(Received for publication, June 29, 1995; and in revised form, October 27, 1995)
From the
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.
Neurofilaments 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, 2, 3) . The genes coding for the
three NF ()proteins have been cloned and
sequenced(4, 5, 6, 7, 8, 9, 10) .
Like other intermediate filaments (IF) genes, NF genes are expressed in
a cell type-specific and developmental
manner(11, 12, 13) . IF genes, from class IV
and VI, are differentially regulated during the development of neuronal
progenitor
cells(5, 9, 14, 15, 16) .
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, 18, 19, 20, 21) . Furthermore, the levels of NF-L mRNA are decreased in dogs with hereditary canine spinal muscular atrophy (22) as well as in motor neurons of patients with amyotrophic lateral sclerosis(23) . 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) . 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, 24) , 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, 26) .
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) . 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.
The
-galactosidase gene (lacZ) was obtained from plasmid
pCH110 (Pharmacia Biotech Inc.). The pCH110 was cut to completion with HindIII followed by EcoRI partial digestion. A
fragment of 4486 bp was then isolated and subcloned into Bluescript
pSK+ linearized with HindIII-EcoRI digestion,
creating plasmid pSKlacZ. The human NF-L basal promoter (from
-292 to +15 bp) was isolated, blunted, and ligated to HindIII linker prior to insertion into HindIII site
in pSKlacZ giving hNF-L/lacZ construct. A XhoI-NotI DNA fragment of 4.8 kb was isolated on
agarose gel for microinjection.
To prepare the hsp-lacZ construct, we subcloned a lacZ enhancer trap vector (28) into the BamHI sites of a Bluescript SK+ plasmid (Stratagene). Each intron was then amplified by PCR and inserted into the unique SmaI site localized at the 5`-end of the cassette. For microinjection, each construct was separated from plasmid sequences on agarose gel after digestion with SalI-NotI.
Figure 1: Schematic representation of NF-L DNA constructs. Internal deletion mutants were produced using a human NF-L gene fragment (hNF-L) (-292 to +4605) in which intragenic regions were replaced by corresponding regions of a mouse NF-L cDNA (see ``Experimental Procedures''). The solid boxes represent the human NF-L exons, while the double-hatched boxes represent mouse NF-L cDNA sequences. The hatched boxes represent the 3`-untranslated regions. Restriction enzyme cutting sites are as follows: B, BamHI; Bg, BglII; RI, EcoRI; Sac, SacI; X, XbaI.
Four transgenic mouse lines have been generated with the intact 4.9-kb hNF-L gene (Fig. 2). In only one mouse line, the transgene remained silent in all tissues examined. In the other three mouse lines, expression of the NF-L transgene, as detected by RNase protection assay, was restricted to the nervous system. Fig. 3A shows the results of a RNase protection experiment carried out with RNA from an adult mouse of line 1 using an antisense RNA probe covering the first hNF-L exon/intron border.
Figure 2: Summary of data generated with five different NF-L gene constructs in 16 transgenic mouse lines. ND signifies that RNA samples were not analyzed; the number of + symbols gives a relative quantification of the intensity of NF-L mRNA expression, while empty boxes indicate absence of expression. The mouse lines presented in Fig. 3and Fig. 4are labeled with a asterisk.
Figure 3: Expression analysis of an NF-L transgene lacking intron 3. Total RNA from various tissues of transgenic mice bearing a complete hNF-L gene (panel A) and the hNF-L/intron1,2 construct (panel B) was analyzed by an RNase protection assay for specific expression of human NF-L transcripts. The antisense RNA probe of 960 bp spanning the first hNF-L exon/intron boundary yields a protected fragment of 316 nucleotides. The control mRNA (lane 15 in B) was obtained from the mouse line 29 expressing a 21.1-kb human NF-L fragment(18) . The negative control (lane 14) corresponds to total cortex mRNA from a normal mouse. B, BamHI; Bg, BglII; RI, EcoRI; Sac, SacI; X, XbaI.
Figure 4: Expression analysis of NF-L transgenes by an RT-PCR assay. Total mRNA from various tissues of transgenic mice was reverse transcribed prior to PCR assay. Two sets of oligonucleotides were used. The first set corresponds to oligonucleotides specific for the first exon of the human NF-L gene, and they do not amplify mouse NF-L transcripts. The second pair corresponds to the mouse G3PDH gene, which is used as an internal control. Lane 1, amplified product from mRNA of normal mouse cortex; lanes 2-11, mRNA from a hNF-L transgenic mouse; lanes 12-21, mRNA from mice with hNF-L/intron3 transgene; lanes 22-31, mRNA from mice with hNF-L/intronless; and lanes 32-41, mRNA from mice with intronless-UTR.
With the hNF-L/intron1,2 transgene, two out of three mouse lines expressed the transgene (Fig. 2). RNase protection analysis of the RNA from offspring revealed, in both mouse lines, a restricted expression to nervous system (Fig. 3B). In one transgenic line, ectopic expression occurred in the heart (Fig. 2).
For
analysis of the hNF-L/intron3, hNF-L/intronless, and the
intronless/UTR constructs, the 960-bp human RNA probe
could not be used since the human exon 1 sequences have been replaced
by the corresponding mouse sequences. Therefore, to analyze transgene
expression, we carried out an RT-PCR assay with a set of
oligonucleotides specific for the 5`-part of the hNF-L first exon (Fig. 4A). The corresponding endogenous NF-L mRNA is
not amplified with these primers (Fig. 4B, lane
1) because of species differences in the 5`-untranslated
sequences. For the hNF-L/intron3, hNF-L/intronless, and the
intronless-UTR
, a total of nine different transgenic
mouse lines were produced, of which seven expressed their transgene (Fig. 2). All three DNA constructs yielded a restricted
expression to nervous tissue. Only line 12, generated with the
hNF-L/intronless construct, showed a signal in the heart (Fig. 4B, lane 30).
The removal of introns 1 and 2 resulted in decreased levels of transgene expression in the cortex and cerebellum as detected by RT-PCR (Fig. 4A, compare lane 12 with lane 2). Further deletion of intron 3 and 3`-UTR had little effect on expression pattern and intensity of the bands (Fig. 4B). These results suggested that introns 1 and 2 do contain regulatory elements that contribute to the enhancement of hNF-L expression in the cortex and cerebellum of adult mouse.
Figure 5:
Neuron-specific regulatory elements in the
NF-L gene direct heterologous lacZ expression. Embryos were
analyzed 13.5 days after microinjection of lacZ DNA
constructs. At left is shown a schematic representation of
each lacZ fusion construct. An indicates the stained
tissues, and the arrow at the end of the line indicates the
embryos from which pictures were taken. At right are photographs of
embryos from side, back, and front.
Figure 6:
Histological detection of
-galactosidase in an hNF-L/lacZ transgenic mouse. The
brain was dissected from adult F1 transgenic of mouse line 1 (see Fig. 5) after perfusion with 4% paraformaldehyde in PBS. The lacZ staining was performed overnight on 3-mm-thick tissue
slices. Frozen sections (15-20 µm) were collected in a
cryostat, counter-stained with neutral red, mounted, and pictured.
Overview of a near-midline sagital and transversal brain section is
shown (A and B, respectively). Panels showing a
higher magnification are as follows: C, the cingulum region
(transversal section); D, the lateral septum (transversal
section); E, the hyppocampus (sagital section); and F, the cerebellum (sagital section). Transversal sections
through the spinal cord dorsal horn (G) and the dorsal root
ganglia (H) are shown; olfactory nuclei (I) is also
shown. Pl, Purkinje cell layer; Ml, molecular layer; Gl, granule cell layer; V, ventricule; LS,
lateral septum; Cg, cingulum; Ep,
ependimis.
Whole mount transgenic embryos were analyzed for expression of those constructs at 13.5 days post-fertilization. With the int-1 construct, four distinct founders were generated. All four lines expressed the lacZ transgene in the nervous tissue (Fig. 5). A mild variation in the pattern of nervous system expression was detected from one line to another. One embryo showed ectopic expression in the limb buds, while another yielded a very weak signal in the somites. Similarly, the five transgenic embryos bearing the int-2 construct presented a variegated staining in the nervous system. Only one embryo revealed ectopic expression to the apical ectoderm ridge. Of the six transgenic embryos generated with the int-3 construct, none yielded lacZ expression in the nervous system. Ectopic expression was detected at low levels in three lines (Fig. 5).
These results demonstrated the existence of neuron-specific enhancers located in the first and second NF-L introns that can contribute to enhance NF-L expression in the telencephalon, diencephalon, and mesencephalon that correspond to anterior structure of the embryonic nervous system (Fig. 5). The third intron is apparently devoid of such elements. To further define enhancer regions in the first intron, we generated a construct named 5`int-1 (Fig. 5), in which the first 560-bp region of intron 1 was subcloned in front of the hsp68/lacZ gene construct in an opposite orientation (Fig. 5). Two transgenic embryos were obtained, and they both expressed lacZ in the nervous system. Our conclusion is that the 5`-region of intron 1 contains elements to enhance expression in the anterior region of the nervous system.
We report here that the minimal human NF-L promoter (-292 bp) is sufficient to direct neuronal expression both of an hNF-L/intronless and an hNF-L/lacZ reporter gene ( Fig. 4and Fig. 5). Although a variegated staining occurred in transgenic embryos generated with the hNF-L/lacZ transgene, the lacZ activity was limited to the nervous tissue. This variegation is likely due to an influence of the chromosomal integration site, as chromatin assembly and/or neighboring regulatory elements could affect transcriptional activity. The neuronal expression of the hNF-L/lacZ fusion construct was an unexpected finding, as this same promoter was previously found to be unable to direct neuron-specific expression of the chloramphenicol acetyltransferase reporter gene(27) . How could different reporter genes yield different results? It is well known that folding of the DNA in a nucleosomal structure generally decreases basal transcriptional activity (for review, see (39) ). Elements capable to initiate loop domain formation, such as matrix attachment regions, can alleviate this phenomenon. We recently demonstrated the presence of matrix attachment regions with DNA unwinding element in the 3`-untranslated region of the hNF-L and lacZ genes but not in the chloramphenicol acetyltransferase gene(40) . Following deletion of matrix attachment region sequences from the lacZ gene, the hNF-L/lacZ construct became susceptible to position effect, and its expression was no longer tissue specific(40) .
The removal of introns 1 and 2 in the human NF-L gene resulted in a decrease of transgene expression in the cortex and cerebellum of adult mouse. The presence of regulatory elements in introns 1 and 2 was further confirmed using the hsp68/lacZ enhancer trap vector. Both introns acted as enhancers inducing neuronal expression of the hsp68 minimal promoter in a position- and orientation-independent fashion. As shown in Fig. 5, intron 1 directed hsp68/lacZ expression mainly to the telencephalon, diencephalon, and mesencephalon. The intron 1 enhancer responsible for expression to anterior structure of embryonic nervous system is located in 5`-region of this intron (Fig. 5, construct 5`int-1). The presence of regulatory elements within introns of the human NF-L gene is consistent with the mapping of the DNase I hypersensitive sites (HS) reported by Yazdanbaksh et al.(41) . These investigators located three different neuron-specific DNase I hypersensitive sites in the 5`-flanking region (HS1, -2, -3) and four within the human NF-L gene (HS4, -5a, -5b, -6). Except for HS4, located in exon 1, all other intragenic hypersensitive sites are located within introns. The HS5a and 5b are positioned at the 5`-region of intron 1 shown here to contain elements for neuronal expression, while HS6 is located at the end of intron 2.
It is noteworthy that the positions of introns 1 and 2 are conserved among all intermediate filament genes of class IV and VI. It has been postulated that genes of class IV and VI, which correspond to the neuronal branch of the IF family, arose by a reverse transcription of an mRNA from an ancestral lamin-like gene(42) . Recently, Zimmerman et al.(43) demonstrated that elements regulating mesodermal and neuronal transcription of the nestin gene, an IF protein of class VI, reside in the first and second introns at positions equivalent to intron 1 and 2 in the NF-L gene. It is thus conceivable that the acquisition of introns by an intronless progenitor gene may have led to the emergence of a lineage of IF genes expressed in the nervous system.
A computer search was carried out to identify known cis-regulatory elements within the promoter and introns of the
human NF-L gene. Elements like NF-E1, HOX type 1, and the PEA3
sequences are present in both introns 1 and 2 (Fig. 7). None of
these cis regulatory elements can explain neuronal
specificity. The mRNA coding for the ETS transcription factor PEA3 is
detected in mouse brain, but its expression is not exclusive to this
tissue(44) . We note, however, that the sequence CAGGA, which
includes part of the core consensus AGGAA recognized by the family of
ETS transcription factors(45) , is present in numerous
neuron-specific genes including the genes for
NF-M(8, 46) , NF-H, ()
-internexin(15) , peripherin(47) , Drosophila dopa decarboxylase(48, 49) ,
chicken
7-nicotinic acetylcholine receptor(50) , mouse
olfactory marker protein(51) , rat neuron-specific enolase
encoding gene(52) , Purkinje-cell-protein-2 encoding
gene(53) , the gene encoding the growth cone-associated protein
SCG10(54) , and type II sodium channel encoding
gene(55) . Most known members of the ETS family are expressed
in lymphoid tissue, but other ETS-related factors are also expressed in
neurons(56) . We recently reported that an ETS-like element
located at -908 in the human NF-L 5`-region is recognized by a
factor expressed in P19 cells differentiated into neurons(56) .
Moreover, two neuron-specific DNase I hypersensitive sites border this
element(41) . Our combined results suggest an implication for
ETS-related transcription factors in the control of neuron-specific
expression. Mutational analysis of the ETS-like elements in the NF-L
gene should establish their specific contributions to expression of
this gene in the nervous system.
Figure 7: Potential regulatory elements in the human NF-L and transcription factor binding sites found in the regulatory region of the human NF-L. The analysis was done on hNF-L regions -292 to +15 (basal promoter), +1225 to +2323 (intron 1), and +2450 to 2834 (intron 2) using the GCC program with the TFD data base from GenBank. No mismatches were allowed with consensus sequences.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X05608[GenBank].