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Expression of the POU Transcription Factor Brn-5 Inhibits Proliferation of NG108-15 Cells
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
236, 693–696 (1997)
RC976996
Expression of the POU Transcription Factor Brn-5 Inhibits Proliferation of NG108-15 Cells Hong Cui and Robert F. Bulleit1 Department of Pharmacology, University of Maryland School of Medicine, Baltimore, Maryland 21201
Received June 9, 1997
The POU domain transcription factors are a subgroup of homeodomain proteins that appears to control cellular phenotypes. The expression of the POU protein Brn-5 occurs selectively in postmitotic CNS neurons. Ectopic expression of Brn-5 in dividing NG108-15 cells reduces the level of RNA encoding the proliferating cell nuclear antigen (PCNA). This ectopic expression also inhibits DNA synthesis as measured by the incorporation of bromodeoxyuridine (BrdU). Thus, Brn-5 may inhibit the continued proliferation of these cells. A potential function of Brn-5 may be to suppress the action of proliferative signals in postmitotic neurons and thus prevents them from reentering the cell cycle. q 1997 Academic Press
Neurogenesis in the mammalian central nervous system (CNS) involves a transition between cell proliferation and terminal differentiation. During CNS development dividing neuronal progenitors will initiate a process by which they permanently withdraw from the cell cycle and begin terminal differentiation. The molecular mechanisms that direct neuronal progenitors to permanently stop proliferating are not clearly understood, but are likely to include changes in DNA replication and the cell cycle machinery. The progression of cells through the cell cycle is controlled by a complex interaction between the cyclin proteins and the cyclin-dependent kinases (Cdks) (1,2). The expression level of a number of Cdk inhibitors can provide critical signals to stop cell proliferation, and in some cell types may initiate terminal differentiation (2,3). However, to permanently hold neurons out of the cell cycle it may also be important to initiate stable changes in their transcriptional machinery. These changes might include expression of transcription factors that directly or indi1 Send correspondence to: Dr. Robert F. Bulleit, Department of Pharmacology, University of Maryland School of Medicine, 655 W. Baltimore St. Rm 4-018, Baltimore MD 21201. Fax: (410)706-0032. E-mail: [email protected].
rectly repress the expression of genes required for DNA replication or cell cycle progression. Transcription factors that define the final phenotypic fate of neurons may be involved in this process. Previous studies suggest that homeobox and POU/ homeobox genes encode transcriptional regulatory proteins that are essential for defining the characteristic properties of cells in metazoans. In both invertebrates and vertebrates these proteins play key roles in controlling development and regulating cellular phenotypes, including the phenotype of CNS neurons (4, 5, 6, 7, 8, 9, 10, 11). We and others have previously reported the identification of a new member of the POU domain transcription factor family (Brn-5/Cns-1/Emb), that is expressed selectively in postmitotic CNS neurons (12, 13, 14, 15). This pattern of expression may suggest a role for Brn-5 in defining the differentiated phenotype of CNS neurons. In this report We present evidence that ectopic expression of Brn-5 in NG108-15 cells, a neuroblastoma/glioma hybrid cell line, suppresses DNA synthesis. Thus, during terminal differentiation, Brn-5 may exert a neuron specific function that permanently maintains neurons in a non-proliferative state. MATERIALS AND METHODS NG108-15 cell transfection. Brn-5, b-galactosidase (b-Gal) or Oct1 cDNAs containing the entire coding regions were inserted into the pcDL-SRa expression vector (16). Expression of the inserted genes was driven by a SV40/HTLV LTR hybrid promotor. NG108-15 cells, a neuroblastoma-glioma hybrid cell line cells (17, a gift of M Nirenberg), were transiently transfected with either Brn-5, b-Gal, or Oct1 expression vectors using lipofectamine (Gibco-BRL) at a ratio of 2 mg DNA/15 ml lipofectamine in 1 ml DMEM containing 11 HAT (100 mM hypoxanthine, 400 nM aminopterin, 16 mM thymidine), 50 unit penicillin, and 50 mg streptomycin. After 5 hours of transfection the medium was replaced with fresh DMEM containing 10% FBS, 11 HAT, and antibiotics. The culture was continued at 377C in 5% CO2 . The transfection efficiency in these experiments varied from 30-50% as determined by histochemical staining for b-Gal. Immunocytochemistry. Transfected NG108-15 cells were labeled with 10 mM BrdU for 2 hours, and dissociated using trypsin. Harvested cells were fixed in suspension using 70% ethanol for 1 hr, followed by 1% paraformaldehyde for 5 min, and 100% methanol for
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10 min. Cells were treated with 0.5 N HCl in 0.5 % Triton X-100 for 10 min on ice, resuspended in distilled water, and boiled for 10 min. We performed double-staining for BrdU and Brn-5 or Oct-1 by incubating cells with the Brn-5 antiserum described previously (15, 1:1500 dilution) or an Oct-1 monoclonal antibody (Santa Cruz, 1:500 dilution) in 0.1% BSA/PBS overnight at 47C, followed by 2 mg/ml a BrdU monoclonal antibody (Boehringer Mannheim) in 0.1% BSA/ PBS for 2 hours at room temperature. Staining was visualized by FITC-conjugated anti-rabbit IgG antibody for Brn-5 or Oct-1, and rhodamine-conjugated anti-mouse IgG antibody for BrdU. Brn-5 or Oct-1 positive cells could be clearly distinguished by bright nuclear staining. No bright nuclear staining of cells was observed in either the non-transfected cells or transfected cells stained with preimmune serum. BrdU positive cells showed bright rhodamine fluorescence. This positive reaction was not observed in cultured cells that had not been exposed to BrdU. An evaluation of specificity of the Brn-5 antiserum is presented in a separate manuscript (15). This evaluation showed that immunostaining with the Brn-5 antiserum was blocked by the addition of excess GST/Brn-5 fusion but not by the addition of other POU domain fusion proteins, GST/Oct-1 or GST/ RPF-1. The POU proteins Oct-1 and RPF-1 shares homology with Brn-5 (12, 13, 14, 18). RNA blot analysis. Total RNA was isolated form cultured NG10815 cells by acid guanidinium thiocyanate-phenol-chloroform extraction (19). RNA was fractionated by formaldehyde agarose gel electrophoresis and transferred to nylon membranes (20). RNA blots were prehybridized for 2-4 hours at 427C in H buffer [5.6 1 SSPE (0.84 M NaCl, 64 mM Na2PO4 , 6 mM EDTA), 50% formamide, 51 Denhardt’s solution (0.1% ficoll, 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin), 1% SDS and 200 mg/ml herring sperm DNA]. The hybridization was continued in H buffer containing 1-3 1 106 cpm/ml of probe (specific activity of 1-2 1 109 cpm/mg) for 48 hr at 427C. The probes were generated from PCNA cDNAs by a random priming reaction. Blots were washed in 0.1 1 SSC, 1% SDS at 657C and exposed to X-ray film.
RESULTS AND DISCUSSION The temporal correspondence between the exit of CNS progenitors from the cell cycle and the expression of Brn-5 suggests a potential role of Brn-5 in terminal neuronal differentiation (15). To test whether Brn-5 expression can regulate neuronal differentiation, we transiently expressed Brn-5 in NG108-15 cells, a neuroblastoma 1 glioma hybrid cell line which can differentiate into cells with properties of mature neurons (17). We expressed Brn-5 under the control of a hybrid SV40/HTLV LTR promoter (16). Cells expressing Brn5 were identified using a Brn-5 antiserum (15). The antiserum showed faint immunoreactivity with nontransfected NG108-15 cells suggesting proliferating NG108-15 cells expressed very low levels of Brn-5. In our initial experiments, cells expressing Brn-5, under the control of the SV40/HTLV LTR promoter, did not have noticeably altered morphology such as the presence of long neurite processes. This observation suggests that Brn-5 might not direct the early morphological differentiation of neurons. However, cultures transfected with Brn-5 appeared to have fewer cells 48 hours after the transfection compared with cultures transfected with same expression vector containing a b-galactosidase cDNA. We also observed a reduced level
FIG. 1. Transfection of cultured NG108-15 cells with a Brn-5 expression vector reduces the level of PCNA mRNA. PCNA mRNA levels were measured using RNA blot analysis 48 hours after NG108-15 cells were transfected with either a b-galactosidase (b-Gal) or a Brn5 expression vector. An RNA blot of PCNA mRNA from cells transfected with either a b-galactosidase (b-Gal) or Brn-5 expression vector. The negative image of the ethidium bromide stained 18S RNA gel indicates that similar amounts of RNA were in each lane of the gel.
of proliferating cell nuclear antigen (PCNA) RNA in cultures transfected with the Brn-5 expression vector when compared to those cultures transfected with the b-galactosidase expression vector (Fig. 1). PCNA provides a marker for proliferating cells. It is an auxiliary protein for DNA polymerase delta and is expressed at high level in cells actively synthesizing DNA (21, 22, 23, 24, 25). Thus, ectopic expression of Brn-5 may alter the ability of cells to synthesize DNA and continue to proliferate. To evaluate whether Brn-5 inhibited DNA synthesis in NG108-15 cell, we labeled transfected NG108-15 cells with BrdU to determine the number of cells synthesizing DNA and thus proliferating. Forty hours after transfection we labeled the cells with BrdU for 2 hours, and observed that few cells expressing Brn-5 were also labeled with BrdU (Fig. 2A). The number of transfected cells that expressed Brn-5 remained constant throughout the culture period suggesting that the increased expression of Brn-5 was not overtly toxic to these cells. To exclude the possibility that the reduced BrdU labeling was due to a non-specific effect of overexpressing a transcription factor, we transfected NG108-15 cells with the same expression vector containing a cDNA for the POU transcription factor Oct1. We observed that many Oct-1 positive cells were also positive for BrdU (Fig. 2B). The percent of Brn-5 positive cells that incorporated BrdU was only 4.7 %, while in the same cultures the percent of Brn-5 negative cells that incorporated BrdU was 27.6 % (Fig. 3). In cultures transfected with the Oct-1 expression vector, the percent of Oct-1 positive cells labeled with BrdU was 24.2 % similar to control cells (Fig. 3). These results suggest that in proliferating NG108-15 cells the
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FIG. 2. Ectopic expression of Brn-5 in NG108 cells. NG108-15 cells were transfected with either a Brn-5 or Oct-1 expression vector. Forty hours after transfection the cells were labeled with 10 mM BrdU for 2 hrs. Double immunostaining with either a Brn-5 (A) or Oct-1 (B) antibody, and the BrdU antibody was use to determine whether Brn-5 or Oct-1 immunopositive cells were labeled with BrdU.
over expression of Brn-5 may directly or indirectly suppress continued cell proliferation. The observation that Brn-5 expression occurs in many different CNS neurons suggests that it may have a common function in postmitotic CNS neurons. Brn5 is a member of the POU domain transcription factor family. It is one of the class VI subgroup of POU domain factors (12, 13, 14, 18). Most of the vertebrate POU genes are expressed in the developing and mature CNS (12, 13, 14, 18, 26, 27, 28) and some of these genes may play a role in maintaining the phenotypic properties of selective neuronal populations (7, 9, 29). We observed that ectopic expression of Brn-5 in NG108-15 cells arrests their continued proliferation. However, the temporal pattern of Brn-5 expression suggests that in the developing CNS its expression normally occurs after neuronal progenitors exit the cell cycle (15). Thus, it is unlikely Brn-5 plays an active role in cell cycle withdrawal. An alternative function may be to provide a permanent mechanism for maintaining neurons in a postmitotic state by directly or indirectly suppressing factors required for cell cycle progression. The observation that ectopic expression of the POU gene Oct-1 did not significantly reduce proliferation of NG108-15 cells may indicate that this function is specific to Brn-5. Expression of other members of the vertebrate POU gene family occur in proliferative cells of the developing brain (30) and at high levels in certain tumors (31). These observations may suggest that these other POU proteins function differently then Brn-5. Other transcription factors when ectopically expressed in cells can suppress proliferation and in some cases induces differentiation. Ectopic expression of the basis helix-loop-helix (bHLH) transcription factor MyoD produces growth arrest in many fibroblast cell
lines and in some lines induces muscle differentiation (32, 33). The neurogenic bHLH transcription factors, NeuroD and Neurogenin, when ectopically expressed in xenopus embryos can produce premature neuronal differentiation (34, 35). NeuroD, like Brn-5, is normally expressed postmitotically but can still arrest growth of cells when ectopically expressed (34). It is not clear from our studies whether Brn-5 can also induce neuronal differentiation of NG108-15 cells. We did not observe morphologic changes following ectopic expression of Brn-5 which might suggest that Brn-5 alone can not
FIG. 3. Ectopic expression of Brn-5 inhibits proliferation of NG108 cells. NG108-15 cells were transfected with either a Brn-5 or Oct-1 expression vector. Forty hours after transfection the cells were labeled with 10 mM BrdU for 2 hrs. Double immunostaining with either a Brn-5 or Oct-1 antibody and the BrdU antibody was use to determine the percent of Brn-5 or Oct-1 immunopositive cells that were labeled with BrdU. We also determined the percent of cells not immunopositive for Brn-5 or Oct-1 that were BrdU positive (Ctrl). The mean percent of Brn-5 positive cells that are BrdU positive are statistically different than either the mean percent of control cells or Oct-1 positive cells that were Brdu positive, * P value õ 0.005.
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direct morphologic differentiation. However, a more extensive study of changes in gene expression is needed to define whether Brn-5 can instruct other aspects of neuronal differentiation. The present study suggests a potential function of Brn-5 may include the suppression of genes required for proliferation. This suppression could provide an assurance that postmitotic neurons can not respond to proliferative signals. ACKNOWLEDGMENTS We thank W. Herr for the gift of the Oct-1 cDNA clone, H. Zhou and J. Nathans for the gift of the RPF-1 fusion protein, M. Nirenberg (NIH) for the gift of NG108-15 cells, and M. Vogel and P. Yarowsky for critical comments on the manuscript. This work was supported in part by the Bressler Research Fund of the University of Maryland.
REFERENCES 1. Elledge, S. J. (1996) Science 274, 1664–1672. 2. Sherr, C. J. (1996) Science 274, 1672–1677. 3. Harper, J. W., and Elledge, S. J. (1996) Curr. Opinion Genet. Dev. 6, 56–64. 4. Chisaka, O., Musci, T. S., and Capecchi, M. R. (1992) Nature 355, 516–520. 5. Doe, C. Q., Hiromi, Y., Gehring, W. J., and Goodman, C. S. (1988) Science 239, 170–175. 6. Doe, C. Q., Smouse, D., and Goodman, C. S. (1988) Nature 333, 376–378. 7. Erkman, L., McEvilly, R. J., Lin, L., Ryan, A. K., Hooshmand, F., O’Connell, S. M., Keithley, E. M., Rapaport, D. H., Ryan, A. F., and Rosenfeld, M. G. (1996) Science 381, 603–606. 8. Ingham, P. W. (1988) Nature 335, 25–34. 9. Nakai, S., Kawano, H., Yudate, T., Nishi, M., Kuno, J., Nagata, A., Jishaga, K., Fujii, H., Hamada, H., Kawamura, K., Shiba, K., and Noda, T. (1995) Genes Dev. 9, 3109–3121. 10. Scott, M. P., and Carroll, S. B. (1987) Cell 51, 689–698. 11. Yang, X., Yeo, S., Dick, T., and Chia, W. (1993) Genes Dev. 7, 504–516. 12. Andersen, B., Schonemann, M. D., Pearse, R. V., Jenne, K., Sugarman, J., and Resenfeld, M. G. (1993) J. Biol. Chem. 268, 23390–23398. 13. Bulleit, R. F., Cui, H., Wang, J., and Lin, X. (1994) J. Neurosci. 14, 1584–1595.
14. Okamoto, K., Wakamiya, M., Noji, S., Koyama, E., Taniguchi, S., Takemura, R., Copeland, N. G., Gilbert, D. J., Jenkins, N. A., Muramatsu, M., and Hamada, H. (1993) J. Biol. Chem. 268, 7449–7457. 15. Cui, H., and Bulleit, R. F. (1997) submitted. 16. Takebe, T., Seiki, M., Fujisawa, J., Hoy, P., Yokota, K., Arai, K., Yoshida, M., and Arai, N. (1988) Mol. Cell Biol. 8, 466–472. 17. Nelson, P., Christian, C., and Nirenberg, M. (1976) Proc. Natl. Acad. Sci. USA 73, 123–127. 18. Zhou, H., Yoshioka, T., and Nathans, J. (1996) J. Neurosci. 16, 2261–2274. 19. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156– 159. 20. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. 21. Bravo, R., Frank, P. A., Blundell, P. A., and MacDonald-Bravo, H. (1987) Nature 326, 515–517. 22. Prelich, G., Kostura, M., Marshak, D. R., Mathews, M. B., and Stillman, B. (1987) Nature 326, 471–475. 23. Prelich, G., Tan, C. K., Kostura, M., Mathews, M. B., So, A. G., Downey, K. M., and Stillman, B. (1987) Nature 326, 517–520. 24. Prelich, G., and Stillman, B. (1988) Cell 53, 117–126. 25. Zuber, M., Tan, E. M., and Ryoji, M. (1989) Mol. Cell Biol. 9, 57–66. 26. He, X., Treacy, M. N., Simmons, D. M., Ingraham, H. A., Swanson, L. W., and Rosenfeld, M. G. (1989) Nature 340, 35–42. 27. Mathis, J. M., Simmons, D. M., He, X., Swanson, L. W., and Rosenfeld, M. G. (1992) EMBO J. 11, 2551–2561. 28. Xiang, M., Zhou, L., Macke, J. P., Yoshioka, T., Hendry, S. H. C., Eddy, R. L., Shows, T. B., and Nathans, J. (1995) J. Neurosci. 15, 4762–4785. 29. Schonemann, M. D., Ryan, A. K., McEvilly, R. J., O’Connell, S. M., Arias, C. A., Kalla, K. A., Li, P., Sawchenko, P. E., and Rosenfeld, M. G. (1995) Genes Dev. 9, 3122–3135. 30. Alvarez-Bolado, G., Rosenfeld, M. G., and Swanson, L. W. (1995) J. Comp. Neurol. 355, 237–295. 31. Eisen, T., Easty, D. J., Bennett, D. C., and Goding, C. R. (1995) Oncogene 11, 2157–2164. 32. Crescenzi, M., Fleming, T., Lassar, A. B., Weintraub, H., and Aaronson, S. A. (1990) Proc. Natl. Acad. Sci. USA 87, 8442– 8446. 33. Davis, R. L., Weintraub, H., and Lassar, A. B. (1987) Cell 51, 987–1000. 34. Lee, J. E., Hollenberg, S. M., Snider, L., Turner, D. L., Lipnick, N., and Weintraub, H. (1995) Science 268, 836–844. 35. Ma, Q., Kintner, C., and Anderson, D. J. (1996) Cell 87, 43–52.
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236, 693–696 (1997)
RC976996
Expression of the POU Transcription Factor Brn-5 Inhibits Proliferation of NG108-15 Cells Hong Cui and Robert F. Bulleit1 Department of Pharmacology, University of Maryland School of Medicine, Baltimore, Maryland 21201
Received June 9, 1997
The POU domain transcription factors are a subgroup of homeodomain proteins that appears to control cellular phenotypes. The expression of the POU protein Brn-5 occurs selectively in postmitotic CNS neurons. Ectopic expression of Brn-5 in dividing NG108-15 cells reduces the level of RNA encoding the proliferating cell nuclear antigen (PCNA). This ectopic expression also inhibits DNA synthesis as measured by the incorporation of bromodeoxyuridine (BrdU). Thus, Brn-5 may inhibit the continued proliferation of these cells. A potential function of Brn-5 may be to suppress the action of proliferative signals in postmitotic neurons and thus prevents them from reentering the cell cycle. q 1997 Academic Press
Neurogenesis in the mammalian central nervous system (CNS) involves a transition between cell proliferation and terminal differentiation. During CNS development dividing neuronal progenitors will initiate a process by which they permanently withdraw from the cell cycle and begin terminal differentiation. The molecular mechanisms that direct neuronal progenitors to permanently stop proliferating are not clearly understood, but are likely to include changes in DNA replication and the cell cycle machinery. The progression of cells through the cell cycle is controlled by a complex interaction between the cyclin proteins and the cyclin-dependent kinases (Cdks) (1,2). The expression level of a number of Cdk inhibitors can provide critical signals to stop cell proliferation, and in some cell types may initiate terminal differentiation (2,3). However, to permanently hold neurons out of the cell cycle it may also be important to initiate stable changes in their transcriptional machinery. These changes might include expression of transcription factors that directly or indi1 Send correspondence to: Dr. Robert F. Bulleit, Department of Pharmacology, University of Maryland School of Medicine, 655 W. Baltimore St. Rm 4-018, Baltimore MD 21201. Fax: (410)706-0032. E-mail: [email protected].
rectly repress the expression of genes required for DNA replication or cell cycle progression. Transcription factors that define the final phenotypic fate of neurons may be involved in this process. Previous studies suggest that homeobox and POU/ homeobox genes encode transcriptional regulatory proteins that are essential for defining the characteristic properties of cells in metazoans. In both invertebrates and vertebrates these proteins play key roles in controlling development and regulating cellular phenotypes, including the phenotype of CNS neurons (4, 5, 6, 7, 8, 9, 10, 11). We and others have previously reported the identification of a new member of the POU domain transcription factor family (Brn-5/Cns-1/Emb), that is expressed selectively in postmitotic CNS neurons (12, 13, 14, 15). This pattern of expression may suggest a role for Brn-5 in defining the differentiated phenotype of CNS neurons. In this report We present evidence that ectopic expression of Brn-5 in NG108-15 cells, a neuroblastoma/glioma hybrid cell line, suppresses DNA synthesis. Thus, during terminal differentiation, Brn-5 may exert a neuron specific function that permanently maintains neurons in a non-proliferative state. MATERIALS AND METHODS NG108-15 cell transfection. Brn-5, b-galactosidase (b-Gal) or Oct1 cDNAs containing the entire coding regions were inserted into the pcDL-SRa expression vector (16). Expression of the inserted genes was driven by a SV40/HTLV LTR hybrid promotor. NG108-15 cells, a neuroblastoma-glioma hybrid cell line cells (17, a gift of M Nirenberg), were transiently transfected with either Brn-5, b-Gal, or Oct1 expression vectors using lipofectamine (Gibco-BRL) at a ratio of 2 mg DNA/15 ml lipofectamine in 1 ml DMEM containing 11 HAT (100 mM hypoxanthine, 400 nM aminopterin, 16 mM thymidine), 50 unit penicillin, and 50 mg streptomycin. After 5 hours of transfection the medium was replaced with fresh DMEM containing 10% FBS, 11 HAT, and antibiotics. The culture was continued at 377C in 5% CO2 . The transfection efficiency in these experiments varied from 30-50% as determined by histochemical staining for b-Gal. Immunocytochemistry. Transfected NG108-15 cells were labeled with 10 mM BrdU for 2 hours, and dissociated using trypsin. Harvested cells were fixed in suspension using 70% ethanol for 1 hr, followed by 1% paraformaldehyde for 5 min, and 100% methanol for
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10 min. Cells were treated with 0.5 N HCl in 0.5 % Triton X-100 for 10 min on ice, resuspended in distilled water, and boiled for 10 min. We performed double-staining for BrdU and Brn-5 or Oct-1 by incubating cells with the Brn-5 antiserum described previously (15, 1:1500 dilution) or an Oct-1 monoclonal antibody (Santa Cruz, 1:500 dilution) in 0.1% BSA/PBS overnight at 47C, followed by 2 mg/ml a BrdU monoclonal antibody (Boehringer Mannheim) in 0.1% BSA/ PBS for 2 hours at room temperature. Staining was visualized by FITC-conjugated anti-rabbit IgG antibody for Brn-5 or Oct-1, and rhodamine-conjugated anti-mouse IgG antibody for BrdU. Brn-5 or Oct-1 positive cells could be clearly distinguished by bright nuclear staining. No bright nuclear staining of cells was observed in either the non-transfected cells or transfected cells stained with preimmune serum. BrdU positive cells showed bright rhodamine fluorescence. This positive reaction was not observed in cultured cells that had not been exposed to BrdU. An evaluation of specificity of the Brn-5 antiserum is presented in a separate manuscript (15). This evaluation showed that immunostaining with the Brn-5 antiserum was blocked by the addition of excess GST/Brn-5 fusion but not by the addition of other POU domain fusion proteins, GST/Oct-1 or GST/ RPF-1. The POU proteins Oct-1 and RPF-1 shares homology with Brn-5 (12, 13, 14, 18). RNA blot analysis. Total RNA was isolated form cultured NG10815 cells by acid guanidinium thiocyanate-phenol-chloroform extraction (19). RNA was fractionated by formaldehyde agarose gel electrophoresis and transferred to nylon membranes (20). RNA blots were prehybridized for 2-4 hours at 427C in H buffer [5.6 1 SSPE (0.84 M NaCl, 64 mM Na2PO4 , 6 mM EDTA), 50% formamide, 51 Denhardt’s solution (0.1% ficoll, 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin), 1% SDS and 200 mg/ml herring sperm DNA]. The hybridization was continued in H buffer containing 1-3 1 106 cpm/ml of probe (specific activity of 1-2 1 109 cpm/mg) for 48 hr at 427C. The probes were generated from PCNA cDNAs by a random priming reaction. Blots were washed in 0.1 1 SSC, 1% SDS at 657C and exposed to X-ray film.
RESULTS AND DISCUSSION The temporal correspondence between the exit of CNS progenitors from the cell cycle and the expression of Brn-5 suggests a potential role of Brn-5 in terminal neuronal differentiation (15). To test whether Brn-5 expression can regulate neuronal differentiation, we transiently expressed Brn-5 in NG108-15 cells, a neuroblastoma 1 glioma hybrid cell line which can differentiate into cells with properties of mature neurons (17). We expressed Brn-5 under the control of a hybrid SV40/HTLV LTR promoter (16). Cells expressing Brn5 were identified using a Brn-5 antiserum (15). The antiserum showed faint immunoreactivity with nontransfected NG108-15 cells suggesting proliferating NG108-15 cells expressed very low levels of Brn-5. In our initial experiments, cells expressing Brn-5, under the control of the SV40/HTLV LTR promoter, did not have noticeably altered morphology such as the presence of long neurite processes. This observation suggests that Brn-5 might not direct the early morphological differentiation of neurons. However, cultures transfected with Brn-5 appeared to have fewer cells 48 hours after the transfection compared with cultures transfected with same expression vector containing a b-galactosidase cDNA. We also observed a reduced level
FIG. 1. Transfection of cultured NG108-15 cells with a Brn-5 expression vector reduces the level of PCNA mRNA. PCNA mRNA levels were measured using RNA blot analysis 48 hours after NG108-15 cells were transfected with either a b-galactosidase (b-Gal) or a Brn5 expression vector. An RNA blot of PCNA mRNA from cells transfected with either a b-galactosidase (b-Gal) or Brn-5 expression vector. The negative image of the ethidium bromide stained 18S RNA gel indicates that similar amounts of RNA were in each lane of the gel.
of proliferating cell nuclear antigen (PCNA) RNA in cultures transfected with the Brn-5 expression vector when compared to those cultures transfected with the b-galactosidase expression vector (Fig. 1). PCNA provides a marker for proliferating cells. It is an auxiliary protein for DNA polymerase delta and is expressed at high level in cells actively synthesizing DNA (21, 22, 23, 24, 25). Thus, ectopic expression of Brn-5 may alter the ability of cells to synthesize DNA and continue to proliferate. To evaluate whether Brn-5 inhibited DNA synthesis in NG108-15 cell, we labeled transfected NG108-15 cells with BrdU to determine the number of cells synthesizing DNA and thus proliferating. Forty hours after transfection we labeled the cells with BrdU for 2 hours, and observed that few cells expressing Brn-5 were also labeled with BrdU (Fig. 2A). The number of transfected cells that expressed Brn-5 remained constant throughout the culture period suggesting that the increased expression of Brn-5 was not overtly toxic to these cells. To exclude the possibility that the reduced BrdU labeling was due to a non-specific effect of overexpressing a transcription factor, we transfected NG108-15 cells with the same expression vector containing a cDNA for the POU transcription factor Oct1. We observed that many Oct-1 positive cells were also positive for BrdU (Fig. 2B). The percent of Brn-5 positive cells that incorporated BrdU was only 4.7 %, while in the same cultures the percent of Brn-5 negative cells that incorporated BrdU was 27.6 % (Fig. 3). In cultures transfected with the Oct-1 expression vector, the percent of Oct-1 positive cells labeled with BrdU was 24.2 % similar to control cells (Fig. 3). These results suggest that in proliferating NG108-15 cells the
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FIG. 2. Ectopic expression of Brn-5 in NG108 cells. NG108-15 cells were transfected with either a Brn-5 or Oct-1 expression vector. Forty hours after transfection the cells were labeled with 10 mM BrdU for 2 hrs. Double immunostaining with either a Brn-5 (A) or Oct-1 (B) antibody, and the BrdU antibody was use to determine whether Brn-5 or Oct-1 immunopositive cells were labeled with BrdU.
over expression of Brn-5 may directly or indirectly suppress continued cell proliferation. The observation that Brn-5 expression occurs in many different CNS neurons suggests that it may have a common function in postmitotic CNS neurons. Brn5 is a member of the POU domain transcription factor family. It is one of the class VI subgroup of POU domain factors (12, 13, 14, 18). Most of the vertebrate POU genes are expressed in the developing and mature CNS (12, 13, 14, 18, 26, 27, 28) and some of these genes may play a role in maintaining the phenotypic properties of selective neuronal populations (7, 9, 29). We observed that ectopic expression of Brn-5 in NG108-15 cells arrests their continued proliferation. However, the temporal pattern of Brn-5 expression suggests that in the developing CNS its expression normally occurs after neuronal progenitors exit the cell cycle (15). Thus, it is unlikely Brn-5 plays an active role in cell cycle withdrawal. An alternative function may be to provide a permanent mechanism for maintaining neurons in a postmitotic state by directly or indirectly suppressing factors required for cell cycle progression. The observation that ectopic expression of the POU gene Oct-1 did not significantly reduce proliferation of NG108-15 cells may indicate that this function is specific to Brn-5. Expression of other members of the vertebrate POU gene family occur in proliferative cells of the developing brain (30) and at high levels in certain tumors (31). These observations may suggest that these other POU proteins function differently then Brn-5. Other transcription factors when ectopically expressed in cells can suppress proliferation and in some cases induces differentiation. Ectopic expression of the basis helix-loop-helix (bHLH) transcription factor MyoD produces growth arrest in many fibroblast cell
lines and in some lines induces muscle differentiation (32, 33). The neurogenic bHLH transcription factors, NeuroD and Neurogenin, when ectopically expressed in xenopus embryos can produce premature neuronal differentiation (34, 35). NeuroD, like Brn-5, is normally expressed postmitotically but can still arrest growth of cells when ectopically expressed (34). It is not clear from our studies whether Brn-5 can also induce neuronal differentiation of NG108-15 cells. We did not observe morphologic changes following ectopic expression of Brn-5 which might suggest that Brn-5 alone can not
FIG. 3. Ectopic expression of Brn-5 inhibits proliferation of NG108 cells. NG108-15 cells were transfected with either a Brn-5 or Oct-1 expression vector. Forty hours after transfection the cells were labeled with 10 mM BrdU for 2 hrs. Double immunostaining with either a Brn-5 or Oct-1 antibody and the BrdU antibody was use to determine the percent of Brn-5 or Oct-1 immunopositive cells that were labeled with BrdU. We also determined the percent of cells not immunopositive for Brn-5 or Oct-1 that were BrdU positive (Ctrl). The mean percent of Brn-5 positive cells that are BrdU positive are statistically different than either the mean percent of control cells or Oct-1 positive cells that were Brdu positive, * P value õ 0.005.
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direct morphologic differentiation. However, a more extensive study of changes in gene expression is needed to define whether Brn-5 can instruct other aspects of neuronal differentiation. The present study suggests a potential function of Brn-5 may include the suppression of genes required for proliferation. This suppression could provide an assurance that postmitotic neurons can not respond to proliferative signals. ACKNOWLEDGMENTS We thank W. Herr for the gift of the Oct-1 cDNA clone, H. Zhou and J. Nathans for the gift of the RPF-1 fusion protein, M. Nirenberg (NIH) for the gift of NG108-15 cells, and M. Vogel and P. Yarowsky for critical comments on the manuscript. This work was supported in part by the Bressler Research Fund of the University of Maryland.
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