- Research article
- Open Access
A short upstream promoter region mediates transcriptional regulation of the mouse doublecortin gene in differentiating neurons
BMC Neuroscience volume 11, Article number: 64 (2010)
Doublecortin (Dcx), a MAP (Microtubule-Associated Protein), is transiently expressed in migrating and differentiating neurons and thereby characterizes neuronal precursors and neurogenesis in developing and adult neurogenesis. In addition, reduced Dcx expression during development has been related to appearance of brain pathologies. Here, we attempt to unveil the molecular mechanisms controlling Dcx gene expression by studying its transcriptional regulation during neuronal differentiation.
To determine and analyze important regulatory sequences of the Dcx promoter, we studied a putative regulatory region upstream from the mouse Dcx coding region (pdcx 2kb) and several deletions thereof. These different fragments were used in vitro and in vivo to drive reporter gene expression. We demonstrated, using transient expression experiments, that pdcx 2kb is sufficient to control specific reporter gene expression in cerebellar cells and in the developing brain (E14.5). We determined the temporal profile of Dcx promoter activity during neuronal differentiation of mouse embryonic stem cells (mESC) and found that transcriptional activation of the Dcx gene varies along with neuronal differentiation of mESC. Deletion experiments and sequence comparison of Dcx promoters across rodents, human and chicken revealed the importance of a highly conserved sequence in the proximal region of the promoter required for specific and strong expression in neuronal precursors and young neuronal cells. Further analyses revealed the presence in this short sequence of several conserved, putative transcription factor binding sites: LEF/TCF (Lymphoid Enhancer Factor/T-Cell Factor) which are effectors of the canonical Wnt pathway; HNF6/OC2 (Hepatocyte Nuclear Factor-6/Oncecut-2) members of the ONECUT family and NF-Y/CAAT (Nuclear Factor-Y).
Studies of Dcx gene regulatory sequences using native, deleted and mutated constructs suggest that fragments located upstream of the Dcx coding sequence are sufficient to induce specific Dcx expression in vitro: in heterogeneous differentiated neurons from mESC, in primary mouse cerebellar neurons (PND3) and in organotypic slice cultures. Furthermore, a region in the 3'-end region of the Dcx promoter is highly conserved across several species and exerts positive control on Dcx transcriptional activation. Together, these results indicate that the proximal 3'-end region of the mouse Dcx regulatory sequence is essential for Dcx gene expression during differentiation of neuronal precursors.
The DCX gene is located on the X chromosome (Xq22.3-q23) and encodes a 40 kDa phosphoprotein of 360 amino acids. The DCX protein is a microtubule-associated protein (MAP) that interacts with and stabilizes the microtubule cytoskeleton [1, 2]. This gene is specifically and transiently expressed in proliferating neuronal progenitors and in post-mitotic neuronal precursors during embryonic development and in neurogenic regions of the adult brain [2–5]. DCX expression occurs during corticogenesis and is absent during regenerative axonal growth, suggesting that DCX is a selective marker of cells committed to the neuronal lineage in both developing and adult brain [6, 7]. One or more mutations in the DCX gene cause X-Linked Subcortical Laminar Band Heterotopia (X-SCLH)/Lissencephaly (LIS) [2, 8]. This developmental brain malformation syndrome is caused by abnormal neuronal migration leading to a profound cerebral cortical layer disorganization resulting in mental retardation and epilepsy.
Such alterations in cortical lamination observed in humans were not detected in mice with Dcx gene deletion; lamination defects were only observed in the hippocampus . In contrast, RNA interference (RNAi)-mediated knock-down of Dcx in rodents caused impairment in radial migration of cortical neurons [10, 11]. Similarly, mice with mutations of both Dcx and Dclk (Doublecortin-like kinase gene, homolog of the Dcx gene) genes presented both disorganized neocortical lamination and severe cytoarchitectural defects of the hippocampus, suggesting redundant functions of Dcx and Dclk during neuronal migration [12–14].
Neuronal differentiation is a tightly orchestrated time- and location-dependent process in which many extracellular and intrinsic factors are involved [15–17]. Whereas the temporal and spatial Dcx expression patterns and Dcx post-translational regulations are well known, Dcx transcriptional regulation is poorly understood. However, to understand the mechanisms involved in neuronal differentiation during embryogenesis and more precisely in stages before neuronal determination, it is crucial to investigate the transcriptional gene control in action during neuronal differentiation. In view of the relevance of Dcx as a marker for neurogenesis and considering the importance of understanding Dcx gene regulation, we analyzed a putative regulatory region upstream of the mouse Dcx gene (pdcx 2kb) and used it to drive reporter gene expression. We characterized the Dcx promoter activity at different time-points during neuronal differentiation of mESC (mouse embryonic stem cells) and we defined a small region as an element required to provide specific and strong expression in neuronal precursors and young neuronal cells.
To study transcriptional regulation of the mouse doublecortin gene (Accession number NT_039718), we selected a 2kb-long fragment upstream of the transcription initiation site of the longest reported Dcx mRNA transcript (Accession number NM_001110222). This 2kb-long fragment (pdcx 2kb) contained TATA and CAAT boxes and was cloned into promoterless reporter (eGFP or Luciferase) vectors for analysis (Figure 1a).
Cell type-specific activity of the 2kb-long Dcx promoter fragment
We first wanted to determine whether pdcx 2kb was sufficient to induce transcriptional activity and to drive it specifically in cells expressing the endogenous Dcx gene, namely neuronal precursor cells. Two mouse cell types were used: cerebellar neurons from post-natal day 3 mice (PND3) in which endogenous Dcx expression has already been reported  and mouse embryonic stem (ESR1) cells.
Cell specificity of pdcx 2kb transcriptional activity was observed in both heterogeneous cell cultures by transient expression of the eGFP protein upon transfection with pdcx 2kb-eGFP constructs (Figure 1b). In primary cerebellar cells, eGFP under the control of pdcx 2kb was detected only in cells expressing the endogenous Dcx gene and not in other cell types, such as GFAP-expressing astrocytes (Figure 1b: A-H). Control experiments using the widely active pCMV-eGFP construct revealed that eGFP fluorescence was detected indifferently in all cell types, neurons and glial cells alike. Both GFAP- and Dcx-positive cells were fluorescent (Figure 1b: I-P), showing that the transfection process was not cell-specific. Similarly, eGFP expression was only detected in ESR1 cells after neuronal differentiation and was limited to Dcx-positive or βIII-Tubulin-positive cells, confirming pdcx 2kb selectivity to Dcx-positive neuronal precursor cells in vitro (data not shown).
Ex vivo electroporation of embryonic mouse brains was also conducted to confirm cell selectivity of pdcx 2kb activity in a more physiological setting. pdcx 2kb-eGFP was transfected in brains of E14.5-E15.5 mice, a specific time point of corticogenesis . Dcx expression was observed in migrating cortical neurons originating from progenitor cells located near the ventricular proliferative zone. Following pdcx 2kb-eGFP transfection, fluorescence was observed around the electroporation site in the proliferative zone of the cerebral cortex. Fluorescent cells displayed morphology similar to that of migrating neuronal precursors (Figure 1c). Moreover, all fluorescent cells also expressed Dcx. Taken together, these qualitative analyses show that the 2kb-long fragment upstream of the Dcx gene possesses regulatory sequences sufficient to mimic the activity of the endogenous Dcx gene in mouse embryos and to limit its activity to Dcx-positive neuronal precursors.
Transcriptional activity of the Dcx promoter in differentiating ES cells
To study more efficiently the transcriptional regulation of the mouse Dcx gene, we chose to use mouse embryonic stem (ESR1) cells: these cells are able to proliferate indefinitely in vitro while retaining pluripotency, and also to differentiate into a large variety of cell types in vitro. Hence, mouse ES cells can differentiate into neural precursors able to generate functional neurons , astrocytes and oligodendrocytes [21, 22]. Using our protocol (see Material and Methods), induction of neuronal differentiation leads to a progressive change in ESR1 cell morphology to a characteristic cobblestone structure and, by neuronal differentiation day 8 (DD8), ESR1 cells begin to form long cellular processes. From DD8/10 on, bipolar cells proliferate and form rosettes.
Dcx protein was detected at DD6 of ESR1 differentiation (Figure 2) and was still present in immature neuronal cells-derived from ESR1 cells at DD8 and DD18. Based on these observations and on the detection of Dcx mRNA at DD4, we decided to use ESR1 at DD6 for further analysis of the transcriptional activity of the mouse Dcx promoter.
To characterize relevant regions of pdcx, we dissected the 2kb-long fragment into shorter fragments of 1.2 kb, 1 kb (obtained by PCR) and 249 bp (obtained by enzymatic restriction), and inserted them upstream of the luciferase reporter gene.
Comparison of the respective activities of all constructs (Figure 3a) in ESR1 cells at DD8 revealed a higher activity in cells transfected with pdcx 2kb than in control cells (figure 3b). Transcriptional activities were also higher in cells transfected with shorter fragments (pdcx 1kb and pdcx 249bp) than with pdcx 2kb (Figure 3b). In contrast, the 1.2kb-long pdcx fragment did not induce a transcriptional activity different from control, suggesting the presence of a transcriptional repressor region in this 200 bp fragment. In addition, deletion of 79 bp at the 3'end of pdcx 1kb (pdcx 1kb-) prevented the pdcx 1kb-induced increase of activity. This last result suggested the presence of important regulatory domains at the 3'end of pdcx, especially in a 79bp-long fragment. Similar effects on the transcriptional activities of the different constructs were observed in PND3 cerebellar cells (Figure 3c), confirming the presence of similar regulatory control mechanisms in neuronal precursors from both origins (ESR1 cells and primary cerebellar cells). However, a discrepancy is observed between the relative activity of pdcx 1kb and pdcx 249bp between both cell cultures (Figure 3b and 3c), maybe reflecting the fact that the target regulatory elements necessary for the Dcx gene expression are different in two cell types. Indeed, it is quite possible that activators or inhibitor elements, located in pdcx 1kb and pdcx 249bp respectively, are most needed during the differentiation of CGN compared to neuroblasts from ESC. At that stage (DD6), ESR1 cells are plated on gelatin-coated plates while cerebellar cells are plated on poly-ornithine substrate (necessary for cell adhesion) (see also below).
The same relative activities of the promoter fragments were maintained throughout the entire differentiation program in ESR1 cells (Figure 4). Indeed, when ESR1 cells were transfected every second day throughout the entire differentiation period and the luciferase activity measured 48 hours later, increasing levels of activities were observed along the process with a peak at the transfer onto poly-ornithine/lamin substrate for constructs pdcx 2kb, pdcx 1kb and pdcx 249bp. The time-course studies confirmed the inhibition or the significant reduction of the transcriptional activity of pdcx at all stages by deletion of 79b at the 3' end of the pdcx sequences in constructs pdcx 1kb and pdcx 249bp (pdcx 1kb- and pdcx 249bp-, respectively). Finally, we also examined the cellular specificity of pdcx 1kb and pdcx 249bp in differentiated ESR1 and PND3 cerebellar cells and confirmed that, similar to the pdcx 2kb construct (Figure 1b), both pdcx 1kb and pdcx 249bp fragments restricted the expression of the eGFP reporter gene in Dcx-expressing cells (data not shown).
Transcription factors regulating the mouse Dcx promoter
Comparative analyses were performed on the sequences of mouse, rat, human and chicken 2kb-long Dcx promoter fragments using the VISTA software (Figure 5). A high sequence similarity was found between mouse and rat pdcx 2kb sequences. Comparison between mouse and human sequences revealed that sequence conservation was not detected throughout the 2kb-long sequence, but was limited to a ± 500 bp portion located at the pdcx 2kb 3'-end. Similarly, sequence conservation between the mouse pdcx 2kb and the chicken pdcx 2kb was only observed in a 183bp-long fragment located at the 3'-end. In addition, the strongest sequence conservation between mouse, rat, human and chicken pdcx 2kb sequences was detected within this 183bp-long fragment (Figure 5). Further analysis, using the MatInspector software, revealed that many putative binding sites for members of several transcription factor families known to participate in neuronal differentiation/migration such as NEUROD, NEUROG, BRN, PAX, HOX, SOX and DLX are present in the 249bp-, 1kb-, 2kb-long Dcx promoter fragments (Additional file 1: Table S1). In particular, this analysis revealed four conserved consensus binding sites for known transcription factors in the 79b-long fragment located at the 3'end of the Dcx promoter (Figure 6): two perfect consensus sites for NF-Y/CAAT, one perfect site for LEF/TCF which are effectors of the canonical Wnt pathway and one imperfect site for HNF6/OC2 (75% conserved relative to consensus), members of the ONECUT family [23–25].
Expression of putative transcription factors during neuronal differentiation of mESC
To investigate which of these transcription factors, present in the 79 bp fragment and potentially involved in Dcx gene regulation, are expressed during neuronal differentiation of ESR1 cells, the levels of mRNA coding for Dcx and for each factor (Lef, Tcf1, Tcf3 and Tcf4, Hnf6 and Oc2, Nf-ya, Nf-yb and Nf-yc) were measured at different time-points during neuronal differentiation (Figure 7).
The abundance of each transcript at each stage was calculated relative to the Gapdh housekeeping gene mRNA, based on published results [26–28]. The observed pattern of Dcx transcript expression is consistent with that of Dcx protein synthesis during neuronal differentiation of ESR1 cells and with the observed transcriptional activity of the Dcx promoter (see above). No Dcx transcript was detected in undifferentiated ESR1 cells. Levels of Dcx transcripts progressively increased to reach a maximum at DD18 followed by a reduction at DD28. Each transcription factor transcript was detected in ESR1 cells. The temporal profile of Lef, Hnf6 and Tcf4 transcription factors presented patterns of expression similar to that of Dcx, suggesting that expression of these transcription factors is also dependent on neuronal differentiating time. Their levels were relatively low during the early phase of ESR1 cell differentiation (DD0-8), progressively increased during the late phase of the differentiation program to peak at DD18 when Dcx transcript levels were maximum. In contrast, Nf-ya, Nf-yb, Nf-yc and Oc2 transcripts displayed relatively constant steady state levels, with little variation during the entire differentiation program. The relative amounts of Tcf1 and Tcf3, seem to depend on the differentiation process, with Tcf3 decreasing during differentiation  and Tcf1 being apparently sensitive to the culture matrix substrate.
Since mRNA for each transcription factor was present in ESR1 cells and since no transcription factor could be discarded on the sole basis of the temporal expression profile of its transcript, selective mutagenesis experiments were undertaken.
To determine the relative importance of each DNA binding site present in the 79bp-long pdcx-luciferase fragment, we individually altered each putative binding site for LEF/TCF, HNF6/OC2 or NF-Y/CAAT in the Dcx promoter using site-directed mutagenesis to generate pdcx 249bp/Lef*, pdcx 249bp/Hnf6* and pdcx 249bp/Nf-y* (Figure 8). Then we compared the luciferase activities induced by each construct to that of pdcx 249bp by transient expression experiments in ESR1 cells at three stages of neuronal differentiation. At the beginning of the differentiation program (DD2), mutation of the NF-Y or HNF6 binding sites did not affect pdcx 249bp activity (Figure 8a). In contrast, mutation of the LEF/TCF site completely inhibited pdcx 249bp activity. Such inhibition was not detected in ESR1 cells at DD8 (Figure 8b). At that stage, the relative activity of every mutated pdcx 249b was below that of the wild type pdcx 249bp but higher than that of the 79bp-truncated pdcx 249bp. Finally, at DD20, when many ESR1 cells expressed the Dcx gene and displayed a neuronal phenotype, mutation of any of the four binding sites reduced the activity of pdcx 249bp to that of the 79bp-truncated pdcx 249bp (Figure 8c). Similar results were found using pdcx 1kb (data not shown) and in PND3 cerebellar cells (Figure 8d). Simultaneous mutation of all three binding sites was also performed (pdcx 249bp/Lef*/Hnf6*/Nf-y*). Transfection experiments revealed that the transcriptional activation of pdcx 249bp mutated for LEF, HNF6 and NF-Y binding sites is similar to the constructs with any one mutated site (Additional file 2: Figure S1) at all stages, suggesting that an additional, not yet identified factor is responsible for the observed residual activity at DD8. Altogether, these results demonstrate first of all the importance of LEF/TCF, HNF6/OC2 or NF-Y/CAAT DNA binding sites for Dcx promoter activity at various stages of neuronal differentiation and that the corresponding factors are part of the regulatory mechanisms controlling Dcx promoter activity. However, these transcription factors are not sufficient to induce full transcriptional activation of the Dcx gene. In addition, the results obtained with the LEF/TCF binding site reveal that this binding site could be more specifically involved in activating pdcx at early stages of neuronal differentiation of ESR1 cells.
This present study shows that 249bp-, 1kb-, 2kb-long DNA fragments located upstream of the Dcx coding sequence, are sufficient for in vitro specific Dcx expression: in heterogeneous differentiated neurons from mouse embryonic stem (ESR1) cells, in primary mouse cerebellar neurons (PND3) and in organotypic slice cultures. The regulatory activity of all three constructs, visualized by eGFP expression, overlapped endogenous expression of Dcx or βIII-Tubulin (data not shown) in immature neuronal cells. In addition, no eGFP expression was observed in non-neuronal cells. These results strongly suggest that the fragments isolated here are sufficient for specific neuronal expression in differentiating neural cells.
During our analysis of transcriptional activities of the deletion constructs, we observed a very weak activity of the 1.2 kb fragment relative to the 1 kb construct, suggesting that the 0.2 kb sequence upstream from pdcx 1kb could contain transcriptional repressor elements. To determine the regulatory element(s) susceptible to participate in transcriptional repression of Dcx expression, we analyzed this sequence with the MatInspector software http://www.genomatix.de, but no unique repressor element was identified. However, this 0.2 kb sequence holds a short tandem repeat (STR) of twenty-five CA repeats (Figure 6), that could act as positive or negative regulator of gene expression. Short tandem repeats have been identified and are widespread in coding and non-coding regions of eukaryotic genomes from yeast to humans [30, 31]. CA repeats are also potential Z-DNA-forming sequences that could affect gene expression. Indeed, STRs were shown to activate or repress gene expression depending on the length of the repeats [32, 33]. This potential repressive element was not further investigated in this work, but it certainly deserves further attention also in the context of Dcx-related disease.
Dcx is expressed in differentiating/migrating immature neurons of embryonic and adult CNS and PNS. Moreover, Dcx presents a maximum expression during corticogenesis (E14-E18 in mouse) and neurogenesis [18, 34]. Using a mouse ESC model of neuronal differentiation, we demonstrated that the transcriptional activity of the Dcx promoter increased during the differentiation program, with a maximum activity shortly after the cells were plated on poly-ornithine/laminin substrates . Both of these extracellular matrix (ECM) components were shown to promote the active extension of neuronal cell protrusions as well as their maturation [36–38]. In the present study, we further show that ECM components also promote expression of the endogenous Dcx protein, in a pattern fully consistent with the transcriptional activity of the identified promoter region during the same differentiation process. This interesting observation suggests that it is possible to reproduce the Dcx expression profile in an in vitro