Human teneurin-1 is a direct target of the homeobox transcription factor EMX2 at a novel alternate promoter
- Jan Beckmann†1, 2,
- Antonio Vitobello†1, 2,
- Jacqueline Ferralli1,
- Daniela Kenzelmann Brož3,
- Filippo M Rijli1, 2 and
- Ruth Chiquet-Ehrismann1, 2Email author
© Beckmann et al; licensee BioMed Central Ltd. 2011
Received: 27 September 2010
Accepted: 8 June 2011
Published: 8 June 2011
Teneurin-1 is a member of a family of type II transmembrane proteins conserved from C.elegans to vertebrates. Teneurin expression in vertebrates is best studied in mouse and chicken, where the four members teneurin-1 to -4 are predominantly expressed in the developing nervous system in area specific patterns. Based on their distinct, complementary expression a possible function in the establishment of proper connectivity in the brain was postulated. However, the transcription factors contributing to these distinctive expression patterns are largely unknown. Emx2 is a homeobox transcription factor, known to be important for area specification in the developing cortex. A study of Emx2 knock-out mice suggested a role of Emx2 in regulating patterned teneurin expression.
5'RACE of human teneurin-1 revealed new alternative untranslated exons that are conserved in mouse and chicken. Closer analysis of the conserved region around the newly identified transcription start revealed promoter activity that was induced by EMX2. Mutation of a predicted homeobox binding site decreased the promoter activity in different reporter assays in vitro and in vivo in electroporated chick embryos. We show direct in vivo binding of EMX2 to the newly identified promoter element and finally confirm that the endogenous alternate transcript is specifically upregulated by EMX2.
We found that human teneurin-1 is directly regulated by EMX2 at a newly identified and conserved promoter region upstream of the published transcription start site, establishing teneurin-1 as the first human EMX2 target gene. We identify and characterize the EMX2 dependent promoter element of human teneurin-1.
Many transmembrane proteins mediate cell-cell interactions and thereby regulate key developmental processes. Teneurins are a unique family of type II transmembrane proteins conserved from Drosophila melanogaster and Caenorhabditis elegans to vertebrates, where four paralogues exist called teneurin 1-4 . This protein class was discovered in a screen for the Drosophila homologue of the extracellular matrix protein tenascin-C . Structure and domain architecture are highly conserved across phyla. All proteins of the teneurin family share a large extracellular domain with eight tenascin-type EGF-like repeats followed by a region of conserved cysteines and YD repeats . Recently, several publications suggested that the C-terminal parts of the teneurin proteins contain peptides with similarities to corticotrophin-releasing factor (CRF) and might have a function in modulating CRF-mediated behavior . All vertebrate teneurins have an N-terminal intracellular domain with two polyproline motifs, EF-hand-like metal ion binding sites and several putative phosphorylation sites. This intracellular domain was shown to be cleaved from the membrane and translocates into the nucleus where it can interact with transcription factors and alter gene expression [5–7].
In C. elegans, RNAi knockdown and deletion of its single teneurin gene (Ten-1) results in a broad range of phenotypes, including defects in axon guidance and neuronal pathfinding, as well as gonadal disintegration and protrusion of the vulva [8–10]. Drosophila harbors two teneurin genes, Ten-a  and Ten-m/Odz [11, 12]. Mutations in either of these genes result in embryonic lethality and Ten-a mutants enhance the segmentation phenotype of weak alleles of Ten-m/Odz . It was also shown that teneurin expression is required for the proliferation and cellular identity in the Drosophila eye . Extensive localization studies in mouse [15–17] and chicken [5, 18–20] embryos, as well as in rat  and zebrafish  revealed that the different members of the teneurin protein family are expressed with overlapping patterns by distinct subpopulations of neurons. Experiments in vitro and in vivo showed that the different members of the teneurin family form disulfide-linked dimers [16, 23] and promote homophilic cell-cell adhesions and neurite outgrowth [18, 24]. These functions of the protein are believed to mediate correct pathfinding and area recognition of neurons. This was shown in the teneurin-3 knockdown mouse, which exhibits dramatic changes in the mapping of ipsilateral retinal inputs causing mismatches in binocular mapping. This is associated with major deficits in the performance of visually mediated behavioral tasks .
Recent findings suggest an important role for the teneurin protein family in establishing cortical arealization and patterning in the developing embryo. Teneurin-2 was found to be expressed in developing limbs, somites and craniofacial mesenchyme in a pattern strikingly similar to that of fibroblast growth factor 8 (Fgf8) and Fgf8 coated beads implanted into chicken limb buds induced ectopic teneurin-2 expression in situ . Furthermore, teneurin-4 transcripts are down regulated, and the expression patterns of teneurins are shifted in the cortices of mice deficient in Emx2 . These findings link the regulation of teneurin expression to Fgf8 and Emx2, two proteins that are part of a complex network of growth and transcription factors regulating arealization of the developing brain, a crucial event regulating sensory perception, the control of our movements and behavior (reviewed in ). The best studied protein in this network is Emx2. Emx2 is the vertebrate homologue of the Drosophila empty spiracles (ems) protein, which is involved in the development of the fly head . This protein is a homeobox-containing transcription factor implicated in mouse cerebral cortex development . It is expressed in a graded manner from rostral (low) to caudal (high) [30–33]. Knock-out and overexpression studies of Emx2 showed the function of this transcription factor in establishing the correct size and positioning of cortical areas [reviewed in 34]. Comparing expression analyses of different embryonic stages to the adult for both Emx2 [32, 33] and teneurins [5, 7, 35] showed that areas of Emx2 expression (e.g., the cortical plate, dentate gyrus and the olfactory bulb) strongly correlate with areas of teneurin expression, suggesting a possible role of teneurins in mediating arealization.
The human teneurin-1 gene resides on the × chromosome at position Xq25, a locus with low gene density [reviewed in 36]. Beside severe mental retardation, patients suffering from a syndrome mapped to this locus also suffer from motor sensory neuropathy, deafness and severely impaired vision [37–41]. Given the predominant expression in the developing brain and its function in establishing proper connectivity in the brain, teneurin-1 is a potential target gene for causing XLMR.
In order to provide the basis for an investigation of possible deletions and mutations in teneurin-1 of XLMR patients, we decided to delineate the gene locus and determined the transcription start site(s) of human teneurin-1. We identified a novel promoter upstream of the published transcription start, which is conserved in chicken and mice. We show that EMX2 directly binds to and regulates human teneurin-1 expression at this alternate promoter.
Identification of alternate transcription start sites of the teneurin-1 gene
Sequences obtained in 5'RACE (Translation start site in bold and underlined)
EMX2 transactivates teneurin-1 promoter reporter constructs in cell culture
EMX2 transactivates a teneurin-1 promoter construct in chick embryos electroporated in ovo
To confirm the influence of the putative EMX2 binding motif on reporter gene activation in vivo, we co-electroporated the mutant promoter 2-lacZ construct with EMX2 and compared the staining with that of embryos co-electroporated with the wild-type promoter 2-lacZ construct and EMX2. Similar to the cell culture based reporter gene assay (Figure 2), reporter gene activity was also strongly reduced in vivo. Indeed, the vast majority of the embryos co-electroporated with EMX2 and the mutated promoter 2-lacZ construct showed a much fainter lacZ staining than those co-electroporated with EMX2 and the wild type promoter 2-lacZ construct (Figure 3b). These findings show that EMX2 is able to induce reporter gene activity at the alternate teneurin-1 promoter and that this activation is greatly dependent on an intact homeobox binding motif.
EMX2 binds a homeobox core element in the alternate teneurin-1 promoter
To reduce the unspecific background obtained with the nuclear extract, we tested EMX2 produced by in vitro transcription and translation in the gel shift assay. Whereas no shift of the labeled probe was detected with mock extracts, the same shifted band as with the nuclear extract could be detected with in vitro transcribed and translated EMX2 (Figure 4b, lanes 1 and 2), while unspecific background was greatly reduced. The binding of the protein to the probe was successfully competed with an excess of unlabeled wildtype oligo-nucleotides (Figure 4b, lane 3), whereas no competition was detected for unlabeled mutated oligo-nucleotides (Figure 4b, lane 4). Adding c-myc antibody to the binding reaction resulted in a super-shifted band (Figure 4b, lane 5), indicating a direct binding of EMX2 to the homeobox motif in the alternate teneurin-1 promoter.
To test whether an interaction between EMX2 and the binding site in the teneurin-1 promoter can also occur in vivo without overexpression of the EMX2 protein, we tested nuclear extracts of brains from E12.5 embryos known to express high EMX2 levels in the EMSA assay (Figure 4c). We were able to detect a shift of the band with the embryo extract, which runs lower than the complex of the overexpressed tagged protein in the control (compare Figure 4c, lanes 1, 2). We were able to compete the binding to the probe with wildtype unlabeled oligo-nucleotides, whereas no competition was detected using the unlabeled mutated oligo-nucleotide (Figure 4c, lanes 3-4). As a final proof of direct binding of EMX2 to the endogenous teneurin-1 promoter at the homeobox binding site in vivo, we performed chromatin immunoprecipitation (ChIP) in chicken embryos electroporated with the FLAG-myc-tagged EMX2 construct. Electroporations were performed in developing telencephalic regions in order to test the ability of Flag-myc-tagged EMX2 to bind the target region in its physiological cell context. Indeed, we detected specific enrichment of the target region containing the homeobox binding site after ChIP with the anti-FLAG antibody, recognizing the electroporated tagged EMX2 protein compared to a negative control region, which was not the case in control embryos (Figure 4d).
Teneurin-1 expression pattern correlates with that of EMX2in E14.5 embryos
EMX2 specifically induces the transcription of the alternate transcript
In this work, we characterized the teneurin-1 gene locus and found novel upstream exons which are conserved between species. These new exons expand the size of the Odz1 locus to more than 800 kb, harboring one intron which is more than 200 kb in size. Genes with large introns have been reported before . A continuous transcription of the entire gene, given a polymerization rate of 3800 nucleotides per minute by RNA polymerase II, would take 3.5 h . This might add another level of regulation of the defined expression in time and space. Here we show that there are at least two promoters regulating teneurin-1 expression with one alternate promoter upstream of the published transcription start. Only this alternate promoter was inducible by EMX2 in reporter gene assays and cells stably overexpressing EMX2 exhibited an increase of the resulting alternate transcript. A single homeobox binding site seems to be critical for the promoter activity and is bound directly by EMX2, as shown by gel shift assay and ChIP in chicken embryos. Although one has to take into account that the teneurin-1 expression, especially in later developmental stages, opposes the expression pattern of EMX2, a direct regulation of teneurin-1 expression by EMX2 is likely to occur at earlier stages. First, we and others  showed that total teneurin-1 expression, as well as the expression of the alternate transcript, correlates well with EMX2 expression at E14.5. Secondly, the expression of teneurin-1 is highly dynamic and its patterned expression and the overall expression level collapses in EMX2-deficient mice . Notably, we found teneurin-1 being expressed in the marginal, but not in the ventricular zone of the cortex. This suggests a possible function of EMX2 in post-mitotic neurons via the control of teneurin-1. Based on these findings, it is conceivable that the promoter at the published transcription start is responsible for the basal expression of teneurin-1, whereas the novel promoter is responsible for the graded expression dependent on EMX2. This finding suggests that this promoter region of the teneurin-1 gene is essential in establishing correct patterning of teneurin-1 expression. Although the transcription factors involved in correct patterning and arealization are well known, and their expression patterns are well characterized, very little is known about the downstream mechanisms contributing in establishing proper arealization and pathfinding [reviewed in 45]. A number of reports describe screens to find genes which are differentially expressed within the cortex [24, 46, 47] or which are potential target genes of differentially expressed transcription factors [11, 26, 48–50]. Interestingly, in both types of approaches members of the teneurin protein family were revealed as differentially expressed genes, supporting the evidence for a role of teneurin in arealization. Lists of potential target genes of the transcription factors involved in arealization, like Emx2 or Pax6, have also been described in knock-out gene expression studies [26, 31, 48], but indirect effects on transcription cannot be ruled out and interesting targets need to be validated. In this study, we validated teneurin-1 as the first direct target gene of EMX2 in human. As a transmembrane protein, teneurin-1 is well-suited to convey nuclear signals to the level of cell-cell interactions. However, the molecular mechanisms of how teneurins mediate their proposed function in brain development and patterning of the cortex remain to be elucidated.
Many cases of XLMR have been mapped to Xq25, the locus of the teneurin-1 gene [37–41]. Interestingly, many of these individuals suffer from motor sensory neuropathy , and teneurin-1 is predominantly expressed in patterns that relate to anterior sensorimotor areas . Taking into account the regulation of teneurin-1 by EMX2 at the novel promoter, setting up proper arealization of the developing cortex, and the well established functions of teneurins in correct pathfinding and neurite growth, we consider teneurin-1 as a potential target gene for XLMR. When analyzing patient samples, attention should be given to the newly established promoter region, as mutations or deletions in this area could lead to a shift in expression of teneurin-1 early in the developing brain leading to improper connectivity and consequently to XLMR.
In this work, we show that teneurin-1 expression is regulated by EMX2 at a novel and conserved upstream promoter. We present teneurin-1 as the first direct target gene in humans and characterize the binding site in the newly identified promoter region.
Rapid amplification of 5' complementary DNA ends (5' RACE)
hten PCR1 rev
hten nested XbaI rev
mouse PCR1 rev
mouse nested XbaI rev
chicken PCR1 rev
chicken nested XbaI rev
oligo dT anchor primer
hten1 promotor 1 XhoI fw
hten1 promotor 1 HindIII rev
hten1 promoter 2 NheI fw
hten1 promoter 2 EcoRI rev
mutate promoter 2 fw
mutate promoter 2 rev
total ten1 qPCR fw
total ten1 qPCR rev
alt exon qPCR fw
alt exon qPCR rev
hGAPDH qPCR fw
hGAPDH qPCR rev
ChIP target region fw
ChIP target region rev
ChIP negative control fw
ChIP negative control rev
The promoter constructs for human teneurin 1 were amplified from human genomic DNA using the Expand High Fidelity system (Roche) with primer hten1 promoter 1 XhoI fw (all primer sequences are given in Table 2) and hten1 promoter 1 HindIII rev using the highlighted restriction sites for directional cloning into vector pSEAP2-Basic (Clontech, Mountain View, CA, USA) of the promoter 1 construct. For the hten1 promoter 2 construct, we used hten1 promoter 2 NheI fw and hten1 promoter 2 EcoRI rev using the highlighted restriction sites for directional cloning into the same vector. The promoter 1 construct contains a sequence of just over 2 kb from nt124097602 to nt124099666 of chromosome × and the promoter 2 construct around 4 kb from nt124336306 to nt124340205 on chromosome × of assembly GRCh37. Analysis of the promoter sequences for potential binding sites was done using the JASPAR database (http://jaspar.cgb.ki.se) and the ems matrix. Mutation of the potential homeobox-binding sequence (nt124338584 to nt124338589 on chromosome X) in the promoter 2 was achieved using overlapping PCR with the primer set for hten1 promoter2 and mutated promoter 2 fw and rev. HEK293-EBNA cells were plated at 1 × 105 cells per well in six-well plates 18 h before transfection. Cells were transfected in DMEM containing 0.3% FCS with Fugene 6 Transfection reagent (Roche) using 1 μg promoter construct DNA and co-transfected with either empty 1 μg pcDNA3 or flag- and myc-tagged EMX2 in pCMV-Entry (OriGene, Rockville, MD, USA) as indicated in the Result section. Twenty-Four hours after transfection, the medium was collected and SEAP reporter gene activity was measured and normalized for the co-transfected plasmid pGL3, expressing firefly luciferase (Promega, Madison, WI, USA) as previously described .
HEK293-ECR cells were transfected as described before with the flag-myc-tagged EMX2 construct in pCMV and cells were selected for stably expressing clones with G418 (Roche) for 2 weeks. Clones were pooled and expression of the construct was tested by Western blot (data not shown). From these cells and untransfected HEK293-ECR cells RNA was isolated with QiaShredder and the RNA Easy kit (Qiagen) following the manufacturer's protocol. From this preparation, cDNA was generated using the Superscript III (Invitrogen) polymerase and random primers following the standard protocol. Real-time Q-PCR was performed on these samples with teneurin-1 specific primers and normalized to GAPDH values (sequences Table 2) using SYBR QPCR Supermix with ROX (Invitrogen) on an AbiPrism 7000 system. Three independent experiments were performed and the averaged results are shown and p-values were calculated using the one-way ANOVA.
For reporter assay experiments, chicken eggs were incubated in a humidified chamber at 38°gC and DNA constructs were injected into the lumen of the neural tube of stage Hamburger Hamilton (HH) 10-12 embryos. Construct concentrations were: 1 μg/μl lacZ reporter construct (BGZ40; ), 1 μg/μl EMX2 expression vector, and 0.2 μg/μl co-injected EGFP in pCMV as positive control of electroporated cells. Embryos were harvested 24 hours after electroporation and processed for β-galactosidase staining. For EMX2 overexpression, 1 μg/μl of Myc/FLAG-tagged EMX2 expression vector and 0.2 μg/μl of pCMV-EGFP construct were co-injected into the lumen of forebrain of stage HH 14 embryos. Positive tissues (n = 20 brains) were collected 72 hours after electoporation and immediately processed for chromatin cross-linking. As negative control, the same amount of unelectroporated tissue was collected and processed for ChIP experiments. Electroporations were performed as described previously using a square wave electroporator .
Chromatin immunoprecipitation Assay
Brains were chopped and then cross-linked in 1% Formaldehyde (F8775, Sigma) for 10 minutes at room temperature. Cross-linking was stopped in 125 mM Glycine for 5 minutes and the material was washed three times in ice cold PBS containing EDTA-free Protease Inhibitor Cocktail (Complete, 04693132001, Roche). DNA shearing was performed in lysis buffer (50 mMTris-HCl pH8.0, 10 mM EDTA, 1%SDS, 1 × Protease Inhibitor Cocktail) using the following parameters: 20 cycles of 30 seconds ON/30 seconds OFF (Diagenode bioruptor sonicator, high power setting).
Chromatin immunoprecipitation was performed by using Dynabeads protein G (100.04D, Invitrogen) as described elsewhere . The following antibodies were used: Mouse monoclonal anti-FLAG M2 (F1804, Sigma), Mouse control IgG (AB18413, Abcam).
Electrophoretic Mobility Gel Shift Assay (EMSA)
EMX2 binding to the promoter construct was examined by Electrophoretic Mobility Gel Shift Assay (EMSA) using DIG-labeled double-stranded oligo-nucleotides (5'CAGGAGAAAGTAATTAAAAAA3' or with mutated binding site 5'CAGGAGAAAGTGGTTAAAAAA3', putative binding site underlined). For probe preparation, 5 μg of sense and anti-sense oligo-nucleotides were diluted in 90 μl TE buffer, incubated for 10 min at 95°C and cooled down for 30 min at room temperature for annealing. DIG-labeling of the probes was achieved using the DIG Gel Shift Kit, 2nd Generation (Roche) according to the manufacturer's instructions. For the gel shift assay, nuclear extracts from stably EMX2 expressing HEK293 cells, in-vitro translated extracts or nuclear extracts of E12.5 embryo brains containing 20 μg of total protein were incubated with 4 μl of 5 × binding buffer of the Gel Shift Kit, 1 μg double-stranded poly(dIdC) and 0.1 μg poly-L-lysine in a 19 μl reaction mix. For the competition assay, unlabeled wild-type or mutant annealed oligo-nucleotide were added with a 150-fold excess. This mix was incubated for 20 min at room temperature. Afterwards, 1 μl of labeled probe was added and the mix was incubated for another 20 min at 30°C. For supershifts, 1 μl of c-myc antibody (Sigma) was added after 10 min incubation with the labeled probe. Following another 10 min of incubation, the reaction mix was loaded onto a precast 6% DNA retardation gel (Invitrogen, Carlsbad, CA, USA), which was pre-run in 0.5 × TBE for 20 min at 80 V and 4°C. The gel was run for 1.5 h at 80 V and 4°C. After separation, the complexes were blotted on a positively charged nylon membrane in 0.5 × TBE for 45 min at 280 mA and DIG detection was performed as described in the manufacturer's instructions.
In-vitrotranscription and translation of EMX2
In-vitro transcription and translation of EMX2 was achieved using the TNT® Coupled Reticulocyte Lysate System (Promega). 25 μl of TNT® rabbit reticulocyte lysate, 2 μl TNT® reaction buffer, 1 μl TNT® T7 RNA polymerase, 0.5 μl of each Amino Acid mixture without Leucine and without Methionine and 1 μg of EMX2-pCMV-Entry were mixed in a 50 μl reaction mix and incubated for 90 min at 30°C, quick frozen in dry ice/ethanol and stored at -80°C until used in EMSA.
In-situ hybridizations on sections were performed as previously described . The following RNA probes were used: For EMX2 we used the entire CDS of EMX2 (NM_010132.2), for total teneurin-1 we used the probe previously published  and for the probe specific for the alternate transcript we used the sequence described in Table 1 (m1_cl_2) plus the first 100 bp of the CDS of mouse teneurin-1 (NM_011855.3).
X-linked mental retardation
Electrophoretic Mobility Shift Assays
rapid amplification of cDNA ends
secreted embryonic alkaline phosphatase
We thank Hans-Rudolf Hotz for the help with the UCSC genome browser, Richard P. Tucker for critical reading of the manuscript and Jean-François Spetz for providing the mouse embryos. This work was supported by the Novartis Research Foundation.
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