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• W1506351306 abstract "Neuron-restrictive silencer element (NRSE) has been identified in multiple neuron-specific genes. This element has been shown to mediate repression of neuronal gene transcription in nonneuronal cells. A palindromic NRSE (NRSEBDNF) is present in the proximal region of brain-derived neurotrophic factor (BDNF) promoter II. Using in vitro binding assays, we establish that the upper half-site is largely responsible for the NRSEBDNF activity. To delineate the in vivorole of NRSE in the regulation of rat BDNF gene, promoter constructs with intact and mutated NRSEBDNF were introduced into transgenic mice. Our data show that NRSEBDNF is controlling the activity of BDNF promoters I and II in the brain, thymus, and lung,i.e. in the tissues in which the intact reporter gene and endogenous BDNF mRNAs are expressed. Mutation of NRSEBDNF did not lead to the ectopic activation of the reporter gene in any other nonneural tissues. In the brain, NRSEBDNF is involved in the repression of basal and kainic acid-induced expression from BDNF promoters I and II in neurons. However, NRSEBDNF does not control the activity of the BDNF gene in nonneuronal cells of brain. Neuron-restrictive silencer element (NRSE) has been identified in multiple neuron-specific genes. This element has been shown to mediate repression of neuronal gene transcription in nonneuronal cells. A palindromic NRSE (NRSEBDNF) is present in the proximal region of brain-derived neurotrophic factor (BDNF) promoter II. Using in vitro binding assays, we establish that the upper half-site is largely responsible for the NRSEBDNF activity. To delineate the in vivorole of NRSE in the regulation of rat BDNF gene, promoter constructs with intact and mutated NRSEBDNF were introduced into transgenic mice. Our data show that NRSEBDNF is controlling the activity of BDNF promoters I and II in the brain, thymus, and lung,i.e. in the tissues in which the intact reporter gene and endogenous BDNF mRNAs are expressed. Mutation of NRSEBDNF did not lead to the ectopic activation of the reporter gene in any other nonneural tissues. In the brain, NRSEBDNF is involved in the repression of basal and kainic acid-induced expression from BDNF promoters I and II in neurons. However, NRSEBDNF does not control the activity of the BDNF gene in nonneuronal cells of brain. Neurotrophins (NTs) 1The abbreviations used are: NT, neurotrophin; BDNF, brain-derived neurotrophic factor; bp, base pair(s); kb, kilobase pair(s); NRSF, neuron-restrictive silencer factor; EMSA, electrophoretic mobility shift assay; nACh, nicotinic acetylcholine receptor; NaCh, sodium channel; mut, mutant; CAT, chloramphenicol acetyltransferase; RPA, RNase protection assay; KA, kainic acid; RE, repressor element; REST, RE-1 silencing transcription factor. 1The abbreviations used are: NT, neurotrophin; BDNF, brain-derived neurotrophic factor; bp, base pair(s); kb, kilobase pair(s); NRSF, neuron-restrictive silencer factor; EMSA, electrophoretic mobility shift assay; nACh, nicotinic acetylcholine receptor; NaCh, sodium channel; mut, mutant; CAT, chloramphenicol acetyltransferase; RPA, RNase protection assay; KA, kainic acid; RE, repressor element; REST, RE-1 silencing transcription factor. are secreted polypeptides that regulate the survival of selective populations of developing neurons and maintenance of the characteristic functions of mature neurons (1Bothwell M. Annu. Rev. Neurosci. 1995; 18: 223-253Crossref PubMed Scopus (764) Google Scholar, 2Levi-Montalcini R. Science. 1987; 237: 1154-1162Crossref PubMed Scopus (2687) Google Scholar, 3Lewin G.R. Barde Y.-A. Annu. Rev. Neurosci. 1996; 19: 289-317Crossref PubMed Scopus (1779) Google Scholar). The family of NTs includes nerve growth factor, brain-derived neurotrophic factor (BDNF), NT-3, and NT-4/5. Each factor is differentially involved in neurogenesis as well as in neuronal adaptive responses, implying well controlled regulatory mechanisms underlying their expression. Multipromoter structure, common to NT genes (4Bishop J.F. Mueller G.P. Mouradian M.M. Mol. Brain Res. 1994; 26: 225-232Crossref PubMed Scopus (63) Google Scholar, 5Leingärtner A. Lindholm D. Eur. J. Neurosci. 1994; 6: 1149-1159Crossref PubMed Scopus (51) Google Scholar, 6Nakayama M. Gahara Y. Kitamura T. Ohara O. Mol. Brain Res. 1994; 21: 206-218Crossref PubMed Scopus (77) Google Scholar, 7Salin T. Timmusk T. Lendahl U. Metsis M. Mol. Cell Neurosci. 1997; 9: 264-275Crossref PubMed Scopus (19) Google Scholar, 8Timmusk T. Palm K. Metsis M. Reintam T. Paalme V. Saarma M. Persson H. Neuron. 1993; 10: 475-489Abstract Full Text PDF PubMed Scopus (725) Google Scholar), has apparently evolved to confer complex regulation of their expression. We have previously shown that four promoters direct tissue-specific expression of the rat BDNF gene (8Timmusk T. Palm K. Metsis M. Reintam T. Paalme V. Saarma M. Persson H. Neuron. 1993; 10: 475-489Abstract Full Text PDF PubMed Scopus (725) Google Scholar). Promoter IV is active in nonneural tissues (lung, heart, and muscle), whereas promoters I, II, and III are predominantly used in the brain and regulated by glutamatergic and GABAergic neurotransmitter systems (8Timmusk T. Palm K. Metsis M. Reintam T. Paalme V. Saarma M. Persson H. Neuron. 1993; 10: 475-489Abstract Full Text PDF PubMed Scopus (725) Google Scholar, 9Metsis M. Timmusk T. Arenas E. Persson H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8802-8806Crossref PubMed Scopus (204) Google Scholar). Recent studies have shown that changes in BDNF expression are linked to synaptic plasticity during development (10Cellerino A. Maffei L. Prog. Neurobiol. 1996; 49: 53-71Crossref PubMed Google Scholar, 11Katz L.C. Shatz C.J. Science. 1996; 274: 1133-1138Crossref PubMed Scopus (2373) Google Scholar) and also to the process of memory and learning (12Berninger B. Poo M.M. Curr. Opin. Neuorbiol. 1996; 6: 324-330Crossref PubMed Scopus (160) Google Scholar, 13Bonhoeffer T. Curr. Opin. Neurobiol. 1996; 6: 119-126Crossref PubMed Scopus (266) Google Scholar, 14Lo D.C. Neuron. 1995; 15: 979-981Abstract Full Text PDF PubMed Scopus (244) Google Scholar, 15Thoenen H. Science. 1995; 270: 593-598Crossref PubMed Scopus (1715) Google Scholar). Mice heterozygous for BDNF gene null-mutation have severe impairment in hippocampal long term potentiation (16Korte M. Carroll P. Wolf E. Brem G. Thoenen H. Bonhoeffer T. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8856-8860Crossref PubMed Scopus (1201) Google Scholar, 18Patterson S.L. Abel T. Deuel T.A.S. Martin K.S. Rose J.C. Kandel E.R. Neuron. 1996; 16: 1137-1145Abstract Full Text Full Text PDF PubMed Scopus (1033) Google Scholar) and in normal spatial learning (19Linnarson S. Björklund A. Ernfors P. Eur. J. Neurosci. 1998; 9: 2581-2587Crossref Scopus (403) Google Scholar). An inverted repeat within the first intron of rat BDNF gene shows substantial similarity to the neuron-restrictive silencer element (NRSE). NRSE, also known as repressor element-1 (RE1), has been defined as a negative-acting DNA regulatory element to prevent the expression of neuronal genes in nonneuronal cell types or in inappropriate neuronal subtypes (20Kraner S.D. Chong J.A. Tsai H.J. Mandel G. Neuron. 1992; 9: 37-44Abstract Full Text PDF PubMed Scopus (283) Google Scholar, 21Mori N. Schoenherr C. Vandenbergh D.J. Anderson D.J. Neuron. 1992; 9: 45-54Abstract Full Text PDF PubMed Scopus (346) Google Scholar, 22Schoenherr C.J. Anderson D.J. Science. 1995; 267: 1360-1363Crossref PubMed Scopus (926) Google Scholar). The first evidence that NRSE is able to mediate repression of transcription came from the analysis of the promoters of rat SCG10 (21Mori N. Schoenherr C. Vandenbergh D.J. Anderson D.J. Neuron. 1992; 9: 45-54Abstract Full Text PDF PubMed Scopus (346) Google Scholar) and rat type II sodium channel (NaChII) (20Kraner S.D. Chong J.A. Tsai H.J. Mandel G. Neuron. 1992; 9: 37-44Abstract Full Text PDF PubMed Scopus (283) Google Scholar) genes. To date, several studies have proposed that NRSE-like sequences, present in the regulatory regions of multiple neuronal genes, are important for their neuron-specific expression (23Bai G. Norton D.D. Prenger M.S. Kusiak J.W. J. Biol. Chem. 1998; 273: 1086-1091Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 24Gan L. Perney T.M. Kaczmarek L.K. J. Biol. Chem. 1996; 271: 5859-5865Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar, 25Kallunki P. Jenkisnon S. Edelman G.M. Jones F.S. J. Biol. Chem. 1995; 270: 21291-21298Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 26Lee S. Williamson J. Lothman E.W. Szele F.G. Chesselet M.F. Von Hagen S. Sapolsky R.M. Mattson M.P. Christakos S. Mol. Brain Res. 1997; 47: 183-194Crossref PubMed Scopus (41) Google Scholar, 27Lönnerberg P. Schoenherr C.J. Anderson D.A. Ibanez C.F. J. Biol. Chem. 1996; 271: 33358-33365Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 28Schoch S. Cibelli G. Thiel G. J. Biol. Chem. 1996; 271: 3317-3323Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 29Schoenherr C.J. Paquette A.J. Anderson D.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9881-9886Crossref PubMed Scopus (360) Google Scholar). The identification of a NRSE-binding protein revealed that the neuron-restrictive silencer factor (NRSF) (22Schoenherr C.J. Anderson D.J. Science. 1995; 267: 1360-1363Crossref PubMed Scopus (926) Google Scholar), also known as RE-1 silencing transcription factor (REST) (30Chong J.A. Tapia R.J. Kim S. Toledo A.J. Zheng Y. Boutros M.C. Altshuller Y.M. Frohman M.A. Kraner S.D. Mandel G. Cell. 1995; 80: 949-957Abstract Full Text PDF PubMed Scopus (927) Google Scholar), is a novel member of the Krüppel family of zinc-finger transcription factors. The same factor, called as X2-box repressor, was identified to bind to the X2-box of the immune system-specific major histocompatibility complex class II gene, DPA, and repress its activity in the terminally differentiated B-cell lineage (31Scholl T. Stevens M.B. Mahanta S. Strominger J.L. J. Immunol. 1996; 156: 1448-1457PubMed Google Scholar). REST/NRSF/XBR mRNA is expressed at high levels in most nonneural tissues throughout development (22Schoenherr C.J. Anderson D.J. Science. 1995; 267: 1360-1363Crossref PubMed Scopus (926) Google Scholar,30Chong J.A. Tapia R.J. Kim S. Toledo A.J. Zheng Y. Boutros M.C. Altshuller Y.M. Frohman M.A. Kraner S.D. Mandel G. Cell. 1995; 80: 949-957Abstract Full Text PDF PubMed Scopus (927) Google Scholar, 31Scholl T. Stevens M.B. Mahanta S. Strominger J.L. J. Immunol. 1996; 156: 1448-1457PubMed Google Scholar). In the nervous system, REST/NRSF/XBR mRNA levels are high in the undifferentiated neuronal progenitors (22Schoenherr C.J. Anderson D.J. Science. 1995; 267: 1360-1363Crossref PubMed Scopus (926) Google Scholar, 30Chong J.A. Tapia R.J. Kim S. Toledo A.J. Zheng Y. Boutros M.C. Altshuller Y.M. Frohman M.A. Kraner S.D. Mandel G. Cell. 1995; 80: 949-957Abstract Full Text PDF PubMed Scopus (927) Google Scholar) and decrease during development. Our previous data revealed that REST/NSRF/XBR mRNA expression proceeds at varying levels also in the neurons of adult brain (32Palm K. Belluardo N. Metsis M. Timmusk T. J. Neurosci. 1998; 15: 1280-1296Crossref Google Scholar). The present study explores molecular mechanisms underlying BDNF gene expression, focusing on the function of the palindromic NRSE (NRSEBDNF) in the proximal region of promoter II. We have previously shown that BDNF promoter-reporter gene construct, covering promoter I, promoter II, and flanking regions, recapitulates endogenous BDNF expression in transgenic mice (33Timmusk T. Lendahl U. Funakoshi H. Arenas E. Persson H. Metsis M. J. Cell Biol. 1995; 128: 185-199Crossref PubMed Scopus (107) Google Scholar). Currently, we have generated transgenic mice carrying the BDNF promoter construct with mutations in the NRSEBDNF sequence, and our results strongly suggest that the transcriptional regulation of BDNF gene in vivo is under the control of NRSEBDNF. RNA isolation, chloramphenicol acetyltransferase (CAT) assay, RNase protection assay (RPA), andin situ hybridization were performed as described (8Timmusk T. Palm K. Metsis M. Reintam T. Paalme V. Saarma M. Persson H. Neuron. 1993; 10: 475-489Abstract Full Text PDF PubMed Scopus (725) Google Scholar, 33Timmusk T. Lendahl U. Funakoshi H. Arenas E. Persson H. Metsis M. J. Cell Biol. 1995; 128: 185-199Crossref PubMed Scopus (107) Google Scholar,34Belluardo N. Wu G. Mudo G. Hansson A.C. Petterson R. Fuxe K. J. Comp. Neurol. 1997; 379: 226-246Crossref PubMed Scopus (127) Google Scholar). A 0.3-kb XbaI/HindIII fragment encompassing BDNF exon II and its flanking regions was cloned into pBSSK vector (Stratagene), and a single-stranded uracil-rich template was prepared using Escherichia coli strain CJ 236. Site-directed mutagenesis was performed using the Kunkel method (35Kunkel T.A. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 488-492Crossref PubMed Scopus (4900) Google Scholar). Mutation of NRSE in the 0.3-kb XbaI/HindIII fragment was carried out in three steps using the following synthetic oligonucleotides. Mutated nucleotides are shown in lowercase letters. NRSEmut1, 5′-CGAGCAGAGTCCATTCAGCAgaTTttACAGAGCCAGCGGATTTGT-3′; NRSEmut1+2 , 5′-ACAGAGCCAGCGGATTTGTttGAcaTGGTAGTACTTCATCCAG-3′; NRSEmut5′-GGGCGAGCAGAGTCCgggacGCAgaTTtACAGAGCCAGCGGATTTGT-3′. Finally, the 0.3-kb XbaI/HindIII fragment carrying the mutated NRSE (NRSEmut) sequence was used to generate BDNFI+IImutCAT construct applying the same cloning strategy as earlier for constructing BDNFI+IICAT (33Timmusk T. Lendahl U. Funakoshi H. Arenas E. Persson H. Metsis M. J. Cell Biol. 1995; 128: 185-199Crossref PubMed Scopus (107) Google Scholar). Transgenic mice were generated and analyzed for transgene integration as described previously (33Timmusk T. Lendahl U. Funakoshi H. Arenas E. Persson H. Metsis M. J. Cell Biol. 1995; 128: 185-199Crossref PubMed Scopus (107) Google Scholar). Transgenic founders were analyzed using dot blot analysis with a CAT-specific probe (33Timmusk T. Lendahl U. Funakoshi H. Arenas E. Persson H. Metsis M. J. Cell Biol. 1995; 128: 185-199Crossref PubMed Scopus (107) Google Scholar).Transgene copy number was estimated using BDNF exon I-specific probe (8Timmusk T. Palm K. Metsis M. Reintam T. Paalme V. Saarma M. Persson H. Neuron. 1993; 10: 475-489Abstract Full Text PDF PubMed Scopus (725) Google Scholar) by comparing the intensity of BDNF exon I-specific signal in the wild-type animals to that in the transgenic animals. Quantification was performed with PhosphorImager using ImageQuant software (Molecular Dynamics). Adult male and female transgenic animals (body weight, 20–30 g) were used in all experiments. KA (20 mg/kg in saline buffer) was injected intraperitoneally, and the animals were sacrificed at 3, 6, or 24 h after the injections. All animal experiments were approved by the local ethical committee. Cellular extracts of thymus and hippocampus were prepared as described (36Andrews N.C. Faller D.V. Nucleic Acids Res. 1991; 19: 2499Crossref PubMed Scopus (2211) Google Scholar). Mobility shift assays were performed as described (37Chiaramello A. Neuman K. Palm K. Metsis M. Neuman T. Mol. Cell. Biol. 1995; 15: 6036-6044Crossref PubMed Scopus (42) Google Scholar). Oligonucleotides corresponding to the AP1 (Promega, Madison, WI) and cAMP response element (Promega) sequences, and to the NRSE derived of rat type II sodium channel gene (20Kraner S.D. Chong J.A. Tsai H.J. Mandel G. Neuron. 1992; 9: 37-44Abstract Full Text PDF PubMed Scopus (283) Google Scholar) and rat SCG10 gene (21Mori N. Schoenherr C. Vandenbergh D.J. Anderson D.J. Neuron. 1992; 9: 45-54Abstract Full Text PDF PubMed Scopus (346) Google Scholar) were used as positive controls. In competition studies, the amount of added unlabeled oligo-competitor is presented as molar excess. In the supershift assay, c-Fos supershifting antibody was used (sc-447; Santa Cruz Biotechnology, Santa Cruz, CA). The inverted repeat (NRSEBDNF) in the proximal region of BDNF promoter II is composed of two NRSEs. The 5′ element has 89% identity with the NRSE consensus sequence (29Schoenherr C.J. Paquette A.J. Anderson D.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9881-9886Crossref PubMed Scopus (360) Google Scholar), whereas the 3′ element shows only 57% identity, suggesting it to be an atypical NRSE. The results of previous deletion and mutation analyses have established that not all 21 residues of the NRSE consensus are critical for NRSE-mediated negative regulatory effects on transcription (29Schoenherr C.J. Paquette A.J. Anderson D.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9881-9886Crossref PubMed Scopus (360) Google Scholar). Mutations introduced to certain positions of the consensus sequence have been suggested to be important for the silencing activity of NRSE (20Kraner S.D. Chong J.A. Tsai H.J. Mandel G. Neuron. 1992; 9: 37-44Abstract Full Text PDF PubMed Scopus (283) Google Scholar, 21Mori N. Schoenherr C. Vandenbergh D.J. Anderson D.J. Neuron. 1992; 9: 45-54Abstract Full Text PDF PubMed Scopus (346) Google Scholar). In the upper element of NRSEBDNF, the nucleotides in the critical positions are identical with the consensus sequence, and in the lower element, they show 80% identity, suggesting that both elements in NRSEBDNF have the potential to mediate repression. To determine whether this inverted repeat-like element participates in the regulation of BDNF gene, mutations at potentially relevant residues were made (mut-NRSEBDNF) (Fig.1) and analyzed in transgenic mice. Our previous studies established that a 9.5-kb genomic fragment including BDNF promoters I and II (BDNFI+IICAT) confers the appropriate tissue-specific pattern of the reporter gene expression (33Timmusk T. Lendahl U. Funakoshi H. Arenas E. Persson H. Metsis M. J. Cell Biol. 1995; 128: 185-199Crossref PubMed Scopus (107) Google Scholar). In the present study, we replaced the intact NRSEBDNF in the BDNFI+IICAT construct with the mut-NRSEBDNF and introduced the mutant construct (BDNF I+IImutCAT) (Fig. 1) into mice. Altogether, nine founders were obtained, and six of them were bred to obtain F1 transgenic lines for further analyses. In all six lines, transgene activity was the highest in the brain, whereas outside the central nervous system, high levels of CAT activity were observed in thymus and lung (Fig. 2). Analysis of CAT activity in different brain regions revealed slight variations in the pattern of expression from one founder line to another; however, the highest levels of CAT activity were always detected in the hippocampus, midbrain, and thalamus. As the spatial expression pattern of the reporter gene in BDNFI+IImutCAT mice was indistinguishable from the transgene expression pattern in BDNFI+IICAT animals, we concluded that mutation of NRSEBDNF is not sufficient to allow ectopic expression from BDNF promoters I and II in tissues that do not express the intact promoter construct and also endogenous BDNF mRNA. However, quantitative differences in the levels of reporter gene expression were seen in transgenic mice with the mutated promoter construct as compared with the animals carrying the intact promoter construct. In the brain, thymus, and lung, total CAT activity and relative CAT activity per transgene copy number was significantly higher in all six transgenic lines carrying mutated transgene as compared with CAT levels in any of the nine founder lines possessing intact reporter construct (Fig. 2). The levels of CAT activity in the thymus and lung of individual BDNFI+IImutCAT lines were on average about 50-fold higher as compared with the levels of CAT activity in thymus and lung of mice carrying BDNFI+IICAT transgene (Fig. 2). In the brain, all the brain regions showed elevated levels of CAT activity in transgenic animals with BDNFI+IImutCAT as compared with CAT activity levels in mice expressing BDNFI+IICAT (Fig.2). In the hippocampus, which exhibited the highest levels of the reporter gene expression in the brain, CAT activity was on average 15-fold higher in transgenic animals with BDNFI+IImutCAT than in animals with BDNFI+IICAT (Fig. 2). The differences in CAT activity in the hippocampus ranged to more than 100-fold between the individual transgenic founder lines with BDNFI+IImutCAT as compared with the individual lines carrying BDNFI+IICAT. These data indicate that NRSEBDNF is controlling the activity of BDNF promoters I and II in the brain, thymus, and lung. Quantitative RPAs were performed to establish whether NRSEBDNF located 0.7 kb downstream of BDNF exon I and 120 bp upstream of BDNF exon II could affect the activity of BDNF promoter I, promoter II, or both. The analysis revealed that in the hippocampus, CAT mRNAs with BDNF exon I (exon I-CAT) were on average about 15-fold higher, and CAT mRNAs with BDNF exon II (exon II-CAT) about 12-fold higher as compared with the levels in the animals with intact promoter construct (Fig. 3). Upon mutation of NRSEBDNF, the levels of exon I-CAT mRNA in thymus and lung were on average about 40-fold higher, and the levels of exon II-CAT mRNA were on average about 50-fold higher as compared with the levels in mice carrying BDNFI+IICAT transgene (Fig. 3). These data demonstrate that disruption of NRSEBDNF affects the activity of both BDNF promoter I and promoter II and leads to the enhanced expression from both promoters. Earlier studies suggested that NRSE is required to prevent expression of neuronal genes in nonneural tissues and cell populations (38Schoenherr C.J. Anderson D.J. Curr. Opin. Neurobiol. 1995; 5: 566-571Crossref PubMed Scopus (142) Google Scholar). Next we examined whether high levels of CAT mRNA expression and CAT activity detected in the brain of BDNFI+IImutCAT animals resulted from the ectopic activation of the transgene in nonneuronal cells. Using in situhybridization, CAT mRNA expression was seen in only three of nine founder lines with the intact promoter construct. In contrast to this, the expression of CAT mRNA was detected in all six founder lines with mutant promoter construct (Fig. 4). In all transgenic lines with BDNFI+IImutCAT, CAT mRNA expression was the highest in the neurons of the hilar region and CA3 pyramidal layer of hippocampus. As CAT mRNA was expressed in the same neuronal subpopulations in mice carrying the intact or the mutated promoter construct, we concluded that NRSEBDNF is not restricting the transgene expression to specific subsets of neurons (Fig. 4 and data not shown). Mutation of NRSEBDNF did not lead to the ectopic expression of transgene in nonneuronal cell populations of brain. Our previous data revealed that the neurons of hippocampus exhibit altered levels of BDNF expression from promoters I and II following KA-induced seizures. BDNF transcripts with exon I were induced more than 50-fold, whereas transcripts with exon II were increased about 5-fold, at 3 h following KA treatment (9Metsis M. Timmusk T. Arenas E. Persson H. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8802-8806Crossref PubMed Scopus (204) Google Scholar). We have also shown that BDNFI+IICAT recapitulates the KA-induced expression from promoters I and II (33Timmusk T. Lendahl U. Funakoshi H. Arenas E. Persson H. Metsis M. J. Cell Biol. 1995; 128: 185-199Crossref PubMed Scopus (107) Google Scholar). These data suggested that kainate responsivecis-acting elements are present in the 9.5-kb BDNF genomic region of BDNFI+IICAT. To determine whether NRSEBDNF may be involved in the KA-induced regulation of BDNF gene, we studied the expression levels of CAT mRNA in the hippocampus of transgenic mice with the intact or mutated promoter constructs at different time points following KA administration. Using in situ hybridization, a marked increase in transgene expression was detected in the pyramidal neurons of CA3 and in the neurons of hilar region in three out of nine founder lines with BDNFI+IICAT and in all six analyzed lines with BDNFI+IImutCAT at 3 h following KA treatment (Figs. 4and 5). In the dentate gyrus, no alterations in transgene expression levels were detected in any of BDNFI+IICAT animals; however, significantly increased levels of CAT mRNA were seen in scattered neurons in three of six founder lines with BDNFI+IImutCAT, at 3 h after KA administration (Fig. 5). Quantitative RPA analysis revealed that in the hippocampus of mice with the intact promoter construct transgene expression levels increased on average 4-fold, whereas in the mutant animals the levels increased on average 15-fold following 3 and 6 h of KA treatment (Fig. 6 and data not shown). As established by in situ hybridization, these higher induction levels resulted from the increased CAT mRNA expression per neuron and also from the increased number of neurons expressing CAT mRNA. At 24 h after drug injection, CAT mRNA levels had returned back to control levels in the hippocampus of both, BDNFI+IICAT and BDNFI+IImutCAT mice, as measured by RPA (data not shown). These data suggest that in the neurons of hippocampus, the induction level of BDNF mRNA transcribed from promoters I and II following KA treatment is under the control of NRSEBDNF.Figure 6NRSEBDNF mediates repression of the KA-induced transgene expression in the neurons of hippocampus.Expression of CAT mRNA and endogenous BDNF mRNA in the founder lines carrying BDNFI+IICAT (Q4 and P4) and BDNFI+IImutCAT (m1 and m26). The increases of CAT mRNA at 6 h following KA treatment were normalized relative to the increases of endogenous BDNF mRNA and to the level of GAPDH mRNA. c., total RNA derived from the hippocampus of untreated transgenic animals; +KA, total RNA derived from the hippocampus of transgenic mice at 6 h following KA treatment; tRNA, yeast tRNA.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Finally, we examined whether both elements of NRSEBDNF are required for its functional activity within and outside the nervous system. EMSA analysis revealed several unique NRSEBDNF-protein complexes that exhibited tissue-specific pattern of migration (Fig.7). Competition experiments showed that the thymus-specific complex could be disrupted with a 50-fold excess of unlabeled oligonucleotides corresponding to the upper element (NRSEBDNF11) (Fig. 7, left). The lower element (NRSEBDNF21) appeared to be a less effective competitor. The same amount of unlabeled NRSEBDNF21 oligos was not sufficient to disrupt this complex (Fig. 7, left) and a 250-fold excess of unlabeled NRSEBDNF21 was needed to abolish the thymus-specific complex (data not shown). These data reveal that thymus-specific factors discriminate between the two elements and suggest that the upper element of NRSEBDNF is critical for its activity in thymus. Previous studies have shown that the NRSE-binding transcription factor REST/NRSF/XBR is predominately expressed in nonneural tissues (22Schoenherr C.J. Anderson D.J. Science. 1995; 267: 1360-1363Crossref PubMed Scopus (926) Google Scholar, 30Chong J.A. Tapia R.J. Kim S. Toledo A.J. Zheng Y. Boutros M.C. Altshuller Y.M. Frohman M.A. Kraner S.D. Mandel G. Cell. 1995; 80: 949-957Abstract Full Text PDF PubMed Scopus (927) Google Scholar, 31Scholl T. Stevens M.B. Mahanta S. Strominger J.L. J. Immunol. 1996; 156: 1448-1457PubMed Google Scholar, 32Palm K. Belluardo N. Metsis M. Timmusk T. J. Neurosci. 1998; 15: 1280-1296Crossref Google Scholar). Next, we examined whether REST/NRSF/XBR-like activity is present in the large complex detected from thymus extracts. A 50-fold excess of unlabeled oligonucleotides containing the NRSE sequences of NaChII (NRSENaChII) (Fig.7, left) and SCG10 genes (data not shown) resulted in the disappearance of the thymus-specific complex formed on NRSEBDNF, suggesting that these NRSEs compete for the same factor as does the upper element of NRSEBDNF. Several other studies have shown that NRSEBDNF11, NRSENaChII, and NRSESCG10 bind REST/NRSF/XBR protein and form a complex with slow migration in EMSA analysis (22Schoenherr C.J. Anderson D.J. Science. 1995; 267: 1360-1363Crossref PubMed Scopus (926) Google Scholar, 29Schoenherr C.J. Paquette A.J. Anderson D.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9881-9886Crossref PubMed Scopus (360) Google Scholar, 30Chong J.A. Tapia R.J. Kim S. Toledo A.J. Zheng Y. Boutros M.C. Altshuller Y.M. Frohman M.A. Kraner S.D. Mandel G. Cell. 1995; 80: 949-957Abstract Full Text PDF PubMed Scopus (927) Google Scholar, 32Palm K. Belluardo N. Metsis M. Timmusk T. J. Neurosci. 1998; 15: 1280-1296Crossref Google Scholar). Accordingly, we suggest that the NRSEBDNF-specific complex detected from thymus extracts contains REST/NRSF/XBR. The mutant NRSEBDNF(mut-NRSEBDNF) oligonucleotide, as expected, did not form any specific DNA-protein complexes with thymus extracts because excess of any of the specific unlabeled competitors failed to affect the weak unspecific complexes detected (Fig. 7, left). Using cell extracts of hippocampus, EMSA analysis detected two NRSEBDNF-specific complexes. Complex 1 is fast migrating, whereas complex 2 migrates similarly to the complex from the thymus extracts (Fig. 7). Competition studies of complex 1 revealed that this complex could be disrupted with the addition of 50-fold excess of unlabeled oligos containing the 3′ element of NRSEBDNF and not with the similar excess of unlabeled NRSEBDNF11, NRSENaChII, and NRSESCG10 oligos. A 250-fold excess of unlabeled NRSEBDNF11 oligos was needed to disrupt this complex (Fig. 7, middle, and data not shown). Complex 1 was also detected using protein extracts from other regions of brain (cerebral cortex, striatum, olfactory bulb, thalamus, and brainstem) (data not shown). These data suggest that complex 1 is a brain-specific complex that" @default.
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• W1506351306 title "Brain-derived Neurotrophic Factor Expression in VivoIs under the Control of Neuron-restrictive Silencer Element" @default.
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