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Environmental Health Science Laboratory, Sumitomo Chemical Co., Ltd., Konohana-ku, Osaka 554-8558, Japan
Received 12 September 2002/ Returned for modification 28 February 2003/ Accepted 2 October 2003
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ABSTRACT |
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 Top Abstract IntroductionMaterials and MethodsResults and DiscussionReferences |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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The mechanisms of Sim2 action in disease pathogenesis and its direct target genes remain to be elucidated. Assuming the existence of a signaling pathway which can be overrepressed by Sim2 in DS neurons, we searched the human genome for the hypothetical subtype or the relative of Sim2 that could compete with native Sim2 repression to activate a downstream signaling pathway with an activation domain, like Drosophila Sim. While such a subtype of Sim2 could not be found, we identified another mammalian bHLH-PAS factor, termed NXF. Here, we report the characterization of this new member of the bHLH-PAS family.
NXF is a novel factor, and its primary structure showed high identity among bHLH-PAS family. On the other hand, NXF possessed a DNA binding domain and a transcriptional activation domain as a bHLH-PAS-type transcriptional activator. Its localized expression in neurons at neuronal tissues such as the neuronal layer of the hippocampus pointed to the possible role in the transcriptional regulation of some neural genes in brain. NXF formed dimers with Arnt members, and the target DNA preference of the heterodimer partially overlapped with that of the Sim2 heterodimer.
We investigated NXF target genes and identified Drebrin as a candidate in brain. This neuronal gene has been reported to engage in dendritic-cytoskeleton modulation at synapses expressed in the neuronal layer of the hippocampus (14) and to have a role in synaptic plasticity (13, 16). We show that Drebrin has regulatory DNA elements on its promoter which NXF can directly activate and which Sim2 can repress directly or indirectly.
The NXF signaling system is a novel transcriptional pathway in neurons, and we discuss the possibility that such an NXF pathway may be a molecular target of adverse Sim2 effects in the mental retardation of DS.
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MATERIALS AND METHODS |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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RNA blotting analysis and in situ hybridization. Commercially available human RNA-blotted membranes (MTN blot and MTN blot IV; Clontech) were analyzed by using a cDNA probe for the full-length coding region of human NXF under highest-stringency conditions. Rat NXF cDNA (HindIII C-terminal fragment) was subcloned into pGEM (Promega), and T7 polymerase or SP6 polymerase was used to make the sense probe and the antisense probe, respectively. In situ hybridization of tissue sections from 8-week-old Sprague-Dawley rats was performed with these probes as described previously (10). For analysis of NXF expression in developing embryos, RT-PCR was performed with commercially available mouse embryo cDNAs (Multiple-Tissue cDNA; Clontech) in parallel with 4-week C57BL mouse brain cDNA.
Gal4 or VP16 fusion proteins and assays. Gal-NXF(C), Gal-NXF(256-596), Gal-NXF(597-802), Gal-NXF(N), Gal-Sim2(N), and Gal-Clock(N) were constructed with fragments introduced into pRC/RSV-Gal4 (termed Gal4-DBD in Fig. 3). The Gal4 part was the Gal4 DNA binding domain derived from pM (Clontech). The other part in each construct was as follows: Gal-NXF(256-596), amino acids 256 to 596; Gal-NXF(597-802), amino acids 597 to 802; NXF(N), amino acids 1 to 596 (containing bHLH-PAS); Sim2(N), human N-terminal BamHI fragment (bHLH-PAS); and Clock(N), human HincII-NcoI fragment (bHLH-PAS). VP16-CP was pVP-CP (Clontech). VP16-X target plasmids (various bHLH-PAS regions) were constructed with pVP16 (Clontech): Arnt1 fragment, human N-terminal NaeI fragment (bHLH-PAS); Arnt2 fragment, N-terminal BglII fragment (bHLH-PAS). The BMAL1 and BMAL2 inserts were full length. The Sim2, Clock, and NXF fragments were the same as Sim2(N), Clock(N), and NXF(N). All factors were cloned by RT-PCR, and the lack of introduced mutations was confirmed by sequencing. A Gal4 reporter with four copies of the Gal4 binding element was constructed with two copies of the oligonucleotide 5'-CGCGTCGAGCTCGGGTCGGAGGACTGTCCTCCGACTGCTCGAGTCGAGCTCGGGTCGGAGGACTGTCCTCCGACTGCTCGAGA-3' introduced into pGL3 (Promega). Transcriptional activity of the NXF C terminus was analyzed in the IMR32 neuroblastoma (ATCC CCL127) with a pRL control (Promega) according to the manufacturer's instructions. Essentially the same results were also obtained with 293 and HeLa cells. The interaction assay was performed in 293 cells by the mammalian two-hybrid assay with a standard procedure according to the instructions of the manufacturer (Clontech).
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Chromatin immunoprecipitation assay. By use of standard protocols, the SK-N-MC cells transfected with an N-terminal Flag tag-fused NXF and Arnt2 expression plasmid were maintained in neomycin selection medium for 2 months, and one stable SK-N-MC transformant cell line among the resultant neomycin-resistant colonies was selected by anti-Flag Arnt2 immunoblotting analysis. Chromatin immunoprecipitation assay was performed with anti-Flag-M2 agarose (Sigma) and a ChIP kit (Upstate Biotechnology) with PCR primer pair A (5'-GAGGCTGGCCCTGAGCGTCC-3' and 5'-CCACTTATTCCAGGCCATTC-3'), which amplifies the NXF/Arnt2 binding region on the Drebrin promoter, and primer pair B (5'-CAGGTGGTGCTTCTGGTACC-3' and 5'-CCGCTTTCCTCCACGGGCAG-3'), which amplifies Arnt2 gene exon 2, as the negative control primer. The buffers and procedure used were as described by the manufacturer.
Nucleotide sequence accession numbers. The human, rat, and mouse NXF cDNA sequences and the mouse NXF genomic sequence have been deposited in GenBank under accession numbers AB049469, AB050103, AB049835, and AB054577, respectively.
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RESULTS AND DISCUSSION |
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TopAbstractIntroductionMaterials and MethodsResults and DiscussionReferences |
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NXF has a transcriptional activation domain and forms heterodimers with Arnt family members. The Gal4 fusion protein with the NXF C terminus [Gal-NXF(C)] (Fig. 3a) could activate a Gal4 reporter construct that contained Gal4 DNA binding elements upstream of the promoter and a luciferase reporter gene. In addition, overexpression of the general transcriptional coactivator p300 enhanced the activity of the NXF C terminus (Fig. 3a, bars 3 and 4). p300 can associate with many unrelated transcription factors, such as nuclear hormone receptor family members, AP1, ETS-1, ETS-2, Arnt, etc. Considering these results, we concluded that NXF had a typical type of transcriptional activation domain that utilizes the general p300 coactivator mechanism as a transcription factor. This domain lay in the C-terminal last half (amino acids 597 to 802) (Fig. 3a, bars 5 and 6), while we do not identify specific sequence feature in the region.
The bHLH domains of bHLH-PAS DNA binding factors may function as heterodimers or homodimers for DNA binding, as suggested by X-ray crystallographic data of the bHLH part from, for example, the bHLH-LZ Max protein (11). To examine whether NXF could form the dimer, we performed mammalian two-hybrid assays. NXF showed specific interaction with Arnt subclass bHLH-PAS factors (Arnt1, Arnt2, and BMALI) in this assay, using a Gal4 fusion protein with the N terminal structure of NXF (amino acids 1 to 595, composed mainly of the bHLH-PAS motif) for bait and the VP16 activation domain fused with various known bHLH-PAS factors as targets (Fig. 3b, panel 1). We did not observe any NXF-NXF homodimerization. In addition, no interactions with other bHLH-PAS factors (BMAL2, Sim2, and Clock) could be observed (Fig. 3b, panel 1, bars 5 to 8). In experiments performed for confirmation, using Gal4 fusion proteins with Arnt1, Arnt2, BMAL1, or BMAL2 substituting for NXF as the bait, all Arnt subclass members except BMAL2 showed specific interaction with the VP16-NXF fusion target (data not shown). The interaction specificity in this assay was validated by control experiments showing specific associations of Sim2 with Arnt1 and Arnt2 and of Clock with BMAL1 and BMAL2, using Gal-Sim2(N) or Gal-Clock(N) as a substitute for NXF(N) (Fig. 3b, panels 2 and 3). These results indicate that NXF functions as a heterodimer with Arnt subclass members (Arnt1, Arnt2, and BMAL1), preferably with Arnt2, which is known to be abundant and restricted in brain (15).
NXF can bind and activate the Sim2-repressive CME element and its variants. To identify potential NXF/Arnt target DNA elements, we performed a screening against randomly selected oligonucleotides with known bHLH-PAS factor-responsive sequences in the DNA binding assay. Recombinant NXF or Arnt2 as a protein source was expressed with the baculovirus vector. When we used CME (30), a Sim/Arnt-responsive DNA element, as a probe, we could detect a specific binding signal depending on both NXF and Arnt2 (Fig. 4a, lane1). The CME element is known to consist of two half sites, a Sim2 recognition half site and the flanking GTG (26) recognized by Arnts. To optimize the element for an NXF/Arnt2 complex, we systematically made a series of CME variants that were mutated one by one at the first and second positions next to the GTG Arnt binding sequence in the CME core (Fig. 4a, upper panel) and additional variants that were mutated at third position to GTG, containing parental ACGTG, GCGTG, TCGTG, or CCGTG as core sequences (Fig. 4a, lower panels). These DNA binding assays revealed a critical CGTG nucleotide, each TCGTG core-containing element as the best NXF/Arnt2 binding sequence, other tolerated NXF/Arnt2 binding elements such as GCGTG core-containing sequences (which are also another Sim2-responsive element) (26), and a weak CCGTG core sequence for NXF/Arnt2 binding. These results indicate that the sequence preference of NXF could partially overlap with that of Sim2, while most preferable element of NXF is distinct from CME, the Sim2 most preferable element. Besides the best element, as shown by confirmation experiments (Fig. 4b and c), NXF could indeed recognize CME as a direct binding element and its responsive element (Fig. 4b, lane 5). Neither NXF nor Arnt2 recognized this CME element alone. The binding signal was competed out with a nonlabeled CME probe but not with an unrelated nonlabeled probe (E-box element), supporting the binding specificity in this assay (Fig. 4b). Consistent with such a result of the DNA binding experiment, significant transcriptional induction by the NXF/Arnt2 complex was observed on four tandem copies of CME in a reporter gene assay with the IMR32 neuroblastoma cell line (Fig. 4c, bar 6). This induced activity was impaired in a dose-dependent manner by Sim2 (Fig. 4c, bars 7 and 8). Antisense Sim2 as a negative control failed to repress the NXF/Arnt2. Essentially the same results were also obtained with 293, PC12, and other cell lines tested. These results may indicate possible cross talking between an NXF positive signal and a Sim2 negative (repressive) signal in some gene transcriptional regulation.
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Identification of potential NXF target genes. To identify the target genes that respond to NXF expression, we applied DNA array technology to compare endogenous gene expression pattern in the SK-N-MC neuroblastoma host cell line with that in transfectants overexpressing NXF cDNA. In the transfectants, several neuronal function-related genes showed alteration of mRNA expression relative to parental NXF-negative SK-N-MC cells (data not shown). Interestingly, the endogenous NXF gene itself was found to be autoregulated by NXF (Fig. 5a, bottom). This is reminiscent of the Drosophila Sim gene autoregulation loop (24). Besides NXF, one of the most induced genes with NXF overexpression was Drebrin (14). RT-PCR analysis confirmed induction of the endogenous Drebrin gene by NXF overexpression in the cell (Fig. 5a, top), while the Sim2 gene for negative control was not responsive to NXF and its endogenous mRNA was constantly expressed with or without NXF (Fig. 5a, middle). Drebrin is reported to be abundant in the hippocampus of the human brain in immunocytochemical studies (13). We also confirmed the prominent Drebrin expression in the neuronal layer of the hippocampus by in situ hybridization (Fig. 2e). It is interesting that the Drebrin gene expression pattern in the brain closely overlaps with that of NXF, which is consistent with our results described here.
NXF can activate the expression of the Drebrin gene or NXF itself through each promoter, and its activity is competed with Sim2. To determine whether NXF directly regulates the transcription of the Drebrin gene and the NXF gene itself through their promoters, we performed the reporter gene assay with a luciferase reporter construct fused with the NXF promoter (-2.7 kbp) or the Drebrin promoter (-2.7 kbp) (Fig. 5b). On both promoters, the NXF/Arnt2 complex caused significant transcriptional induction. Furthermore, Sim2-dependent transcriptional repression of the induced activity in a dose-dependent manner was observed on each promoter. The Sim2-VP16 chimera could again activate both promoters (Fig. 5b, bar 7), suggesting that the direct binding of Sim2/Arnt2 on the promoter sequence partly contributes the repression in addition to functional interference. These results indicate that both NXF/Arnt2 and Sim2 act on the gene expression directly through the promoter sequence of Drebrin or NXF itself.
The NXF gene and the Drebrin gene have NXF-activating DNA elements on their promoters. In a search of the promoter sequences of NXF and Drebrin, putative NXF-responsive elements were found. The positions on each promoter are shown and numbered in Fig. 5c. To examine these numbered elements, we performed DNA binding analysis with each labeled element as a probe, and we found some candidate elements on both promoters (sequences 1, 4, 6, 7, 10, 13, 14, and 16) to be good NXF/Arnt2 binding elements, although this was not the case for the remainder (Fig. 5d, left). We also performed reporter gene analysis with chimera gene constructs having each DNA element (Fig. 5d, right). In this in vitro assay, some of these elements from the Drebrin or NXF gene promoter were activated by NXF/Arnt2 and repressed by Sim2, and such transcriptional activities corresponded to their binding to NXF/Arnt2.
To examine NXF/Arnt2 binding elements on the NXF and Drebrin promoter regions further, we constructed several mutated reporter plasmids. One is a mutated NXF promoter with several point mutations at the strong NXF/Arnt2 binding elements in the promoter (positions 1, 4, and 6). Another is a Drebrin promoter A that is mutated only at the false NXF/Arnt2 binding elements (positions 9, 12, and 15), which do not show a significant NXF/Arnt2 binding signal in our DNA binding experiments. The last is a Drebrin promoter B which has mutations at the strong NXF/Arnt2 binding elements (positions 10, 13 and 14). We performed additional reporter analysis with these constructs and observed dramatic decreases of NXF/Arnt2-dependent inductivity of the mutated NXF promoter and mutated Drebrin promoter B (Fig. 6a). In contrast, the Drebrin promoter A which is mutated only at the false NXF/Arnt2 binding elements showed no significant decrease of promoter activity.
Moreover, to strengthen the conclusion that the Drebrin promoter has direct NXF/Arnt2 binding elements, we performed chromatin immunoprecipitation assays. Anti-Flag antibody-Flag-fused NXF/Arnt2 protein complex could specifically pull down the Drebrin promoter element on chromatin in Flag-NXF/Arnt2-overexpressed transformants (Fig. 6b). In this analysis, control preprecipitated input (sonicated chromatin fragment) and immunoprecipitated chromatin fragment were amplified with PCR primer A for the Drebrin promoter or with PCR primer B for the negative control Arnt2 gene exon 2 region. Only primer A could amplify the chromatin that precipitated with anti-Flag antibody-Flag-fused NXF/Arnt2 protein complex, and the amplified product was competed with excess Flag peptide in immunoprecipitation, showing the specificity of the assay (Fig. 6b). These results indicate that Flag-NXF specifically associated endogenous Drebrin gene promoter region in vivo.
Conclusion. Here we have reported the identification of novel bHLH-PAS-type transcription factor NXF. With several experiments, we concluded that the Drebrin gene and NXF itself were direct targets of NXF and that their promoter activity could be repressed by Sim2 (Fig. 7a).
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We propose a possible mechanism of Sim2 action in the pathogenesis of DS, describing the contributions of NXF and Drebrin in the model (Fig. 7b). Sim2 would affect two points in this model, i.e., the positive feedback system of NXF gene expression and the regulatory mechanism of Drebrin expression. We have reported here that both of these were regulated and balanced by NXF and Sim2. In the DS brain, with a 1.5-fold (28) dose of Sim2 due to Sim2 gene trisomy, the positive feedback loop of the autoregulation system of NXF gene expression would be turned down exponentially, and the effect of the 1.5-fold dose of Sim2 might be amplified at this point. The resulting low level of NXF protein would be additionally competed by the excess Sim2 in DS, and that should synergistically affect the expression of certain target genes such as Drebrin.
The identification of NXF and its novel signaling with Sim2 may give the initial clues and tools to dissect some of the signaling pathways underlying the pathogenesis in DS or other neural diseases, besides contributing to the understanding of neural gene transcriptional regulation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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