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One of the best-characterized transcription factors for controlling neuronal gene expression is the repressor element 1 (RE-1)–silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) repressor protein that is involved in suppressing several neuronal gene expressions in non-neuronal cells (11,12,26–28). We have previously demonstrated that the NRSE of MOR functions as a critical regulator to repress MOR gene expression in specific neuronal cells depending on NRSF expression level (11). In this study, we explored the role of the putative Sp transcription factor binding element (G/C box) in MOR promoter region located 3′ end of NRSE and demonstrated that Sp3 transcription factor represses the expression of MOR gene by binding to this element and by interacting with NRSF.\nFrom the sequence data analysis, we initially found the conserved Sp family binding site adjacent to NRSE in mouse, rat and human MOR gene that could regulate MOR gene expression. When treated with mithramycin A, a G/C box specific binding inhibitor, the mRNA level of MOR gene was increased in NRSF containing NS20Y cells but not in NRSF negative PC12 cells (Figure 1). The result suggested that this GC box element may mainly act as a repressor in MOR gene regulation and explained why the MOR transcription level is very low in NS20Y cell line.\nTo explore the repression mechanism by this GC box in MOR gene regulation, several MOR-luciferase constructs with mutations in either NRSE or the G/C box or both were made and tested for their promoter activities. Mutations in both NRSE and Sp-binding site showed 14-fold increase in the activity whereas either NRSE or G/C box mutation had 4- to 5-fold increased activity compared with that of wild-type sequences in NS20Y cells (Figure 2). Therefore, the negative effect of the putative Sp-binding element for the MOR gene expression could be considered as additive to or preferably synergic with NRSE. A recent study reported that the transcription levels of the several NRSF target genes containing GC-rich regions were decreased with mithramycin A in a dose-dependent manner in both βTC3 and PC12 cells indicating that Sp1 acts as an activator for the expression of NRSF target genes (29). Two positive Sp family binding elements in MOR promoter were previously identified which is located at ∼300 bp upstream from the GC box identified in the current study (25). In those upstream elements, Sp1 acts as a major activator by binding to both elements whereas Sp3 has minor activator effect.\nThe identity of the transcriptional repressor that binds to the newly identified GC box was first hinted by co-immunoprecipitation of the Sp3 factor with NRSF in both HeLa and NS20Y nuclear extracts (Figure 4). Then, the specific and functional binding of Sp3 factor to the GC box was confirmed by gel-shift assays using either in vitro translated NRSF and Sp3 factor (Figure 5) or nuclear extract (Figure 6), and in vivo ChIP assay (Figure 7). The results clearly demonstrated that Sp3 factor binds the G/C motif and interacts with NRSF in MOR promoter region, suggesting that transcription factor Sp3 is required for silencing MOR expression together with NRSF.\nWithin the Sp family transcription factors, Sp1 and Sp3 are ubiquitously expressed in mammalian cells. Sp3 is structurally similar to Sp1, with similar affinities for Sp1-binding site. Although Sp1 and Sp3 share \u003e90% sequence homology in the DNA-binding domain and they bind to the same cognate DNA element, they have strikingly different functions (30). Although Sp1 mainly acts as transcriptional activator, Sp3 can repress activity of the promoter by recruiting HDAC1 or HDAC2. HDAC1 and HDAC2 have been shown to potently repress luteinizing hormone receptor gene transcription. The mSin3A, a component of the Sin3–HDAC complex, potentiates the inhibition mediated by HDAC1 (31,32). It has been well established that the Zn2+-finger-containing protein REST/NRSF binds the 21 bp NRSE DNA element found in many neuronal genes including MOR and then nucleates the formation of a multiprotein complex that represses gene transcription by deacetylating histones, methylating both DNA and histone H3, and dephosphorylation of RNA polymerase II (15,18,33). In conjunction with our current data, especially from the co-immunoprecipitation of Sp3 with NRSF, it can be postulated that the involvement of Sp3 is needed for the full repression of MOR gene in addition to NRSF.\nAn interesting feature of the Sp3 protein family is that, in addition to a full-length isoform, Sp3 has two short isoforms which are products of differential translational initiation. It has been suggested that the short Sp3 isoforms may be responsible for the transcriptional repression because they do not have any of the transactivation domains located at the N-terminus of Sp3 factor (34). However, our current and previous data strongly suggest that the regulatory action of Sp3 factors is promoter- and cellular context dependent at least in the MOR gene expression because (i) only the full-length Sp3 can interact with NRSF in co-immunoprecipitation experiment whereas all three isoforms of Sp3 are expressed in NS20Y cells (Figure 4C), (ii) two short isoforms of Sp3 act as repressors by binding to the promoter element located at −219 to −189 of MOR gene (4), (iii) Sp3 acts as a weak positive element by binding to Sp1 sites at the proximal region (−450 to −249) of MOR promoter (25).\nTo our knowledge, this is the first report showing that the sequence-specific transcription factor (NRSF) has a differential interaction among Sp3 isoforms to regulate gene expression. Because two short isoforms are generated by internal start codon, it can be speculated that the N-terminal region (112 amino acid residues) of Sp3, which is absent from the two short isoforms, takes responsibility for the interaction of the full-length Sp3 with NRSF and for determining the role of Sp3 factors for MOR gene expression. In addition, differential post-translational modification states among Sp3 factors, such as sumoylation, phosphorylation and acetylation, may have an important role in determining the fate of Sp3 factors in the regulation of gene expression (35,36).\nAlthough the exact mechanism of interdependency (or cooperativity) between NRSF and Sp3 for binding to NRSE/GC box remains to be elucidated, it is tempting to speculate that the function of Sp3 could be determined by the interaction with NRSF. In other words, if NRSF is abundant in the nucleus, Sp3 interacts with NRSF mainly acting as a repressor while, when NRSF is depleted in the nucleus or inactive, Sp3 might bind to the positive elements and act as a part of Sp1/Sp3 activator complex.\nMany researchers eagerly awaited the results of NRSF loss-of-function experiments in the hope that they would shed light on the role of NRSF in the regulation of neural induction. Previous studies clearly indicated that knock-out of NRSF function is not sufficient to induce either precocious neurogenesis in neural precursors or transformation of non-neural cells into neurons (26). Our data may suggest that NRSF is not a master regulator of neural induction and that its function is orchestrated by a symphony of transcription factors for the control of target genes. A recent report identified that huntingtin protein is mutated in Huntington's disease (37). Wild-type huntingtin acts as a positive transcriptional regulator for many NRSE-containing genes involved in the maintenance of the neuronal phenotype (38). Consistently, loss of expression of NRSE-controlled neuronal genes is shown in cells, mice and human brain with Huntington's disease. In Huntington's disease, it has been proposed that mutant huntingtin led to accumulate the NRSF in nucleus, resulting in the strong repression of the BDNF which is controlled by NRSE (39). It is probable that the silencing of NRSF target genes in Huntinton's disease could also be the result of additive or synergic repression through increasing interaction with Sp3 or other transcriptional factors resulting from abnormal presence of NRSF in neuronal cells.\nEmerging evidence indicates that the genetic context is extremely important in determining the function of NRSE/NRSF-dependent repression mechanism. In this study, we demonstrated that Sp3 transcription factor represses the MOR gene expression by binding to a GC box adjacent to NRSE and by interacting with NRSF providing evidence that there is another level of complexity in regulating the MOR transcription. Detailed elucidation of such synergic repression mechanism will provide an insight understanding about the complexity of the transcriptional regulation of MOR gene as their expression varies in density in different regions of the central nervous system."}

{"project":"2_test","denotations":[{"id":"17130167-15322094-76660571","span":{"begin":448,"end":450},"obj":"15322094"},{"id":"17130167-7871435-76660572","span":{"begin":451,"end":453},"obj":"7871435"},{"id":"17130167-9771705-76660573","span":{"begin":454,"end":456},"obj":"9771705"},{"id":"17130167-12417311-76660573","span":{"begin":454,"end":456},"obj":"12417311"},{"id":"17130167-9454838-76660573","span":{"begin":454,"end":456},"obj":"9454838"},{"id":"17130167-15322094-76660574","span":{"begin":643,"end":645},"obj":"15322094"},{"id":"17130167-15528196-76660575","span":{"begin":2375,"end":2377},"obj":"15528196"},{"id":"17130167-9765304-76660576","span":{"begin":2549,"end":2551},"obj":"9765304"},{"id":"17130167-15284899-76660577","span":{"begin":3694,"end":3696},"obj":"15284899"},{"id":"17130167-12091390-76660578","span":{"begin":4022,"end":4024},"obj":"12091390"},{"id":"17130167-12972613-76660579","span":{"begin":4025,"end":4027},"obj":"12972613"},{"id":"17130167-12032298-76660580","span":{"begin":4386,"end":4388},"obj":"12032298"},{"id":"17130167-10570134-76660581","span":{"begin":4389,"end":4391},"obj":"10570134"},{"id":"17130167-15681389-76660582","span":{"begin":4392,"end":4394},"obj":"15681389"},{"id":"17130167-11773047-76660583","span":{"begin":5001,"end":5003},"obj":"11773047"},{"id":"17130167-15703380-76660584","span":{"begin":5478,"end":5479},"obj":"15703380"},{"id":"17130167-9765304-76660585","span":{"begin":5603,"end":5605},"obj":"9765304"},{"id":"17130167-15494207-76660586","span":{"begin":6371,"end":6373},"obj":"15494207"},{"id":"17130167-16781829-76660587","span":{"begin":6374,"end":6376},"obj":"16781829"},{"id":"17130167-9771705-76660588","span":{"begin":7252,"end":7254},"obj":"9771705"},{"id":"17130167-8458085-76660589","span":{"begin":7531,"end":7533},"obj":"8458085"},{"id":"17130167-8790425-76660590","span":{"begin":7688,"end":7690},"obj":"8790425"},{"id":"17130167-12881722-76660591","span":{"begin":8011,"end":8013},"obj":"12881722"}],"text":"DISCUSSION\nCombinatorial interactions between cis-elements and trans-acting factors are required for the regulation of gene expression. One of the best-characterized transcription factors for controlling neuronal gene expression is the repressor element 1 (RE-1)–silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) repressor protein that is involved in suppressing several neuronal gene expressions in non-neuronal cells (11,12,26–28). We have previously demonstrated that the NRSE of MOR functions as a critical regulator to repress MOR gene expression in specific neuronal cells depending on NRSF expression level (11). In this study, we explored the role of the putative Sp transcription factor binding element (G/C box) in MOR promoter region located 3′ end of NRSE and demonstrated that Sp3 transcription factor represses the expression of MOR gene by binding to this element and by interacting with NRSF.\nFrom the sequence data analysis, we initially found the conserved Sp family binding site adjacent to NRSE in mouse, rat and human MOR gene that could regulate MOR gene expression. When treated with mithramycin A, a G/C box specific binding inhibitor, the mRNA level of MOR gene was increased in NRSF containing NS20Y cells but not in NRSF negative PC12 cells (Figure 1). The result suggested that this GC box element may mainly act as a repressor in MOR gene regulation and explained why the MOR transcription level is very low in NS20Y cell line.\nTo explore the repression mechanism by this GC box in MOR gene regulation, several MOR-luciferase constructs with mutations in either NRSE or the G/C box or both were made and tested for their promoter activities. Mutations in both NRSE and Sp-binding site showed 14-fold increase in the activity whereas either NRSE or G/C box mutation had 4- to 5-fold increased activity compared with that of wild-type sequences in NS20Y cells (Figure 2). Therefore, the negative effect of the putative Sp-binding element for the MOR gene expression could be considered as additive to or preferably synergic with NRSE. A recent study reported that the transcription levels of the several NRSF target genes containing GC-rich regions were decreased with mithramycin A in a dose-dependent manner in both βTC3 and PC12 cells indicating that Sp1 acts as an activator for the expression of NRSF target genes (29). Two positive Sp family binding elements in MOR promoter were previously identified which is located at ∼300 bp upstream from the GC box identified in the current study (25). In those upstream elements, Sp1 acts as a major activator by binding to both elements whereas Sp3 has minor activator effect.\nThe identity of the transcriptional repressor that binds to the newly identified GC box was first hinted by co-immunoprecipitation of the Sp3 factor with NRSF in both HeLa and NS20Y nuclear extracts (Figure 4). Then, the specific and functional binding of Sp3 factor to the GC box was confirmed by gel-shift assays using either in vitro translated NRSF and Sp3 factor (Figure 5) or nuclear extract (Figure 6), and in vivo ChIP assay (Figure 7). The results clearly demonstrated that Sp3 factor binds the G/C motif and interacts with NRSF in MOR promoter region, suggesting that transcription factor Sp3 is required for silencing MOR expression together with NRSF.\nWithin the Sp family transcription factors, Sp1 and Sp3 are ubiquitously expressed in mammalian cells. Sp3 is structurally similar to Sp1, with similar affinities for Sp1-binding site. Although Sp1 and Sp3 share \u003e90% sequence homology in the DNA-binding domain and they bind to the same cognate DNA element, they have strikingly different functions (30). Although Sp1 mainly acts as transcriptional activator, Sp3 can repress activity of the promoter by recruiting HDAC1 or HDAC2. HDAC1 and HDAC2 have been shown to potently repress luteinizing hormone receptor gene transcription. The mSin3A, a component of the Sin3–HDAC complex, potentiates the inhibition mediated by HDAC1 (31,32). It has been well established that the Zn2+-finger-containing protein REST/NRSF binds the 21 bp NRSE DNA element found in many neuronal genes including MOR and then nucleates the formation of a multiprotein complex that represses gene transcription by deacetylating histones, methylating both DNA and histone H3, and dephosphorylation of RNA polymerase II (15,18,33). In conjunction with our current data, especially from the co-immunoprecipitation of Sp3 with NRSF, it can be postulated that the involvement of Sp3 is needed for the full repression of MOR gene in addition to NRSF.\nAn interesting feature of the Sp3 protein family is that, in addition to a full-length isoform, Sp3 has two short isoforms which are products of differential translational initiation. It has been suggested that the short Sp3 isoforms may be responsible for the transcriptional repression because they do not have any of the transactivation domains located at the N-terminus of Sp3 factor (34). However, our current and previous data strongly suggest that the regulatory action of Sp3 factors is promoter- and cellular context dependent at least in the MOR gene expression because (i) only the full-length Sp3 can interact with NRSF in co-immunoprecipitation experiment whereas all three isoforms of Sp3 are expressed in NS20Y cells (Figure 4C), (ii) two short isoforms of Sp3 act as repressors by binding to the promoter element located at −219 to −189 of MOR gene (4), (iii) Sp3 acts as a weak positive element by binding to Sp1 sites at the proximal region (−450 to −249) of MOR promoter (25).\nTo our knowledge, this is the first report showing that the sequence-specific transcription factor (NRSF) has a differential interaction among Sp3 isoforms to regulate gene expression. Because two short isoforms are generated by internal start codon, it can be speculated that the N-terminal region (112 amino acid residues) of Sp3, which is absent from the two short isoforms, takes responsibility for the interaction of the full-length Sp3 with NRSF and for determining the role of Sp3 factors for MOR gene expression. In addition, differential post-translational modification states among Sp3 factors, such as sumoylation, phosphorylation and acetylation, may have an important role in determining the fate of Sp3 factors in the regulation of gene expression (35,36).\nAlthough the exact mechanism of interdependency (or cooperativity) between NRSF and Sp3 for binding to NRSE/GC box remains to be elucidated, it is tempting to speculate that the function of Sp3 could be determined by the interaction with NRSF. In other words, if NRSF is abundant in the nucleus, Sp3 interacts with NRSF mainly acting as a repressor while, when NRSF is depleted in the nucleus or inactive, Sp3 might bind to the positive elements and act as a part of Sp1/Sp3 activator complex.\nMany researchers eagerly awaited the results of NRSF loss-of-function experiments in the hope that they would shed light on the role of NRSF in the regulation of neural induction. Previous studies clearly indicated that knock-out of NRSF function is not sufficient to induce either precocious neurogenesis in neural precursors or transformation of non-neural cells into neurons (26). Our data may suggest that NRSF is not a master regulator of neural induction and that its function is orchestrated by a symphony of transcription factors for the control of target genes. A recent report identified that huntingtin protein is mutated in Huntington's disease (37). Wild-type huntingtin acts as a positive transcriptional regulator for many NRSE-containing genes involved in the maintenance of the neuronal phenotype (38). Consistently, loss of expression of NRSE-controlled neuronal genes is shown in cells, mice and human brain with Huntington's disease. In Huntington's disease, it has been proposed that mutant huntingtin led to accumulate the NRSF in nucleus, resulting in the strong repression of the BDNF which is controlled by NRSE (39). It is probable that the silencing of NRSF target genes in Huntinton's disease could also be the result of additive or synergic repression through increasing interaction with Sp3 or other transcriptional factors resulting from abnormal presence of NRSF in neuronal cells.\nEmerging evidence indicates that the genetic context is extremely important in determining the function of NRSE/NRSF-dependent repression mechanism. In this study, we demonstrated that Sp3 transcription factor represses the MOR gene expression by binding to a GC box adjacent to NRSE and by interacting with NRSF providing evidence that there is another level of complexity in regulating the MOR transcription. Detailed elucidation of such synergic repression mechanism will provide an insight understanding about the complexity of the transcriptional regulation of MOR gene as their expression varies in density in different regions of the central nervous system."}