- Email: [email protected]
Functional Evolution of the Myb Oncogene Family
Blood Cells, Molecules, and Diseases (2001) 27(2) Mar/Apr: 456 – 458 doi:10.1006/bcmd.2001.0404, available online at http://www.idealibrary.com on
Lipsick et al.
Functional Evolution of the Myb Oncogene Family Submitted 01/31/01 (Communicated by M. Lichtman, M.D., 02/08/01)
Joseph S. Lipsick,1,2 John Manak,1 Nesanet Mitiku,1 Chao-Kung Chen,1 Pat Fogarty,1 and Erin Guthrie1 ABSTRACT: Three Myb-related genes (A-Myb, B-Myb, and c- Myb) have been found in all vertebrates examined thus far including mammals, birds, and amphibians. Two invertebrates, the sea urchin and the fruit fly, have only one Myb-related gene. Our laboratory has used Drosophila as a model system to explore the function of its sole Myb gene. We have also reintroduced the three different vertebrate Myb genes into Drosophila in order to begin to understand how their different functions may have arisen following gene duplication during evolution. © 2001 Academic Press Key Words: A-Myb; B-Myb; c-Myb; Dm-myb; evolution.
INTRODUCTION
Myb and A-Myb, the B-Myb gene appears to be expressed in all dividing cells in vertebrates. Homozygous disruption of the B-Myb gene results in a very early failure of embryonic development in the laboratory mouse prior to implantation (5). Similar to other multigene families in vertebrates, the Myb gene family has only a single representative in various invertebrates including the fruit fly and the sea urchin (6, 7). The proteins encoded by these animal Myb proteins all contain a highly conserved DNA-binding domain composed of three tandem amino-terminal Myb domains and a conserved carboxy-terminal negative regulatory domain (Fig. 1). In addition, the c-Myb and A-Myb proteins all contain a highly conserved central transcriptional activation domain that includes an acidic region and a heptad leucine repeat (“leucine zipper”). B-Myb and the invertebrate Myb proteins lack this central domain, suggesting that these proteins are perhaps most similar to one another in both structure and function. In this regard, transcriptional activation can readily be demonstrated for the c-Myb and AMyb proteins in a variety of experimental systems. However, B-Myb and Drosophila Myb ap-
The Myb oncogene family was first discovered because of the retroviral transduction of the normal c-Myb proto-oncogene into the avian myeloblastosis virus that causes acute monoblastic leukemia in chickens (reviewed in Ref. 1). c-Myb is expressed at high levels in immature blood cells and also in a variety of proliferating epithelial cells. Further studies revealed that the normal c-Myb gene is essential for viability and is absolutely required for fetal hematopoiesis in the laboratory mouse (2). Two additional Myb-related genes, A-Myb and B-Myb, were discovered by low stringency nucleic acid hybridization (3). Extensive analysis of expression sequence tags (ESTs) and emerging genomic sequence data suggest that these three genes are likely to comprise the entire repertoire of closely related genes in mammals. Gene disruption experiments in the laboratory mouse have shown that A-Myb is required for spermatogenesis and mammary gland epithelium proliferation during pregnancy (4). In contrast to the tissue-specific expression and functions of c-
This paper summarizes a presentation made at the Second International Workshop on Myb Genes, held in Melbourne on November 22–24, 2000, sponsored in part by the Leukemia & Lymphoma Society (U.S.A.). 1 Department of Pathology, Stanford University School of Medicine, Stanford, California 94305-5324. 2 Correspondence and reprint requests to: Joseph S. Lipsick. E-mail: [email protected]. 1079-9796/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved
456
Lipsick et al.
Blood Cells, Molecules, and Diseases (2001) 27(2) Mar/Apr: 456 – 458 doi:10.1006/bcmd.2001.0404, available online at http://www.idealibrary.com on
proteins in Drosophila. In addition to providing specific information about the function and evolution of the Myb genes and proteins, we believe that our work may provide a more general model for understanding the functional evolution of multigene families. In particular, many families of closely related transcription factors have evolved in a similar fashion in which a highly conserved DNA-binding domain has been juxtaposed to a variety of other regulatory modules following gene duplication. FIG. 1. Comparison of structural components of animal myb and Drosophila Myb (Dm-Myb) proteins.
RESULTS AND DISCUSSION
pear to score as transcriptional activators only under more limited experimental conditions. We previously identified a gene in the cellular slime mold Dictyostelium discoideum that contains a very closely related DNA-binding domain, however, no other regions similar to those of the animal Myb proteins were identified (8). Surprisingly, an extensive series of experiments and subsequent searches of the genomic and cDNA sequences have failed to identify a closely related Myb gene in the nematode Caenorhabditis elegans, although members of more distantly related Myb gene families are present (Cdc5 and SNAPc) (9) (S. McCann, Ph.D. thesis). Taken together, these observations suggest an evolutionary model in which a single gene encoding a c-Myb-like DNA-binding domain (similar to that in Dictyostelium Myb) acquired a negative regulatory domain (similar to that in Drosophila Myb, sea urchin Myb, and vertebrate B-Myb). This gene then underwent a single duplication, following which one copy acquired a transcriptional activation domain. This latter gene then underwent an additional duplication to give rise to two closely related, tissue-specific transcriptional regulators (A-Myb and c-Myb). We have recently begun to use Drosophila as a model system for two purposes. First, to better understand the function of its protein product in normal growth and development we have sought to generate a null mutant of Drosophila Myb. Second, we have used the binary GAL4 –UAS system to explore the consequences of ectopic expression of the three different vertebrate Myb
We have used P element mobilization to generate two null alleles of the Drosophila Myb gene. The resulting animals die as late third instar larvae/prepupae with delayed imaginal disc development, small testes, and a marked decrease in blood cell number and function (J. Manak, N. Mitiku, C. K. Chen, and J. Lipsick, unpublished data). This phenotype is reminiscent of that previously described for a series of mutants that control essential cell cycle functions in Drosophila (10). We have therefore begun to examine the consequences of the absence of Myb protein in the proliferating cells of the imaginal discs. In wing discs, we have used FACS analysis to demonstrate an increased number of cells with a G1like DNA content and a decreased number of cells with a G2/M-like DNA content. Further analysis revealed an increased number of cells arrested in what appears to be an aberrant M phase with incompletely condensed chromosomes, abnormal ploidy, and centrosomal defects (J. Manak, N. Mitiku, and J. Lipsick, unpublished data). These results differ somewhat from a previous report that hypomorphic temperature-sensitive Myb mutants cause a block in the G2/M transition in pupal wings (11). Presumably these differences are due to the nature of the mutations studied and/or the cell types examined. We have used the binary GAL4 –UAS system (12) to express oncogenic v-Myb, chicken A-Myb, B-Myb and c-Myb, or Drosophila Myb (Dm-Myb) in developing Drosophila eyes (P. Fogarty, E. Guthrie, and J. Lipsick). In brief, when the very early eyeless promoter was used to drive expres457
Blood Cells, Molecules, and Diseases (2001) 27(2) Mar/Apr: 456 – 458 doi:10.1006/bcmd.2001.0404, available online at http://www.idealibrary.com on
sion of these genes, eye development was essentially normal. However, the eyes were markedly smaller in the presence of A-Myb and c-Myb, and to a lesser degree B-Myb. Our interpretation of these results is that the vertebrate Myb proteins interfere to varying degrees with the normal function of Dm-Myb that is required for the extensive proliferation that gives rise to a normal eye imaginal disc. In contrast, we have found that those Myb proteins that contain the conserved central transcriptional activation domain (v-Myb, c-Myb, and A-Myb) cause severe developmental defects when produced relatively late during eye development using a glass-responsive promoter. These defects are reminiscent of those caused by lossof-function mutants of lozenge, a Drosophila homologue of vertebrate AML1 (13). No similar defects were observed when B-Myb or Dm-Myb were expressed using the same promoter. Our interpretation of these results is that relatively late during eye development the transcriptional activation domain of A-Myb and c-Myb dominantly interferes with highly conserved machinery required for the normal function of lozenge. This is intriguing because AML1, the human homologue of lozenge, is frequently translocated in human acute myelogenous leukemia, resulting in conversion of a transcriptional activator into a transcriptional repressor (14).
Lipsick et al.
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REFERENCES 1.
Ganter, B., and Lipsick, J. S. (1999) Myb and oncogenesis. Adv. Cancer Res. 76, 21– 60. 2. Mucenski, M. L., McLain, K., Kier, A. B., Swerdlow, S. H., Schreiner, C. M., Miller, T. A., Pietryga, D. W., Scott, W. J., Jr., and Potter, S. S. (1991) A functional c-myb gene is required for normal murine fetal hepatic hematopoiesis. Cell 65, 677– 689. 3. Nomura, N., Sasamoto, S., Ishii, S., Date, T., Matsui, M., and Ishizaki, R. (1988) Isolation of human cDNA clones of myb-related genes, A-myb and B-myb [published erratum appears in Nucleic Acids Res. 17, 1282, 1989]. Nucleic Acids Res. 16, 11075–11089.
13.
14.
458
Toscani, A., Mettus, R. V., Coupland, R., Simpkins, H., Litvin, J., Orth, J., Hatton, K. S., and Reddy, E. P. (1997) Arrest of spermatogenesis and defective breast development in mice lacking A-myb. Nature 386, 713–717. Tanaka, Y., Patestos, N. P., Maekawa, T., and Ishii, S. (1999) B-myb is required for inner cell mass formation at an early stage of development. J. Biol. Chem. 274, 28067–28070. Katzen, A. L., Kornberg, T. B., and Bishop, J. M. (1985) Isolation of the proto-oncogene c-myb from D. melanogaster. Cell 41, 449 – 456. Coffman, J. A., Kirchhamer, C. V., Harrington, M. G., and Davidson, E. H. (1997) SpMyb functions as an intramodular repressor to regulate spatial expression of CyIIIa in sea urchin embryos. Development 124, 4717– 4727. Stober-Grasser, U., Brydolf, B., Bin, X., Grasser, F., Firtel, R., and Lipsick, J. S. (1992) The Myb DNAbinding domain is highly conserved in Dictyostelium discoideum. Oncogene 7, 589 –596. Ohi, R., Feoktistova, A., McCann, S., Valentine, V., Look, A. T., Lipsick, J. S., and Gould, K. L. (1998) Myb-related Schizosaccharomyces pombe cdc5p is structurally and functionally conserved in eukaryotes. Mol. Cell. Biol. 18, 4097– 4108. Gatti, M., and Baker, B. S. (1989) Genes controlling essential cell-cycle functions in Drosophila melanogaster. Genes Dev. 3, 438 – 453. Katzen, A. L., Jackson, J., Harmon, B. P., Fung, S. M., Ramsay, G., and Bishop, J. M. (1998) Drosophila myb is required for the G2/M transition and maintenance of diploidy. Genes Dev. 12, 831– 843. Brand, A., and Perrimon, N. (1993) Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118, 401– 415. Daga, A., Karlovich, C. A., Dumstrei, K., and Banerjee, U. (1996) Patterning of cells in the Drosophila eye by Lozenge, which shares homologous domains with AML1 genes. Development 10, 1194 –1205. Lutterbach, B., Westendorf, J. J., Linggi, B., Patten, A., Moniwa, M., Davie, J. R., Huynh, K. D., Bardwell, V. J., Lavinsky, R. M., Rosenfeld, M. G., Glass, C., Seto, E., and Hiebert, S. W. (1998) ETO, a target of t(8;21) in acute leukemia, interacts with the N-CoR and mSin3 corepressors. Mol. Cell. Biol. 18, 7176 – 7184.
Lipsick et al.
Functional Evolution of the Myb Oncogene Family Submitted 01/31/01 (Communicated by M. Lichtman, M.D., 02/08/01)
Joseph S. Lipsick,1,2 John Manak,1 Nesanet Mitiku,1 Chao-Kung Chen,1 Pat Fogarty,1 and Erin Guthrie1 ABSTRACT: Three Myb-related genes (A-Myb, B-Myb, and c- Myb) have been found in all vertebrates examined thus far including mammals, birds, and amphibians. Two invertebrates, the sea urchin and the fruit fly, have only one Myb-related gene. Our laboratory has used Drosophila as a model system to explore the function of its sole Myb gene. We have also reintroduced the three different vertebrate Myb genes into Drosophila in order to begin to understand how their different functions may have arisen following gene duplication during evolution. © 2001 Academic Press Key Words: A-Myb; B-Myb; c-Myb; Dm-myb; evolution.
INTRODUCTION
Myb and A-Myb, the B-Myb gene appears to be expressed in all dividing cells in vertebrates. Homozygous disruption of the B-Myb gene results in a very early failure of embryonic development in the laboratory mouse prior to implantation (5). Similar to other multigene families in vertebrates, the Myb gene family has only a single representative in various invertebrates including the fruit fly and the sea urchin (6, 7). The proteins encoded by these animal Myb proteins all contain a highly conserved DNA-binding domain composed of three tandem amino-terminal Myb domains and a conserved carboxy-terminal negative regulatory domain (Fig. 1). In addition, the c-Myb and A-Myb proteins all contain a highly conserved central transcriptional activation domain that includes an acidic region and a heptad leucine repeat (“leucine zipper”). B-Myb and the invertebrate Myb proteins lack this central domain, suggesting that these proteins are perhaps most similar to one another in both structure and function. In this regard, transcriptional activation can readily be demonstrated for the c-Myb and AMyb proteins in a variety of experimental systems. However, B-Myb and Drosophila Myb ap-
The Myb oncogene family was first discovered because of the retroviral transduction of the normal c-Myb proto-oncogene into the avian myeloblastosis virus that causes acute monoblastic leukemia in chickens (reviewed in Ref. 1). c-Myb is expressed at high levels in immature blood cells and also in a variety of proliferating epithelial cells. Further studies revealed that the normal c-Myb gene is essential for viability and is absolutely required for fetal hematopoiesis in the laboratory mouse (2). Two additional Myb-related genes, A-Myb and B-Myb, were discovered by low stringency nucleic acid hybridization (3). Extensive analysis of expression sequence tags (ESTs) and emerging genomic sequence data suggest that these three genes are likely to comprise the entire repertoire of closely related genes in mammals. Gene disruption experiments in the laboratory mouse have shown that A-Myb is required for spermatogenesis and mammary gland epithelium proliferation during pregnancy (4). In contrast to the tissue-specific expression and functions of c-
This paper summarizes a presentation made at the Second International Workshop on Myb Genes, held in Melbourne on November 22–24, 2000, sponsored in part by the Leukemia & Lymphoma Society (U.S.A.). 1 Department of Pathology, Stanford University School of Medicine, Stanford, California 94305-5324. 2 Correspondence and reprint requests to: Joseph S. Lipsick. E-mail: [email protected]. 1079-9796/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved
456
Lipsick et al.
Blood Cells, Molecules, and Diseases (2001) 27(2) Mar/Apr: 456 – 458 doi:10.1006/bcmd.2001.0404, available online at http://www.idealibrary.com on
proteins in Drosophila. In addition to providing specific information about the function and evolution of the Myb genes and proteins, we believe that our work may provide a more general model for understanding the functional evolution of multigene families. In particular, many families of closely related transcription factors have evolved in a similar fashion in which a highly conserved DNA-binding domain has been juxtaposed to a variety of other regulatory modules following gene duplication. FIG. 1. Comparison of structural components of animal myb and Drosophila Myb (Dm-Myb) proteins.
RESULTS AND DISCUSSION
pear to score as transcriptional activators only under more limited experimental conditions. We previously identified a gene in the cellular slime mold Dictyostelium discoideum that contains a very closely related DNA-binding domain, however, no other regions similar to those of the animal Myb proteins were identified (8). Surprisingly, an extensive series of experiments and subsequent searches of the genomic and cDNA sequences have failed to identify a closely related Myb gene in the nematode Caenorhabditis elegans, although members of more distantly related Myb gene families are present (Cdc5 and SNAPc) (9) (S. McCann, Ph.D. thesis). Taken together, these observations suggest an evolutionary model in which a single gene encoding a c-Myb-like DNA-binding domain (similar to that in Dictyostelium Myb) acquired a negative regulatory domain (similar to that in Drosophila Myb, sea urchin Myb, and vertebrate B-Myb). This gene then underwent a single duplication, following which one copy acquired a transcriptional activation domain. This latter gene then underwent an additional duplication to give rise to two closely related, tissue-specific transcriptional regulators (A-Myb and c-Myb). We have recently begun to use Drosophila as a model system for two purposes. First, to better understand the function of its protein product in normal growth and development we have sought to generate a null mutant of Drosophila Myb. Second, we have used the binary GAL4 –UAS system to explore the consequences of ectopic expression of the three different vertebrate Myb
We have used P element mobilization to generate two null alleles of the Drosophila Myb gene. The resulting animals die as late third instar larvae/prepupae with delayed imaginal disc development, small testes, and a marked decrease in blood cell number and function (J. Manak, N. Mitiku, C. K. Chen, and J. Lipsick, unpublished data). This phenotype is reminiscent of that previously described for a series of mutants that control essential cell cycle functions in Drosophila (10). We have therefore begun to examine the consequences of the absence of Myb protein in the proliferating cells of the imaginal discs. In wing discs, we have used FACS analysis to demonstrate an increased number of cells with a G1like DNA content and a decreased number of cells with a G2/M-like DNA content. Further analysis revealed an increased number of cells arrested in what appears to be an aberrant M phase with incompletely condensed chromosomes, abnormal ploidy, and centrosomal defects (J. Manak, N. Mitiku, and J. Lipsick, unpublished data). These results differ somewhat from a previous report that hypomorphic temperature-sensitive Myb mutants cause a block in the G2/M transition in pupal wings (11). Presumably these differences are due to the nature of the mutations studied and/or the cell types examined. We have used the binary GAL4 –UAS system (12) to express oncogenic v-Myb, chicken A-Myb, B-Myb and c-Myb, or Drosophila Myb (Dm-Myb) in developing Drosophila eyes (P. Fogarty, E. Guthrie, and J. Lipsick). In brief, when the very early eyeless promoter was used to drive expres457
Blood Cells, Molecules, and Diseases (2001) 27(2) Mar/Apr: 456 – 458 doi:10.1006/bcmd.2001.0404, available online at http://www.idealibrary.com on
sion of these genes, eye development was essentially normal. However, the eyes were markedly smaller in the presence of A-Myb and c-Myb, and to a lesser degree B-Myb. Our interpretation of these results is that the vertebrate Myb proteins interfere to varying degrees with the normal function of Dm-Myb that is required for the extensive proliferation that gives rise to a normal eye imaginal disc. In contrast, we have found that those Myb proteins that contain the conserved central transcriptional activation domain (v-Myb, c-Myb, and A-Myb) cause severe developmental defects when produced relatively late during eye development using a glass-responsive promoter. These defects are reminiscent of those caused by lossof-function mutants of lozenge, a Drosophila homologue of vertebrate AML1 (13). No similar defects were observed when B-Myb or Dm-Myb were expressed using the same promoter. Our interpretation of these results is that relatively late during eye development the transcriptional activation domain of A-Myb and c-Myb dominantly interferes with highly conserved machinery required for the normal function of lozenge. This is intriguing because AML1, the human homologue of lozenge, is frequently translocated in human acute myelogenous leukemia, resulting in conversion of a transcriptional activator into a transcriptional repressor (14).
Lipsick et al.
4.
5.
6.
7.
8.
9.
10.
11.
12.
REFERENCES 1.
Ganter, B., and Lipsick, J. S. (1999) Myb and oncogenesis. Adv. Cancer Res. 76, 21– 60. 2. Mucenski, M. L., McLain, K., Kier, A. B., Swerdlow, S. H., Schreiner, C. M., Miller, T. A., Pietryga, D. W., Scott, W. J., Jr., and Potter, S. S. (1991) A functional c-myb gene is required for normal murine fetal hepatic hematopoiesis. Cell 65, 677– 689. 3. Nomura, N., Sasamoto, S., Ishii, S., Date, T., Matsui, M., and Ishizaki, R. (1988) Isolation of human cDNA clones of myb-related genes, A-myb and B-myb [published erratum appears in Nucleic Acids Res. 17, 1282, 1989]. Nucleic Acids Res. 16, 11075–11089.
13.
14.
458
Toscani, A., Mettus, R. V., Coupland, R., Simpkins, H., Litvin, J., Orth, J., Hatton, K. S., and Reddy, E. P. (1997) Arrest of spermatogenesis and defective breast development in mice lacking A-myb. Nature 386, 713–717. Tanaka, Y., Patestos, N. P., Maekawa, T., and Ishii, S. (1999) B-myb is required for inner cell mass formation at an early stage of development. J. Biol. Chem. 274, 28067–28070. Katzen, A. L., Kornberg, T. B., and Bishop, J. M. (1985) Isolation of the proto-oncogene c-myb from D. melanogaster. Cell 41, 449 – 456. Coffman, J. A., Kirchhamer, C. V., Harrington, M. G., and Davidson, E. H. (1997) SpMyb functions as an intramodular repressor to regulate spatial expression of CyIIIa in sea urchin embryos. Development 124, 4717– 4727. Stober-Grasser, U., Brydolf, B., Bin, X., Grasser, F., Firtel, R., and Lipsick, J. S. (1992) The Myb DNAbinding domain is highly conserved in Dictyostelium discoideum. Oncogene 7, 589 –596. Ohi, R., Feoktistova, A., McCann, S., Valentine, V., Look, A. T., Lipsick, J. S., and Gould, K. L. (1998) Myb-related Schizosaccharomyces pombe cdc5p is structurally and functionally conserved in eukaryotes. Mol. Cell. Biol. 18, 4097– 4108. Gatti, M., and Baker, B. S. (1989) Genes controlling essential cell-cycle functions in Drosophila melanogaster. Genes Dev. 3, 438 – 453. Katzen, A. L., Jackson, J., Harmon, B. P., Fung, S. M., Ramsay, G., and Bishop, J. M. (1998) Drosophila myb is required for the G2/M transition and maintenance of diploidy. Genes Dev. 12, 831– 843. Brand, A., and Perrimon, N. (1993) Targeted gene expression as a means of altering cell fates and generating dominant phenotypes. Development 118, 401– 415. Daga, A., Karlovich, C. A., Dumstrei, K., and Banerjee, U. (1996) Patterning of cells in the Drosophila eye by Lozenge, which shares homologous domains with AML1 genes. Development 10, 1194 –1205. Lutterbach, B., Westendorf, J. J., Linggi, B., Patten, A., Moniwa, M., Davie, J. R., Huynh, K. D., Bardwell, V. J., Lavinsky, R. M., Rosenfeld, M. G., Glass, C., Seto, E., and Hiebert, S. W. (1998) ETO, a target of t(8;21) in acute leukemia, interacts with the N-CoR and mSin3 corepressors. Mol. Cell. Biol. 18, 7176 – 7184.