Multiple Neuron-specific Enhancers in the Gene Coding for the Human Neurofilament Light Chain

Abstract

To define DNA regions involved in the neuron-specific expression of the neurofilament light (NF-L) gene, we generated transgenic mice bearing different NF-L constructs. A 4.9-kilobase human NF-L fragment including −292 base pairs of 5′-flanking sequences contained sufficient elements for nervous system expression in transgenic mice. Deletion of introns 1 and 2 from this 4.9-kilobase DNA fragment resulted in reduced levels of transgene expression in the cortex, while deletion of intron 3 had little effect. Both introns 1 and 2 could act independently as enhancers to confer neuronal expression of the basal heat shock promoter (hsp68) fused to lacZ in transgenic mice. The hNF-L basal promoter (−292 base pairs) was found to contain elements for directing neuronal expression of either the lacZ reporter gene or an intronless hNF-L construct. Sequence comparison revealed that intron 1, intron 2, and the basal human NF-L promoter all contain an ETS-like motif, CAGGA, present in a variety of genes expressed in the nervous system. To define DNA regions involved in the neuron-specific expression of the neurofilament light (NF-L) gene, we generated transgenic mice bearing different NF-L constructs. A 4.9-kilobase human NF-L fragment including −292 base pairs of 5′-flanking sequences contained sufficient elements for nervous system expression in transgenic mice. Deletion of introns 1 and 2 from this 4.9-kilobase DNA fragment resulted in reduced levels of transgene expression in the cortex, while deletion of intron 3 had little effect. Both introns 1 and 2 could act independently as enhancers to confer neuronal expression of the basal heat shock promoter (hsp68) fused to lacZ in transgenic mice. The hNF-L basal promoter (−292 base pairs) was found to contain elements for directing neuronal expression of either the lacZ reporter gene or an intronless hNF-L construct. Sequence comparison revealed that intron 1, intron 2, and the basal human NF-L promoter all contain an ETS-like motif, CAGGA, present in a variety of genes expressed in the nervous system. INTRODUCTIONNeurofilaments are formed by the copolymerization of three neuron-specific proteins with an apparent molecular mass on SDS gel of 70 kDa (NF-L), 150 kDa (NF-M), and 200 kDa (NF-H)(1.Hoffman P.N. Lasek R.J. J. Cell Biol. 1975; 66: 351-366Crossref PubMed Scopus (713) Google Scholar, 2.Liem R.K.H. Yen S.-H. Salomon G.D. Shelanski M.L. J. Cell Biol. 1978; 79: 637-645Crossref PubMed Scopus (364) Google Scholar, 3.Geisler N. Weber K. J. Mol. Biol. 1981; 151: 565-571Crossref PubMed Scopus (212) Google Scholar). The genes coding for the three NF 1The abbreviations used are: NFneurofilamentNF-Lneurofilament lightbpbase pair(s)kbkilobase(s)hNF-Lhuman neurofilament lightUTRuntranslated regionPCRpolymerase chain reactionRT-PCRreverse transcriptase PCRHShypersensitive sitesPBSphosphate-buffered saline. proteins have been cloned and sequenced(4.Lewis S.A. Cowan N.J. Mol. Cell. Biol. 1986; 6: 1529-1534Crossref PubMed Google Scholar, 5.Julien J.-P. Meijer D. Hurst J. Grosveld F. Mol. Brain Res. 1986; 1: 243-250Crossref Scopus (157) Google Scholar, 6.Julien J.-P. Grosveld F. Yazdanbaksh K. Flavell D. Meijer D. Mushynski W. Biochim. Biophys. Acta. 1987; 909: 10-20Crossref PubMed Scopus (100) Google Scholar, 7.Julien J.-P. Côté F. Beaudet L. Flavell D. Grosveld F. Mushynski W.E. Gene (Amst.). 1988; 68: 307-314Crossref PubMed Scopus (82) Google Scholar, 8.Levy E. Liem R.K.H. D'Eustachio P. Cowan N.J. Eur. J. Biochem. 1987; 166: 71-77Crossref PubMed Scopus (106) Google Scholar, 9.Myers M.W. Lazzarini R.A. Lee V.M.-Y. Schlaepfer W.W. Nelson D.L. EMBO J. 1987; 6: 1617-1626Crossref PubMed Scopus (215) Google Scholar, 10.Lees J.F. Shneidman P.S. Skuntz S.F. Carden M.J. Lazzarini R.A. EMBO J. 1988; 7: 1947-1955Crossref PubMed Scopus (183) Google Scholar). Like other intermediate filaments (IF) genes, NF genes are expressed in a cell type-specific and developmental manner(11.Osborn M. Weber K. Cell. 1982; 31: 303-306Abstract Full Text PDF PubMed Scopus (278) Google Scholar, 12.Cochard P. Paulin D. J. Neurosci. 1984; 4: 2080-2094Crossref PubMed Google Scholar, 13.Liesi P. Julien J.-P. Vilja P. Grosveld F. Rechardt L. J. Histochem. Cytochem. 1986; 34: 923-929Crossref PubMed Scopus (45) Google Scholar). IF genes, from class IV and VI, are differentially regulated during the development of neuronal progenitor cells(5.Julien J.-P. Meijer D. Hurst J. Grosveld F. Mol. Brain Res. 1986; 1: 243-250Crossref Scopus (157) Google Scholar, 9.Myers M.W. Lazzarini R.A. Lee V.M.-Y. Schlaepfer W.W. Nelson D.L. EMBO J. 1987; 6: 1617-1626Crossref PubMed Scopus (215) Google Scholar, 14.Lendahl U. Zimmerman L.B. McKay R.D.G. Cell. 1990; 60: 585-595Abstract Full Text PDF PubMed Scopus (2758) Google Scholar, 15.Ching G.Y. Liem R.K.H. J. Biol. Chem. 1991; 266: 19459-19468Abstract Full Text PDF PubMed Google Scholar, 16.Lieberburg I. Spinner N. Snyder S. Anderson J. Goldgaber D. Smulowitz M. Carroll Z. Emanuel B. Breitner J. Rubin L. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2463-2467Crossref PubMed Scopus (54) Google Scholar).Neurofilaments are expressed in most neurons of the nervous system, and their expression coincides with terminal neuronal differentiation. There is growing evidence that deregulation of neurofilament expression may play a central role in motor neuron disease. Transgenic mice that overexpress neurofilament proteins show motor neuron degeneration (17.Côté F. Collard J.-F. Julien J.-P. Cell. 1993; 73: 35-46Abstract Full Text PDF PubMed Scopus (493) Google Scholar, 18.Xu Z. Cork L. Griffin J.W. Cleveland D.W. Cell. 1993; 73: 23-33Abstract Full Text PDF PubMed Scopus (486) Google Scholar, 19.Lee M.K. Marszalek J.R. Cleveland D.W. Neuron. 1994; 13: 975-988Abstract Full Text PDF PubMed Scopus (338) Google Scholar, 20.Collard J.-F. Côté F. Julien J.-P. Nature. 1995; 375: 61-64Crossref PubMed Scopus (409) Google Scholar, 21.Julien J.-P. Côté F. Collard J.-F. Neurobiol. Aging. 1995; 16: 487-492Crossref PubMed Scopus (0) Google Scholar). Furthermore, the levels of NF-L mRNA are decreased in dogs with hereditary canine spinal muscular atrophy (22.Muma N. Cork L. Lab. Invest. 1993; 69: 436-442PubMed Google Scholar) as well as in motor neurons of patients with amyotrophic lateral sclerosis(23.Bergeron C. Beric-Maskarel K. Muntasser S. Weyer L. Somerville M.J. Percy M.E. J. Neuropathol. Exp. Neurol. 1994; 53: 221-230Crossref PubMed Scopus (145) Google Scholar). Yet, little is known about the mechanisms that regulate expression of neurofilament genes in the nervous system. This is in part due to the lack of suitable in vitro systems to study their expression (15.Ching G.Y. Liem R.K.H. J. Biol. Chem. 1991; 266: 19459-19468Abstract Full Text PDF PubMed Google Scholar). For instance, high levels of NF-L expression occurred after transfection of a complete genomic NF-L gene in non-neuronal cells such as cultured fibroblasts(6.Julien J.-P. Grosveld F. Yazdanbaksh K. Flavell D. Meijer D. Mushynski W. Biochim. Biophys. Acta. 1987; 909: 10-20Crossref PubMed Scopus (100) Google Scholar, 24.Monteiro M.J. Cleveland D.W. J. Cell Biol. 1989; 108: 579-593Crossref PubMed Scopus (92) Google Scholar), even though the endogenous NF-L gene remained silent. In contrast, DNA fragments containing either the complete human or mouse NF-L genes were correctly expressed in transgenic mice(25.Julien J.-P. Tretjakoff I. Beaudet L. Peterson A. Genes & Dev. 1987; 1: 1085-1095Crossref PubMed Scopus (87) Google Scholar, 26.Monteiro M.J. Hoffman P.N. Gearhart J.D. Cleveland D.W. J. Cell Biol. 1990; 111: 1543-1557Crossref PubMed Scopus (145) Google Scholar).We have shown previously that a human NF-L fragment including −292 bp of 5′-flanking sequences and intron sequences contained sufficient elements to drive NF-L expression in the nervous tissues of adult transgenic mice(27.Beaudet L. Charron G. Houle D. Tretjakoff I. Peterson A. Gene (Amst.). 1992; 116: 205-214Crossref PubMed Scopus (39) Google Scholar). To further clarify the potential role of introns in modulating expression of the human NF-L gene, we used the transgenic mouse approach to test the transcriptional activity of NF-L DNA regions. We report here the existence of neuron-specific enhancers in both introns 1 and 2, which contribute to a wider expression pattern of the NF-L gene. In addition, we show that the regulatory elements included in the NF-L basal promoter (−292 bp) are sufficient to target neuronal expression.EXPERIMENTAL PROCEDURESPlasmid ConstructTo delete introns of the hNF-L gene, we took advantage of the existence of conserved restriction sites between the human and mouse NF-L genes. First, a 4.9-kb BamHI-XbaI fragment including the human NF-L gene was subcloned into the corresponding sites of a Bluescript L. Charron G. Houle D. Tretjakoff I. Peterson A. Gene (Amst.). 1992; 116: 205-214Crossref PubMed Scopus (39) Google Scholar). The was to a by a in deletion of a DNA fragment intron 1 and 2 all with part of 1 and The was on A corresponding DNA fragment of bp from the mouse J.-P. Meijer D. Hurst J. Grosveld F. Mol. Brain Res. 1986; 1: 243-250Crossref Scopus (157) Google Scholar) was subcloned in that to a NF-L gene with complete coding sequences introns 1 and 2 A approach was used to delete intron that a was The fragment from the mouse was used to the For the and the the was to by by a the mouse J.-P. Meijer D. Hurst J. Grosveld F. Mol. Brain Res. 1986; 1: 243-250Crossref Scopus (157) Google Scholar, 13.Liesi P. Julien J.-P. Vilja P. Grosveld F. Rechardt L. J. Histochem. Cytochem. 1986; 34: 923-929Crossref PubMed Scopus (45) Google Scholar), a fragment of bp was and to the to all deletion were on gel after complete BamHI-XbaI for the the DNA fragment was after with The restriction is bp after the of the human NF-L gene was from The was to with by A fragment of bp was and subcloned into Bluescript with The human NF-L basal promoter −292 to bp) was and to to into in construct. A DNA fragment of was on gel for the we subcloned a lacZ J. D. M. Genes & Dev. 1991; PubMed Scopus Google Scholar) into the sites of a Bluescript intron was by and into the the of the For was from sequences on gel after with into DNA fragments were and to of 1 in to into of were to the of M. Cell. 1981; Full Text PDF PubMed Scopus Google Scholar). of transgenic mice was by of of genomic DNA as L. Charron G. Houle D. Tretjakoff I. Peterson A. Gene (Amst.). 1992; 116: 205-214Crossref PubMed Scopus (39) Google Scholar, J.-P. Beaudet L. Tretjakoff I. Peterson A. J. 1990; Google was from tissues of transgenic mice by the P. N. Biochem. 1987; PubMed Scopus Google Scholar). A fragment 1 and intron 1 sequences of the human NF-L gene was subcloned into the of the with an of high was generated with in the of of was with of and was as K. Res. 1984; PubMed Scopus Google Scholar). fragments were on as of mRNA was in of to were in of A The was by of and of and in of of and of was during 1 and for was to for the hNF-L and and the to the and The was to the of the as of and 1 of DNA 1 1 an of both was on and and are for the hNF-L sequences and A of for the mouse mRNA to of in sequences are and for were in in for were in three of and in a containing 1 2 and in were for by for 1 in containing and in with were in and in with were on and on of NF-L the of sequences on transcriptional of the hNF-L gene, we generated transgenic mice with DNA in which introns were in The in of conserved restriction sites between the human and mouse NF-L genes to genomic regions of the human NF-L gene with fragments of the mouse NF-L deletion included the basal human NF-L promoter (−292 bp) and the endogenous hNF-L transgenic mouse have been generated with the 4.9-kb hNF-L gene In mouse the transgene remained in all tissues In the other three mouse expression of the NF-L as by was to the nervous system. the of a with from an adult mouse of 1 an the hNF-L of generated with different NF-L gene in transgenic mouse that were the of a of the of NF-L mRNA while of The mouse in are with a of an NF-L transgene intron from tissues of transgenic mice bearing a complete hNF-L gene and the was by an for expression of human NF-L The of bp the hNF-L a fragment of The mRNA in was from the mouse a human NF-L Z. Cork L. Griffin J.W. Cleveland D.W. Cell. 1993; 73: 23-33Abstract Full Text PDF PubMed Scopus (486) Google Scholar). The to mRNA from a the of three mouse expressed the transgene of the from in both mouse a expression to nervous system In transgenic expression occurred in the of the and the the human could used the human 1 sequences have been by the corresponding mouse to transgene we an with a of for the of the hNF-L The corresponding endogenous NF-L mRNA is with of in the For the and the a of different transgenic mouse were of which expressed their transgene three DNA a expression to nervous generated with the a in the of NF-L by an mRNA from tissues of transgenic mice was to of were The to for the of the human NF-L gene, and mouse NF-L The to the mouse gene, which is used as an 1, from mRNA of mouse mRNA from a hNF-L transgenic mRNA from mice with mRNA from mice with and mRNA from mice with of introns 1 and 2 resulted in decreased levels of transgene expression in the and as by with deletion of intron 3 and had little on expression pattern and of the that introns 1 and 2 contain regulatory elements that contribute to the of hNF-L expression in the and of adult of a lacZ Gene by the hNF-L with the that basal NF-L was to expression in the nervous system. we previously that the promoter was to neuronal expression of the reporter gene. To further clarify the activity of this promoter (−292 to we on the used reporter gene transgenic were with the of which three expressed the The were with A of lacZ was found in the nervous system We a in the expression from to an of the This which been for other C. G. P. J.F. Proc. Natl. Acad. Sci. U. S. A. 1990; PubMed Scopus Google Scholar, M. D. C. Mol. Dev. 1992; PubMed Scopus (45) Google Scholar), from the of elements that confer expression found in the F. G. G. Cell. 1987; Full Text PDF PubMed Scopus Google Scholar). on adult of 1 expression to of neurons In the cortex, lacZ was in the and of the in neurons of the the lateral the and in the was found in neurons of the cell of the neurons of the molecular were cells were lacZ expression was found in the of the spinal and in the and In all regions of the nervous system, a of neurons was The that the basal hNF-L promoter contain transcriptional elements to neuron-specific expression in both and adult regulatory elements in the NF-L gene lacZ were after of lacZ DNA constructs. is shown a of lacZ construct. the and the the of the the from which were are of from and of in an transgenic The was from adult transgenic of mouse 1 after with in The lacZ was on were in a with and of a and is shown and a are as the the lateral the and the the spinal and the are is cell molecular cell lateral in NF-L the existence of regulatory elements introns of the human NF-L gene. To further the of regulatory elements in the DNA fragments intron sequences were for their activity a This is of the basal promoter of the gene for the heat shock by the E. gene. The promoter basal activity in transgenic mice S. A. Peterson A. J. Nature. 1988; PubMed Scopus Google Scholar) a was in J. D. M. Genes & Dev. 1991; PubMed Scopus Google Scholar, J. L. C. M. Genes & Dev. 1990; 4: PubMed Scopus Google Scholar, C. D. 1993; Google Scholar). the transcriptional of the basal promoter the activity of elements by a The hNF-L introns 1, 2, and 3 were by and subcloned independently in of the to and transgenic were for expression of the were expressed the lacZ transgene in the nervous A in the pattern of nervous system expression was from to expression in the while a in the the transgenic bearing the a in the nervous system. revealed expression to the the transgenic generated with the lacZ expression in the nervous system. expression was levels in three the existence of neuron-specific enhancers in the and NF-L introns that contribute to NF-L expression in the and that to of the nervous system The intron is of such To further define regions in the we generated a in which the of intron 1 was subcloned in of the gene in an transgenic were and both expressed lacZ in the nervous system. is that the of intron 1 elements to expression in the of the nervous report here that the human NF-L promoter (−292 bp) is sufficient to neuronal expression both of an and an reporter gene a occurred in transgenic generated with the the lacZ activity was to the nervous This is due to an of the as regulatory elements could transcriptional The neuronal expression of the was an as this promoter was previously found to to neuron-specific expression of the reporter L. Charron G. Houle D. Tretjakoff I. Peterson A. Gene (Amst.). 1992; 116: 205-214Crossref PubMed Scopus (39) Google Scholar). could different reporter genes different is well known that of the DNA in a basal transcriptional activity M. 1990; 6: Full Text PDF PubMed Scopus Google to such as this We the of regions with DNA in the of the hNF-L and lacZ genes in the G. Julien J.-P. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). deletion of sequences from the lacZ gene, the to and expression was G. Julien J.-P. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google of introns 1 and 2 in the human NF-L gene resulted in a of transgene expression in the and of adult The of regulatory elements in introns 1 and 2 was further the Both introns as enhancers neuronal expression of the promoter in a and shown in intron 1 expression to the and The intron 1 for expression to of nervous system is in of this intron The of regulatory elements introns of the human NF-L gene is with the of the sites by Yazdanbaksh K. P. D. M. Grosveld F. M. Res. 1993; PubMed Scopus Google Scholar). three different neuron-specific sites in the 5′-flanking and the human NF-L gene for in 1, all other sites are The and are the of intron 1 shown here to contain elements for neuronal while is the of intron is that the of introns 1 and 2 are conserved all intermediate genes of class IV and been that genes of class IV and VI, which to the neuronal of the IF by a of an mRNA from an E. Weber K. Biochem. 1994; PubMed Scopus Google Scholar). Zimmerman L. U. M. McKay B. B. J. G. A. Neuron. 1994; Full Text PDF PubMed Scopus Google Scholar) that elements and neuronal of the gene, an IF of class VI, in the and introns to intron 1 and 2 in the NF-L gene. is that the of introns by an intronless progenitor gene may have to the of a of IF genes expressed in the nervous was to known elements the promoter and introns of the human NF-L gene. 1, and the sequences are present in both introns 1 and 2 of regulatory elements neuronal The mRNA coding for the is in mouse expression is to this A. P. Genes & Dev. 1992; 6: PubMed Scopus Google Scholar). We that the CAGGA, which part of the by the of A. Biochim. Biophys. Acta. 1993; PubMed Scopus Google Scholar), is present in neuron-specific genes including the genes for E. Liem R.K.H. D'Eustachio P. Cowan N.J. Eur. J. Biochem. 1987; 166: 71-77Crossref PubMed Scopus (106) Google Scholar, D. B. Res. 1990; PubMed Scopus Google Scholar), G.Y. Liem R.K.H. J. Biol. Chem. 1991; 266: 19459-19468Abstract Full Text PDF PubMed Google Scholar), D. M. L. A. EMBO J. 1992; PubMed Scopus (45) Google Scholar), J. Genes & Dev. 1989; PubMed Scopus Google Scholar, J. U. EMBO J. 1988; 7: PubMed Scopus Google Scholar), L. M. J.-P. EMBO J. 1992; PubMed Scopus Google Scholar), mouse E. C. M. F. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: PubMed Scopus Google Scholar), neuron-specific K. E. Gene (Amst.). 1987; 60: Scopus Google Scholar), S. Tretjakoff Peterson Genes & Dev. 1991; PubMed Scopus Google Scholar), the gene the N. Anderson Neuron. 1990; 4: Full Text PDF PubMed Scopus Google Scholar), and R.A. G. Neuron. 1990; 4: Full Text PDF PubMed Scopus Google Scholar). known of the are expressed in other are expressed in Julien J.-P. Cell & 1994; Google Scholar). We that an ETS-like in the human NF-L is by a expressed in cells into Julien J.-P. Cell & 1994; Google Scholar). neuron-specific sites this K. P. D. M. Grosveld F. M. Res. 1993; PubMed Scopus Google Scholar). an for in the of neuron-specific of the ETS-like elements in the NF-L gene their to expression of this gene in the nervous regulatory elements in the human NF-L and sites found in the regulatory of the human The was on hNF-L regions −292 to to and to the with the base from were with INTRODUCTIONNeurofilaments are formed by the copolymerization of three neuron-specific proteins with an apparent molecular mass on SDS gel of 70 kDa (NF-L), 150 kDa (NF-M), and 200 kDa (NF-H)(1.Hoffman P.N. Lasek R.J. J. Cell Biol. 1975; 66: 351-366Crossref PubMed Scopus (713) Google Scholar, 2.Liem R.K.H. Yen S.-H. Salomon G.D. Shelanski M.L. J. Cell Biol. 1978; 79: 637-645Crossref PubMed Scopus (364) Google Scholar, 3.Geisler N. Weber K. J. Mol. Biol. 1981; 151: 565-571Crossref PubMed Scopus (212) Google Scholar). The genes coding for the three NF 1The abbreviations used are: NFneurofilamentNF-Lneurofilament lightbpbase pair(s)kbkilobase(s)hNF-Lhuman neurofilament lightUTRuntranslated regionPCRpolymerase chain reactionRT-PCRreverse transcriptase PCRHShypersensitive sitesPBSphosphate-buffered saline. proteins have been cloned and sequenced(4.Lewis S.A. Cowan N.J. Mol. Cell. Biol. 1986; 6: 1529-1534Crossref PubMed Google Scholar, 5.Julien J.-P. Meijer D. Hurst J. Grosveld F. Mol. Brain Res. 1986; 1: 243-250Crossref Scopus (157) Google Scholar, 6.Julien J.-P. Grosveld F. Yazdanbaksh K. Flavell D. Meijer D. Mushynski W. Biochim. Biophys. Acta. 1987; 909: 10-20Crossref PubMed Scopus (100) Google Scholar, 7.Julien J.-P. Côté F. Beaudet L. Flavell D. Grosveld F. Mushynski W.E. Gene (Amst.). 1988; 68: 307-314Crossref PubMed Scopus (82) Google Scholar, 8.Levy E. Liem R.K.H. D'Eustachio P. Cowan N.J. Eur. J. Biochem. 1987; 166: 71-77Crossref PubMed Scopus (106) Google Scholar, 9.Myers M.W. Lazzarini R.A. Lee V.M.-Y. Schlaepfer W.W. Nelson D.L. EMBO J. 1987; 6: 1617-1626Crossref PubMed Scopus (215) Google Scholar, 10.Lees J.F. Shneidman P.S. Skuntz S.F. Carden M.J. Lazzarini R.A. EMBO J. 1988; 7: 1947-1955Crossref PubMed Scopus (183) Google Scholar). Like other intermediate filaments (IF) genes, NF genes are expressed in a cell type-specific and developmental manner(11.Osborn M. Weber K. Cell. 1982; 31: 303-306Abstract Full Text PDF PubMed Scopus (278) Google Scholar, 12.Cochard P. Paulin D. J. Neurosci. 1984; 4: 2080-2094Crossref PubMed Google Scholar, 13.Liesi P. Julien J.-P. Vilja P. Grosveld F. Rechardt L. J. Histochem. Cytochem. 1986; 34: 923-929Crossref PubMed Scopus (45) Google Scholar). IF genes, from class IV and VI, are differentially regulated during the development of neuronal progenitor cells(5.Julien J.-P. Meijer D. Hurst J. Grosveld F. Mol. Brain Res. 1986; 1: 243-250Crossref Scopus (157) Google Scholar, 9.Myers M.W. Lazzarini R.A. Lee V.M.-Y. Schlaepfer W.W. Nelson D.L. EMBO J. 1987; 6: 1617-1626Crossref PubMed Scopus (215) Google Scholar, 14.Lendahl U. Zimmerman L.B. McKay R.D.G. Cell. 1990; 60: 585-595Abstract Full Text PDF PubMed Scopus (2758) Google Scholar, 15.Ching G.Y. Liem R.K.H. J. Biol. Chem. 1991; 266: 19459-19468Abstract Full Text PDF PubMed Google Scholar, 16.Lieberburg I. Spinner N. Snyder S. Anderson J. Goldgaber D. Smulowitz M. Carroll Z. Emanuel B. Breitner J. Rubin L. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2463-2467Crossref PubMed Scopus (54) Google Scholar).Neurofilaments are expressed in most neurons of the nervous system, and their expression coincides with terminal neuronal differentiation. There is growing evidence that deregulation of neurofilament expression may play a central role in motor neuron disease. Transgenic mice that overexpress neurofilament proteins show motor neuron degeneration (17.Côté F. Collard J.-F. Julien J.-P. Cell. 1993; 73: 35-46Abstract Full Text PDF PubMed Scopus (493) Google Scholar, 18.Xu Z. Cork L. Griffin J.W. Cleveland D.W. Cell. 1993; 73: 23-33Abstract Full Text PDF PubMed Scopus (486) Google Scholar, 19.Lee M.K. Marszalek J.R. Cleveland D.W. Neuron. 1994; 13: 975-988Abstract Full Text PDF PubMed Scopus (338) Google Scholar, 20.Collard J.-F. Côté F. Julien J.-P. Nature. 1995; 375: 61-64Crossref PubMed Scopus (409) Google Scholar, 21.Julien J.-P. Côté F. Collard J.-F. Neurobiol. Aging. 1995; 16: 487-492Crossref PubMed Scopus (0) Google Scholar). Furthermore, the levels of NF-L mRNA are decreased in dogs with hereditary canine spinal muscular atrophy (22.Muma N. Cork L. Lab. Invest. 1993; 69: 436-442PubMed Google Scholar) as well as in motor neurons of patients with amyotrophic lateral sclerosis(23.Bergeron C. Beric-Maskarel K. Muntasser S. Weyer L. Somerville M.J. Percy M.E. J. Neuropathol. Exp. Neurol. 1994; 53: 221-230Crossref PubMed Scopus (145) Google Scholar). Yet, little is known about the mechanisms that regulate expression of neurofilament genes in the nervous system. This is in part due to the lack of suitable in vitro systems to study their expression (15.Ching G.Y. Liem R.K.H. J. Biol. Chem. 1991; 266: 19459-19468Abstract Full Text PDF PubMed Google Scholar). For instance, high levels of NF-L expression occurred after transfection of a complete genomic NF-L gene in non-neuronal cells such as cultured fibroblasts(6.Julien J.-P. Grosveld F. Yazdanbaksh K. Flavell D. Meijer D. Mushynski W. Biochim. Biophys. Acta. 1987; 909: 10-20Crossref PubMed Scopus (100) Google Scholar, 24.Monteiro M.J. Cleveland D.W. J. Cell Biol. 1989; 108: 579-593Crossref PubMed Scopus (92) Google Scholar), even though the endogenous NF-L gene remained silent. In contrast, DNA fragments containing either the complete human or mouse NF-L genes were correctly expressed in transgenic mice(25.Julien J.-P. Tretjakoff I. Beaudet L. Peterson A. Genes & Dev. 1987; 1: 1085-1095Crossref PubMed Scopus (87) Google Scholar, 26.Monteiro M.J. Hoffman P.N. Gearhart J.D. Cleveland D.W. J. Cell Biol. 1990; 111: 1543-1557Crossref PubMed Scopus (145) Google Scholar).We have shown previously that a human NF-L fragment including −292 bp of 5′-flanking sequences and intron sequences contained sufficient elements to drive NF-L expression in the nervous tissues of adult transgenic mice(27.Beaudet L. Charron G. Houle D. Tretjakoff I. Peterson A. Gene (Amst.). 1992; 116: 205-214Crossref PubMed Scopus (39) Google Scholar). To further clarify the potential role of introns in modulating expression of the human NF-L gene, we used the transgenic mouse approach to test the transcriptional activity of NF-L DNA regions. We report here the existence of neuron-specific enhancers in both introns 1 and 2, which contribute to a wider expression pattern of the NF-L gene. In addition, we show that the regulatory elements included in the NF-L basal promoter (−292 bp) are sufficient to target neuronal