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• W2085450161 abstract "Neuregulin 1 (NRG1) is essential for the development and function of multiple organ systems, and its dysregulation has been linked to diseases such as cancer and schizophrenia. Recently, altered expression of a novel isoform (type IV) in the brain has been associated with schizophrenia-related genetic variants, especially rs6994992 (SNP8NRG243177). Here we have isolated and characterized full-length NRG1 type IV cDNAs from the adult and fetal human brain and identified novel splice variants of NRG1. Full-length type IV spans 1.8 kb and encodes a putative protein of 590 amino acids with a predicted molecular mass of ∼66 kDa. The transcript consists of 11 exons with an Ig-like domain, an epidermal growth factor-like (EGF) domain, a β-stalk, a transmembrane domain, and a cytoplasmic “a-tail,” placing it in the β1a NRG1 subclass. NRG1 type IV was not detected in any tissues except brain and a putative type IV NRG1 protein of 66 kDa was similarly brain-specific. Type IV transcripts are more abundantly expressed in the fetal brain, where, in addition to the full-length structure, two novel type IV variants were identified. In vitro luciferase-reporter assays demonstrate that the 5′ promoter region upstream of type IV is functional, with differential activity associated with genetic variation at rs6994992, and that promoter competition may impact on type IV expression. Our data suggest that type IV is a unique brain-specific NRG1 that is differentially expressed and processed during early development, is translated, and its expression regulated by a schizophrenia risk-associated functional promoter or single nucleotide polymorphism (SNP). Neuregulin 1 (NRG1) is essential for the development and function of multiple organ systems, and its dysregulation has been linked to diseases such as cancer and schizophrenia. Recently, altered expression of a novel isoform (type IV) in the brain has been associated with schizophrenia-related genetic variants, especially rs6994992 (SNP8NRG243177). Here we have isolated and characterized full-length NRG1 type IV cDNAs from the adult and fetal human brain and identified novel splice variants of NRG1. Full-length type IV spans 1.8 kb and encodes a putative protein of 590 amino acids with a predicted molecular mass of ∼66 kDa. The transcript consists of 11 exons with an Ig-like domain, an epidermal growth factor-like (EGF) domain, a β-stalk, a transmembrane domain, and a cytoplasmic “a-tail,” placing it in the β1a NRG1 subclass. NRG1 type IV was not detected in any tissues except brain and a putative type IV NRG1 protein of 66 kDa was similarly brain-specific. Type IV transcripts are more abundantly expressed in the fetal brain, where, in addition to the full-length structure, two novel type IV variants were identified. In vitro luciferase-reporter assays demonstrate that the 5′ promoter region upstream of type IV is functional, with differential activity associated with genetic variation at rs6994992, and that promoter competition may impact on type IV expression. Our data suggest that type IV is a unique brain-specific NRG1 that is differentially expressed and processed during early development, is translated, and its expression regulated by a schizophrenia risk-associated functional promoter or single nucleotide polymorphism (SNP). Neuregulin 1 (NRG1) 2The abbreviations used are: NRG1, neuregulin 1; EGF, epidermal growth factor; SNP, single nucleotide polymorphism; TSS, transcription start site; RT-PCR, reverse transcription-PCR; qRT-PCR, quantitative real-time RT-PCR; PBGD, porphobilinogen deaminase; bis-Tris, 2-[bis(2-hydroxyethyl)-amino]-2-(hydroxymethyl)propane-1,3-diol; ORF, open reading frame; CTF, C-terminal fragment; CT, cycle at threshold; HEK293, human embryonic kidney cell line 293. 2The abbreviations used are: NRG1, neuregulin 1; EGF, epidermal growth factor; SNP, single nucleotide polymorphism; TSS, transcription start site; RT-PCR, reverse transcription-PCR; qRT-PCR, quantitative real-time RT-PCR; PBGD, porphobilinogen deaminase; bis-Tris, 2-[bis(2-hydroxyethyl)-amino]-2-(hydroxymethyl)propane-1,3-diol; ORF, open reading frame; CTF, C-terminal fragment; CT, cycle at threshold; HEK293, human embryonic kidney cell line 293. is a signaling protein that mediates cell-cell interactions and plays critical roles in the growth of the nervous system, heart, breast, and other organ systems. The gene is located on 8p12.21, and ∼15 “classic” NRG1 isoforms are generated through alternative promoter usage and splicing (1Buonanno A. Fischbach G.D. Curr. Opin. Neurobiol. 2001; 11: 287-296Crossref PubMed Scopus (431) Google Scholar, 2Falls D.L. J. Neurocytol. 2003; 32: 619-647Crossref PubMed Scopus (89) Google Scholar). In terms of their structural organization, all NRG1 isoforms have an epidermal growth factor-like domain (EGF), which is necessary and sufficient for the biological activities of NRG1 (3Holmes W.E. Sliwkowski M.X. Akita R.W. Henzel W.J. Lee J. Park J.W. Yansura D. Abadi N. Raab H. Lewis G.D. Shepherd H.M. Kuang W.J. Wood W.I. Goeddal D.V. Vanolen R.L. Science. 1992; 256: 1205-1210Crossref PubMed Scopus (926) Google Scholar, 4Wen D. Suggs S.V. Karunagaran D. Liu N. Cupples R.L. Luo Y. Janssen A.M. Ben Baruch N. Trollinger D.B. Jacobsen V.L. Meng S.Y. Lu H.S. Hu S. Chang D. Yang W. Yanigahara D. Koshi R.A. Yarden Y. Mol. Cell. Biol. 1994; 14: 1909-1919Crossref PubMed Scopus (233) Google Scholar). Upstream of the EGF domain, NRG1s contain either an immunoglobulin-like (Ig) domain or a cysteine-rich domain (CRD). The Ig-like domain is thought to mediate binding to the extracellular matrix and potentiate the NRG1 signal (5Loeb J.A. Fischbach G.D. J. Cell Biol. 1995; 130: 127-135Crossref PubMed Scopus (114) Google Scholar, 6Rimer M. J. Neurocytol. 2003; 32: 665-675Crossref PubMed Scopus (25) Google Scholar). Downstream of the EGF domain, C termini contain a transmembrane (TM) domain and a common cytoplasmic “a”-tail and a rare “b”-tail, or they are synthesized as soluble peptides (2Falls D.L. J. Neurocytol. 2003; 32: 619-647Crossref PubMed Scopus (89) Google Scholar, 4Wen D. Suggs S.V. Karunagaran D. Liu N. Cupples R.L. Luo Y. Janssen A.M. Ben Baruch N. Trollinger D.B. Jacobsen V.L. Meng S.Y. Lu H.S. Hu S. Chang D. Yang W. Yanigahara D. Koshi R.A. Yarden Y. Mol. Cell. Biol. 1994; 14: 1909-1919Crossref PubMed Scopus (233) Google Scholar). Recently, additional 5′ exons have been identified in the NRG1 gene, giving rise putatively to novel families of NRG1, types IV, V, and VI (7Steinthorsdottir V. Stefansson H. Ghosh S. Birgisdottir B. Bjornsdottir S. Fasquel A.C. Olafsson O. Stefansson K. Gulcher J.R. Gene. 2004; 342: 97-105Crossref PubMed Scopus (129) Google Scholar). Genetic variation in NRG1 has been linked to risk for schizophrenia (for review see Ref. 8Harrison P.J. Law A.J. Biol. Psychiatry. 2006; 60: 132-140Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar). The association with schizophrenia was first reported in an Icelandic population in which a NRG1 haplotype (Hapice) consisting of five single nucleotide polymorphisms (SNPs) and two microsatellites covering the 5′-end of the gene doubled the risk for the disorder (9Stefansson H. Sigurdsson E. Steinthorsdottir V. Bjornsdottir S. Sigmundsson T. Ghosh S. Brynjolfsson J. Gunnarsdottir S. Ivarsson O. Chou T.T. Hjaltason O. Birgisdottir B. Jonsson H. Gudnadottir V.G. Gudmundsdottir E. Bjornsson A. Ingvarsson B. Ingason A. Sigfusson S. Hardardottir H. Harvey R.P. Lai D. Zhou M. Brunner D. Mutel V. Gonzalo A. Lemke G. Sainz J. Johannesson G. Andresson T. Gudbjartsson D. Manolescu A. Frigge M.L. Gurney M.E. Kong A. Gulcher J.R. Petursson H. Stefansson K. Am. J. Hum. Genet. 2002; 71: 877-892Abstract Full Text Full Text PDF PubMed Scopus (1428) Google Scholar). Four of these SNPs (SNP8NRG221132, SNP8NRG221533, SNP8NRG241930, and SNP8NRG243177 (rs6994992)) represent a 22-kb surrogate haplotype that resides in the 5′ flanking putative promoter region of NRG1 directly upstream of the novel E187 exon, which is unique to the NRG1 type IV isoform (7Steinthorsdottir V. Stefansson H. Ghosh S. Birgisdottir B. Bjornsdottir S. Fasquel A.C. Olafsson O. Stefansson K. Gulcher J.R. Gene. 2004; 342: 97-105Crossref PubMed Scopus (129) Google Scholar, 10Law A.J. Lipska B.K. Weickert C.S. Hyde T.M. Straub R.E. Hashimoto R. Harrison P.J. Kleinman J.E. Weinberger D.R. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 6747-6752Crossref PubMed Scopus (360) Google Scholar). Follow-up studies in multiple ethnic populations and a meta-analysis have confirmed genetic association between schizophrenia and NRG1 using markers within the same core haplotype (8Harrison P.J. Law A.J. Biol. Psychiatry. 2006; 60: 132-140Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar, 11Li D. Collier D.A. He L. Hum. Mol. Genet. 2006; 15: 1995-2002Crossref PubMed Scopus (263) Google Scholar) or with overlapping markers in the 5′ region, making NRG1 a leading schizophrenia susceptibility gene. Recent studies have focused on possible molecular mechanisms of NRG1-mediated susceptibility for schizophrenia by studying the regulation of NRG1 expression in the human brain. Hashimoto et al. (12Hashimoto R. Straub R.E. Weickert C.S. Hyde T.M. Kleinman J.E. Weinberger D.R. Mol. Psychiatry. 2004; 9: 299-307Crossref PubMed Scopus (238) Google Scholar) examined type I-III NRG1 transcripts in the dorsolateral prefrontal cortex and found increased NRG1 type I mRNA in schizophrenia, a finding that was subsequently confirmed in a separate and larger sample in the hippocampus (10Law A.J. Lipska B.K. Weickert C.S. Hyde T.M. Straub R.E. Hashimoto R. Harrison P.J. Kleinman J.E. Weinberger D.R. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 6747-6752Crossref PubMed Scopus (360) Google Scholar). However, evidence for a relationship between genetic risk in the gene and increased expression of type I mRNA in the brain was inconclusive. Subsequently, Law et al. (10Law A.J. Lipska B.K. Weickert C.S. Hyde T.M. Straub R.E. Hashimoto R. Harrison P.J. Kleinman J.E. Weinberger D.R. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 6747-6752Crossref PubMed Scopus (360) Google Scholar) identified that genetic variation in the Hapice risk region; particularly a single SNP, rs6994992, is associated with NRG1 type IV mRNA levels in the normal human brain and in individuals with schizophrenia, with individuals carrying the risk allele having higher NRG1 type IV levels. This finding suggests a novel molecular mechanism for the genetic association of the Hapice region of NRG1 with schizophrenia, implicating specifically the type IV isoform. Moreover, based on the location of rs6994992 (in the proximal promoter of NRG1, 1.2 kb upstream of the transcription start site (TSS)) and on the in silico prediction that the SNP maps to a serum response element, Law et al. (10Law A.J. Lipska B.K. Weickert C.S. Hyde T.M. Straub R.E. Hashimoto R. Harrison P.J. Kleinman J.E. Weinberger D.R. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 6747-6752Crossref PubMed Scopus (360) Google Scholar) proposed that rs6994992 represents a functional promoter site involved in NRG1 type IV-specific transcriptional control. Subsequently, other studies have demonstrated that Hapice, and especially rs6994992, is linked to aspects of altered brain function (13Hall J. Whalley H.C. Job D.E. Baig B.J. McIntosh A.M. Evans K.L. Thomson P.A. Porteous D.J. Cunningham-Owens D.G. Johnstone E.C. Lawrie S.M. Nat. Neurosci. 2006; 9: 1477-1478Crossref PubMed Scopus (213) Google Scholar, 14Stefanis N.C. Trikalinos T.A. Avramopoulos D. Smyrnis N. Evdokimidis I. Ntzani E.E. Ioannidis J.P. Stefanis C.N. Biol. Psychiatry. 2007; (, in press)Google Scholar) providing convergent evidence for an important biological role for genetic regulation of the NRG1 gene, in particular the type IV isoform, in normal brain function and the development of schizophrenia. Unfortunately, nothing is known about the biology of NRG1 type IV or even the structure of its full-length transcripts. Given the discovery of the potential relevance of NRG1 type IV to genetic risk for schizophrenia, gaining knowledge of its full-length structure, its cell-type specific and transcriptional regulation, and whether it is translated into a protein is a critical step toward understanding its role in human brain development and disease. In the present study, we have isolated and comprehensively characterized full-length cDNA clones of NRG1 type IV from the fetal and adult human brain and identified novel splice variants of the NRG1 gene belonging to the type IV family. We have also performed expression profiling of a number of human tissues and show that NRG1 type IV undergoes cell-type specific regulation (unlike NRG1 types I-III), in the context of it being exclusively expressed in the brain at both the mRNA and protein level. NRG1 type IV is also more abundant in the fetal brain, where novel type IV splice variants were identified, indicating potentially unique functions of this gene during human brain development. In addition, we have isolated and characterized ∼1.5 kb of the type IV promoter and demonstrate allelic-specific differences related to rs6994992 in promoter activity and promoter competition with a neighboring NRG1 promoter. Site-directed mutagenesis of rs6994992 in the context of the 1.5-kb promoter confirmed differential allele-specific promoter activity. Overall, these data demonstrate that the schizophrenia risk-associated SNP, rs6994992, is a functional promoter variant associated with schizophrenia genetic predisposition and NRG1 type IV expression. Molecular Cloning and Characterization of NRG1 Type IV cDNA Clones: RT-PCR and cDNA Cloning and Sequencing—To isolate full-length cDNA clones encoding type IV splice isoforms of the human NRG1 gene, RT-PCR and primer-specific amplifications were performed using human brain and human fetal brain cDNAs. Marathon-Ready cDNA libraries were purchased from Clontech (Mountain View, CA). Adult human brain cDNA libraries were generated using total RNA from the hippocampus and prefrontal cortex (Clontech, BD Biosciences, and Ambion (Austin, TX), respectively). For construction of human cDNA libraries, 5 μg of total RNA was reverse-transcribed to cDNA in a total volume of 20 μl by using SuperScript™III (Invitrogen) primed with oligo(dT)20 according to manufacturer’s instructions. After reverse transcription, the cDNA product was digested with 2 units of RNase H (Invitrogen) at 37 °C for 30 min. Two microliters of the first-strand cDNA was employed in PCR amplification using Platinum TaqDNA Polymerase High Fidelity (Invitrogen). RT-PCR primers for full-length NRG1 type IV transcript amplification were designed specific to the unique 5′ exon E187 of NRG1 type IV (E187_s3, 5′-GGCAGCAGCATGGGGAAAGGA-3′) overlapping the putative translation start site and reverse primers specific for the conserved termination exon of the NRG1 gene, the a-tail (E846;Atail_anti2, 5′-AGGTTTTATACAGCAATAGGGTCTT-3′). To identify potential transcripts containing a cytoplasmic b-tail (E778), we used a reverse primer located in the a-b-tail junction (a/bjunc anti 1, 5′-TAGCAGGGAGGCTGTTACTGTCAT-3′). Reverse primers for full-length cloning of type IV were designed specific to exon E846 based on previous data, suggesting that NRG1 type IV represents a class of Ig-containing NRG1 variants (7Steinthorsdottir V. Stefansson H. Ghosh S. Birgisdottir B. Bjornsdottir S. Fasquel A.C. Olafsson O. Stefansson K. Gulcher J.R. Gene. 2004; 342: 97-105Crossref PubMed Scopus (129) Google Scholar). In the vast majority of Ig-NRG1 variants (types I and II) E846 represents the conserved termination exon. To search for the potential existence of rare b-cytoplasmic tail variants (2Falls D.L. J. Neurocytol. 2003; 32: 619-647Crossref PubMed Scopus (89) Google Scholar), we used a reverse primer in the a-b-tail junction that would delineate a b-tail from the common a-tail. Long range PCR was performed as follows: 94 °C for 5 min followed by 35 cycles of 94 °C for 1 min, 55 °C for 2 min, and 68 °C for 6 min. After the last cycle, extension was conducted at 68 °C for 10 min. PCR products were resolved on 1% agarose gels in 0.5× Tris borate-EDTA buffer. Gels were stained with ethidium bromide, and the DNA bands were visualized with a Kodak EDAS 290 imaging setup that consists of an orange band pass filter, a Kodak DC290 Camera, and a 302 nm UVA transilluminator. A fragment of ∼1.8 kb was excised from the gel for each individual sample and purified using the QIAquick gel extraction kit (Qiagen, Valencia, CA). The purified fragment was cloned into either pCR®-XL-TOPO or pCR®2.1-TOPO vector (Invitrogen) and subsequently transformed into TOP10F-competent cells (Invitrogen). Five-to-ten-well isolated colonies were picked up and grown at 37 °C, 225 rpm overnight. The plasmid miniprep DNAs were prepared from 1.5 ml of bacterial cultures by using QIAprep®Spin miniprep kit (Qiagen). The insert in selected clones was bidirectionally sequenced by the BigDye terminator kit (Applied Biosystems, Foster City, CA) with M13 primers (M13forward, 5′-GTAAAACGACGGCCAG-3′; M13reverse, 5′-CAGGAAACAGCTATGAC-3′). For deeper sequencing of the inserts and confirmation of clone sequences generated with M13 primers, additional purified plasmid DNAs were resequenced using a “primer walking” strategy with a set of NRG1 sequencing primers designed to the conserved exons within the NRG1 gene (supplemental data, Table 1). The combination of these primers covered the full-length coding region of NRG1 type IV. Sequence data were constructed using the Sequencher software (Gene Codes Corp., Ann Arbor, MI), and the exon structure of each clone was determined by aligning the cDNA sequence to the genomic DNA sequence of the human NRG1 gene (Gen-Bank™ BK000383). Nucleotide Sequence Accession—The cDNA sequences of the NRG1 type IV splice variants have been submitted to the Gen-Bank™ data base under accession numbers EF372273-EF372277 and EF517295-EF517297. The NRG1 genomic DNA sequence used in comparison with the NRG1 type IV cDNA sequences has the GenBank™ accession number BK000383. Quantitative Real-time RT-PCR (qRT-PCR) Analysis of NRG1 Type IV Expression in Human Tissues—To investigate whether NRG1 type IV is expressed in a panel of selected human tissues, qRT-PCR assays were performed using cDNAs constructed from poly(A)+ mRNA or total RNA from human heart, skeletal muscle, breast tumor, liver, lung, testis, adult hippocampus, fetal brain (Clontech BD Biosciences), human B lymphoblast, and human monocyte cell lines. The human cell lines were derived from normal volunteers participating in studies at the National Institutes of Health. NRG1 type IV mRNA expression levels were measured by qRT-PCR using an ABI Prism 7900 sequence detection system with 384-well format (Applied Biosystems) as described previously (10Law A.J. Lipska B.K. Weickert C.S. Hyde T.M. Straub R.E. Hashimoto R. Harrison P.J. Kleinman J.E. Weinberger D.R. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 6747-6752Crossref PubMed Scopus (360) Google Scholar). The forward primer for qRT-PCR amplification overlaps with that used for RT-PCR amplification of NRG1 type IV in the cloning experiments. Co-amplification and normalization of NRG1 type IV mRNA levels were to the internal control gene, porphobilinogen deaminase (PBGD). Fold changes in expression were calculated as 2-ΔΔ CT (cycle at threshold) relative to adult human hippocampus. For comparison, NRG1 types I, II, and III were measured in the same panel of cDNAs as described previously (10Law A.J. Lipska B.K. Weickert C.S. Hyde T.M. Straub R.E. Hashimoto R. Harrison P.J. Kleinman J.E. Weinberger D.R. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 6747-6752Crossref PubMed Scopus (360) Google Scholar). Western Blot Analysis—Western blot analysis was performed using an antibody to the a-tail cytoplasmic domain of NRG1 (neuregulin-1a/b1/2 (C-20) (sc-348, Santa Cruz Biotechnology). 20 μg of protein from human dorsolateral prefrontal cortex, hippocampus, fetal brain, human heart, and skeletal muscle (Clontech, Protein Medleys) and lymphocytes and monocytes was denatured in 4× NuPAGE LDS sample buffer (Invitrogen) and 0.75 μl of 1.0 m dithiothreitol at 95 °C for 5 min. Samples were separated by gel electrophoresis using NuPAGE 4-12% bis-Tris gels (Invitrogen) and transferred to nitrocellulose membranes. The sc-348 antibody dilution was 1:200. Following overnight incubation at 4 °C, membranes were washed three times with TBS-T (Tris-buffered saline with Tween 0.1%) and incubated for 1 h with 1:2000 goat anti-rabbit IgG-horseradish peroxidase (sc-2004, Santa Cruz Biotechnology). Membranes were washed five times (for 5 min each) with TBS-T. Horseradish peroxidase-immunoreactive protein bands were detected by enhanced chemiluminescence (ECL) Western blotting analysis system (RPN2109, Amersham Biosciences) and exposed to Kodak scientific imaging film. Prestained Kaleidoscope standards were used for product size determination (Bio-Rad, 161-0324) Blots were stripped using Restore Western blot stripping buffer (21062, Pierce Biotechnology) and reprobed with 1:5000 anti-β-actin antibody (A5441, Sigma). Construction of Promoter-Luciferase Reporter Plasmids, DNA Sequencing, Cell Culture, Transfection, and Measurement of NRG1 Promoter Activity—To investigate whether a mechanism of the genetic association of the 5′ region of NRG1 with schizophrenia involves transcriptional regulation, we used a combined approach of bioinformatic modeling and informed promoter-luciferase fusion experiments. To test whether the 5′ flanking region of the NRG1 gene upstream of E187 represents a functional promoter and rs6994992 represents a cis-acting regulatory element, a 1.5-kb fragment of the human NRG1 gene 5′ to the type IV transcription initiation site was amplified by PCR and used in the construction of luciferase fusion plasmids. PCR primers were designed based on human GenBank™ sequence BK000383 (forward primer F-1500, 5′-GCAGAGCCATCAATGAGGTCA-3′; reverse primer R2, 5′-CTGGGAGTGAGAGGTGACGCTTCA-3′). To examine the effects of genetic variation specifically at rs6994992, promoter fragments were amplified from separate control individuals based on genotype at this SNP. Each construct was compared in single experiments. The recombinants pGL4.SNPC and pGL4.SNPT represent the ancestral and derived alleles, respectively, at rs6994992. Furthermore, to confirm that rs6994992 per se is the functional SNP responsible for differential promoter activity, we used the 1.5-kb pGL4.SNPT construct as a template for site-directed mutagenesis of rs6994992. Single mutants were generated with the QuikChange® II site-directed mutagenesis kit (Strategene) with mutagenic primers to convert T to C at rs6994992 (supplemental Table 1). All mutagenic reactions were performed according to manufacturer’s instructions, and all constructs were verified by direct sequencing. Because gene transcription can be regulated by promoter competition (15Choi O.R. Engel J.D. Cell. 1988; 55: 17-26Abstract Full Text PDF PubMed Scopus (220) Google Scholar, 16Conte C. Dastugue B. Vaury C. EMBO. J. 2002; 21: 3908-3916Crossref PubMed Scopus (26) Google Scholar), and the NRG1 type IV promoter may be comparatively inefficient (based on low expression levels in the brain (10Law A.J. Lipska B.K. Weickert C.S. Hyde T.M. Straub R.E. Hashimoto R. Harrison P.J. Kleinman J.E. Weinberger D.R. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 6747-6752Crossref PubMed Scopus (360) Google Scholar)) compared with the neighboring promoter for the type II isoform, we sought insight into neighboring NRG1 promoter localization and activity through the use of a Markov model-based statistical tool for promoter detection (17Ohler U. Nucleic Acids Res. 2006; 34: 5943-5950Crossref PubMed Scopus (81) Google Scholar). Based on bioinformatic predictions we also amplified promoter constructs containing DNA sequence upstream of E187 and into the type II exon TSS including: 1) 1.5 kb upstream of the type IV TSS combined with the downstream tandem promoter for the type II isoform (forward primer F-1500, 5′-GCAGAGCCATCAATGAGGTCA-3′; reverse primer -215B, 5′-CTTCTGGCTGTCGGTTCGGACT-3′); 2) 2 kb upstream of the type IV TSS combined with the downstream tandem promoter for the type II isoform (forward primer F-2000, 5′-TTGGAGAGATGGGAGTAAAACT-3′; reverse primer -215B, 5′-CTTCTGGCTGTCGGTTCGGACT-3′); and 3) 3 kb upstream of the type IV TSS combined with the downstream tandem promoter for the type II isoform (forward primer F-3000, 5′-GGAAGATTCAAGATGGAGGGGA-3′; reverse primer -215B, 5′-CTTCTGGCTGTCGGTTCGGACT-3′). Type IV/II promoter fusion constructs were generated from the SNP-T individual above and were used for comparison with the NRG1 type IV-only promoter constructs to determine the relative strengths of these regions to drive gene expression in vitro. All promoter PCR products were cloned into pCRII-TOPO vectors (Invitrogen) and then subcloned into the polylinker region of pGL4.10 (Promega) using standard molecular techniques. The promoter region sequences obtained by PCR were verified by direct sequence analysis using M13 forward and reverse primers. Orientations of all promoter sequence constructs were confirmed by restriction endonuclease digestion and DNA sequencing. Sequencing results were analyzed with BLAST. For transfection, a human embryonic kidney cell line (HEK293) was isolated and cultured in Dulbecco’s modified Eagle’s medium/F-12 (Invitrogen) plus 10% fetal bovine serum and 1% penicillin-streptomycin in a humidified 5% CO2 incubator at 37 °C. 3-5 × 105 cells/well were seeded into 6- or 12-well plates, respectively, for transfection. NRG1 promoter-luciferase fusion plasmids (4 μg, 6-well, and 1.6 μg, 12-well; and 400 ng, 6-well, and 160 ng, 12-well) of PRL-TK plasmid (Renilla luciferase expression vector, an internal control for transfection efficiency) were co-transfected with Lipofectamine 2000 (Invitrogen). Plasmid pGL4-SV40 was used as a positive control promoter vector. Negative controls comprised transfection with empty pGL4.10 and no vector-transfected cell controls. All experiments were performed in triplicate and then repeated. 48 h post-transfection, cells were lysed. Luciferase and Renilla activity was measured in 20-μl reactions using the Dual-Luciferase® reporter assay system kit (Promega) on a Lumat LB9507 luminometer. Levels of luciferase activity are expressed as relative light units. The ratio of firefly to Renilla relative light units was determined, and data are normalized to the pGL4.10 empty vector. To address the potential tissue and species specificity of NRG1 type IV promoter activity, we also transfected under similar conditions the following additional cell lines: Neuro 2A, SH-SY5Y, and SK-N-SH-neuroblastoma cell lines. Rat primary cortical neurons were cultured and transfected as described previously (18Sala C. Piech V. Wilson N.R. Passafaro M. Liu G. Sheng M. Neuron. 2001; 31: 115-130Abstract Full Text Full Text PDF PubMed Scopus (579) Google Scholar). qRT-PCR analysis of NRG1 type IV expression in these additional cell lines also was examined. Cloning, Sequencing, and Characterization of Human Full-length NRG1 Type IV cDNAs—Human NRG1 type IV full-length cDNAs were cloned from adult and fetal human brain cDNA libraries. Sequencing of cDNA clones derived from the adult human brain (hippocampus and prefrontal cortex) revealed that they contained an ∼1800-bp insert and an open reading frame (ORF) of 1770 bp encoding a putative NRG1 type IV proprotein of 590 amino acids with a calculated molecular mass of ∼66 kDa. This variant corresponds to full-length NRG1 of the novel type IV family, and we termed this variant “type IV-β1a” (Fig. 1). We report that E187 has an AUG codon in the context of a Kozak sequence (CAGCATGG), potentially encoding 13 unique amino acids upstream of E178 (Fig. 1). Detailed characterization of the sequenced products aligned to the assembled genomic sequence (BK000383) revealed the presence of 11 exons (Fig. 1). Each of the 11 exons identified have been reported previously as part of the NRG1 gene and the 5′ exon structure upstream of the EGFc domain (E130) confirms identification of partial NRG1 type IV transcripts utilizing 5′-RACE (rapid amplification of cDNA ends) in the human brain (7Steinthorsdottir V. Stefansson H. Ghosh S. Birgisdottir B. Bjornsdottir S. Fasquel A.C. Olafsson O. Stefansson K. Gulcher J.R. Gene. 2004; 342: 97-105Crossref PubMed Scopus (129) Google Scholar). Characterization of the full-length NRG1 type IV transcript revealed that the 3′-exons downstream of the EGF-like domain (E130c) comprise E103, E127, E131, E207, and E846 with a termination codon, placing the transcript in the β1a′ family of NRG1 proteins (Fig. 1). These findings categorize the adult full-length type IV as a NRG1 proprotein belonging to the Ig class of EGF β-1 containing transmembrane variants with a cytoplasmic a-tail. In the fetal brain, the main full-length β1a variant of NRG1 type IV is identical to that found in the adult human hippocampus and prefrontal cortex (Fig. 1, Fetal A, IV-β1a). In addition two other full-length variants were identified with variability in the “spacer region” (s1/E51a and s2/E51b) downstream of the Ig-like domains (E178/122) with either the inclusion of exons E51a and E51b (Fig. 1, Fetal B) or just E51a alone (Fetal C). The ORF was maintained in both transcripts. These observations suggest that these variants represent full-length spliced versions of the adult and fetal NRG1 type IV-β1a. In addition, a transcript was identified (Fig. 1, Fetal D, IV-β1a) in the fetal brain that is identical to Fetal C but harbors a nonsense mutation (CAG/TAG) in the TMc domain (E103), creating a premature stop codon. This transcript is predicted to produce a putative truncated NRG1 type IV" @default.
• W2085450161 created "2016-06-24" @default.
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• W2085450161 title "Molecular Cloning of a Brain-specific, Developmentally Regulated Neuregulin 1 (NRG1) Isoform and Identification of a Functional Promoter Variant Associated with Schizophrenia" @default.
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