A 640 bp proximal promoter in the Dll3 gene directs neural tube specific expression
In order to discover regulatory sequences important in controlling the expression of Dll3
, we examined the sequence surrounding the open reading frame for regions of conservation between the mouse and human genomes. The Dll3
gene has a high degree of evolutionary divergence that exposed a region of 640 bp strongly conserved between the mouse and human genomes (Evoprinter, Odenwald et al., 2005
). This 640 bp region is the only sequence outside of the Dll3
open reading frame that showed significant conservation within 20 kb surrounding the gene. The 640 bp sequence is located immediately upstream of the start codon and extends beyond the predicted transcriptional initiation site (). Within the 640 bp are three blocks of homology: Homology A is 78% identical over 250 bp, Homology B is 77 % identical over 13 bp, and Homology C is 70% identical over 100 bp. To test the activity of this 640 bp promoter, we assayed its ability to drive GFP expression in transgenic mouse embryos. Six of six embryos expressing the (Dll3wt-GFP)
transgene had strong, consistent GFP signal at E11.5 in the neural tube, dorsal root ganglia, hindbrain, ventral telencephalon, somites, and limbs (, Dll3-WT
). These domains accurately reflect the expression pattern reported for Dll3
(Dunwoodie et al., 1997
; ), although GFP mRNA and protein persist in more differentiated cells likely owing to differences in stability relative to Dll3. These differences are visualized by comparing Dll3
mRNA in the neural tube of E11.5 Dll3wt-GFP
embryos (). Importantly, the 640 bp Dll3wt
promoter retains activity for many aspects of Dll3
expression including initiation of expression in the ventricular zone and enriched expression at the lateral edges of the ventricular zone (), as well as restriction to neural tissue.
A proximal Dll3 promoter conserved between mouse and human directs Dll3 like expression in transgenic mice
This analysis was extended to multiple embryonic stages by generating a stable transgenic line and assaying for GFP expression (Supplemental Fig. 1
). Expression of GFP in the somites first appears at E9.5. At E13.5, expression was seen in the distal lateral muscle in the limbs, telencephalon (striatum) and diencephalon (hypothalamus), dorsal spinal cord, and retina. This spatial and temporal pattern of expression for the GFP reporter also mimics expression of the endogenous Dll3
gene (Dunwoodie et al., 1997
). These observations demonstrate that the 640 bp promoter contains sufficient information for transcriptional regulation of Dll3
Efficient activity of the 640 bp Dll3 promoter requires E-box sites
Contained within the conserved 640 bp sequence block are seven E-boxes (), the consensus binding site for the Class II neural bHLH transcription factors (Murre et al., 1994
). The functional significance of the E-box sequences was assessed by mutating all seven of the E-box sites in the Dll3wt-GFP
transgene and assaying GFP expression at E11.5 in transgenic embryos (, Dll3-mET
). In the absence of all E-boxes, there was a dramatic reduction in the activity of the promoter (3 transgenic embryos had detectable but low expression). The low level GFP expression remaining was restricted to the neural tube largely in the wild-type pattern (see , Dll3-mET
, inset). This result clearly establishes an important role for E-box sequences in Dll3
regulation; however, it also indicates that the cell-type specific activity of the promoter is not solely dependent on these E-box sequences.
E-box sites are required for activity of the 640 bp Dll3 promoter
Ascl1 and Neurog2 regulate Dll3 expression in the developing dorsal neural tube
The identification of a Dll3
promoter whose activity is dependent on E-box sequences suggested that the E-box binding bHLH factors present in the developing neural tube may directly regulate Dll3
levels through these sequences. To begin to address this possibility we examined Dll3
expression in embryos mutant for the bHLH factors Ascl1, Neurog2 and Neurog1. In wild-type mouse embryonic neural tube at E11.5, Dll3
is expressed strongly at the lateral edge of the ventricular zone (VZ) and in scattered cells in the dorsal VZ along the entire dorsal/ventral axis (). mRNA in situ hybridization for Dll3
in null mutants of Ascl1
(Guillemot et al., 1993
(Fode et al., 1998
), and Neurog1
(Ma et al., 1998
) was assessed (). Dll3
was most dramatically affected in the Ascl1
mutant. In this mutant, Dll3
was not detected specifically within and adjacent to the normal expression domain of Ascl1
(). A subset of the Dll3
expression pattern was also lost in the Neurog2
mutant. In this case, the strong lateral expression seen in the dorsal neural tube is clearly lost (compare ). This is consistent with Neurog2
expression in the dorsal neural tube being enriched in the lateral, more differentiated cells (; Helms et al., 2005
). No perturbation in Dll3
was detected in the Neurog1
mutant (). Thus, the activity of Ascl1 and Neurog2, but not Neurog1, is required for proper expression of Dll3
specifically in the dorsal neural tube. Furthermore, the sequential nature of Ascl1 and Neurog2 expression in the dorsal neural tube (; Helms et al., 2005
), and the discrete pattern of Dll3
perturbation in the two mutants, suggests Dll3
is regulated by integrating activities of multiple bHLH transcription factors.
Dll3 expression and Dll3 promoter activity in the dorsal neural tube requires Ascl1 and Neurog2
The requirement for Ascl1 in the activity of the 640 bp Dll3
promoter was also tested in transgenic mice. Dll3wt-GFP
transgenic mice were bred onto the Ascl1
mutant background. In the presence of normal levels of Ascl1, Dll3wt-GFP
expresses GFP in scattered cells within the dorsal VZ, with intense GFP in more differentiated cells at the lateral edges of the neural tube (). In the absence of Ascl1, the scattered GFP cells in the VZ are absent and there are fewer differentiated cells at the lateral edges of the dorsal neural tube (). It is likely that much of the remaining GFP containing cells at the lateral edge are from dI1 and dI2 interneurons streaming ventrally from their origin in more dorsal regions. These populations do not require Ascl1, rather they require the other bHLH factors Atoh1 and Neurog1 (Gowan et al., 2001
). The lack of GFP signal in the dorsal VZ is consistent with a role for Ascl1 in activation of expression through the Dll3
promoter. Similar experiments were attempted with Neurog2
mutant mice but no Dll3wt-GFP+;Neurog2-/-
embryos were obtained from over 10 litters suggesting the transgene randomly inserted into the genome near the Neurog2
Ascl1 binds the Dll3 promoter in vivo
The dramatic loss of Dll3
expression in the Ascl1
mutant neural tubes and the presence of binding consensus sites for bHLH factors in the Dll3
promoter suggested that Ascl1 functions directly through at least some of these sites. We used Chromatin Immuno-Precipitation (ChIP) analysis to determine whether Ascl1 is localized to the Dll3
promoter in vivo. Chromatin was immunoprecipitated from formaldehyde cross-linked E12.5 neural tubes with antibodies specific to Ascl1. To determine if Ascl1 localized to the Dll3
promoter, qPCR analysis was performed using primers to this region. DNA immunoprecipitated with Ascl1 antibodies showed significant enrichment for the Dll3
promoter target similar to a regulatory region for Dll1
(Dll1-M), an enhancer that was previously shown to be directly regulated by Ascl1 (Castro et al. 2006
). Negative controls including the Dll1
open reading frame (ORF) or Gapdh
had no enrichment. In addition, no enrichment was seen with chromatin isolated from Ascl1
null neural tubes (, top panel). Chromatin from wild-type and Ascl1
mutant neural tubes was immunoprecipitated similarly with antibodies to RNA polymerase II, demonstrating the chromatin from the mutant neural tubes was competent in this assay (, bottom panel). These results demonstrate Ascl1 directly binds to the Dll3
promoter in vivo in E12.5 neural tubes.
Ascl1 occupies the regulatory regions of Dll1 and Dll3 in embryonic neural tube
In a similar set of experiments we utilized ChIP assays to test whether Neurog2 directly binds to the Dll3 promoter in vivo. Although the Dll3 promoter was enriched after ChIP with Neurog2 antibodies relative to negative controls, the efficiency of the pull-downs from embryonic neural tube was low, and thus, these experiments were not definitive (data not shown).
Individual E-box sites have distinct properties with respect to Dll3-promoter activity
The results above demonstrate that at least Ascl1 is directly regulating the expression of Dll3 through the 640 bp Dll3
promoter. Furthermore, the activity of the promoter requires intact E-box sites. To more precisely define the contribution of each E-box to the activity of the Dll3
promoter, the requirement for each individual E-box was assayed in transgenic mice. The results reveal a complex use of the E-box sequences for both activation and suppression of transgene expression. Mutation of four of the E-boxes, E1, E3, E5, and E6, had minor, if any, detectable effects on promoter activity when mutated individually (Supplementary Fig. S2
). However, when mutated in combination, such as in Dll3-m3,5,6
, enhancer activity was markedly decreased (, Dll3-mE3,5,6
). These data suggest a model in which multiple, redundant E-box sites are important for Dll3
expression. In contrast, individual mutations of E0, E2, and E4 revealed their individual importance to the activity of the Dll3
promoter. The following sections detail the properties of each of these sites for Dll3
E-boxes E0 and E4 serve major activator function in the Dll3 promoter
Of the seven E-boxes present in the promoter, only E-boxes E0 and E4 were required to maintain activity of the Dll3 promoter when tested individually. With each single mutation, a profound loss of expression was seen in all embryos assayed (, Dll3-mE0 and Dll3-mE4). To test whether E0 and E4 are sufficient within the context of the Dll3 promoter to drive the wild-type Dll3 pattern, a reconstructive approach was taken. Starting with the E-box null mutant (Dll3-mET), E0 and/or E4 were mutated back to wild-type creating three new constructs-- Dll3-mET+E0 (E0 only), Dll3-mET+E4 (E4 only), and Dll3-mET+E0,4 (E0 and E4 only) (). When tested in transgenic mice, E0 or E4 alone could rescue efficient GFP expression throughout the neural tube in all embryos expressing the transgene in a pattern consistent with wild-type, albeit at a consistently reduced intensity compared to the wild-type promoter (, compare Dll3-mET+E0 and Dll3-mET+E4 with Dll3-WT). The inability of E0 or E4 individually to restore the high level of GFP seen with the wild-type promoter suggests their function may be additive. This was directly tested by assaying Dll3-mET+E0,4. This construct directed efficient expression of GFP at levels exceeding the constructs with the individual E0 or E4 and approaching those seen with the wild-type promoter (, Dll3-mET+E0,4). However, relative to wild-type, this construct also showed expanded expression within the brain and ectopic expression in the mesenchyme, suggesting at least one of the other E-boxes has repressor activity. Thus, in the wild-type promoter, the combined activator activity of E0 and E4 must be attenuated by the presence of the other E-boxes.
E-box E2 serves major repressor function in the Dll3 promoter
E-box E2, in contrast to E0 and E4, appears to play a major role as a repressor. Mutation of E2 within the Dll3 promoter resulted in two types of ectopic expression of the reporter gene that we term temporal and tissue ectopic expression (, Dll3-mE2). Temporal ectopic expression appears in the VZ, indicating that expression initiates in cells more immature than in those seen with the wild-type promoter (, Dll3-mE2, arrowhead). In contrast, tissue ectopic expression appears in mesenchymal tissue surrounding the neural tube (, Dll3-mE2, arrow). E2 thus appears to serve an important function in Dll3 regulation by restricting its expression to neural progenitors of the appropriate stage.
The presence of an N-box, the consensus binding site for Hairy/En(S)/HES factors, in the promoter provided another candidate repressor pathway to examine since these factors typically suppress neurogenesis (Kageyama et al., 1997
; Sasai et al., 1992
). Mutation of the N-box resulted in temporal ectopic expression (), consistent with the presence of Hes1 and Hes5 in the neural tube VZ at this time (Ohtsuka et al., 1999
). Notably, the tissue ectopic expression seen when E2 was mutated was not detected with the N-box mutation demonstrating that multiple mechanisms restrict activity of the Dll3
The ectopic expression seen when the activator E-boxes E0 and E4 were the only E-boxes present (, Dll3-mET+E0,4) strongly mimics the individual E2 mutant (, Dll3-mE2), implicating E2 function in attenuating E0/E4 activity. We tested this hypothesis by constructing a transgene containing E2 plus E0 and E4 (, Dll3-mE3,5,6). The presence of E2 dramatically repressed the ectopic expression seen with the E0/E4 only mutant. The overwhelming loss of expression in Dll3-mE3,5,6 suggests that in the context of the wild-type promoter, the repressive activity of E2 must be modulated not only by the activator E-boxes E0 and E4 but also by a combination of the other E-boxes (E3, E5, and E6). In summary, of the seven E-boxes tested, a dramatic affect on enhancer activity was detected for three; E0 and E4 have enhancer activity, and E2 has repressor activity.
Dll3 promoter E-boxes are differentially bound by bHLH factors in vitro
Although ChIP analysis established that Ascl1 is bound to the Dll3 promoter in vivo, it is unable to spatially resolve interactions with specific E-boxes or to provide insight into the specific complexes that are involved. To determine the ability of Ascl1 and Neurog2 to interact with specific E-boxes, we used EMSA with in vitro translated Ascl1, Neurog2, and Tcfe2a-E12 (E12) proteins, as well as nuclear extracts from E10.5 neural tube. A summary of the data obtained with in vitro translated protein lysates is presented in . There was surprising variability in the binding of bHLH heterodimer complexes to each E-box. An example of a typical experiment showing the classical behavior of an E-box/ClassII bHLH interaction, using the E5 E-box probe, is shown in . E5 can be bound efficiently by E12 homodimer (lane 2) and Ascl1/E12 heterodimer (lane 4), much less efficiently with Neurog2/E12 heterodimer (lane 7), and not at all by Ascl1/Ascl1 homodimer (lane 3). Using this assay, we demonstrate that each E-box has distinct properties with respect to the bHLH/E-box complexes that can form in vitro.
Ascl1/E12 (, black bars) and to a lesser extent Neurog2/E12 (, dark gray bars) bound five of the seven E-boxes with varying efficiencies. E-box E2 stood out as a strong Ascl1/E12 binding site. In contrast, E0 and E4, the major enhancer E-boxes, were not efficiently bound by Ascl1/E12 or Neurog2/E12. These findings were surprising since Ascl1/E12 and Neurog2/E12 are known activators of transcription (Gradwohl et al., 1996
; Johnson et al., 1992
In an attempt to identify an E-box binding bHLH transcription activator that might act through the enhancer E-boxes E0 and E4, the bHLH factor Nhlh1 (previously Nscl1, Hen1) was tested. We tested Nhlh1 since it is expressed in the neural tube just lateral to the VZ as cells become post-mitotic (Begley et al., 1992
), an expression pattern similar to Dll3wt-GFP
. Nhlh1 bound efficiently as a heterodimer with E12 specifically to E4, but not the other E-boxes (). Thus, Nhlh1 is one candidate that might upregulate Dll3
through this E-box sequence.
Evidence for novel DNA binding complexes of Ascl1 including homodimers and multi-factor complexes with Neurog2
The requirement of E-boxes E0, E2 and E4 for wild-type activity of the Dll3
promoter was demonstrated in transgenic mice (). The efficient binding of Ascl1 and Neurog2 heterodimers to the E-box with an apparent repressor activity (E2) but not to E-box E0 and E4 with enhancer activity () presents an apparent contradiction since Ascl1 and Neurog2 are transcriptional activators (Gradwohl et al., 1996
; Johnson et al., 1992
). To gain further insight into the complexes that can form on these E-box sequences, we used EMSA with proteins from E10.5 neural tube nuclear extracts and specific antibodies to Ascl1, Neurog2, and Tcfe2a-E12 (). This analysis revealed novel Ascl1 and Neurog2 DNA binding complexes can form at least in vitro, particularly to E2, the repressor E-box, and E4, an activator E-box.
EMSA using nuclear extracts reveal the formation of Ascl1/Neurog2 DNA binding complexes
Protein complexes with E-box E0
EMSA with nuclear extracts revealed a protein-DNA complex formed on E0, but it did not require an intact E-box since competition with a cold E-box mutant oligonucleotide efficiently competed for binding (, lanes 1-3). Furthermore, the complex was only slightly blocked with pretreatment of the nuclear extracts with antibodies to Ascl1 or Neurog2, and not at all with antibodies to Tcfe2a-E12 (, lanes 4-11). These results are consistent with the EMSA with in vitro translated proteins where no bHLH was found to bind E0. Thus, although E0 is required for Dll3 promoter activity, the proteins involved in this activity were not identified ().
Protein complexes with E-box E2
E-box E2 has strong negative activity that keeps the Dll3 promoter restricted to the neural tube and keeps it from turning on prematurely. Using in vitro translated proteins, E2 can be bound efficiently by E12/E12, Ascl1/E12, and Neurog2/E12 (). Surprisingly, E2, but none of the other E-boxes tested, was also efficiently bound by an Ascl1/Ascl1 homodimer, a complex whose existence has not been previously reported (, lanes 2-5).
EMSA performed with E10.5 nuclear extracts also revealed E-box dependent complexes binding E2, but the complex identified includes both Ascl1 and Neurog2 (, lanes 1-3). Pretreating the extracts with antibodies specific to Ascl1 or to Neurog2 completely blocked the formation of the same band demonstrating the existence of a novel Ascl1/Neurog2 E-box binding complex (, lanes 6 and 8). To verify that we were detecting a specific interaction of the antisera to Ascl1 and Neurog2 in the complex, we heat inactivated antisera prior to use (Δ), and we tested an unrelated anti-GFP antiserum. In both cases, there was no attenuation of the protein-DNA complex (, lanes 4-9). In addition, cross detection of Ascl1 by Neurog2 antisera was not seen using in vitro transcribed and translated protein (, lanes 5-6). Antibodies to Tcfe2a-E12 had little if any effect on the formation of the complexes (, lanes 10-11). The ability of an Ascl1/Neurog2 heterodimer to bind E2 E-box DNA was confirmed using in vitro translated proteins (, lanes 8-12). Thus, E2 can be bound by multiple Ascl1 containing complexes, including the classical Ascl1/E12 heterodimer as well as an Ascl1/Ascl1 homodimer and Ascl1/Neurog2 heterodimer.
Protein complexes with E-box E4
E-box E4 has strong enhancer activity in the Dll3 promoter (, Dll3-mE4). Using in vitro translated proteins, only the heterodimer Nhlh1/E12 bound E4 efficiently (). EMSA with nuclear extracts, however, revealed a novel E-box binding transcription factor complex that again includes Ascl1 and Neurog2, and also suggest it requires at least one additional unidentified factor (). The presence of Ascl1 and Neurog2 in the protein-E4 complex was demonstrated by the complete disruption of the complex specifically with antibodies to both Ascl1 and Neurog2, but not to Tcfe2a-E12 or to a control GFP (, lanes 4-13). Surprisingly, blocking the Ascl1 and Neurog2 interaction with DNA by addition of specific antisera revealed a new protein-DNA complex with faster mobility than the wild-type complex (, lanes 6,8,10 asterisk). This new complex also requires an intact E-box (data not shown). The Ascl1/Neurog2 independent complex revealed could normally be a component of a higher order complex with these bHLH factors, or it could represent a binding activity only revealed after Ascl1 and Neurog2 are removed from the extract. However, consistent with the interpretation that an additional factor is required in the Ascl1/Neurog2 complex with E4, in vitro translated proteins alone can not form an Ascl1/Neurog2 heterodimer with E4 (). Thus, a novel multimeric complex containing Ascl1, Neurog2, and possibly another unidentified factor, or modification of the heterodimer, may play a role in Dll3 promoter activity.
Ascl1 homodimer and Ascl1/Neurog2 heterodimers function as transcriptional activators
Two novel Ascl1 DNA binding complexes were identified via in vitro EMSA analysis: Ascl1/Ascl1 homodimer and Ascl1/Neurog2 heterodimer. Both complexes can bind E2, the E-box that contains repressor activity. To test a model whereby one or both of these two novel heterodimeric complexes function to repress Dll3 expression, a cell culture based luciferase assay was utilized. Ascl1, Neurog2, and Tcfe2a-E12 were expressed in HEK293 cells with luciferase reporters containing hexamers of either wild-type E2 or mutant mE2. The results are shown as the fold induction of luciferase activity from the wild-type E2 reporter relative to that from the mutant mE2 (). Singly Ascl1, Neurog2, and E12 are all activators, with Neurog2 being by far the strongest. Co-expressing Ascl1 with E12 dramatically increases the transcriptional activation activity through the E2 sequence, consistent with the known function of the Ascl1/E12 heterodimer as an activator complex. To bias the formation of specific Ascl1 complexes, expression constructs were designed to tether Ascl1 with a peptide to either Ascl1 itself to favor the homodimer, to Neurog2 to favor the Ascl1/Neurog2 heterodimer, or to E12 to favor the Ascl1/E12 heterodimer. The Ascl1 tethered homodimer (Ascl1tAscl1) and the Ascl1 tethered to E12 (Ascl1tE12) were both strong activators in this assay. Ascl1 tethered to Neurog2 (Ascl1tNeurog2) also activated transcription but to a much lesser extent. Taken together, in these reporter assays, all Ascl1 complexes appear to act as activators, not repressors, but with varying efficiencies. Thus, the repressor activity of E2 can not easily be explained by binding of the novel Ascl1 complexes, suggesting other factors bind E2 to repress ectopic expression of Dll3.
Ascl1/Ascl1 homodimers and Ascl1/Neurog2 heterodimers function as transcriptional activators