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• W2034658858 abstract "The POU domain transcription factor Brn-3a is able to stimulate neurite outgrowth when overexpressed in the neuronal ND7 cell line, whereas the closely related Brn-3b factor does not have this effect. We show that Brn-3a overexpression also enhances the expression of the three neurofilament genes at both the mRNA and protein levels, whereas Brn-3b overexpression has no effect. In addition Brn-3a activates the three neurofilament gene promoters in co-transfection assays in both neuronal and non-neuronal cells. As observed for enhanced neurite outgrowth, the stimulation of neurofilament gene expression and activation of the neurofilament gene promoters is observed with the isolated POU domain of Brn-3a. A single amino acid change in the POU homeodomain of Brn-3a to the equivalent amino acid in Brn-3b abolishes its ability to activate the neurofilament promoters, whereas the reciprocal change converts Brn-3b to an activator of these promoters. The POU domain transcription factor Brn-3a is able to stimulate neurite outgrowth when overexpressed in the neuronal ND7 cell line, whereas the closely related Brn-3b factor does not have this effect. We show that Brn-3a overexpression also enhances the expression of the three neurofilament genes at both the mRNA and protein levels, whereas Brn-3b overexpression has no effect. In addition Brn-3a activates the three neurofilament gene promoters in co-transfection assays in both neuronal and non-neuronal cells. As observed for enhanced neurite outgrowth, the stimulation of neurofilament gene expression and activation of the neurofilament gene promoters is observed with the isolated POU domain of Brn-3a. A single amino acid change in the POU homeodomain of Brn-3a to the equivalent amino acid in Brn-3b abolishes its ability to activate the neurofilament promoters, whereas the reciprocal change converts Brn-3b to an activator of these promoters. The POU (Pit-Oct-Unc) family of transcription factors (for reviews see Refs. 1Verrijzer C.P. van der Vliet P.C. Biochim. Biophys. Acta. 1993; 1173: 1-21Crossref PubMed Scopus (243) Google Scholar and 2Wegner M. Drolet D.W. Rosenfeld M.G. Curr. Opin. Cell Biol. 1993; 5: 488-498Crossref PubMed Scopus (232) Google Scholar) was originally defined on the basis of a common 150–160-amino acid DNA binding domain held in common between the mammalian transcription factors Pit-1, Oct-1, and Oct-2 and the regulatory protein Unc-86 which plays a key role in the development of neuronal cell types, particularly sensory neurons in the nematode (3Desai C. Garriga G. McIntire S.L. Horvitz H.R. Nature. 1988; 336: 638-646Crossref PubMed Scopus (369) Google Scholar,4Finney M. Ruvkin G. Horvitz H.R. Cell. 1988; 55: 757-769Abstract Full Text PDF PubMed Scopus (253) Google Scholar). A number of subsequent studies (see for example Ref. 5He X. Treacy M.N. Simmons D.M. Ingraham H.A. Swanson L.S. Rosenfeld M.G. Nature. 1989; 340: 35-42Crossref PubMed Scopus (666) Google Scholar) have defined a large number of other POU family transcription factors in a variety of organisms that have been classified into subgroups according to their relationships to one another (for review see Ref. 2Wegner M. Drolet D.W. Rosenfeld M.G. Curr. Opin. Cell Biol. 1993; 5: 488-498Crossref PubMed Scopus (232) Google Scholar). Among the mammalian POU factors identified in this way, the Brn-3 factors show the closest homology to the nematode protein Unc-86 and together with the Drosophila factors I-POU and tI-POU (6Treacy M.N. He X. Rosenfeld M.G. Nature. 1991; 350: 577-584Crossref PubMed Scopus (145) Google Scholar, 7Treacy M.N. Neilson L.I. Turner E.E. He X. Rosenfeld M.G. Cell. 1992; 68: 491-505Abstract Full Text PDF PubMed Scopus (82) Google Scholar) these factors constitute the POU IV subfamily within the POU family (2Wegner M. Drolet D.W. Rosenfeld M.G. Curr. Opin. Cell Biol. 1993; 5: 488-498Crossref PubMed Scopus (232) Google Scholar). Indeed it has been proposed (8Erkman L. McEvilly J. Luo L. Ryan A.K. Hooshmand F. O'Connell S.M. Keithley E.M. Rapaport D.H. Ryan A.F. Rosenfeld M.G. Nature. 1996; 381: 603-606Crossref PubMed Scopus (433) Google Scholar) that the Brn-3 factors constitute the mammalian homologues of Unc-86 (8Erkman L. McEvilly J. Luo L. Ryan A.K. Hooshmand F. O'Connell S.M. Keithley E.M. Rapaport D.H. Ryan A.F. Rosenfeld M.G. Nature. 1996; 381: 603-606Crossref PubMed Scopus (433) Google Scholar). Unlike the situation in the nematode, however, three distinct Brn-3 factors have been identified in mammals that show close homology in the DNA binding POU domain but are considerably less homologous outside it and are encoded by three different genes (9Theil T. McLean-Hunter S. Zornig M. Möröy T. Nucleic Acids Res. 1993; 21: 5921-5929Crossref PubMed Scopus (82) Google Scholar, 10Theil T. Zechner U. Klett C. Adolph S. Möröy T. Cytogenet. Cell Genet. 1994; 66: 267-271Crossref PubMed Scopus (38) Google Scholar). These factors are Brn-3a (also known as Brn-3 or Brn-3.0) (5He X. Treacy M.N. Simmons D.M. Ingraham H.A. Swanson L.S. Rosenfeld M.G. Nature. 1989; 340: 35-42Crossref PubMed Scopus (666) Google Scholar, 11Gerrero M.R. McEvilly R.J. Tuner E. Lin C.R. O'Connell S. Jenne K.J. Hobbs M.V. Rosenfeld M.G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10841-10845Crossref PubMed Scopus (190) Google Scholar, 12Lillycrop K.A. Budhram-Mahadeo V.S. Lakin N.D. Terrenghi G. Wood J.N. Polak J.M. Latchman D.S. Nucleic Acids Res. 1992; 20: 5093-5096Crossref PubMed Scopus (117) Google Scholar), Brn-3b (also known as Brn-3.2) (12Lillycrop K.A. Budhram-Mahadeo V.S. Lakin N.D. Terrenghi G. Wood J.N. Polak J.M. Latchman D.S. Nucleic Acids Res. 1992; 20: 5093-5096Crossref PubMed Scopus (117) Google Scholar, 13Turner E.E. Jenne K.J. Rosenfeld M.G. Neuron. 1994; 12: 205-218Abstract Full Text PDF PubMed Scopus (159) Google Scholar), and Brn-3c (also known as Brn-3.1) (11Gerrero M.R. McEvilly R.J. Tuner E. Lin C.R. O'Connell S. Jenne K.J. Hobbs M.V. Rosenfeld M.G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10841-10845Crossref PubMed Scopus (190) Google Scholar, 14Ninkina N.N. Stevens G.E.M. Wood J.N. Richardson W.D. Nucleic Acids Res. 1993; 21: 3175-3182Crossref PubMed Scopus (104) Google Scholar). Each of these three factors is expressed in distinct but overlapping sets of neurons in the developing and adult nervous system (5He X. Treacy M.N. Simmons D.M. Ingraham H.A. Swanson L.S. Rosenfeld M.G. Nature. 1989; 340: 35-42Crossref PubMed Scopus (666) Google Scholar, 11Gerrero M.R. McEvilly R.J. Tuner E. Lin C.R. O'Connell S. Jenne K.J. Hobbs M.V. Rosenfeld M.G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10841-10845Crossref PubMed Scopus (190) Google Scholar, 13Turner E.E. Jenne K.J. Rosenfeld M.G. Neuron. 1994; 12: 205-218Abstract Full Text PDF PubMed Scopus (159) Google Scholar, 14Ninkina N.N. Stevens G.E.M. Wood J.N. Richardson W.D. Nucleic Acids Res. 1993; 21: 3175-3182Crossref PubMed Scopus (104) Google Scholar, 15Fedtsova N.G. Turner E.E. Mech. Dev. 1996; 53: 291-304Crossref Scopus (206) Google Scholar) with Brn-3a for example defining the earliest post-mitotic neurons to appear in the developing central nervous system (15Fedtsova N.G. Turner E.E. Mech. Dev. 1996; 53: 291-304Crossref Scopus (206) Google Scholar). This suggests that, like Unc-86, the mammalian Brn-3 factors may play a critical role in the development of specific neuronal cells within the nervous system with each factor having distinct but possibly related roles. In agreement with this, recent experiments (8Erkman L. McEvilly J. Luo L. Ryan A.K. Hooshmand F. O'Connell S.M. Keithley E.M. Rapaport D.H. Ryan A.F. Rosenfeld M.G. Nature. 1996; 381: 603-606Crossref PubMed Scopus (433) Google Scholar, 16Xiang M. Gan L. Zhou L. Klein W.H. Nathans J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11950-11955Crossref PubMed Scopus (202) Google Scholar) have shown that inactivation of the Brn-3b gene results in considerable loss of retinal neurons, whereas mice in which the Brn-3c gene has been inactivated show loss of olfactory neurons, and Brn-3a inactivation results in the loss of neurons in the brain stem and trigeminal ganglia (16Xiang M. Gan L. Zhou L. Klein W.H. Nathans J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 11950-11955Crossref PubMed Scopus (202) Google Scholar). These distinct functional roles and expression patterns of the Brn-3 factors are paralleled by their distinct effects on target genes. Thus it has previously been shown that the Brn-3a factor can activate a wide variety of target genes expressed in neuronal cells including those encoding pro-opiomelanocortin (11Gerrero M.R. McEvilly R.J. Tuner E. Lin C.R. O'Connell S. Jenne K.J. Hobbs M.V. Rosenfeld M.G. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 10841-10845Crossref PubMed Scopus (190) Google Scholar), the neuronal intermediate filament α-internexin (17Budhram-Mahadeo V. Morris P.J. Lakin N.D. Theil T. Ching G.Y. Lillycrop K.A. Möröy T. Liem R.K.H. Latchman D.S. J. Biol. Chem. 1995; 270: 2853-2858Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), and the synaptic vesicle protein SNAP-25 (18Lakin N.D. Morris P.J. Theil T. Sato T.N. Moroy T. Wilson M.C. Latchman D.S. J. Biol. Chem. 1995; 270: 15858-15863Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 19Morris P.J. Lakin N.D. Dawson S.J. Ryabanin A.E. Kilimann M.W. Wilson M.C. Latchman D.S. Mol. Brain Res. 1996; 43: 279-285Crossref PubMed Scopus (28) Google Scholar). In contrast Brn-3b represses the promoters of the α-internexin and the gene encoding SNAP-25 and also interferes with their activation by Brn-3a (17Budhram-Mahadeo V. Morris P.J. Lakin N.D. Theil T. Ching G.Y. Lillycrop K.A. Möröy T. Liem R.K.H. Latchman D.S. J. Biol. Chem. 1995; 270: 2853-2858Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 19Morris P.J. Lakin N.D. Dawson S.J. Ryabanin A.E. Kilimann M.W. Wilson M.C. Latchman D.S. Mol. Brain Res. 1996; 43: 279-285Crossref PubMed Scopus (28) Google Scholar), although it can stimulate the promoter of the neuronal nicotinic acetylcholine receptor α2 subunit gene which is not affected by Brn-3a (20Milton N.G.N. Bessis A. Changeux J.P. Latchman D.S. J. Biol. Chem. 1995; 270: 15143-15147Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). To analyze the functional effects of the Brn-3 factors in more detail, we have used the ND7 neuronal cell line which was generated by the fusion of a mouse neuroblastoma cell line and primary rat dorsal root ganglion neurons (21Wood J.N. Bevan S.J. Coote P. Darn P.M. Hogan P. Latchman D.S. Morrison C. Rougon G. Theveniau M. Wheatley S.C. Proc. R. Soc. Lond. B Biol. Sci. 1990; 241: 187-194Crossref PubMed Scopus (219) Google Scholar). We have previously shown (12Lillycrop K.A. Budhram-Mahadeo V.S. Lakin N.D. Terrenghi G. Wood J.N. Polak J.M. Latchman D.S. Nucleic Acids Res. 1992; 20: 5093-5096Crossref PubMed Scopus (117) Google Scholar, 22Budhram-Mahadeo V.S. Theil T. Morris P.J. Lillycrop K.A. Möröy T. Latchman D.S. Nucleic Acids Res. 1994; 22: 3092-3098Crossref PubMed Scopus (38) Google Scholar) that when these cells are induced to differentiate to a non-dividing phenotype bearing numerous neurite processes (23Suburo A.M. Wheatley S.C. Horn D.A. Gibson S.J. Jahn R. Fischer-Colbrie R. Wood J.W. Latchman D.S. Polak J.M. Neuroscience. 1992; 46: 881-889Crossref PubMed Scopus (56) Google Scholar), the levels of Brn-3a rise dramatically from a low level in the proliferating cells to a much higher level in the non-dividing cells. Conversely, the levels of Brn-3b fall from a high level in the dividing cells to a much lower level in the differentiated cells, whereas the levels of Brn-3c remain unchanged. Moreover, such changes in the expression of Brn-3a and Brn-3b appear to have functional effects. Thus as would be expected, the rise in the level of the Brn-3a activator together with the fall in the level of the Brn-3b repressor which occur during ND7 cell differentiation result in the activation of a transfected synthetic target promoter containing a binding site for these factors, when ND7 cells are induced to differentiate (22Budhram-Mahadeo V.S. Theil T. Morris P.J. Lillycrop K.A. Möröy T. Latchman D.S. Nucleic Acids Res. 1994; 22: 3092-3098Crossref PubMed Scopus (38) Google Scholar, 24Morris P.J. Theil T. Ring C.J.A. Lillycrop K.A. Möröy T. Latchman D.S. Mol. Cell. Biol. 1994; 14: 6907-6914Crossref PubMed Scopus (72) Google Scholar). Similarly, the expression of the endogenous gene encoding SNAP-25 (whose promoter is activated by Brn-3a and repressed by Brn-3b in co-transfection experiments) (18Lakin N.D. Morris P.J. Theil T. Sato T.N. Moroy T. Wilson M.C. Latchman D.S. J. Biol. Chem. 1995; 270: 15858-15863Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 19Morris P.J. Lakin N.D. Dawson S.J. Ryabanin A.E. Kilimann M.W. Wilson M.C. Latchman D.S. Mol. Brain Res. 1996; 43: 279-285Crossref PubMed Scopus (28) Google Scholar) is considerably enhanced during ND7 cell differentiation. Indeed we have recently obtained evidence that the rise in Brn-3a and the fall in Brn-3b occurring during ND7 cell differentiation play a critical role in the differentiation process itself. Thus preventing the rise in Brn-3a using an antisense approach prevents ND7 cell differentiation and the rise in SNAP-25 expression which normally occurs in response to differentiation-inducing stimuli such as serum removal (18Lakin N.D. Morris P.J. Theil T. Sato T.N. Moroy T. Wilson M.C. Latchman D.S. J. Biol. Chem. 1995; 270: 15858-15863Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Moreover, artificial overexpression of Brn-3a in ND7 cells results in clonal cell lines which extend neurite processes when grown in full serum-containing medium which does not normally induce the differentiation of these cells (25Smith M.D. Dawson S.J. Latchman D.S. Mol. Cell. Biol. 1997; 17: 345-354Crossref PubMed Scopus (63) Google Scholar). Conversely ND7-derived cell lines overexpressing Brn-3b fail to show neurite outgrowth when removed from serum which would normally induce such outgrowth in parental ND7 cells (26Smith M.D. Dawson S.J. Latchman D.S. J. Biol. Chem. 1997; 272: 1382-1388Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). These findings indicate therefore that the balance between Brn-3a and Brn-3b is likely to play a critical role in the differentiation of ND7 cells, and they allow the use of these cells as a model system to understand the role of these factors in neuronal differentiation and to elucidate the mechanism by which they act. In view of the finding that SNAP-25 is essential for neurite outgrowth by a variety of different neuronal cells in vitro and in vivo (27Osen-Sand A. Catsicas M. Staple J.K. Jones K.A. Ayala G. Knowles J. Grenningloh G. Catsicas S. Nature. 1993; 364: 445-448Crossref PubMed Scopus (379) Google Scholar), it is likely that the ability of Brn-3a to induce SNAP-25 expression plays a role in its ability to induce neurite outgrowth. However, it is likely that this dramatic effect of Brn-3a on the phenotype of ND7 cells involves more than its ability to induce SNAP-25 and that other target genes encoding proteins involved in neurite process formation are also likely to be activated by Brn-3a. We previously found, however, that the growth-associated protein GAP-43 shows no change in its expression in the Brn-3a or Brn-3b overexpressing ND7 cells, in agreement with our finding that its promoter is unaffected by co-transfection with Brn-3a (25Smith M.D. Dawson S.J. Latchman D.S. Mol. Cell. Biol. 1997; 17: 345-354Crossref PubMed Scopus (63) Google Scholar). In view of the importance of the neurofilament proteins in neuronal process formation and particularly in specifying axonal calibre (for review see Ref. 28Liem R.K.H. Curr. Opin. Cell Biol. 1990; 2: 86-90Crossref PubMed Scopus (24) Google Scholar) and the simultaneous appearance of neurofilament and Brn-3a expression during early post-mitotic neuronal differentiation (15Fedtsova N.G. Turner E.E. Mech. Dev. 1996; 53: 291-304Crossref Scopus (206) Google Scholar), we have investigated the expression of the neurofilament heavy, medium, and light chain genes in our ND7 cells overexpressing different Brn-3 transcription factors and have also studied the effect of the Brn-3 factors on the promoters of these genes in co-transfection experiments carried out in both neuronal and non-neuronal cells. BHK-21 cells (29Macpherson I. Stoker M. Virology. 1962; 16: 147-151Crossref PubMed Scopus (595) Google Scholar) and ND7 cells (21Wood J.N. Bevan S.J. Coote P. Darn P.M. Hogan P. Latchman D.S. Morrison C. Rougon G. Theveniau M. Wheatley S.C. Proc. R. Soc. Lond. B Biol. Sci. 1990; 241: 187-194Crossref PubMed Scopus (219) Google Scholar) were routinely cultured in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and L15 medium containing 10% fetal calf serum, respectively. The ND7-derived cell lines overexpressing Brn-3a, Brn-3b, or Brn-3c were constructed as described previously (25Smith M.D. Dawson S.J. Latchman D.S. Mol. Cell. Biol. 1997; 17: 345-354Crossref PubMed Scopus (63) Google Scholar, 26Smith M.D. Dawson S.J. Latchman D.S. J. Biol. Chem. 1997; 272: 1382-1388Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) by stable transfection of ND7 cells with cDNA clones for Brn-3a, Brn-3b, or Brn-3c under the control of the glucocorticoid-inducible mouse mammary tumor virus promoter in the vector PJ5 (30Morgenstern J.P. Land H. Nucleic Acids Res. 1990; 18: 1068Crossref PubMed Scopus (256) Google Scholar). These cells were grown in L15 medium containing 10% fetal calf serum supplemented with G418 to a final concentration of 800 μg/ml to maintain expression of the transgene. Treatment with dexamethasone at a final concentration of 1 μm was used to induced expression of the murine mammary tumor virus promoter. The neurofilament reporter gene constructs containing various regions of the NF-H, NF-M, and NF-L promoter linked to the readily assayable chloramphenicol acetyltransferase gene have been described previously (31Shneidman P.S. Bruce J. Schwartz M.L. Schlaepfer W.W. Mol. Brain Res. 1992; 13: 127-138Crossref PubMed Scopus (28) Google Scholar, 32Schwartz M.L. Katagi C. Bruce J. Schlaepfer W.W. J. Biol. Chem. 1994; 269: 13444-13450Abstract Full Text PDF PubMed Google Scholar). In the transient transfections, the Brn-3a and Brn-3b expression vectors contain full-length cDNA or cDNAs encoding various regions of the molecule cloned under the control of the Moloney murine leukemia virus promoter in the vector pJ4 (30Morgenstern J.P. Land H. Nucleic Acids Res. 1990; 18: 1068Crossref PubMed Scopus (256) Google Scholar). The mutant forms of Brn-3a and Brn-3b containing an altered amino acid at position 22 in the homeodomain were constructed as described previously using the pALTER-1 vector (Promega) and were then subcloned into the pJ4 vector for use in transient transfection experiments (33Dawson S.J. Morris P.J. Latchman D.S. J. Biol. Chem. 1996; 271: 11631-11633Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Transient transfection of DNA was carried out according to the method of Gorman (34Gorman C.M. Glover D.M. DNA Cloning, A Practical Approach. IRL Press at Oxford University Press, Oxford1985: 143-190Google Scholar). Routinely 1 × 106 BHK-21 cells (29Macpherson I. Stoker M. Virology. 1962; 16: 147-151Crossref PubMed Scopus (595) Google Scholar) or ND7 cells (21Wood J.N. Bevan S.J. Coote P. Darn P.M. Hogan P. Latchman D.S. Morrison C. Rougon G. Theveniau M. Wheatley S.C. Proc. R. Soc. Lond. B Biol. Sci. 1990; 241: 187-194Crossref PubMed Scopus (219) Google Scholar) were transfected with 10 μg of the reporter plasmid and 10 μg of the Brn-3 expression vectors. In all cases cells were harvested 72 h later. The amount of DNA taken up by these cells in each case was measured by slot blotting the extract and hybridization with a probe derived from the ampicillin resistance gene in the plasmid vector (35Abken H. Reifenrath B. Nucleic Acids Res. 1992; 20: 3527Crossref PubMed Scopus (82) Google Scholar). This value was then used to equalize the values obtained in the CAT 1The abbreviation used is: CAT, chloramphenicol acetyltransferase. assay as a control for differences in uptake of plasmid DNA in each sample. In parallel experiments, we also included an internal control plasmid in which the cytomegalovirus promoter drives β-galactosidase expression allowing equalization of the samples on the basis of β-galactosidase activity prior to assay of CAT activity. Assays of chloramphenicol acetyltransferase activity were carried out according to the method of Gorman (34Gorman C.M. Glover D.M. DNA Cloning, A Practical Approach. IRL Press at Oxford University Press, Oxford1985: 143-190Google Scholar) using samples that had been equalized for protein content as determined by the method of Bradford (36Bradford M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (211941) Google Scholar). RNA was isolated from stably transfected cells by the guanidinium thiocyanate method (37Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62898) Google Scholar). The expression of the neurofilament RNAs was then quantitated using a reverse transcriptase/polymerase chain reaction assay. Initially all samples were amplified using primers for the constitutively expressed mRNA encoding the L6 ribosomal protein. Subsequently the neurofilament mRNAs were amplified using specific primers for each mRNA. Primers were as follows: NFL 5′-CCGTACTTTTCGACCTCCTA-3′ and 5′-TCTCTTGTGTGCGGATAGAC-3′; NFM 5′-ACACCATCCAGCAGTTGGAA-3′ and 5′-TGTGTTGGACCTTGAGCTTG-3′; NFH 5′-CCCAAGAGGAGATAACTGAG-3′ and 5′-TCAATGTCCAGGGCCATCTT-3′; L6 5′-ATCGCTCCTCAAACTTGACC-3′ and 5′-AACTACAACCACCTCATGCC-3′. All amplifications were carried out using a number of cycles and amount of mRNA that had been determined in preliminary experiments to be in the range of the assay where the amount of product formed was linearly related to the amount of input RNA. Protein was isolated from cells by freeze-thaw extraction in protein buffer (20 mm HEPES, pH 7.9, 0.45 m NaCl, 25% glycerol, 0.2 mm EDTA contains 1 μg/ml leupeptin, 1 μg/ml pepstatin, and 1 mm dithiothreitol) and submitted to analysis by SDS-polyacrylamide gel electrophoresis. Gels were transferred to nitrocellulose filter by Western blotting, and the neurofilament proteins were detected by probing with the mouse monoclonal antibodies NM-08-304, NM-08-307, and NM-08-306 (Genosys). To ensure equal loading of protein samples, gels were stripped and reprobed using a control antibody, in this case pGp9.5. The ND7-derived cell lines overexpressing individual forms of Brn-3 were constructed (25Smith M.D. Dawson S.J. Latchman D.S. Mol. Cell. Biol. 1997; 17: 345-354Crossref PubMed Scopus (63) Google Scholar, 26Smith M.D. Dawson S.J. Latchman D.S. J. Biol. Chem. 1997; 272: 1382-1388Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) by stably transfecting ND7 cells with expression vectors containing each form of Brn-3 under the control of the dexamethasone-inducible murine mammary tumor virus promoter in the vector pJ5 (30Morgenstern J.P. Land H. Nucleic Acids Res. 1990; 18: 1068Crossref PubMed Scopus (256) Google Scholar). Both by RNA analysis (25Smith M.D. Dawson S.J. Latchman D.S. Mol. Cell. Biol. 1997; 17: 345-354Crossref PubMed Scopus (63) Google Scholar, 26Smith M.D. Dawson S.J. Latchman D.S. J. Biol. Chem. 1997; 272: 1382-1388Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar) and DNA mobility shift assays using an oligonucleotide containing a binding site for the Brn-3 factors (data not shown), all the cell lines showed some overexpression of the appropriate form of Brn-3 in the absence of dexamethasone with enhanced expression being inducible upon addition of 1 μmdexamethasone. Although expression is therefore somewhat leaky and induced severalfold by hormone, it is possible to use these cells to examine the effects of overexpressing different forms of Brn-3 by studying these effects both in the absence or presence of dexamethasone and comparing the results to similarly treated control cells. We therefore prepared mRNA and protein from the various ND7-derived cell lines proliferating in full serum-containing medium either in the presence or absence of dexamethasone. In each case mRNA and protein was prepared from three independent cell lines of each type which had previously been shown to overexpress Brn-3a, Brn-3b, or Brn-3c (25Smith M.D. Dawson S.J. Latchman D.S. Mol. Cell. Biol. 1997; 17: 345-354Crossref PubMed Scopus (63) Google Scholar,26Smith M.D. Dawson S.J. Latchman D.S. J. Biol. Chem. 1997; 272: 1382-1388Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). Initially, the expression of each of the neurofilament proteins was quantitated using a Western blotting assay. As shown in Fig. 1, each of the neurofilament proteins showed a somewhat enhanced level in the Brn-3 overexpressing cells in the absence of steroid compared with control cells stably transfected with the pJ5 vector alone. Moreover, upon steroid induction of exogenous Brn-3a gene expression, the levels of each of the neurofilament proteins increased in the Brn-3a cell lines but not in the control cells. This resulted in overexpression in the Brn-3a cell lines of 3.7-fold for NF-H (p < 0.01) 2.2-fold for NF-M (p < 0.05), and 3-fold for NF-L (p < 0.01) relative to control cells. Hence, the expression of all three neurofilament proteins is enhanced in the Brn-3a expressing cells. In contrast no increase in expression of any of the neurofilament proteins was observed in the Brn-3b expressing cells, whereas only small (less than 2-fold) increases were observed in the Brn-3c expressing cells. Having established that Brn-3a can enhance neurofilament protein levels, we wished to determine whether this was paralleled by increased neurofilament mRNA levels as would be expected if Brn-3a directly activates the neurofilament gene promoters. The relative expression of each of the neurofilament mRNAs was therefore quantified in the cells using a reverse transcriptase/polymerase chain reaction assay. In these experiments (Fig. 2) the neurofilament heavy chain mRNA was overexpressed approximately 2-fold in the Brn-3a overexpressing cells compared with control cells stably transfected with the pJ5 vector alone (p < 0.01). Upon steroid induction this overexpression of the neurofilament heavy chain gene rose to approximately 6-fold over that observed in similarly treated control cells (p < 0.01). Similar overexpression of the neurofilament light chain of approximately 2-fold before steroid treatment (p < 0.01) and of approximately 5-fold after steroid induction (p < 0.01) was also observed in the Brn-3a overexpressing cells, whereas the neurofilament medium chain mRNA showed only marginal overexpression in the Brn-3a cells before steroid treatment but was overexpressed approximately 3-fold (p < 0.01) after steroid treatment (Fig. 2). In contrast no significant overexpression of any of the neurofilament genes was observed in the Brn-3b or Brn-3c overexpressing cells either before or after steroid treatment. These findings indicate therefore that the overexpression of Brn-3a in the ND7 cells can induce the overexpression of all three of the neurofilament genes at both the mRNA and protein levels. Thus prior to dexamethasone treatment when some overexpression of Brn-3a is observed in the ND7 cells (25Smith M.D. Dawson S.J. Latchman D.S. Mol. Cell. Biol. 1997; 17: 345-354Crossref PubMed Scopus (63) Google Scholar), there is also some overexpression of the different neurofilament genes. Moreover, when enhanced expression of Brn-3a is induced by steroid treatment, this results in significant overexpression of all three neurofilament genes above the level observed in comparably treated control cells with the neurofilament heavy chain showing the greatest effect at both the mRNA and protein levels, followed by the light chain and then the medium chain. In previous experiments we have shown that the ability of Brn-3a to induce target genes in co-transfection experiments is dependent upon two activation domains, one of which is located at the N terminus of the molecule (17Budhram-Mahadeo V. Morris P.J. Lakin N.D. Theil T. Ching G.Y. Lillycrop K.A. Möröy T. Liem R.K.H. Latchman D.S. J. Biol. Chem. 1995; 270: 2853-2858Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), and the other is located at the C terminus coincident with the POU domain. The relative importance of these different activation domains varies with the target promoter tested (17Budhram-Mahadeo V. Morris P.J. Lakin N.D. Theil T. Ching G.Y. Lillycrop K.A. Möröy T. Liem R.K.H. Latchman D.S. J. Biol. Chem. 1995; 270: 2853-2858Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 22Budhram-Mahadeo V.S. Theil T. Morris P.J. Lillycrop K.A. Möröy T. Latchman D.S. Nucleic Acids Res. 1994; 22: 3092-3098Crossref PubMed Scopus (38) Google Scholar, 24Morris P.J. Theil T. Ring C.J.A. Lillycrop K.A. Möröy T. Latchman D.S. Mol. Cell. Biol. 1994; 14: 6907-6914Crossref PubMed Scopus (72) Google Scholar). In our previous experiments (25Smith M.D. Dawson S.J. Latchman D.S. Mol. Cell. Biol. 1997; 17: 345-354Crossref PubMed Scopus (63) Google Scholar) we showed that transfection of ND7 cells with a naturally occurring form of Brn-3a (Brn-3a short) that lacks the N-terminal activation domain (9Theil T. McLean-Hunter S. Zornig M. Möröy T. Nucleic Acids Res. 1993; 21: 5921-5929Crossref PubMed Scopus (82) Google Scholar, 38Liu Y.Z. Dawson S.J. Latchman D.S. J. Mol. Neurosci. 1996; 7: 77-85Crossref PubMed Scopus (30) Google Scholar) still results in the promotion of neurite outgrowth, although to a somewha" @default.
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• W2034658858 title "Coordinate Induction of the Three Neurofilament Genes by the Brn-3a Transcription Factor" @default.
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