Introduction

The sodium channel gene SCN8A encodes NaV1.6, one of the major voltage-gated sodium channels responsible for generating action potentials during neuronal firing. SCN8A is expressed throughout the central and peripheral nervous systems, with particularly high expression in cerebellum, and is the major sodium channel at adult nodes of Ranvier (Caldwell et al. 2000; Schaller et al. 1995). Mutations of SCN8A are associated with neurologic disorders in human and mouse. Missense and nonsense mutations of mouse Scn8a result in movement disorders, including tremor, ataxia, dystonia, and paralysis (Burgess et al. 1995; Meisler et al. 2004). Conditional knockout of mouse Scn8a in Purkinje cells is sufficient to generate tremor and ataxia (Levin et al. 2006). Heterozyotes for a null allele of human SCN8A exhibit ataxia and/or impaired cognition (Trudeau et al. 2006). SCN8A orthologs with expression in brain have been identified in several species of fish (Lopreato et al. 2001; Novak et al. 2006).

The transcriptional regulation of SCN8A is not well characterized. We recently described the promoter of the mouse and human SCN8A genes, which contain a cluster of four mutually exclusive 5′ noncoding exons, exon 1a to 1d, each of which is spliced directly to the first coding exon (Drews et al. 2005). A 4.8-kb genomic fragment containing all of the noncoding exons demonstrated tissue-specific expression in transgenic mice (Drews et al. 2005). An 0.85-kb subfragment containing exon 1b and exon 1c was expressed at a high level in a neuronal cell line.

We now report the use of evolutionary sequence comparison to identify highly conserved noncoding sequences in the promoter region of SCN8A. Large-scale comparisons between mammalian, chicken, and fish genomes have been used to identify highly conserved regulatory regions that are several hundred nucleotides in length (Dodou et al. 2003; Ghanem et al. 2003; Nobrega et al. 2003; Spitz et al. 2003; Zerucha et al. 2000). Genomic sequence comparisons alone are not sufficient for identifying short conserved elements, 10–20 bp in length, the typical length of binding sites for regulatory proteins. To bypass this limitation, we performed a two-step approach: (1) 5′ RACE to identify the start sites of transcription initiation in chicken, fish, and mammals, and (2) sequence comparison of the surrounding genomic DNA. This approach enabled us to identify a cluster of four short noncoding elements that are largely invariant in vertebrate genomes. In this article we describe the sequence and preliminary functional characterization of these evolutionarily conserved noncoding elements.

Materials and methods

5′ RACE

5′ RACE was performed using the GeneRacer Kit (Invitrogen, Carlsbad, CA) with 250 ng of chicken brain poly(A)+ RNA (Ambion, Austin, TX) as template and reverse primers complementary to the first coding exon of SCN8A (exon 1). Three primers were used in succession: primer 1 for reverse transcription (5′GGTTT GCTGT CTTCA TCGTC GTC), primer 2 for PCR (5′TCTGC AATGC GTTTC TCAAT GTTAG), and primer 3 for nested PCR (5′AGCCG TGCTG CCATC TTTTC ATC). PCR amplification was initiated by 2 min of denaturing at 94°C followed by 33 cycles of 30 sec at 94°C, 30 sec at 65°C, and 1 min at 72°C, with a final extension step of 6 min at 72°C. PCR products were cloned into the pGEMT-Easy vector (Promega, Madison, WI) using the Quick Ligase Kit (New England BioLabs, Ipswich, MA). Inserts were amplified by PCR and visualized by ethidium bromide staining of agarose gels. Inserts of unique size were purified using the QIAquick Gel Extraction Kit (Qiagen, Valencia, CA) and sequenced at the University of Michigan Sequencing Core (http://www.seqcore.brcf.med.umich.edu/).

Multispecies DNA sequence analysis

SCN8A genomic sequences from the following sources were used: Homo sapiens chromosome 12 BAC clone RP11-285E4 (GenBank AC025097), Mus musculus chromosome 15 BAC clone RP23-319B16 from strain C57BL/6J (GenBank AC104833), Fugu rubripes whole-genome shotgun sequence SCAFFOLD 1918 (GenBank CAAB01001918.1), Monodelphis domestica (opossum) whole-genome shotgun sequence (GenBank AAFR03066481), and Gallus gallus whole-genome shotgun sequence assembly (GenBank NW060828). The genomic sequence for the two duplicated Scn8a genes in Danio rerio was obtained from clone DKEY-9P24 (GenBank CR376824.2) (Scn8aa) and RP71-15H20 (GenBank BX470135.6) (Scn8ab). Mouse exon 1c sequence was reported by Drews et al. (2005) (GenBank AY510081). Human exon 1c sequence was obtained from mRNA sequence (GenBank NM014191), and chicken exon 1c sequence was determined by 5′ RACE (GenBank EF210713). Zebrafish exon 1c sequence was obtained from a Scn8ab mRNA (GenBank NM131628), and subsequent alignment of that sequence to genomic sequence upstream of Scn8aa coding sequence (GenBank NM001045183). Sequences were aligned using Sequencher software (GeneCodes, Ann Arbor, MI). MatInspector was used to identify potential transcription factor binding sites (http://www.genomatix.de/products/MatInspector/index.html). The repeat content of human genomic DNA was analyzed using RepeatMasker (www.repeatmasker.org) and PipMaker (http://www.bio.cse.psu.edu/pipmaker/). The pictogram of conserved sequence elements was assembled using Pictogram software (http://www.genes.mit.edu/pictogram.html).

Luciferase constructs

The 470-bp Scn8a promoter-luciferase construct, p470Luc (Fig. 3, top) was constructed from the previously described 0.85-kb promoter construct (construct 6, Drews et al. 2005) by digestion with AscI and MluI followed by religation. Mutations of the conserved elements were introduced to this construct using two synthetic 90mers containing the restriction sites for Eco47III and BglI at their termini (Sigma, St. Louis, MO). After digestion of the wild-type construct with these two enzymes, the wild-type fragment was replaced with the synthetic fragments. In the mutated construct pGL3ΔB, the conserved sequence CAAGATGGCG in element B was changed to CGGAACCGAG. In the mutated construct pGL3ΔDR, the first copy of the conserved repeat GCAGT in the direct repeat is changed to TACGG and the second copy is changed to TGACG. All constructs were verified by sequencing.

Cell culture and transfection

The mouse neuronal hybrid cell line MN-1 was cultured and transfected as previously described (Drews et al. 2005), using 50 ng of test plasmid, 10 ng of control plasmid (pRL-SV40, Promega), and the Fugene 6 transfection reagent (Roche Molecular Biochemicals, Indianapolis, IN). Cell lysates were collected at 40–48 h post-transfection. Firefly luciferase and Renilla luciferase were assayed using the Dual-Luciferase Reporter Assay System (Promega). Each transfection was performed in triplicate; each construct was analyzed in two to four independent transfection experiments. The mean and standard error for luciferase activity were calculated using the Statistical Package for the Social Sciences (SPSS Inc., Chicago, IL).

Transgenic mice

The wild-type plasmid p470Luc was digested with XbaI and NcoI, and the luciferase cDNA insert was replaced with the nLacZ reporter fragment from Hsp68-LacZ (Nobrega et al. 2003) to generate the transgene p470LacZ. The linearized insert was gel purified and microinjected into (C57BL/6J × SJL)F2 fertilized eggs in the Transgenic Animal Model core at the University of Michigan (http://www.med.umich.edu/tamc/). Mice carrying the transgene were identified by PCR with the forward primer 5′CTAAC GAAGC TGCTG CAGAA TGAG from the Scn8a promoter and the reverse primer 5′GTTTG AGGGG ACGAC GACAG TATC from LacZ. Transgenic founders were crossed with strain C57BL/6J to generate F1 mice for analysis of LacZ expression. Brain, kidney, liver, and spleen were frozen, sectioned, and stained for β-galactosidase activity with the Xgal substrate (Histoserv, Germantown, MD).

Results

Chicken Scn8a transcripts contain a single 5′ noncoding exon related to mammalian exon 1c

To identify the transcription start site for chicken Scn8a, we performed 5′ RACE on poly(A)+ brain RNA, using reverse primers located in the first coding exon. Forty-three clones with inserts of 13 different size classes were analyzed. Sequencing revealed that all of the clones contained the same 5′ noncoding exon, which was spliced directly to the first coding exon (Table 1). The chicken noncoding exon ends with a consensus splice donor site. The longest clone contained 175 bp of upstream noncoding sequence. Sequence comparison of the chicken noncoding exon with the mammalian exons 1a-1d demonstrated 72% sequence identity (74/103 bp) with mammalian exon 1c. This degree of conservation is unusual for 5′ UTR sequences, which exhibit on average only 2% conservation in chicken and mammalian orthologs (ICGS 2004, Fig. 2). The identification of a single chicken 5′-RACE product contrasts with our previous identification of four different noncoding exons by 5′ RACE of human and mouse transcripts (Drews et al. 2005), and suggests that mammalian exon 1c represents the ancestral exon. No matches to the other mammalian noncoding exons were detected in adjacent chicken genomic DNA sequence.

Table 1 Transcript sequence data for the 5′ noncoding exon 1c of SCN8A from five vertebrate species
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Fish Scn8a contains a 5′ noncoding exon related to mammalian exon 1c

Sequence comparison between the chicken noncoding exon and fugu and zebrafish genomic sequences upstream of the first coding exon of Scn8a identified a fish homolog of exon 1c, which was also present in one zebrafish cDNA clone (Table 1). The fish exon terminates in a consensus splice donor site that has diverged by a few base pairs from the mammalian exon (Table 1; Fig. 1, top panel). The identification of an ortholog of exon 1c in the fish further supports the view that exon 1c encodes the ancestral 5′ UTR. The 47% sequence identity between exon 1c in fish and mammals is unusually high for noncoding sequence, suggesting that there has been selection to retain functional elements within this noncoding exon. There are no matches to the other mammalian noncoding exons in the fish genomic sequence.

Fig. 1
figure 1

Alignment of four conserved noncoding elements in the 5′ UTR of vertebrate SCN8A genes. Relative positions within exon 1c are shown in the top panel. The variation in position of the splice donor sites (SD) in these species is indicated below the exon (z, zebrafish Scn8ab; o, opossum; c, chicken; m, mouse; h, human). The YY1 site in the promoter is present in reverse orientation from that shown. Ten copies of the direct repeat were combined to generate the DR1&DR2 consensus, 5 from DR1 in each species and 5 from DR2. In the pictogram representation of consensus elements, the height of each letter represents the nucleotide frequency (http://www.genes.mit.edu/pictogram.html)

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Four short, highly conserved noncoding elements in exon 1c

Sequence alignment of exon 1c from human, mouse, rat, opossum, chicken, zebrafish, and fugu revealed four regions of particularly high conservation. Element A is a 22-bp sequence located near the 5′ end of exon 1c, with 20/22-bp sequence identity between mammals and fish (Fig. 1). (This element is not represented in the chicken gene.) Element B is a 20-bp sequence with 15/20-bp sequence identity between human, fish, and chicken (Fig. 1). Two direct repeats of 18 bp, designated DR1 and DR2, are separated by 14 bp in mammalian SCN8A. DR1 and DR2 are highly conserved in all five species examined (Fig. 1). Sequence conservation was not detected in genomic sequence immediately upstream and downstream of exon 1c (Supplementary Fig. 1).

Genomic organization of the 5′ region of SCN8A

Mouse exon 1c and human exon 1c are located 65 kb and 70 kb upstream of the first coding exon, respectively (Drews et al. 2005). To determine the location of exon 1c in chicken and fish, we aligned the exon sequence with the genomic sequence. The chicken exon aligned to the 5′ end of a gap in the genomic sequence (GenBank NW060828). We carried out PCR with primers upstream and downstream of the gap provided the missing sequence, which was only 100 bp long. This located the chicken exon 1c to a position 9 kb upstream of the first coding exon. In the two zebrafish Scn8a genes, the noncoding exon is located 9 kb and 14 kb upstream of the first coding exon, respectively. In the fugu genome, the noncoding exon is 3 kb upstream of exon 1 (Fig. 2).

Fig. 2
figure 2

Evolution of gene organization in the 5′ region of SCN8A. The length of the interval between the 5′ noncoding exon and the first coding exon increased from 3 kb to 70 kb during vertebrate evolution. In mammalian SCN8A, this interval is largely occupied by repetitive elements (bottom panel). After divergence from chicken, the mammalian lineage acquired three additional 5′ noncoding exons that lack sequence similarity to exon 1c. Exon 1a is derived from LINE element sequence (Drews et al. 2005). The four mammalian 5′ noncoding exons are mutually exclusive; each is spliced directly to the first coding sequence (Drews et al. 2005). Repeated sequences recognized by RepeatMasker software are shown in color in the bottom panel. Exons not drawn to scale

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In the human gene, repetitive elements and simple sequences recognized by the RepeatMasker program account for 58% of the 70-kb interval between the 5′ noncoding exons and the first coding exon (Fig. 2, bottom panel). The increase in size of the SCN8A gene thus appears to have occurred largely by invasion of repetitive elements, as is characteristic of the genome as a whole.

Exon 1c appears to be the only 5′ noncoding exon in chicken and fish, based on our 5′-RACE data for chicken as well as fish ESTs. There is no evidence for conservation of mammalian exons 1a, 1b, and 1d, or substitution of other 5′ exons, from genomic or transcript sequences. Exon 1a overlaps with an inserted LINE element in the human and mouse genes; the absence of a LINE element at this position in the opossum gene indicates a time of LINE insertion between 80 and 135 million years ago. Transcripts containing the four noncoding exons are expressed in multiple regions of the mouse brain and nervous system, indicating that they do not determine distinct expression domains (Drews et al. 2005).

Mutation of conserved elements reduces promoter activity in transfected cells

In previous studies of the mouse Scn8a promoter, we described an 850-bp fragment with strong promoter activity in transfected MN1 neuronal cells (Drews et al. 2005). To further localize the active promoter, we tested a 470-bp subfragment containing exon 1c as the only noncoding exon (Fig. 3A). This fragment retained 70% of the activity of the 850-bp fragment (Fig. 3B). To test the role of conserved elements B and DR1/DR2 in reporter gene expression, we generated two mutant constructs. In the ΔB construct, the ten most conserved nucleotides of element B were changed from CAAGATGGCT to TGGAACCGAG, without changing GC content or introducing new transcription factor binding sites detectable with MatInspector software. This mutation resulted in a 70% reduction in luciferase expression (Fig. 3C). In the second mutant construct, ΔDR1&DR2, the invariant 5-bp core sequence of the direct repeat GCAGT was changed to TACGG and TGACG in DR1 and DR2, respectively. ΔDR1&DR2 exhibited 30% reduction in expression compared with the wild-type fragment (Fig. 3C). Both element B and the direct repeat thus exhibit transcriptional enhancer activity in transfected cells.

Fig. 3
figure 3

Transcription-enhancing activity of conserved element B and direct repeats 1 and 2. A The three-part expression construct p470Luc contains a 470-bp promoter fragment including the noncoding exon 1c, a 150-bp fragment with the splice acceptor and start codon from SCN8A, and the luciferase cDNA. B Firefly luciferase activity was normalized to cotransfected Renilla luciferase. Luciferase activity is presented as percent of the 850-bp promoter, mean ± SE (n = 6). C Luciferase activity as percent of the wild-type p470Luc, mean ± SE (n = 12)

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Brain-specific expression in transgenic mice

The transgene p470LacZ containing the 470-bp promoter fragment directing expression of the nuclear LacZ reporter was analyzed in three independent lines of transgenic mice. β-Galactosidease activity was present throughout the brain in all three lines, with particularly high expression in hippocampal neurons and cerebellar Purkinje cells (Fig. 4). Expression was also high in the cortex, thalamus, and hypothalamus, with less intense staining visible throughout the brain. There was no activity in the olfactory bulb in any of the three lines, although the endogenous gene and the 4.8-kb transgene are strongly expressed there (Drews et al. 2005). An olfactory-specific enhancer thus appears to be missing from the 470-bp construct. No activity was detected in kidney, spleen, and liver of any of the lines (Fig. 4; data not shown). With the exception of the olfactory bulb, the pattern of expression of p470Luc reflects the tissue specificity of the endogenous Scn8a gene.

Fig. 4
figure 4

Expression of p470LacZ in transgenic mice. The transgene structure is the same as Fig. 3 with Lacz reporter. Tissue sections were stained with Xgal to detect LacZ expression. A H, hippocampus; C, cortex; T, thalamus. B Cerebellum. C Hippocampus. D Cerebellar Purkinje cells. E Kidney. F spleen

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In silico analysis of transcription factor binding sites in the conserved noncoding elements of exon 1c

We used the MatInspector program for in silico analysis of the conserved elements in exon 1c to detect similarity to binding sites for known transcription factors. Conserved element A contains the 11-bp sequence GCATAATTGAT which differs only at nucleotide 9 from the experimentally determined consensus for the transcription factor Pou6f1/Brn5 (Rhee et al. 1998), resulting in the high matrix similarity score of 0.918. Conserved element B has strong similarity to the YY1 consensus binding site, with a predictive score of 0.88. Direct repeats DR1 and DR2 match the consensus RE-1 site for binding the transcription factor REST/NRSF, with a lower score of 0.69. This is below the average matrix similarity for known functional RE-1 sites (Bruce et al. 2004; Mortazavi et al. 2006; Schoenherr et al. 1995). It is possible that the close juxtaposition of two weak sites could support functional interaction with the transcription factor. This analysis suggests that Pou6f1/Brn5, YY1, and REST/NRSF should be experimentally tested as candidates for regulation of Scn8a expression. Efforts to assess the occupancy of these sites using ChIP were unsuccessful due to the very high GC content of exon 1c and the surrounding genomic DNA (92% GC).

Discussion

We previously compared the promoter regions of the human and mouse SCN8A genes only with the goal of identifying regulatory elements controlling gene transcription (Drews et al. 2005). The very high level of sequence conservation between human and mouse (93% in exon 1c) prevented the identification of small regulatory elements. In the current study, we extended the sequence comparison to chicken, fish, and opossum. Mammalian sequence could not be directly aligned with fish sequence, but the use of 5′ RACE to locate the promoter region of the chicken gene enabled us to align the fish noncoding sequences. The unusually high sequence conservation of the 5′ UTR of SCN8A (Table 1) is strongly indicative of conserved function. Elements within the 5′ UTR have the potential to function in regulation of transcription or in post-transcriptional regulation of mRNA stability, localization, or translational efficiency. We tested the first possibility and demonstrated transcription-enhancing activity within the 5′ noncoding exon of SCN8A.

Comparison of the organization of the 5′ end of the vertebrate SCN8A genes provided an example of the general increase in gene size and complexity during evolution. More than half of the tenfold increase in length of the intronic sequence between the 5′ noncoding exon and the first coding exon between fish and mammalian SCN8A is accounted for by the insertion of repetitive DNA. Another characteristic evolutionary trend is acquisition of multiple regulatory units controlling individual genes. Mammalian SCN8A appears to represent an intermediate stage in that process: In comparison with the one 5′ noncoding exon in fish and chicken, the mammalian gene has four distinct 5′ noncoding exons, but their expression patterns do not differ (Drews et al. 2005). The evolutionary comparison with chicken, fish, and marsupial genes indicates that mammalian exon 1c is descendent from the ancestral 5′ noncoding sequence. The small proportion of 5′-RACE products containing exon 1c that we previously reported for human and mouse was an underestimate caused by the difficulty of amplification of this 93% GC fragment (Drews et al. 2005). When searching for transcripts containing the 5′ UTRs of Scn8a, we found that 12 of the 13 mammalian ESTs in public databases contain exon 1c. Exon 1c was also present in 2/2 fish Scn8a ESTs, 4/4 Xenopus Scn8a ESTs, and 1/1 chicken EST (www.genome.ucsc.edu). Exon 1c thus appears to be the major first exon.

Within exon 1c four short elements exhibit a high proportion of evolutionarily invariant nucleotides (Fig. 1). Three lines of evidence indicate that exon 1c and its conserved elements are involved in transcriptional regulation. First, a 470-bp fragment containing exon 1c has strong promoter activity in transfected cells. Second, mutation of three of the four sites reduced the expression in transfected cells. Third, the 470-bp fragment directs tissue-specific expression in vivo in transgenic mice. The close juxtaposition of four conserved elements within 120 bp suggests that they may function together to stabilize a protein complex during SCN8A transcription.

The conserved elements in exon 1c are related to binding sites for three transcription factors involved in neuronal gene regulation: Pou6f1, YY1, and REST/NSRF (Ballas and Mandel 2005; Coulson et al. 2005; Cui and Bulleit 1998; Gordon et al. 2006). Pou6f1 has activity as a transcriptional enhancer (Molinari et al. 2004) and inhibitor (Wey et al. 1996) and is regulated by neuregulin in myelinating Schwann cells (Wu et al. 2001). Scn8a and Pou6f1 are closely physically linked on mouse chromosome 15 and human chromosome 12q13. YY1 is a ubiquitously expressed member of the GLI-Kruppel family of zinc-finger transcription factors (Shi et al. 1997; Thomas and Seto 1999) that interacts with cofactors with histone modification activity (Thomas and Seto 1999). A YY1 element in reverse orientation, located at position +13 to +24 downstream of the transcription initiation site, is required for transcription of the LINE-1 retrotransposon (Athanikar et al. 2004). The YY1 element in exon 1c is similarly located in reverse orientation downstream of the initiation site. The REST/NSRF transcription factor binds RE-1 elements and silences gene expression by a chromatin modification mechanism. Although the potential RE-1 sites in DR1 and DR2 do not exhibit a strong match to the consensus, the close juxtaposition of these two weak sites may result in functional binding.

We compared the sequence of exon 1c with other small promoter fragments that support brain -specific expression: a 107-bp fragment of GnRH (Kim et al. 2007), a 200-bp fragment of Cyp19 (Nausch et al. 2007), and a 5-kb fragment of BAI1-AP4 (Kim et al. 2004). None of the other promoters share sequences with exon 1c, indicating that there are alternative mechanisms for generating neuron-specific expression. The enhancers of Sox2, a transcription factor involved in neuronal development, are also distinct from exon 1c (Uchikawa et al. 2004).

We have used evolutionary approaches to localize a cluster of conserved sequences and performed initial functional characterization. Further sequence comparisons with paralogous mammalian sodium channel genes, and with sodium channel genes from lower chordates, may elucidate the molecular basis for the divergence between brain-specific and muscle-specific sodium channels and identify elements that control the common expression patterns of the major mammalian neuronal channels SCN1A, SCN2A, SCN3A, and SCN8A.

Mutations in transcription factor binding sites contribute to the burden of genetic disease in man (Villard 2004). The biological importance of quantitative levels of SCN8A expression is clear from the haploinsufficiency of the human gene (Trudeau et al. 2006) and the phenotypic consequences of small differences in expression level of the mouse gene (Buchner et al. 2003) Altered expression of sodium channel genes is thought to play a role in the pathogenesis of human epilepsy. This report represents a first step toward understanding the molecular regulation of SCN8A.