Oct 11, 1994 - Research and Development Center, Frederick, MD 21702, USA ... essential for the development of the hematopoietic system but appears to ...... Liu,Y.-J., Johnson,G.D., GordonJ. and Mac Lennan,I.C.M. (1992). Immunol.
The EMBO Journal vol.13 no.24 pp.5994-6005, 1994
Mouse A-myb encodes a trans-activator and is expressed in mitotically active cells of the developing central nervous system, adult testis and B lymphocytes Konrad Trauth, Bettina Mutschler, Nancy A.Jenkins1, Debra J.Gilbert1, Neal G.Copeland1 and Karl-Heinz Klempnauer2 Hans-Spemann-Laboratory, Max-Planck-Institute for Immunobiology, Stubeweg 51, D-79108 Freiburg, Germany and 'Mammalian Genetics Laboratory, ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, Frederick, MD 21702, USA 2Corresponding author Communicated by A.Nordheim
C-myb encodes a transcriptional activator that is essential for the development of the hematopoietic system but appears to lack major roles in non-hematopoietic cells. The identification of two conserved mybrelated genes, designated A-myb and B-myb, has raised the possibility that these genes are functional equivalents of c-myb in non-hematopoietic cells. Here, we report the isolation and preliminary characterization of the mouse A-myb gene. Mouse A-myb maps to the proximal region of chromosome 1 and encodes a transcriptional activator with properties similar to those of the c-myb and v-myb proteins. During embryogenesis A-myb is predominantly expressed in several regions of the developing central nervous system (CNS) and the urogenital ridge. Expression in the CNS is confined to the neural tube, the hindbrain, the neural retina and the olfactory epithelium, and coincides with the presence of proliferating immature neuronal precursor cells. In the adult mouse, A-myb is expressed during the early stages of sperm cell differentiation and in B lymphocytes located in germinal centers of the spleen. Taken together, these results suggest a role for A-myb in the proliferation and/or differentiation of neurogenic, spermatogenic and B-lymphoid cells. Key words: mouse A-myb/myb-related gene/neuronal differentiation/spermatogenesis/trans-activation
Introduction The c-myb proto-oncogene, the cellular counterpart of the transforming gene (v-myb) of the avian myeloblastosis virus (AMV), is essential for the development of the hematopoietic system (reviewed in Shen-Ong, 1990; Graf, 1992). C-myb is highly expressed in immature hematopoietic progenitor cells and is turned off during terminal differentiation. Sustained c-myb expression interferes with the terminal differentiation of hematopoietic cells (Clarke et al., 1988; Selvakumaran et al., 1992), whereas inhibition of c-myb expression reduces the proliferative capacity of precursor cells (Gewirtz and Calabretta, 1988). Mice lacking a functional c-myb gene die during embryonic
development from defects in fetal hepatic hematopoiesis (Mucenski et al., 1991). C-myb and its oncogenic derivative v-myb encode nuclear DNA binding proteins (Klempnauer and Sippel, 1986, 1987) specifically recognizing the DNA sequence PyAAC(G/T)G (Biedenkapp et al., 1988). Promotors containing such binding sites are activated by v-myb and c-myb (Klempnauer et al., 1989; Nishina et al., 1989; Weston and Bishop, 1989; Ibanez and Lipsick, 1990). Several myb target genes for v-myb and c-myb have been identified (Ness et al., 1989; Burk and Klempnauer, 1991; Siu et al., 1992). Whether these genes play important roles in hematopoietic cells has not yet been determined. Two myb-related genes, A-myb and B-myb, have been identified in several vertebrate species (Nomura et al., 1988; Bouwmeester et al., 1992; Foos et al., 1992; Sleeman, 1993). The predicted proteins encoded by these genes exhibit substantial homology to the DNA binding domain of the c-myb protein, suggesting that they are involved in sequence-specific DNA binding. In contrast to c-myb, whose role in transcriptional regulation and hematopoietic cell proliferation is well established, very little is known about the functions of A-myb and B-myb. To explore the function of the A-myb gene we have isolated a full-length mouse A-myb cDNA clone. We have used this clone to investigate the trans-activating potential of the A-myb protein, to determine the mouse chromosomal localization of A-myb and to study the spatial and temporal patterns of A-myb expression during embryogenesis and in the adult mouse. Our results raise the interesting possibility that A-myb acts as a functional homolog of cmyb in the developing central nervous system (CNS), in B-cell differentiation and during spermatogenesis.
Results Sequence of mouse A-myb Degenerate oligonucleotides corresponding to conserved regions of human and chicken A-myb were used as PCR primers to amplify A-myb-specific DNA fragments from reverse-transcribed RNA of the mouse B-cell line K46 (Kim et al., 1979). By using the resulting PCR fragments to screen various mouse cDNA libraries, and constructing a specifically primed cDNA library from the A-myboverexpressing mouse B-cell line K46, we obtained a fulllength mouse A-myb cDNA clone. The predicted 754 amino acid A-myb protein has a calculated molecular weight of -82 kDa and is highly related to the human Amyb protein (Figure 1). Comparison of mouse A-myb and c-myb proteins shows that most of the sequences shared by these proteins are clustered within three domains. The homologous region at the N-terminus corresponds to the sequence-specific DNA binding domain found in all myb proteins analyzed (Klempnauer and Sippel, 1987; Howe
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second conserved region, located in the middle of the protein (amino acids 258-280 of A-myb), corresponds to the trans-activation domain of the c-myb protein (Weston and Bishop, 1989). The third region of homology is located in the C-terminal region of the protein and was first identified in a myb-related protein from Drosophila melanogaster (Peters et al., 1987). As yet, a specific function has not been assigned to this domain. A leucine zipper-like sequence (amino acids 375-403 of c-myb), postulated to be involved in negative regulation of c-myb function (Sakura et al., 1989), is only partially conserved. The presence of two proline residues in this region of the A-myb protein makes it unlikely that this sequence forms a leucine zipper in the A-myb protein. Amino acids 366-488 of A-myb cannot be aligned with the c-myb
protein. Interestingly, both ends of this stretch of sequences are homologous to the exon 9A of c-myb, which is involved in differential splicing (Rosson et al., 1987; Shen-Ong et al., 1989; Schuur et al., 1993). It will be interesting to investigate whether A-myb mRNA is also spliced differentially.
Chromosomal localization of the A-myb gene The mouse chromosomal location of A-myb was determined by interspecific backcross analysis using progeny derived from matings of (C57BL/6JXMus spretus)F>X C57BL/6J mice (Copeland and Jenkins, 1991). Restriction digests of C57BL/6J and M.spretus DNAs were analyzed by Southern blot hybridization for informative restriction fragment length polymorphisms (RFLPs) using a mouse A-myb probe. A 9.1 kb M.spretus EcoRV RFLP (see
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Materials and methods) was used to follow the segregation of the A-myb locus in backcross mice. The mapping results indicated that A-myb is located in the very proximal region of mouse chromosome 1 linked to bullous pemphigoid antigen 1 (Bpagl ), interleukin- receptor 1 (Illrl) and cytotoxic T lymphocyte-associated protein 4 (Ctla4). Although 133 mice were analyzed for every marker and are shown in the segregation analysis (Figure 2), up to 192 mice were typed for some pairs of markers. Each locus was scrutinized for pairwise combinations for recombination chromosomes and the total number of mice analyzed for each pair of loci. The most likely gene order is: centromere -A-myb-26/192-Bpagl-S/l 9 l-IlJrl-1 8/1 36-Ctla4. The recombination frequencies (expressed in cM standard error) are: A-myb-13.5 + 2.5-Bpagl-2.6 + 1.2Illrl-13.2 ± 2.9-Ctla4. 5996
Northern blot analysis of A-myb expression during mouse development and in the adult mouse To obtain an overview of the expression of the murine Amyb gene, we analyzed mRNA preparations from embryos and yolk sac from different stages of gestation (E9.S-EI5.5) as well as from adult tissues by Northern blotting. Between days E9.5 and EIS.S, when most body structures are formed, A-myb was expressed at all stages but was most abundant around day El 1 of gestation (Figure 3A). We also found A-myb transcripts in the yolk sac throughout all stages, beginning at days E9.5-El6.5, peaking at days E12.5-E13.5 and declining in the following stages (Figure 3B). We consistently detected two Amyb-specific RNA species of 5.5 and 7.3 kb. The biological significance of these two RNA species is not yet known. In the adult mouse, high levels of A-myb mRNA were found in lymphoid tissues and the reproductive organs, with the highest expression of the A-myb gene being in the testis. We also observed A-myb transcripts in the brain, heart, lung and kidney (Figure 4). The testis contained an additional faster migrating A-myb RNA species of -2.4 kb in length. This smaller RNA species, which was not observed in other tissues, does not appear to be a breakdown product, since other mRNAs, such as GAPDH RNA (visible on longer exposures of the autoradiogram shown in Figure 4) or B-myb RNA (data not shown), showed no sign of degradation. A-myb expression was not found in muscle or liver. A-myb expression in the developing nervous system
Because A-myb was expressed at a time when most of the structures within the mouse embryo are established, we investigated the expression pattern of A-myb in the embryo by in situ hybridization. Between days E10.5 and E14.5 after gestation, we found A-myb to be expressed in the neural tube, brain, eye, olfactory epithelium and genital ridge. A-myb expression in the neural tube. Within the neural tube A-myb was expressed from days ElO.S to E13.5 of gestation. A-myb expression was detected only in the ventricular zone of the basal plate, which contains mitotically active neuronal precursor cells, but not in other parts of the neural tube (Figure 5). Interestingly, A-myb expression was not equally distributed over the ventricular zone of the basal plate, but rather was restricted to two distinct regions; only cells lying immediately dorsal to the floor plate and a cell population located ventral to the sulcus limitans showed A-myb expression. A-myb was expressed throughout the entire length of the neural tube (Figure SE). At day E14.5, when all the neurons of the neural tube are post-mitotic and the ventricular zone has dramatically regressed, A-myb expression was undetectable (Figure SF). A-myb expression in the developing brain. Between days EIO.S and E13.5 A-myb was expressed in the basal plate of the metencephalon and myelencephalon (Figure 6). As in the neural tube, A-myb expression was not uniform over the entire ventral half of the hindbrain but was restricted to a distinct region near the floor plate (data not shown). At all stages A-myb expression was confined to the ventricular zone. At day E14.5, when the ventricular
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The primordium of the olfactory organ is the paired olfactory placode, which appears as a thickening of the cranial ectoderm by day E8.5 of development (Klein and Grazadei, 1983). Each placode subsequently invaginates to form an olfactory pit. Cells of the placode proliferate and at day El0 neuronal differentiation starts (Cuschieri and Bannister, 1975). In the following days of embryonic development the olfactory epithelium consists of mitotically active apical and basal stem cells, separated by a layer of olfactory neurons. As shown in Figure 7G-J, A-myb expression was detected only in the basal layer of proliferating stem cells at all stages of development analyzed (days El1.5-El4.5). There was no A-myb expression in the apical cells or in the olfactory neurons. Interestingly, the A-myb signal was not distributed uniformly over the basal layer, but appeared in a discontinuous patch-like pattern.
A-myb expression in the developing urogenital ridge and adult testis Outside of the CNS A-mvb was expressed in the developing urogenital ridge. The primordial germ cells are found posterior to the primitive streak in the extra-embryonic mesoderm already at day E7.25 of gestation. These cells undergo mitosis and in the following days migrate out to settle in the genital ridge (El 1.0), which is located in the medio-ventral aspect of the urogenital ridge (Snow and Monk, 1983; Ginsburg et al., 1990). As illustrated in Figure 8, A-myb was expressed in the genital ridge at day EI 1.5 of gestation. We also performed in situ hybridization analyses on sections of adult testis, which showed the highest level of A-myb expression among all adult tissues. The epithelium of the seminiferous tubules of the testis consists of two cell types: a proliferating population of spermatogenic cells and a non-proliferating population of Sertoli cells. The spermatogenic cells are arranged in concentric layers representing successive stages of sperm cell differentiation. 5997
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The spermatogonia, the mitotically active stem cells, are found at the periphery, whereas the spermatids, which no longer divide, are located close to the lumen of the tubule. The germ cells are intimately associated with Sertoli cells throughout their development. As illustrated in Figure 8, an intense A-myb-specific signal was detected in the layers of the seminiferous tubules containing mainly spermatogonia and spermatocytes. A-myb was not expressed in the interstitial space between tubules where Leydig cells are located, or in the central portion of the tubule which contains spermatids and spermatozoa. Since Sertoli cells are intermingled between spermatogonia and spermatocytes and because our in situ hybridization procedure does not permit resolution at the cellular level, it is possible that A-myb is also expressed in Sertoli cells.
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A-myb expression during B-cell development In humans, A-myb is expressed in B cells (Golay et al., 1991). Since Northern blots of adult spleen showed an elevated level of A-myb expression, we hybridized crosssections through the spleen to an A-myb-specific probe. A-myb expression was identified in several distinct regions of the spleen (Figure 9A and C). Figure 9B displays a hematoxilin-eosin-stained section of an adult spleen, showing a primary B-cell follicle containing small resting B cells, as well as secondary follicles containing proliferating B cells. As demonstrated in Figure 9C, A-myb was expressed only in the proliferating B cells located within the secondary follicles. We also performed in situ hybridization analyses of the thymus but were unable to detect a clear hybridization signal for A-myb. We presume
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that many cells in the thymus contribute to A-mvb expression and that, therefore, the detection of a specific hybridization signal is difficult.
Transactivation by mouse A-myb To study the transactivation potential of A-mvb we cotransfected an expression vector for mouse A-mvb (pCDNA3mAmyb) with myb-responsive reporter genes. As shown in Figure 10A-C, the expression of plasmids p3xATk-Luc, which contains three copies of a mvb binding site derived
Mouse A-myb from the chicken mim- I gene, and p-240-Luc, which contains chicken mim-1 promoter sequences from -240 to + 150 bp (Ness et al., 1989), was induced by A-myb to a similar or greater extent as by c-myb. Since cooperation of v-myb and c-myb with C/EBP transcription factors is required for mim-1 expression (Burk et al., 1993; Ness et al., 1993), the activation of the mim-l promoter by A-myb suggested that A-myb also co-operates with C/EBP transcription factors. To address this issue, we cotransfected the mim- 1 reporter plasmid with expression vectors for mouse A-myb and C/EBPa, , or 6, using the fibroblast cell line QT6 which has a low content of endogenous C/EBP transcription factors (S.Mink and K.H.Klempnauer, unpublished observations). Figure IOD shows that A-myb was indeed able to synergize with each C/EBP family member. To study the transactivation potential of A-myb under more physiological conditions, we stably introduced the A-myb expression vector into the macrophage cell line HD 11. These cells respond to v-myb or c-myb with changes in the expression of several endogenous genes such as mim- 1, MD- 1 and the lysozyme gene (Burk and Klempnauer, 1991). Several G418-resistant clones with elevated levels of A-myb expression were obtained. The analysis of one of these clones is shown in Figure 11. Overexpression of A-myb resulted in the appearance of several protein species representing either proteolytic degradation products or differentially modified forms of the A-myb protein (Figure 11 A). The appearance of several forms of the A-myb protein was not a peculiarity of the clone analyzed in Figure 11 but was also observed with other clones (data not shown). As shown in Figure 1 IB, mim-1, MD- 1 and lysozyme mRNAs were overexpressed in the A-myb expressing clone in comparison with G418resistant control clones or with the parental HDI1 cell line. In addition to the activation of the known myb target genes, A-myb also caused alterations in cell morphology; the A-myb-expressing cells appeared more rounded and were less adherent than control cells (Figure 1 IC). These changes are similar to the alterations induced by v-myb in HD 11 cells (Burk and Klempnauer, 1991). Morphological alterations as well as changes in myb target gene expression were also observed in other HD 11 clones stably expressing A-myb (data not shown).
Discussion A-myb is a conserved member of the myb family and encodes a transcriptional activator Myb-related genes have been found in many eucaryotes, such as mammals, insects, yeast, slime mold and higher plants (Gonda et al., 1985; Katzen et al., 1985; Gerondakis and Bishop, 1986; Paz-Ares et al., 1987; Peters et al., 1987; Tice-Baldwin et al., 1989; Oppenheimer et al., 1991; Stober-Grasser et al., 1992). The hallmark of the myb family is an amino acid sequence of 50-55 residues that is tandemly repeated two or three times at the Nterminus of each myb protein and serves as a sequencespecific DNA binding domain (Klempnauer and Sippel, 1987; Biedenkapp et al., 1988; Howe et al., 1990; Oehler et al., 1990; Frampton et al., 1991). We refer to the novel gene described here as mouse A-myb, because it encodes a protein that contains the characteristic myb DNA binding
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Fig. 7. A-myb expression in the developing eye and olfactory epithelium. (A-F) Sections through the eye of El 1.5 (A and B),
mXl
domain and closely resembles the human A-myb protein. The isolation of this gene demonstrates that mice, like humans (Nomura et al., 1988) and other vertebrates (Bouwmeester et al., 1992; Foos et al., 1992; Lam et al., 1992; Sleeman, 1993), possess multiple myb family members which have been conserved during evolution. Using the known v-myb and c-myb target genes as tools, we have found that A-myb encodes a potent transcriptional activator whose properties resemble those of the v-myb and c-myb proteins. Promoters containing myb binding sites were activated by A-myb; in addition, in HDl cells A-myb induced expression of the endogenous mim- 1, lysozyme and MD-1 genes, all of which are known target genes for v-myb and c-myb. The activation of these genes by A-myb strongly suggests that the mechanisms by which A-myb, c-myb and v-myb activate target genes are similar. This apparent similarity is substantiated by the observation that A-myb, like c-myb and v-myb, co-operates with different C/EBP transcription factors. Finally, A-myb caused alterations in the morphology of HD 11 cells which were similar to the alterations induced by v-myb in these cells (Burk and Klempnauer, 1991). Since these alterations presumably result from changes in the expression of other as yet unknown genes, it is likely that the set of genes targeted by both v-myb and A-myb consists of more than the three genes analyzed here. Based on these observations we speculate that A-myb and c-myb are functional homologs. 6000
E12.5 (C and D) and E14.5 (E and F) mouse embryos. Arrows in (C-F) mark the boundaries of the presumptive iris/ciliar body region. (G-J) Sections through the olfactory epithelium of El 1.5 (G and H) and E14.5 (I and J) mouse embryos. All sections were hybridized to A-myb antisense probe and are shown under lightfield (A, C, E, G and I) or darkfield (B, D, F, H and J) illumination. bl, basal cell layer; inl, inner nuclear layer; onl, outer nuclear layer; pe, pigment epithelium. Bars: A and C, 200 gm; E, 125 ,um; G, 125 ,um; I, 200 ,um.
The chromosomal location of A-myb A-myb has been mapped to human chromosome 8q22 (Barletta et al., 1991). Regions of homology between human chromosome 8 and mouse chromosomes 3, 4, 8, 9, 14 and 15 have been identified (Copeland et al., 1993a,b). However, homology with mouse chromosome 1 has not been observed previously. The proximal region of mouse chromosome I is homologous to human chromosomes 6 and 2 (summarized in Figure 2). However, Amyb maps 13.5 cM proximal of Bpagl which is one of the most proximal markers previously mapped on mouse chromosome 1 (Figure 2). Given this large distance and
the absence of any other gene markers that
are
also
mapped in humans in this interval, it is not surprising that a new region of homology between mouse and human chromosomes has been identified here.
A-myb is expressed in neuronal progenitor cells Several observations suggests that A-myb is expressed during neuronal differentiation. In the developing neural tube and hindbrain, A-myb-expressing cells
are
confined
to the ventricular zone which contains mitotically active
neuronal progenitor cells. Furthermore, in both the neural tube and the hindbrain, regression of the ventricular zone during embryogenesis is accompanied by a decrease in A-myb expression. This suggests that A-myb exerts its main function during early differentiation of neuronal cells but is dispensable as soon as cells enter the post-
Mouse A-myb
A 2 .I.
I 1 11T
C
Fig. 8. A-myb expression in the urogenital ridge and adult testis. (A and B) Saggital section through the urogenital ridge of an El 1.5 mouse embryo. (C and D) Section through the semiferous tubules of the testis of an adult mouse. All sections were hybridized with A-myb antisense probe and are shown under lightfield (A and C) or darkfield (B and D) illumination. gr, genital ridge; mn, mesonephros; st, spermatids. Bars: A, 100 gm; C, 130 ,m.
mitotic phase. During eye development, A-myb is expressed in the posterior region of the inner layer of the optic cup which gives rise to the neural retina, but is excluded from the anterior region which develops into the ciliary body and iris (Bard and Ross, 1982a,b). The Hox7.1 and Hox-8.1 genes have been described as markers for the presumptive ciliary body/iris domain and the neural retina region, respectively (Monaghan et al., 1991). Amyb expression thus coincides with Hox-8.1 expression, indicating that A-myb expression is confined to cells of the neuronal lineage. As soon as the differentiation of the retina starts, A-myb remains expressed in proliferating neuronal cells of the outer nuclear layer but is turned off in the post-mitotic ganglion cells and amacrine cells on the inner side of the retina. Finally, the pattern of A-myb expression in the olfactory epithelium also supports the idea that A-myb plays a role in neuronal progenitor cells. The olfactory epithelium consists of three cell types: proliferating apical and basal stem cells, and an intermediate cell layer of post-mitotic, predifferentiated olfactory neurons. Apical and basal stem cells are presumed to belong to different lineages, because in Xenopus laevis the olfactory epithelium has been shown to originate from two distinct ectodermal cell layers. The olfactory neurons and basal cells are derived from the cells of the inner nervous layer, whereas the supporting and apical cells are derived from the cells of the non-nervous layer
of the ectoderm (Klein and Grazadei, 1983). Our results clearly show that A-myb is expressed only in the neurally committed basal cells but not in the apical or intermediate cell layers. The discontinuous hybridization pattern of the A-myb probe with cells in the basal layer shows a striking similarity to the pattern of [3H]thymidine incorporation in the regenerating adult olfactory epithelium, suggesting further that A-myb is expressed in mitotically active neuronal precursor cells. Our finding that A-myb is expressed in neuronal precursor cells suggests the interesting possibility that the role of A-myb in these cells may be similar to c-myb function in hematopoietic precursor cells. However, unlike c-myb, which is expressed in all hematopoietic lineages, A-myb is not a general marker of neuronal precursor cells. For example, A-myb is not expressed in the alar plate of the neural tube or in the mid- and forebrain, all of which contain proliferating neuronal cells at the developmental stages analyzed. A-myb expression therefore marks particular lineages of neuronal cells.
A-myb is expressed during spermatogenesis and B-cell development In addition to its expression in nervous tissues, A-myb seems to play a role during spermatogenesis and B-cell development. A-myb is expressed in the genital ridge as well as in testis, which shows the highest level of A-myb 6001
K.Trauth et al.
p3xATk- Lu
i
p 8 1 Tk -L.uc
p-2 40-LLiC
~ ~
L K
pCDNA3mnA-myb DC [NNA CM ' -1(.M
t!
t
..
r
'"
Cl
C.: BP
EB
../
C/EBP,
Fig. 9. A-myb expression in the spleen of an adult mouse. (A) Section through the spleen of an adult mouse hybridized to A-myb antisense probe. (B and C) A-mvb-expressing centers in the spleen shown under higher magnification and stained with hematoxilin-eosin (B) or hybridized to A-myb antisense probe (C). Two secondary follicles in (B) and (C) are marked by arrows. pf, primary follicle. Bars: A, 250 tim; B and C, 100 p.m.
expression among all adult tissues. In the testis, A-myb is strongly expressed in spermatogonia and spermatocytes but not in spermatids, again suggesting a role for A-myb during proliferation and differentiation of germ cells. Amyb is also expressed during early stages of spermatogenesis in X.leavis (Sleeman, 1993), suggesting that its function in germ cell development is conserved among vertebrates. Finally, A-myb is also expressed in B lymphocytes. Primary follicles, which contain small resting B cells, do not express A-myb; by contrast, secondary follicles (germinal centers), generated in the course of a T celldependent immune response and containing proliferating
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Fig. 10. Transactivation of mvb-responsive reporter genes by A-myb and CIEBP transcription factors. QT6 cells (A, B and D) or HDl 1 cells (C) were cotransfected with 3 gg of the reporter plasmids shown at the top and different combinations of expression vectors, as indicated below the columns. In (A-C), 3 gg of each expression vector were used. In (D), the A-myb expression vector was used at I pg and the C/EBP expression vectors were used at 0.5 tg per plate. To control the transfection efficiencies the cells were additionally transfected with 2 ,ug pCHI 10. Cells were harvested 24 h after transfection and analyzed for luciferase and 1-galactosidase activities. Transfection efficiencies were normalized with respect to the cotransfected pCH 110 plasmid. The columns show the average activation factors of the luciferase reporter genes. The activity of each reporter gene in the absence of exogenous transactivator was arbitrarily designated as 1. Thin lines show the standard deviations.
B cells (Liu et al., 1992), show A-myb expression. Thus, A-myb is active in proliferating but not in resting B cells, in agreement with the observation that A-myb is expressed in in vivo-activated tonsillar B cells but not in resting B cells (Golay et al., 1991). Taken together, our results suggest a role for A-myb in the development of the CNS and the visual and olfactory
systems, and in the establishment of the germ cell and B-
cell
lineages.
It is
striking
that all
A-myb-expressing
Mouse A-myb
C
A
B 1
2
1
2
3
mim-1 67
0.
93
..S
Iys
S:,
MD-1
GAPDH
Fig. 11. Stable expression of mouse A-myb in HDl I cells. (A) Immunoprecipitation of [35S]methionine-labeled proteins from HDI 1 cells (lane 1) and a subclone of HDl 1 cells (MA-5) stably expressing mouse A-myb (lane 2), using mouse A-mvb-specific antibodies. The sizes (in kDa) and positions of molecular weight markers are indicated. (B) Polyadenylated RNA from mouse A-myb-expressing HDII subclone MA-5 (lane 1), two G418-resistant HDI I subclones not expressing myb protein (lanes 2 and 3) and the parental HD 11 cell line (lane 4) were analyzed by Northern blotting using probes specific for mim-l, the chicken lysozyme gene, MD-I and GAPDH. (C) Photomicrographs of cells of a G418-resistant control subclone of HD II cells carrying the empty pCDNA3 expression vector (top) and the mouse A-mvb-expressing clone MA-5.
cells identified are proliferating precursors of certain cell lineages and that they cease to express A-myb during terminal differentiation. Since the trans-activation potentials of A-myb and c-myb are very similar, our results suggest that A-myb and c-myb are functional homologs, each acting in a specific spectrum of cell lineage.
Materials and methods Isolation and characterization of a mouse A-myb cDNA clone By comparing human and chicken A-mvb protein sequences we identified domains likely to be conserved among A-myb from different vertebrates. Degenerate oligonucleotide primers 5'-GA(CT)(ACT)TATT(CT)CTCAA(AG)GA-3' and 5'-GT(CT)TT(CT)CCATAAAC(ACGT)AC(ACGT)G(ACGT)TTCCCC-3' (corresponding to amino acids 596-572 and 714-
723 of human A-mvb) were then used as PCR primers on reversetranscribed RNA from the mouse K46 B-cell line. The resulting PCR product was sequenced and used to screen mouse cDNA libraries. In addition, the sequence obtained from the PCR product was used to construct a specifically primed cDNA library from the K46 cell line. Several clones containing the ATG start codon were isolated from this library. The 3' of the A-myb cDNA was initially recovered by screening a commercial mouse bone marrow cDNA library (Clonetech) with the PCR-derived mouse A-myb fragment. Subsequently, using sequence information obtained from the bone marrow-derived A-myb clone, the 3' end was also PCR-amplified from reverse-transcribed K46 RNA. The A-myb sequence shown in Figure I is thus completely derived from the K46 cell line.
Cell lines and mice
HDI I is a line of MC29-transformed chicken macrophages obtained from T.Graf and H.Beug. QT6 is a line of chemically transformed quail fibroblasts (Moscovici et al., 1977). Adult organs and embryos were obtained from NMRI and C57BL/6 mice.
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Northern blotting Preparation of polyadenylated RNA, Northern blotting and detection of the chicken mim- 1, lysozyme, MD- I and GAPDH RNAs were performed as described (Burk and Klempnauer, 1991). Mouse A-myb RNA was detected by using nucleotide sequences downstream of a HindIll restriction site (located at amino acid 290 of A-myb) as probe. In situ hybridization analysis The procedure used in this study followed the protocol of Kessel and Gruss (1991). To prepare [35S]ATP-labeled hybridization probes, an 870 bp HindlIl fragment from the 3' end of A-myb, which corresponds to amino acids 567-657 of the A-myb coding region and does not hybridize to c-myb or B-myb mRNA (data not shown), was cloned into plasmid pBluescriptKSM13- (Stratagene). Sense and antisense probes were generated by in vitro transcription using T7 and T3 polymerase,
respectively.
Eukaryotic expression vectors To construct the A-myb expression vector cDNA3mAmyb, the sequence upstream of the A-myb translational start codon was first modified by PCR to GAA17CAGATCTCACCATGCCC (the ATG codon is in bold). The complete mouse A-myb coding region was cloned between the EcoRI and XbaI sites of the CMV promoter containing plasmid pCDNA3 (Stratagene), using the artificial EcoRI inserted upstream of the start codon and a XbaI site located downstream of the translational stop codon and derived from the polylinker of the pBluescript plasmid. The chicken c-myb expression vectors pCM100 and pCM101 have been described (Foos et al., 1992). Expression vectors for rat C/EBPa and mouse C/EBPf and 8 were obtained from S.McKnight (Friedman et al., 1989; Cao et al., 1991). Reporter genes, transfections, CAT and luciferase assays and G418 selection Reporter plasmids p-8lTk-Luc (Nordeen, 1988), p3xATk-Luc, p-240Luc (Ness et al., 1989), pSV2B2 (Klempnauer et al., 1989) and pCH 10 (Pharmacia) were transfected by the calcium phosphate coprecipitation method (Graham and van der Eb, 1973). Preparation of cell extracts, luciferase and ,B-galactosidase assays were performed as described (Burk et al., 1993). G418-resistant cells were selected in the presence of 400 tg/ml G418 and cultivated further at 200 ,ug/ml G418.
Interspecific mouse backcross mapping Interspecific backcross progeny were generated by mating (C57BL/ 6JXM.spretus)F1 females and C57BL/6J males as described (Copeland and Jenkins, 1991). A total of 205 N2 mice were used to map the Amyb locus (see text for details). DNA isolation, restriction enzyme digestion, agarose gel electrophoresis, Southern blot transfer and hybridization were performed as described (Jenkins et al., 1982). The probe, a 400 bp mouse cDNA clone, was labeled with [x-32P1dCTP using a nick translation labeling kit (Boehringer Mannheim); washing was carried out to a final stringency of O.5X SSCP, 0.1% SDS, 65°C. Fragments of 7.4 and 2.1 kb were detected in EcoRV-digested C57BL/ 6J DNA, and a fragment of 9.1 kb was detected in EcoRV-digested M.spretus DNA. The presence or absence of the 9.1 kb M.spretusspecific EcoRV fragment was followed in backcross mice. A description of the probes and RFLPs for the loci linked to A-myb, including Bpagl, Illr1 and Ctla4, has been reported (Copeland et al., 1993a,b). Recombination distances were calculated as described (Green, 1981)
using the computer program SPRETUS MADNESS. Gene order was determined by minimizing the number of recombination events required to explain the allele distribution patterns.
Acknowledgements We thank M.Kosco-Vilbois (Basel Institute for Immunology) for her advice on spleen staining and in situ hybridization data, R.Balling, E.-M.Fuichtbauer and K.Schughart for helpful discussions, B.Cho, U.Kerber and S.Vucikuja for excellent technical assistance, N.Nomura and S.L.McKnight for providing plasmids and L.Lay for preparing the photographs. This work was supported by the National Cancer Institute, DHHS, under contract NOI-CO-74101 with ABL, by grants from the DFG (Kl 461/5-1 and KI 461/5-2) and by the Max Planck Society.
References Bard,J.B.L. and Ross,A.S. (1982a) Dev. Biol., 92, 73-86. Bard,J.B.L. and Ross,A.S. (1982b) Dev. Biol., 92, 87-96.
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Barletta,C., Druck,T., LaForgia,S., Calabretta,B., Drabkin,H., Patterson, D., Croce,C.M. and Huebner,K. (1991) Ccancer Res., 51, 3821-3824. Biedenkapp,H., Borgmeyer,U., Sippel,A.E. and Klempnauer,K.-H. (1988) Nature, 355, 835-837. Bouwmeester,T., Guehmann,S., El-BaradiT., Kalkbrenner,F., van Wijk,l., Moelling,K. and Pieler,T. (1992) Mech. Dev., 37, 57-68. Burk,O. and Klempnauer,K.-H. (1991) EMBO J., 10, 3713-3719. Burk,O., Mink,S., Ringwald,M. and Klempnauer,K.-H. (1993) EMBO J., 12, 2027-2038. Cao,Z., Umek,R.M. and McKnight,S.L. (1991) Genes Dev., 5, 15381552. Clarke,M.F., Kukowska-Latallo,J.F., Westin,E., Smith,M. and Prochownik, E.V. (1988) Mol. Cell. Biol., 8, 884-892. Copeland,N.G. and Jenkins,N.A. (1991) Trends Genet., 7, 113-118. Copeland,N.G., Gilbert,D.J., Li,K., Sawamura,D., Guidice,G.J., Chu,M.L., Jenkins,N.A. and Uitto,J. (1993a) Genomics, 15, 180-181. Copeland,N.G. et al. (1993b) Science, 262, 57-66. Cuschieri,A. and Bannister,L.H. (1975) J. Anat., 119, 471-498. Foos,G., Grimm,S. and Klempnauer,K.-H. (1992) EMBO J., 11, 46194629. Frampton,J., Gibson,T.J., Ness,S.A., Doderlein,G. and Graf,T. (1991) Protein Engng, 4, 891-901. Friedman,A.D., Landschult,W.H. and McKnight,S.L. (1989) Genes Dev., 3, 1314-1322. Garcia,A., LaMontagne,K., Reavis,D., Stober-Grasser,U. and Lipsick,J.S. (1991) Oncogene, 6, 265-273. Gerondakis,S. and Bishop,J.M. (1986) Mol. Cell. Biol., 6, 3677-3684. Gewirtz,A.M. and Calabretta,B. (1988) Science, 242, 1303-1306. Ginsburg,M., Snow,M.H.L. and McLaren,A. (1990) Development, 110, 521-528. Golay,J., Capucci,A., Arsura,M., Castellano,M., Rizzo,V. and Introna,M. (1991) Blood, 77, 149-158. Gonda,T.J., Gough,N.M., Dunn,A.R. and de Blaquiere,J. (1985) EMBO J., 4, 2003-2008. Graf,T. (1992) Curr Opin. Genet. Dev., 2, 249-255. Graham,F.L. and van der Eb,A.J. (1973) Virology, 52, 456-467. Green,E.L. (1981) In Green,E.L. (ed.), Genetics and Probability in Animal Breeding Experiments. Oxford University Press, New York, pp. 77-113. Hinds,J.W. and Hinds,P.L. (1974) Dev. Biol., 37, 381-416. Hinds,J.W. and Hinds,P.L. (1978) J. Comp. Neurol., 179, 277-300. Howe,K.M., Reakes,C.F.L. and Watson,R.J. (1990) EMBO J., 9,161-169. Ibanez,C.E. and Lipsick,J.S. (1990) Mol. Cell. Biol., 10, 2285-2293. Jenkins,N.A., Copeland,N.G., Taylor,B.A. and Lee,B.K. (1982) J. Virol., 43, 26-36. Katzen,A.L., Kornberg,T.B. and Bishop,J.M. (1985) Cell, 41, 449-456. Kessel,M. and Gruss,P. (1991) Cell, 67, 89-104. Kim,K.J., Kanellopoulos-Langevin,C., Merwin,R.M., Sachs,D.H. and Asofsky,R. (1979) J. Immunol., 122, 549-554. Klein,S.L. and Grazadei,P.P.C. (1983) J. Comp. Neurol., 217, 17-30. Klempnauer,K.-H. and Sippel,A.E. (1986) Mol. Cell. Biol., 6, 62-69. Klempnauer,K.-H. and Sippel,A.E. (1987) EMBO J., 6, 2719-2725. Klempnauer,K.-H., Arnold,H. and Biedenkapp,H. (1989) Genes Dev., 3, 1582-1589. Lam,E.-W.F., Robinson,C. and Watson,R.J. (1992) Oncogene, 7, 18851890. Liu,Y.-J., Johnson,G.D., GordonJ. and Mac Lennan,I.C.M. (1992) Immunol. Today, 13, 17-21. Monaghan,A.P., Davidson,D.R., Sime,C., Graham,E., Baldock,R., Bhattacharya,S.S. and Hill,R.E. (1991) Development, 112, 1053-1061. Moscovici,C., Moscovici,M.G., Jiminez,H., Lai,M.M.C., Hayman,M.J. and Vogt,P.K. (1977) Cell, 11, 95-103. 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) Cell, 65, 677-689. Ness,S.A., Marknell,A. and Graf,T. (1989) Cell, 59, 1115-1125. Ness,S.A., Kowentz-Leutz,E., Casini,T., GrafT. and Leutz,A. (1993) Genes Dev., 7, 749-759. Nishina,Y., Nakagoshi,H., Imamoto,F., Gonda,T.J. and Ishii,S. (1989) Nucleic Acids Res., 17, 107-117. Nomura,N., Takahashi,M., Matsui,M., Ishii,S., Date,T., Sasamoto,S. and Ishizaki,R. (1988) Nucleic Acids Res., 16, 11075-11089. Nordeen,S.K. (1988) Biotechniques, 6, 454-457. Oehler,T., Arnold,H., Biedenkapp,H. and Klempnauer,K.-H. (1990) Nucleic Acids Res., 18, 1703- 17 10. Oppenheimer,D.G., Herman,P.L., Sivakumaran,S., Esch,J. and Marks,M.D. (1991) Cell, 67, 483-493.
Mouse A-myb Paz-Ares,J., Ghosal,D., Wienand,U., Peterson,P.A. and Saedler,H. (1987) EMBO J., 6, 3553-3558. Peters,C.W.B., Sippel,A.E., Vingron,M. and Klempnauer,K.-H. (1987) EMBO J., 6, 3085-3090. Rosson,D., Dugan,D. and Reddy,E.P. (1987) Proc. Natl Acad. Sci. USA, 84, 3171-3175. Sakura,H., Kanei-Ishii,C., Nagase,T., Nakagoshi,H., Gonda,T.J. and Ishii,S. (1989) Proc. Natl Acad. Sci. USA, 86, 5758-5762. Schuur,E.R., Dasgupta,P., Reddy,E.P., Rabinovich,J.M. and Baluda,M.A. (1993) Oncogene, 8, 1839-1847. Selvakumaran,M., Lieberman,D.A. and Hoffmann-Liebermann,B. (1992) Mol. Cell. Biol., 12, 2493-2500. Shen-Ong,G.L.C. (1990) Biochim. Biophvs. Acta, 1032, 39-52. Shen-Ong,G.L.C., Luscher,B. and Eisenman,R.N. (1989) Mol. Cell. Biol., 9, 5456-5463. Siu,G., Wurster,A.L., Lipsick,J.S. and Hedrick,S.M. (1992) Mol. Cell. Biol., 12, 1592-1604. Sleeman,J.P. (1993) Oncogene, 8, 1931-1941. Snow,M.H.L. and Monk,M. (1983) In McLaren,A. and Wylie,C.C. (eds), Current Problems in Germ Cell Differentiation. Cambridge University Press, Cambridge, UK, pp. 115-135. Stober-Grasser,U., Brydolf,B., Bin,X., Grasser,F., Firtel,R.A. and Lipsick,J.S. (1992) Oncogene, 7, 589-596. Tice-Baldwin,K., Fink,G.R. and Arndt,K.T. (1989) Science, 246, 931935. Weston,K. and Bishop,J.M. (1989) Cell, 58, 85-93.
Received on August 24, 1994; revised on October 11, 1994
Note added in proof The accession number for the nucleotide sequence of mouse A-mvb mRNA is X82327.
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