Vol. 11, No. 4

MOLECULAR AND CELLULAR BIOLOGY, Apr. 1991, p. 2189-2199 0270-7306/91/042189-11$02.00/0 Copyright C) 1991, American Society for Microbiology

Developmental Expression of Spl in the Mouse JEFFREY D. SAFFER,1* STEPHEN P. JACKSON,2t AND MARY B. ANNARELLA' The Jackson Laboratory, Bar Harbor, Maine 04609,1 and Department of Biochemistry, University of California, Berkeley, California 947202 Received 30 October 1990/Accepted 3 January 1991

The expression of the trans-acting transcription factor Spl in mice was defined by a combination of RNA analysis and immunohistochemical localization of the Spl protein. Although ubiquitously expressed, there was an unexpected difference of at least 100-fold in the amount of Spl message in different cell types. Spl protein levels showed corresponding marked differences. Substantial variations in Spl expression were also found in some cell types at different stages of development. Spl levels appeared to be highest in developing hematopoietic cells, fetal cells, and spermatids, suggesting that an elevated Spl level is associated with the differentiation process. These results indicate that Spl has a regulatory function in addition to its general role in the transcription of housekeeping genes.

The control of transcription is a key step in the regulation of gene expression. Although specific DNA elements around and within a gene contain the information directing this regulation, proteins that recognize these DNA sequences are the factors actually responsible for modulating RNA polymerase activity. Several sequence-specific DNA-binding transcription factors that are involved in the process of transcriptional regulation have been described, and much has been learned at the molecular level about their structure and function (24, 25). Some transcription factors are characterized by cell type-specific expression and are involved in regulating the specialized functions of that cell. For example, the Oct-2 protein is B cell specific (5, 30), and Myo Dl is muscle cell specific (7). In contrast, other factors appear to be ubiquitously expressed and involved in the transcription of genes expressed in all cell types. A well-characterized example of this type of factor is Spl, which is reportedly found in all mammalian cell nuclei (2). Although originally described as a specific factor required for simian virus 40 (SV40) transcription (9, 10), Spl interacts with GC boxes in the promoters of many other genes. For example, cellular genes that contain one or more GC boxes include adenosine deaminase (33), hypoxanthine guanine phosphoribosyl transferase (23), epidermal growth factor receptor (14), and DNA polymerase f (35). Also, other viral promoters, in addition to SV40, require Spl binding for transcriptional activity, including herpes simplex virus type 1 (19) and the AIDS retrovirus (human immunodeficiency virus type 1 or human T cell lymphotropic virus type III) (18). Several lines of evidence suggest a regulatory role for Spl. First, increasing the level of Spl in vitro (8, 9) or in Drosophila cells (6) leads to increased transcription from the SV40 promoter. More importantly, recent studies have shown that Spl is a limiting factor in cultured mammalian cells and that artifically elevated Spl expression in these cells increases expression from promoters containing GCbox elements (28). Second, the Spl-binding sites of different genes have an at least 10- to 20-fold range of binding affinities (21), indicating that transcription of genes with weak Spl*

binding sites will require larger amounts of Spl than are required for genes with strong binding sites. Thus, some genes will be more sensitive than others to variations in the level of Spl. Third, the observation that Spl levels increase during SV40 infection (15, 28) implies that Spl levels can be regulated. In this system, alteration of the normal Spl expression may support the SV40 viral life cycle and the accompanying cellular changes. To further investigate the regulation and action of Spl, we have determined Spl expression in different cell types during development of the mouse. In this paper, we show that although Spl is ubiquitously expressed, substantial variations that are likely to play an important regulatory role occur in Spl expression. These studies are relevant to defining (i) the physiological role of Spl in growth and development, (ii) the variations in expression of the many genes regulated by Spl, and (iii) the regulation of Spl itself. MATERIALS AND METHODS Mice. BALB/cByJ mice were raised within the research animal facilities at The Jackson Laboratory with a 12-h light cycle and a standard chow diet. Mice were handled according to the regulations of the American Association for the Accreditation of Laboratory Animal Care. The Jackson Laboratory is a fully credited member of this organization and strictly adheres to the regulations for the welfare and humane treatment of its animals. For timed pregnancies, the day on which plugs were observed was considered the first day of gestation. Suckling pups were weaned from their mothers on day 21. Mice younger than 10 days were anesthetized on ice and killed by decapitation; older mice were killed by cervical dislocation. These methods are consistent with the recommendations of the Panel on Euthanasia of the American Veterinary Medical Association. Since diurnal variations occur in the expression of some genes, all animals were killed between 9 and 10 a.m. Preparation of tissue RNA. For each age group, the organs or tissues to be used were rapidly dissected and placed in liquid nitrogen. The organs or tissues from four male mice were pooled and crushed in a mortar and pestle containing liquid nitrogen. Total-RNA preparation was based on the procedure of Jacobsson et al. (17). No more than 1 g of the resulting powder, along with the residual nitrogen, was transferred to a 15-ml polypropylene tube and homogenized

Corresponding author.

t Present address: The Wellcome CRC Institute, University of Cambridge, Cambridge, England CB21QR. 2189

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SAFFER ET AL.

with 1.8 ml of 8 M guanidine hydrochloride-10 mM EDTA for 2 min with a Brinkmann Polytron at a setting of 5. After the addition of 0.2 ml of 10% Sarkosyl, homogenization was continued for 15 s. The homogenate was centrifuged at 12,000 x g for 20 min at 4°C. Then 1 ml of the supernatant was transferred to a clean tube, 0.1 ml of 2 M sodium acetate (pH 6.5) was added, and the RNA was selectively precipitated by the dropwise addition of 0.5 ml ethanol. After exactly 2 h at -20°C, the RNA was pelleted as described above, resuspended in 1 ml of 8 M guanidine hydrochloride-10 mM EDTA-0.2 M sodium acetate, and reprecipitated by the addition of 0.5 ml ethanol and incubation at -20°C for another 2 h. The resulting RNA pellet was washed with a solution containing 8 M guanidine hydrochloride-10 mM EDTA:ethanol (2:1), resuspended in 0.1 ml of 15% formaldehyde-0.2 M sodium acetate, and precipitated by the addition of 0.25 ml of ethanol and incubation in a dry ice-ethanol bath for 10 min. The pellet resulting from centrifugation was washed in 70% ethanol. EDTA (10 mM) was added to the pellet, and the RNA was extracted by heating the pellet in a 70°C water bath for 10 min with vortexing every 2 min. Any remaining insoluble material was removed by centrifugation. For long-term storage, the RNA was precipitated with ethanol in the presence of 0.3 M sodium acetate and 0.1% sodium dodecyl sulfate (SDS). RNA analysis. For slot blot analysis, 2- and 0.5-,ug aliquots of the RNA samples were suspended in 10x SSC (1 x SSC is 0.15 M NaCl-15 mM Na citrate) and fixed to a Zetabind (AMF-Cuno, Meridin, Conn.) nylon membrane with a slot blot manifold (Life Technologies, Bethesda, Md.). The slot blot filters also contained 4 amounts of the Spl cDNA and 4 amounts of poly(A) as standards to compare hybridization signals on different membranes. For Northern (RNA) blot analysis, 5 FIg of RNA was electrophoresed through a 1.2% agarose-2.2 M formaldehyde gel (22) and transferred to a Zetabind membrane with 20x SSC. The filters were then washed in 2x SSC at room temperature. All membranes were UV shadowed (32) to ensure that equal amounts of total RNA had been fixed to the membrane. Hybridization conditions were a modification of those described by Church and Gilbert (4). Filters were washed for 1 h at 65°C in 0.1x SSC-0.5% SDS, prehybridized for at least 1 h in 7% SDS-0.5 M sodium phosphate-2 mM EDTA0.1% sodium pyrophosphate at 65°C, and hybridized in the same buffer containing 10 ,ug of poly(A) per ml, 50 ,ug of sheared salmon DNA per ml, and the radiolabeled probe. Filters were washed three times at room temperature in 2x SSC-0.1% SDS and then once at room temperature and twice at 60°C in 0.1 x SSC-0.1% SDS. Slot blot filters were first hybridized to a mouse Spl cDNA and then stripped and reprobed with an oligo(dT) probe. Hybridization and washing conditions with this second probe were identical to those described above except that no poly(A) was used as a competitor during hybridization and the final washes were at 50°C. Levels of hybridization were quantified on a Bio-Rad model 620 densitometer. The mouse Spl cDNA used for probing was a 3.2-kb insert from the clone pMusSpl-11 (29). The isolated insert was labeled with [a-32P]dCTP to a specific activity of approximately 109 cpm/,lg by the random primer method (11). The oligo(dT) probe was end labeled with [_y-32P]ATP and diluted with unlabeled oligo(dT) to 106 cpm/,g, with 2.5 ,ug/ml used for hybridizations. Immunohistochemistry. Organs and tissues were fixed in Tellyesniczky-Fekete fixative (3.2% formaldehyde, 4.3%

MOL. CELL. BIOL.

acetic acid, 61% ethanol) for 24 h and then with 70% ethanol for at least 24 h. The samples were embedded in paraffin by routine processing with a Miles Tissue Tek VIP processor. Sections (5 ,um) were placed on gelatin-coated slides for analysis. Endogenous peroxidase was blocked with 3% H202 in methanol, and sections were rinsed with ovalbumin to block nonspecific binding. Spl-specific antibodies were raised in rabbits against the polypeptide encoded by the cDNA clone Spl-1 (20) as expressed in Escherichia coli (serum no. 2892) or against HeLa cell Spl (serum no. 2873). A Vector (Burlingame, Calif.) avidin-biotin kit was used for immunoperoxidase staining with diaminobenzidine as the substrate. A light-green counterstain was used to visualize cellular structures. Serial sections were also stained with preimmune serum from each rabbit as a control. Additional sections were stained with hematoxylin-eosin to help identify cell types and structures (data not shown). Immunohistochemistry with a mouse antihistone antibody (Chemicon, Temecula, Calif.) revealed essentially uniform staining in the analyzed tissues, thus ruling out a differential loss of nuclear proteins during fixation and staining. RESULTS Spl is differentially expressed in mouse organs and tissues. To address the questions of whether Spl levels vary in different cell types in vivo and whether some cells show variations during development and differentiation, 11 organs and tissues in mice at eight ages from 5 to 64 days were studied. The expression of Spl in the mouse was determined by two complementary approaches: analysis of Spl mRNA levels by slot blot and Northern techniques and analysis of Spl protein expression by immunocytochemical staining. By analyzing both RNA and protein, regulation at the transcriptional and posttranscriptional levels may be uncovered, and expression in specific cell types can be defined. For the RNA analysis, the expression of Spl message relative to the total amount of poly(A)+ RNA in the organs and tissues was determined. In this way, the results do not reflect general changes in gene transcription. Each slot blot was hybridized with a mouse Spl cDNA probe and, after stripping the filter, rehybridized with labeled oligo(dT) as a measure of the total message present. Although inbred, age-matched mice were used for these experiments, the RNA analysis was carried out on RNA prepared from the organs and tissues pooled from four animals, except as noted. Thus, the results are an average and do not reflect potential individual-to-individual variations. There was a 100-fold variation in the level of Spl message in these samples (Fig. 1). The highest level of expression was observed in the thymus, which peaked at 35 days. High levels of Spl mRNA were also found in the lung and spleen. The lowest level of expression was in mature thigh muscle. For most organs, the amount of hybridization signal resulting from the oligo(dT) probe was fairly constant during development, and thus, the Spl levels presented also reflect expression relative to the total amount of RNA. The exception to this was in testes, for which a substantial increase in poly(A)+ RNA was observed. Thus, although the amount of Spl message actually increased relative to the total amount of RNA, Spl expression decreased relative to overall message levels. The specificity of the Spl cDNA probe was confirmed with Northern blots of total RNA from the different tissues. In each case only an 8.2-kb message was detected (data not shown), except in the testes as discussed below.

VOL. 11, 1991

5

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summarized from several hybridizations, represent Spi expression relative to the total message present and have been standardized to data from the organ expressing the highest levels (thymus from 35-day-old mice). The intensity of the background depicts the relative expression of Spi. Standard errors are shown for each value except when only one sample was analyzed (indicated by (Note that in other experiments using single animals, the Spi

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For immunological detection of Spl in the mouse organs and tissues, two separate antisera were used. One is from a rabbit immunized against purified HeLa cell Spl (serum no. 2873), and the other is from a rabbit immunized against the polypeptide encoded by the Spl-1 cDNA (20) as expressed in E. coli (serum no. 2892). Although each of these reagents reacts primarily against Spl, minor bands were seen for each in Western immunoblot analysis of mouse cell proteins (data not shown). For this reason, immunohistochemical analysis was carried out with each reagent separately. Spl staining was indicated by a nuclearly localized signal, present in both antisera and absent in both preimmune control sera. Also note that immunohistochemical analysis with an antihistone antibody yielded uniform staining (data not shown), so that differential staining observed for Spl was not due to selective fixation or a loss of nuclear proteins from some cells. All cell types analyzed in this report expressed at least some Spl protein. However, there were clear differences in the level of expression in specific cell types. In Fig. 2, representative sections of organs and tissues from 30- and 35-day-old mice are presented. Except as noted below, the staining in these organs and tissues was qualitatively similar at each age examined. In the kidney, staining for Spl in the medulla was most pronounced in the collecting tubules (Fig. 2A), while staining in the cortex was highest in the glomeruli

(Fig. 2B). Spl expression in the lung was most notable in both the type 1 and type 2 cells of the alveoli (Fig. 2C). In the brain, the cerebral cortex (Fig. 2D) showed very light staining of most nuclei, with darker staining associated with the nuclei of cells surmised to be motor neurons; the choroid plexus (Fig. 2E) showed a moderate expression of Spl. In the cerebellum (Fig. 2F), the molecular layer (top) contains very few nuclei and correspondingly showed little staining; the granular layer (left and middle) exhibited a fairly uniform light stain; and the white matter (below the granular layer) contained a few cells, tentatively identified as oligodendrocytes, with intensely staining nuclei. Both the T cells and epithelial cells of the thymus (Fig. 2G) showed high levels of Spl expression. No notable differences were observed between the cortex and the medulla; however, the surrounding capsule of fat cells (bottom) showed minimal staining. In the spleen (Fig. 2H) Spl protein expression was also high, correlating with the mRNA levels; regions of white pulp (as shown) had an apparently higher level of Spl protein than the red pulp (not shown), although this appearance was due in large part to the density of nuclei. At the mRNA level, Spl expression in the heart was considerably greater than in thigh muscle. In contrast, at the protein level similar staining was observed in individual

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FIG. 2. Spl protein expression in mouse tissues. Paraffin sections of each organ or tissue were assayed for Spl protein expression immunologically. Immunohistochemistry was carried out with the two antisera described (see Materials and Methods) on organs or tissues from mice of the ages indicated below. Staining with each reagent was similar, and only representative sections with antiserum no. 2892 are presented (capital letters) along with control sections stained with the corresponding preimmune serum (lowercase letters). Bars, 25 p.m. Tissues and mouse ages: A, kidney medulla, 30 days; B, kidney cortex, 30 days; C, lung, 30 days; D, cerebrum, 35 days; E, choroid plexus, 35 days; F, cerebellum, 35 days; G, thymus, 30 days; H, spleen, 30 days; I, heart, 30 days; J, thigh, 30 days; K, epididymis, 30 days; L, ileum, 35 days.

nuclei in both tissues (Fig. 21 and J). For comparison, the nuclei of capillary cells are present at the bottom of Fig. 2J. Two additional organs not examined at the RNA level were stained for Spl protein. These two, epididymis (Fig. 2K) and ileum (Fig. 2L), demonstrate the extremes of Spl protein expression. The columnar epithelial cells in the epididymis showed striking staining, while Spl expression in all cells of the ileum was barely detectable. Spl protein expression in the stomach changes with development. Development of the digestive tract continues through the weaning age (in mice, about 3 weeks). Although striking biochemical changes occur in the small intestine during this time, no obvious changes in Spl expression were noted (data not shown). In contrast, noticeable changes occurred in the distribution of Spl in the stomach (Fig. 3). The relatively even expression of Spl in the young milk-fed mice shifts to a predominant expression in the fundic glands, with both the oxyntic and peptic cells showing increased Spl

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expression as the animal begins a grain diet. This higher level of Spl protein continues into adulthood. One notable feature of the immunohistochemistry was the nonuniform staining of some nuclei. This can be seen in some other sections (e.g., Fig. 2D) but is more frequent in the stomach. The nature of this distribution or its potential role is not clear. Cell-specific expression in the testes correlates with a novel transcript. In the seminiferous tubules of the testes, spermatogenesis occurs radially inward from the spermatagonia to spermatocytes to spermatids and finally the spermatozoa. Spl protein expression was highest in the spermatids found in sexually mature animals (Fig. 4). In older animals (25 days and older), staining in different tubules varied. For example, in the 64-day sample, a tubule at the left exhibited weaker staining than the two tubules in the center. This variation was not unexpected, since there are longitudinal variations in the activity of the seminiferous tubules. Figure 4 shows the highest expression in the testes at each age.

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In all tissues examined, including the ovary (data not shown), the Spl message was 8.2 kb long. However, as the testes matured, a 2.4-kb message appeared (Fig. 5). Since the epididymis contains only the 8.2-kb Spl mRNA, the novel message size was not attributable to the spermatozoa. Interestingly, the common large Spl message contains more than 5 kb of sequence not required for the Spl protein, while the testis-specific mRNA is barely long enough to code for a full-length protein. Differentiating hematopoietic cells express high levels of Spl. In the immunohistochemical analysis of the liver, some cells with very striking staining were observed (Fig. 6A). Although the hepatocytes showed a low level of Spl expression, cells expressing a very high level of Spl were interspersed throughout the liver with no apparent regional

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specificity. These darkly staining cells were noted in 12.5day fetuses (see Fig. 7e to g), the earliest fetuses examined with regard to developing liver, and in 18-day fetuses (data not shown). However, these cells disappeared rapidly after birth and were absent by 23 days (Fig. 6B). This time course suggested that the cells expressing very high levels of Spl were of hematopoietic origin, since the fetal and neonatal livers are important hematopoietic organs. To define the cell type precisely, 10 serial sections were prepared from a neonatal liver and every other section was stained for Spl expression. The alternate sections were treated with hematoxylin-eosin, Gomori's iron pigment stain, toluidine blue, and May-Gruenwald-Giemsa stain and stained for myeloperoxidase activity. Those results (data not shown) strongly suggested that the cells expressing high levels of Spl were developing granulocytes. As further support for this identification, different populations of hematopoietic cells were obtained by culturing bone marrow cells in the presence of different growth factors. Immunofluorescent analysis of Spl expression in those cells (data not shown) indicated that metamyelocytes, a precursor to granulocytes, had a high level of Spl expression consistent with that observed in the liver. In contrast to the immunohistochemical analyses presented earlier, there was a notable difference in the staining intensity of the developing granulocytes when the two antisera described above were used. The antiserum against the HeLa-cell Spl stained the nuclei of these cells much more darkly than the hepatocytes but not in a manner as striking as that observed in Fig. 6 and 7. We are investigating the

Spl EXPRESSION IN THE MOUSE

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the many trans-acting factors involved, the variations in Spl will be one contribution to that overall regulation. Since elevating Spl expression in tissue culture cells does increase expression from promoters containing a GC box (28), observed differences in Spl levels are likely to result in profound changes in gene transcription. For example, in the testes, expression of Spl is highest in the spermatids. In these cells, the protamine genes are highly transcribed (12) and the protamine 2 gene, Prm-2, contains a putative Spl-

binding site within the 5' sequences required for positive regulation (3). We therefore speculate that the high expression of protamine is due to the increased Spl levels. The elevated levels of Spl in spermatids may, in part, result from translation of the unique 2.4-kb mRNA. This smaller transcript was not found in the epididymis (Fig. 5) or ovary (data not shown), suggesting that it arises from testisspecific processes. Alternately sized transcripts in testes have been described for many genes. For example, the

2198

SAFFER ET AL.

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TABLE 1. Relative abundance of Spl in different cell typesa Cells in which relative abundance of Spl/nucleus was:

Organ or

tissue

Kidney Lung Brain Thymus Spleen Heart Thigh Fat Intestine Testis Epididymis Stomach Liver

Moderate

Low

Purkinje cells, neuroglial cells

High

Collecting tubules, glomeruli Type 1 cells, type 2 cells Motor neurons, oligodendrocytes, fetal (12.5-day) neural cells T cells, epithelial cells Hematopoietic cells

Muscle fibers Muscle fibers Adipocytes All cell types Spermatagonia, Spermatocytes Squamous epithelium, smooth muscle cells Hepatocytes (adult), Kupffer cells

Hepatocytes (fetal)

Spermatids Columnar epithelial cells Oxyntic cells, peptic cells Metamyelocytes (neonate)

a Summary of the data from immunohistochemical localization of Spl. The list does not include every cell type found in each organ or tissue but is intended to give an indication of the variation in Spl abundance in various cell types.

retinoblastoma (Rb) gene encodes a 4.7-kb message, but in mature testes a novel 2.8-kb message is also found (1). The c-mos proto-oncogene encodes a testis-specific 1.7-kb message, larger than the 1.4-kb mRNA found in ovaries (27). The origin and function of the testis-specific Spl transcripts have yet to be defined. For the most part, Spl mRNA expression and protein levels were related. This suggests that the major control of Spl expression is at the mRNA level. However, there were apparent exceptions to this relationship. In the stomach, no significant change in the Spl mRNA level was observed despite the notable increase of Spl protein in the fundic glands. This could reflect changes in posttranscriptional regulation, including protein stability. Alternatively, the increased expression within the fundic glands could be offset by a decrease in the rest of the stomach. In testes, there was an age-associated increase in Spl protein, which was localized in spermatids. Although the amount of Spl message relative to overall poly(A)+ RNA production decreased, the amount of Spl message relative to total RNA did increase. The high expression of Spl in the spermatids and in developing granulocytes suggests an association of elevated Spl with the final stages of differentiation. This could also be a factor contributing to the high levels of Spl in the thymus and spleen, since both contain differentiating hematopoietic cells. Consistent with this hypothesis of elevated Spl levels during differentiation was the high level of Spl expression in the developing tissues of the 8.5- and 12.5-day fetuses. Alternatively, the rapid growth rate of the fetal cells, including the decidua, and of the developing hematopoietic cells could explain the higher Spl levels. Indeed, a correlation between Spl and cell growth could be expected, since Spl regulates the housekeeping functions required for cell growth. Although this model may be true to an extent, the lack of a consistent correlation, as with intestinal crypt cells (Fig. 2L) that divide every 9 to 13 h (26) but contain very little Spl protein, suggests there are other considerations and reflects the complexity of gene regulation. While the cells expressing high levels of Spl are of great interest, it is also important to note the cells expressing low levels of Spl. Well-developed, fully differentiated cells with specialized functions for the most part exhibited the lowest amounts of Spl. These cells, such as hepatocytes, probably require some Spl for housekeeping functions, but transcrip-

tion of the more abundant messages in these cells, e.g., albumin, does not require Spl. Although much needs to be learned about the role of Spl in growth and development, we have demonstrated that substantial differences exist in Spl expression in different cell types through development. These data suggest that Spl is not simply a general factor required for transcription of a multitude of housekeeping genes. Rather, Spl is likely to play an important regulatory role in cellular processes during development and differentiation. ACKNOWLEDGMENTS We thank Robert Evans and Ted Duffy for setting up the bone marrow cultures and for many helpful discussions, Jane Barker for help in identifying hematopoietic cells, and John Sundberg for much help with pathology. Priscilla Jewett and Ann Higgins provided expert assistance with the immunohistochemistry. Special thanks to Sally Thurston for support, encouragement, and helpful discussions throughout the work. This work was funded in part by The Jackson Laboratory grant 89-13. S.P.J. was supported by a Lucille P. Markey fellowship.

REFERENCES 1. Bernards, R., G. M. Schackleford, M. R. Gerber, J. M. Horowitz, S. H. Friend, M. Schartl, E. Bogemnann, J. M. Rapaport, T. McGee, T. P. Dryja, and R. A. Weinberg. 1989. Structure and expression of the murine retinoblastoma gene and characterization of its encoded protein. Proc. Natl. Acad. Sci. USA 86:

6474-6478.

2. Briggs, M. R., J. T. Kadonaga, S. P. Bell, and R. Tjian. 1986. Purification and biochemical characterization of the promoterspecific transcription factor, Spl. Science 234:47-52. 3. Bunick, D., P. A. Johnson, T. R. Johnson, and N. B. Hecht. 1990. Transcription of the testis-specific mouse protamine 2 gene in a homologous in vitro transcription system. Proc. Natl. Acad. Sci. USA 87:891-895. 4. Church, G. M., and W. Gilbert. 1984. Genomic sequencing. Proc. Natl. Acad. Sci. USA 81:1991-1995. 5. Clerc, R. G., L. M. Corcoran, J. H. LeBowitz, D. Baltimore, and P. A. Sharp. 1988. The B-cell-specific Oct-2 protein contains POU box- and homeo box-type domains. Genes Dev. 2:15701581. 6. Courey, A. J., and R. Tjian. 1988. Analysis of Spl in vivo reveals multiple transcriptional domains, including a novel glutamine-rich activation motif. Cell 55:887-898. 7. Davis, R. L., H. Weintraub, and A. B. Lassar. 1987. Expression of a single transfected cDNA converts fibroblasts to myoblasts.

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Cell 51:987-1000. 8. Dynan, W. S., J. D. Saffer, W. S. Lee, and R. Tjian. 1985. Transcription factor Spl recognizes promoter sequences from the monkey genome that are similar to the simian virus 40 promoter. Proc. Natl. Acad. Sci. USA 82:4915-4919. 9. Dynan, W. S., and R. Tjian. 1983. Isolation of transcription factors that discriminate between different promoters recognized by RNA polymerase II. Cell 32:669-680. 10. Dynan, W. S., and R. Tjian. 1983. The promoter-specific transcription factor Spl binds to upstream sequences in the SV40 promoter. Cell 35:79-87. 11. Feinberg, A. P., and B. Vogelstein. 1983. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132:6-13. 12. Hecht, N. B. 1990. Regulation of 'haploid expressed genes' in male germ cells. J. Reprod. Fertil. 88:679-693. 13. Hirai, S.-I., R. P. Ryseck, F. Mechta, R. Bravo, and M. Yaniv. 1989. Characterization of junD: a new member of the jun proto-oncogene family. EMBO J. 8:1433-1439. 14. Ishii, S., Y.-H. Xu, R. H. Stratton, B. A. Roe, G. T. Merlino, and I. Pastan. 1985. Characterization and sequence of the promoter region of the human epidermal growth factor receptor gene. Proc. Natl. Acad. Sci. USA 82:4920-4924. 15. Jackson, S. P., J. J. MacDonald, S. Lees-Miller, and R. Tjian. 1990. GC box binding induces phosphorylation of Spl by a DNA-dependent protein kinase. Cell 63:155-165. 16. Jackson, S. P., and R. Tjian. 1988. 0-glycosylation of eukaryotic transcription factors: implications for mechanisms of transcriptional regulation. Cell 55:125-133. 17. Jacobsson, A., U. Stadler, M. A. Glotzer, and L. P. Kozak. 1985. Mitochondrial uncoupling protein from mouse brown fat. J. Biol. Chem. 260:16250-16254. 18. Jones, K. A., J. T. Kadonaga, P. A. Luciw, and R. Tjian. 1986. Activation of the AIDS retrovirus promoter by the cellular transcription factor, Spl. Science 232:755-759. 19. Jones, K. A., and R. Tjian. 1985. Spl binds to promoter sequences and activates herpes simplex virus 'immediate-early' gene transcription in vitro. Nature (London) 317:179-182. 20. Kadonaga, J. T., K. R. Carner, F. R. Masiarz, and R. Tjian. 1987. Isolation of cDNA encoding transcription factor Spl and functional analysis of the DNA binding domain. Cell 51:10791090. 21. Kadonaga, J. T., K. A. Jones, and R. Tjian. 1986. Promoterspecific activation of RNA polymerase II transcription by Spl. Trends Biochem. Sci. 11:20-23. 22. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory,

Spl EXPRESSION IN THE MOUSE

2199

Cold Spring Harbor, N.Y. 23. Melton, D. W., D. S. Konecki, J. Brennand, and C. T. Caskey. 1984. Structure, expression, and mutation of the hypoxanthine phosphoribosyltranferase gene. Proc. Natl. Acad. Sci. USA 81:2147-2151. 24. Mitchell, P. J., and R. Tjian. 1989. Transcriptional regulation in mammalian cells by sequence-specific DNA binding proteins. Science 245:371-378. 25. Polyanovsky, 0. L., and A. G. Stepchenko. 1990. Eukaryotic transcription factors. Bioessays 12:205-210. 26. Potten, C. S., R. Scholfield, and L. G. Lajtha. 1979. A comparison of cell replacement in bone marrow, testis, and three regions of surface epithelium. Biochim. Biophys. Acta 560:281299. 27. Propst, F., and G. F. Vande Woude. 1985. Expression of c-mos proto-oncogene transcripts in mouse tissues. Nature (London) 315:516-518. 28. Saffer, J. D., S. J. Jackson, and S. J. Thurston. 1990. SV40 stimulates expression of the trans-acting factor Spl at the mRNA level. Genes Dev. 4:659-666. 29. Saffer, J. D., S. J. Thurston, M. B. Annarelia, and J. G. Compton. 1990. Localization of the gene for the trans-acting transcription factor Spl to the distal end of mouse chromosome 15. Genomics 8:571-574. 30. Staudt, L. M., H. Singh, R. Sen, T. Wirth, P. A. Sharp, and P. A. Baltimore. 1986. A lymphoid-specific protein binding to the octamer motif of immunoglobulin genes. Nature (London) 323:640-643. 31. T'Ang, A., K.-J. Wu, T. Hashimoto, W.-Y. Liu, R. Takahashi, X.-H. Shi, K. Mihara, F.-H. Zhang, Y. Y. Chen, C. Du, J. Qian, Y.-G. Lin, A. L. Murphee, W.-R. Qiu, T. Thompson, W. F. Benedict, and Y.-K. T. Fung. 1989. Genomic organization of the human retinoblastoma gene. Oncogene 4:401-407. 32. Thurston, S. J., and J. D. Saffer. 1989. Ultraviolet shadowing nucleic acids on nylon membranes. Anal. Biochem. 178:41-42. 33. Valerio, D., M. G. C. Duyvesteyn, B. M. M. Dekker, G. Weeda, T. M. Berkvens, L. van der Voorn, H. van Ormondt, and A. J. van der Eb. 1985. Adenosine deaminase: characterization and expression of a gene with a remarkable promoter. EMBO J. 4:437-443. 34. Wilkinson, D. G., S. Bhatt, R. R.-P. Ryseck, and R. Bravo. 1989. Tissue-specific expression of c-jun and junB during organogenesis in the mouse. Development 106:465-471. 35. Yamaguchi, M., Y. Hayashi, and A. Matsukage. 1988. Mouse DNA polymerase P gene promoter: fine mapping and involvement of Spl-like mouse transcription factor in its function. Nucleic Acids Res. 16:8773-8787.