intrinsic histone deacetylase activity

Carmen

Griffin

Calaycay

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

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“…Two of the mutants which neither transformed nor transactivated (mutants 281 and 364) mapped to the highly conserved amino-terminal DNA-binding domain of p48vmyb (21,26 DNA-bound protein, one within the DNA-binding domain itself and one in the less conserved carboxyl terminus of the protein. This suggests a greater complexity for the action of p48v-myb than might be expected from studies of the GAL4 and GCN4 proteins, which appear to contain functionally and physically separable DNA-binding and transcriptional activation domains (for a review, see reference 42).…”

Section: Resultsmentioning

confidence: 99%

Transformation by v-myb correlates with trans-activation of gene expression.

Lane¹,

Ibañez²,

Garcia³

et al. 1990

Mol. Cell. Biol.

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The v-myb oncogene of avian myeloblastosis virus causes acute myelomonocytic leukemia in chickens and transforms avian myeloid cells in vitro. Its protein product p48v-myb is a nuclear, sequence-specific, DNA-binding The v-myb oncogene of the avian myeloblastosis virus (AMV) causes acute myelomonocytic leukemia in chickens and transforms avian myeloid cells but not fibroblasts in vitro (for a review, see reference 29). The related E26 avian leukemia virus causes acute erythroid leukemia in chickens and transforms avian erythroid and myeloid cells in vitro (36,43). The c-myb proto-oncogene from which v-myb arose is expressed at high levels predominantly in immature hemopoietic cells including those of the erythroid, lymphoid, and myeloid lineages (8, 16). The expression of c-myb declines as these cells mature (11,48). c-myb itself appears to control this differentiation process because the expression of exogenous c-myb or v-myb can block or reverse the differentiated phenotype of various hemopoietic cells (3,10,12,33,37,47). myb-related genes have also been identified in mammals, Drosophila melanogaster, Zea mays, Saccharomyces cerevisiae, and Dictyostelium discoideum (23,39,41,44,46; R. Firtel, and J. Lipsick, unpublished data).The protein products of v-myb and c-myb are present in the cell nucleus, bind DNA in a sequence-specific fashion, and trans-activate gene expression in transient DNA transfection assays (4-6, 22, 24a, 25, 27, 38, 49). Consistent with these findings, the myb-related BASI gene of S. cerevisiae encodes a genetically defined transcription factor which binds directly to DNA (46). In addition, the myb-related product of the Cl locus ofZ. mays controls several unlinked, developmentally regulated genes involved in the synthesis of anthocyanin pigments (41).A number of oncogenes which encode nuclear proteins (GGGCCC) to the end-filled HindlIlresistant neo fragment of the pUC-NEO plasmid (31). This DNA was digested with SmaI to trim excess linkers, purified by agarose gel electrophoresis, and then cloned into the SmaI site of pUC18 to create the plasmid pANA (Fig. 1). This plasmid was then digested with SmaI to release a 1.4-kilobase neo fragment flanked by single Apal linkers. ApaI was chosen because it does not cut within v-myb; however, the method can easily be adapted for other restriction sites.The plasmid pUC-MYB-dE was used as a target for mutagenesis (21). Plasmid DNA was digested with the desired restriction enzyme, and linear DNA was purified by agarose gel electrophoresis and then ligated to the 1.4-kilobase neo fragment described above. Digestion of the target plasmid with restriction enzymes with multiple recognition sites was limited by titration of ethidium bromide to maximize the yield of linear DNA (40). In rare cases, certain individual sites (e.g., Avall at 960) were never mutagenized by this method, suggesting a strong restriction enzyme site preference during partial digestion in the presence of ethidium bromide. Target DNAs produced by enzymes which generated three-nucleot...

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“…The 48 kD v-Myb protein is a truncated form of the 75 kD c-Myb protein which in addition to the N-and Cterminal deletions contains 11 amino acid substitutions. Both the c-Myb and the v-Myb proteins are localized in the nucleus (Klempnauer et al, 1984;Boyle et al, 1984), have short half-lives and bind to DNA in a sequence-speci®c fashion (Klempnauer and Sippel, 1987;Biedenkapp et al, 1988).…”

Section: Introductionmentioning

confidence: 99%