HUMAN GABAB RECEPTOR 1 PROMOTERS

Abstract: The present invention relates to nucleic acid molecules constituting GABAB receptor 1 promoters P1a and/or P1b, and to methods for screening for compounds which are modulators of GABAB receptor 1 transcription, said methods comprising the use of nucleic acid molecules constituting GABAB receptor P1a and/or P1b promoters.

Description: HUMAN GABAB RECEPTOR 1 PROMOTERS FIELD OF THE INVENTION The present invention relates to nucleic acid molecules constituting GABA B receptor 1 promoters Pla and/or Plb, and to methods for screening for compounds which are modulators of GABA B receptor 1 transcription, said methods comprising the use of nucleic acid molecules constituting GABA B receptor Pl and/or Plb promoters. BACKGROUND GABA β receptor 1 GABA (γ-aminobutyric acid) is an endogenous neurotransmitter in the central and peripheral nervous systems. Receptors for GABA have traditionally been divided into GABA A and GABA B receptor subtypes. GABA B receptors (for a review see Kerr, D.I.B. and Ong, J. (1995) Pharmac. Ther. vol. 67, pp.187-246) belong to the superfamily of G- protein coupled receptors. GABA B receptor agonists are described as being of use in the treatment of CNS disorders, such as muscle relaxation in spinal spasticity, cardiovascular disorders, asthma, gut motility disorders such as irritable bowel syndrome and as prokinetic and anti-tussive agents. GABA receptor agonists have also been disclosed as useful in the treatment of emesis (WO 96/1 1680) and reflux disease (WO 98/1 1885). The cloning of the cDNA encoding the rat GABA B receptors splice isoforms GABA B Rla and GABA B Rlb is disclosed by Kaupmann et al. ( 1997) Nature, vol. 386, 239-246. The mature rat GABA B Rlb differed from GABA B Rla in that the N-terminal 147 residues were replaced by 18 different residues. It was presumed that the rat GABA B Rla and -b receptor variants are derived from the same gene by alternative splicing. The cloning of the cDNA encoding the human GABA B receptor GABA B Rlb is disclosed in WO 97/46675. The cloning of the human GABA B receptor 1 gene and the elucidation of the exon-mtron organization is in part or fully disclosed in PCT/SE98/01947. in EMBL HS271 M21 (GenBank AL031983) EMBL AJ010170 to AJ010191 , in Peters. HC et al . Neurogenetics 2. 47-54 ( 1998) and in Goei VL et al Biological Psychiatry 44. 659-66 (1998) The ■ > human GABA B receptor 1 gene consists of 23 exons, spanning over a distance of 30 kb (Figure 1) The elucidation of the gene organization revealed that the human GABA B Rl and GABAβRlb are splice variants encoded by a single gene The GABA B Rla and GABAβRlb isoforms are differentially expressed, at least in the rat (Kaupmann et al (1997) Nature, vol 386, 239-246) The physiological consequences of multiple GABA B o receptor 1 splice isoforms has not yet been determined, but their existence constitute an opportunity for the development of specific pharmaceutical agents GABA β receptor 2 Based on its homology with the mammalian GABA B R1 cDNA, a second member of the 5 GABA B receptor family was identified (Jones, KA et al , Nature 396, 674-679 ( 1998). White, JA et al , Nature 396, 679-682 (1998), Kaupmann, K et al , Nature 396, 683-687 (1998), WO 99/20751) The corresponding protein, GABA B R2, forms heteromers with GABAβRla and Rib, resulting in cell surface expression of a functional GABA B receptor (Kuner, R et al Science 283, 74-77 ( 1999)) At least in recombinant expression systems, 0 GABAβRl and R2 coexpression is necessary for the formation of a functional GABA B receptor Jones et al (Nature 396, 674-679 ( 1998)) disclosed that a GAB A B R1 GABA B R2 stochiometry of 1 1 is an optimal ratio for successful cell surface expression of a ligand binding and functional GABA B receptor Thus, modulating GABA B R1 expression could alter the stochiometry between GABAβR l nd other interacting proteins and be a means to 5 regulate signaling through GABA B receptors and thereby interfere with various physiological processes Transcrφtional regulation Gene regulation is mediated by specific DNA elements in the promoter that directs binding o of transcription factors, which thereby mediate transcription of the gene Eukaryotic transcription factors can be divided in two main groups /) basal transcription factors that interact w ith promoter sequences proximal to the start of transcription, thereby initiating transcription upon recruitment of RNA polymerase II and //) transcription factors that bind to specific distal promoter elements, thereby modulating the transcription upon contact with the basal transcription machinery The DNA sequence that directs the start of transcription in most eukaryotic genes is the TATA-box which is often located approximately 30 base pairs upstream from the start of transcription However, the TATA- box is not a prerequisite for initiation of transcription as there are many promoters, including the GABA B Rl promoters described in this study, that lack a TATA-box A fundamental physiological process in the eukaryotic organism is that cells can communicate with their environment and respond to extracellular stimuli through signaling molecules, such as hormones and growth factors The final event for such signaling is the binding of transcription factors to specific distal promoter elements leading to for example up-regulated or tissue specific gene expression Because of their regulatory role, signaling molecules are putative targets for screening of therapeutic agents The presence of two distinct and differentially regulated promoters within the human GABA B receptor 1 gene, disclosed in this patent application, makes it possible to screen for therapeutic agents selectively regulating expression of GABA B receptor la and lb-type splice isoforms Indications Compounds which are modulators of GABA B receptor 1 transcription are potentially useful in the treatment of disorders which are related to neurally-controlled physiological responses regulated by GABA B receptors, e g CNS disorders such as muscle relaxation in spinal spasticity, Alzheimer's disease and other dementias, psychiatric and neurological disorders such as depression, anxiety and epilepsy, cardiovascular disorders, asthma, gut motility disorders such as irritable bowel syndrome, emesis and reflux disease In some humans, the lower esophageal sphincter (LES) is prone to relaxing more frequently than in other humans As a consequence, fluid trom the stomach can pass into the esophagus since the mechanical barrier is temporarily lost at such times, an event hereinafter referred to as "reflux" Gastro-esophageal reflux disease (GERD) is the most prevalent upper gastrointestinal tract disease. Current therapy has aimed at reducing gastric acid secretion, or by reducing esophageal acid exposure by enhancing esophageal clearance, lower esophageal sphincter tone and gastric emptying. The major mechanism behind reflux has been considered to depend on a hypotonic lower esophageal sphincter. However, recent research (e.g. Holloway & Dent ( 1990) Gastroenterol. Clin. N. Amer. 19, 517-535) has shown that most reflux episodes occur during transient lower esophageal sphincter relaxations (TLESR), i.e. relaxations not triggered by swallows. It has also been shown that gastric acid secretion usually is normal in patients with GERD. Consequently, there is a need for compounds which reduce the incidence of TLESR and thereby prevent reflux. DESCRIPTION OF THE INVENTION This invention relates to nucleic acid molecules constituting GABA B receptor 1 promoters and fragments of said promoters. By GABA B receptor 1 promoters is understood the nucleic acids sequences upstream of the ATG translation initiation codon of GABA B receptor la of the GABA B receptor 1 gene, designated Pla, and the nucleic acids sequences upstream of the ATG translation initiation codon of GABA B recpetor lb of the GABA B receptor 1 gene, designated Plb, as illustrated in Figure 1. In the present context the term "promoter" is meant to include core promoter sequences proximal to the start of transcription and upstream promoter elements which bind constitutively active transcription factors, as well as distal promoter elements which bind specific transcription factors. Accordingly, the present invention provides a nucleic acid molecule constituting a human GABA B receptor 1 promoter Pla, or a functionally equivalent modified form thereof, or active fragments thereof. The present invention also provides a nucleic acid molecule constituting a human GABA B receptor 1 promoter Plb, or a functionally equivalent modified form thereof or active fragments thereof Bv a functionally equiv alent modified form is understood nucleic acids modified from the original sequence that can bind transcription factors By active Iragments of the promoters is understood nucleic acid fragments that can bind transcription factors In preferred forms of the inv ention the said nucleic acid molecule is selected from (a) a nucleic acid molecule comprising a nucleotide sequence set forth as SEQ ID NO 1 , (b) a nucleic acid molecule comprising a nucleotide sequence capable of hybridizing, under stringent conditions to a nucleotide sequence complementary to the DNA molecule as defined in (a) In other preferred forms of the invention the said nucleic acid molecule is selected from (a) a nucleic acid molecule comprising a nucleotide sequence set forth as SEQ ID NO 2, (b) a nucleic acid molecule comprising a nucleotide sequence capable of hybridizing, under stringent conditions, to a nucleotide sequence complementary to the DNA molecule as defined in (a) In another preferred form of the invention the said nucleic acid molecule may be a nucleic acid molecule constituting a human GABA B receptor 1 promoter Pla, or a functionally equivalent modified form thereof, or active fragments thereof, in combination with a nucleic acid molecule constituting a human GABA B receptor 1 promoter Plb, or a functionally equivalent modified form thereof, or active fragments thereof In another preferred form of the invention the said nucleic acid molecule may be a nucleic acid molecule selected from (a) a nucleic acid molecule comprising a nucleotide sequence set forth as SEQ ID NO 1 (b) a nucleic acid molecule comprising a nucleotide sequence capable of hybridizing, under stringent conditions, to a nucleotide sequence complementary to the DNA molecule as defined in (a), in combination with a nucleic acid molecule is selected from (a) a nucleic acid molecule comprising a nucleotide sequence set forth as SEQ ID NO 2 (b) a nucleic acid molecule comprising a nucleotide sequence capable of hybridizing, under stringent conditions, to a nucleotide sequence complementary to the DNA molecule as defined in (a) It should thus be understood that the nucleic acid molecules according to the invention is not to be limited strictly to molecules comprising the sequences set forth as SEQ ID 1 and 2 Rather the invention encompasses nucleic acid molecules carrying modifications like substitutions, small deletions insertions or inversions, which nevertheless have substantially the biochemical activity ot the GABA B receptor promoters la and/or lb according to the invention Included in the invention are consequently nucleic acid molecules, the nucleotide sequence of which is at least 95% homologous, preferably at least 96%, 97%, 98% or 99% homologous, with the nucleotide sequence shown as SEQ ID NO 1 or 2 in the Sequence Listing The term "stringent hybridization conditions" is known in the art from standard protocols (e g Current Protocols in Molecular Biology, editors F Ausubel et al , John Wiley and Sons, Inc 1994) and could be understood as as stringent or more stringent than those defined by e g hybridization to filter-bound DNA in 0 5 M NaHPO 4 . 7% sodium dodecyl sulfate (SDS), 1 mM EDTA at +65°C, and washing in 0 lxSSC / 0 1 % SDS at +68°C It will be appreciated that the nucleic acid sequences shown in the Sequence Listing is only an example within a large but definite group of nucleic acid sequences, which will have the GABA B receptor promoter activity In yet another aspect, the invention provides a vector transformed with a nucleic acid molecule of the present invention The said vector can e g be a rephcable expression vector which carries a nucleic acid molecule according to the invention In the present context the term "rephcable" means that the vector is able to replicate in a given type of host cell into which is has been introduced Examples of vectors are viruses such as bacteπophages. cosmids, plasmids and other recombination vectors Nucleic acid molecules are inserted into vector genomes by methods well known in the art Another embodiment of the present invention is an expression svstem comprising nucleic acid molecules encoding GABA B receptor promoters Pla and/or Plb. or functionally equivalent modified forms, or active fragments thereof In preferred forms of this embodiment of the invention the said nucleic acid molecule is selected from (a) a nucleic acid molecule comprising a nucleotide sequence set forth as SEQ ID NO 1 and/or SEQ ID NO 2 , (b) a nucleic acid molecule comprising a nucleotide sequence capable of hybridizing, under stringent conditions, to a nucleotide sequence complementary to the polypeptide coding region of a DNA molecule as defined in (a) In another preferred form of this embodiment of the invention the said nucleic acid molecule may be a nucleic acid molecule constituting a human GABA B receptor 1 promoter Pla, or a functionally equivalent modified form thereof, or active fragments thereof, in combination with a nucleic acid molecule constituting a human GABA B receptor 1 promoter Plb, or a functionally equivalent modified form thereof, or active fragments thereof In another preferred form of this embodiment of the invention the said nucleic acid molecule may be a nucleic acid molecule selected from (a) a nucleic acid molecule comprising a nucleotide sequence set forth as SEQ ED NO 1 , (b) a nucleic acid molecule comprising a nucleotide sequence capable of hybridizing, under stringent conditions, to a nucleotide sequence complementary to the DNA molecule as defined in (a), in combination with a nucleic acid molecule is selected from (a) a nucleic acid molecule comprising a nucleotide sequence set forth as SEQ ID NO. 2, (b) a nucleic acid molecule comprising a nucleotide sequence capable of hybridizing, under stringent conditions, to a nucleotide sequence complementary to the DNA molecule as defined in (a) The expression system mav. in addition, comprise a reporter gene, the promoter and the reporter gene being positioned so that the expression of the reporter gene is regulated by the GABA B receptor 1 promoters Pl a and/or Plb Suitable expression systems according to the invention are e g. bacterial or yeast plasmids. wide host range plasmids and vectors derived from combinations of plasmid and phage or virus DNA Furthermore, an origin of replication and/or a dominant selection marker can be present in the vector according to the invention Suitable reporter genes that can be used for the construction of expression systems according to the invention are e g the firefly luciferase gene, the bacterial chloramphenicol acetvl transferase (CAT) gene, the β-galactosidase (β-GAL) gene, and the green fluorescent protein (GFP). A further aspect of this embodiment of the invention is a host cell transfected with an expression system comprising nucleic acid molecules constituting GABA B receptor promoters Pla and/or Plb, or functionally equivalent modified forms thereof, or active fragments thereof. Suitable host cells are cells known to express GABA B receptors or cells known to express transcription factors, which can influence the transcription of GABA B receptors. Host cells transfected with DNA encoding specific transcription factors can preferably be used to study the interaction with defined transcription factors and the GABA B receptor promoter. Another embodiment of the invention is a method for the assay of GAB A B receptor promoter activity said method comprising the use of a host cell transfected with an expression system comprising nucleic acid molecules constituting GABA B receptor promoters Pla and/or Plb, or functionally equivalent modified forms thereof, or active fragments thereof A further embodiment of the present invention is a method for the screening ot compounds which are modulators of GABA B receptor 1 transcription, said method comprising the use of nucleic acid molecules constituting GABA B receptor Pla and/or Plb promoters Accordingly, the present invention provides a method for screening compounds which are modulators of GABA B receptor 1 transcription, comprising the steps of (a) transfecting a cell host with a suitable expression system comprising a nucleic acid molecule constituting human GABA B receptor 1 promoter PI A, and/or a human GABA B receptor 1 promoter P1B or functionally equivalent modified forms, or active fragments thereof coupled to a reporter gene, (b) contacting a test compound with the cell, and (c) determining whether the test compound modulates the level of expression of the reporter gene In one aspect of this embodiment of the invention it is provided a method of screening of compounds which are modulators of GABA B receptor 1 transcription, wherein the cell host endogenously expresses GABA B receptor 1. In another aspect of this embodiment of the invention it is provided a method of screening of compounds which are modulators of GABAβ receptor 1 transcription, wherein the said cell host is further transfected with a suitable expression system comprising a nucleic acid molecule encoding one or more specific transcription factors Prelerabh. the said transcription factors are selected from the group. CREB-1, CREB-2, CREM- 1 , ATF- 1 , ATF-2, ATF-3, ATF-4, Spl, Sp2, Sp3, Sp4, AP- 1. and AP-2. A further embodiment of the invention is a transgemc non-human animal whose genome comprises an expression system comprising nucleic acid molecules constituting GABA B receptor promoters Pla and/or Plb, or functionally equivalent modified forms thereof, or active fragments thereof, coupled to a reporter gene Such transgemc non-human mammals can be generated by insertion of DNA comprising GABA B receptor promoters by microinjection, retroviral infection or other means well known to those skilled in the art, into appropriate fertilized embrvos to produce a transgemc animal (Hogan B et al Manipulating the Mouse Embryo Λ Laboratory Manual Cold Spring Harbor Laboratory ( 1986)) Accordingly, the present invention provides a method for the screening of compounds which are modulators of GABA B receptor 1 transcription, comprising the use of a transgemc non-human animal whose genome comprises an expression system comprising nucleic acid molecules constituting GABA B receptor promoters Pla and/or Plb, or functionally equivalent modified forms thereof or active fragments thereof, coupled to a reporter gene, or tissues or cells isolated from such transgemc animals EXAMPLES The following examples are preferred non-limiting examples embodying preferred aspects of the invention Example 1 Isolation and identification of human GABAR R l promoters Pl a and Plb Genomic DNA containing the human GABAβ receptor gene was isolated from human genomic libraries and genomic DNA Human genomic libraries w ere obtained from Clontech (Palo Alto, CA, USA) The libraries were constructed from female leukocyte DNA (catalogue # HLl 1 1 1 J), cloned into λEMBL-3 vector The average size of inserts are 16 kb and the number of independent clones are 1 7x10^ Human genomic DNA was obtained from Clontech (catalogue # 6550-1) In order to isolate recombinant phases containing exon and intron sequences of the human GABA B receptor gene, 48 individual bacterial plates with a diameter of 150 mm and approximately 4 10"^ individual plaques per plate, were screened The methods and solutions used were as described in the Library Protocol Handbook General Procedures for the Hybridization of Lambda Phage Libraries w/DNA Probes (Clontech) with some modifications as will be apparent from the following The experiment was carried out essentiallv as follow s The numbers are given per plate basis A sample of the phage library diluted in 0 1 ml sterile lambda diluent was prepared in order to obtain an estimated titer of 40.000 pfu (plaque forming units) A 0 6 ml LB- medium culture of the E coh host strain K802 (obtained from Clontech) was infected with 40000 pfu recombinant phages for 15 minutes at 37°C The culture was then mixed with 7 ml top agarose (6 5 g of agarose added per liter LB) and poured onto LB plates The plates were incubated at 37°C for approximate!} 7 hours The plates were then chilled at +4°C Plaque hybridization experiments were as follows Membrane filters, Colony/Plaque Screen (DuPont Wilmington, DE. USA), were placed onto the top of the plates for 3 minutes For denaturation of DNA the filters weie removed and floated in 0 5 M NaOH on a plastic wrap for 2 minutes, with the plaque side up This step was repeated once to ensure efficient denaturation Following neutralization the membrane filters were placed in 1M Tπs-HCl pH 7 5, two times 2 minutes and allowed to dry To obtain probes for DNA hybridization screening of the membrane filters, a GABA B receptor cDNA clone was digested with SacII and a 479 bp fragment, separated by agarose electrophoresis, excised and transferred to a polypropylene microcentπfuge tube Additional probes were obtained by PCR amplification of various regions of the GABA B receptor cDNA The isolated cDNA fragment was 32 P-labeled using Megapπme DNA labeling system (Amersham Pharmacia Biotech, Uppsala, Sweden) by the following procedure Water was added at a ratio of 3 ml per gram of gel. and placed in a boiling water bath for 7 minutes to melt the gel and denature the DNA A volume of DNA/agarose solution containing 25 ng of DNA was added to the labeling reaction, according to the supplier s instructions Labeled nucleotides were removed from DNA labeling reactions using MicroSpin™ G-50 Columns (Amersham Pharmacia Biotech. Uppsala. Sweden ) The DNA hybridization reaction was performed under stringent conditions according to the method described below The filter membranes were prehvbndized at 65°C for at least 1 hour in a solution composed of 1 % SDS. 1M NaCl, and 10% dextran sulfate using a hybridization oven (Hybaid Ltd. Ashford, UK). Following prehybridization a solution containing denatured herring sperm DNA of a final concentration of 100 μg/ml and the 32p-iabeled DNA probe at a concentration <10 ng/ml (for optimal signal to background ratio) was added to the prehybridization solution and the membrane filters were incubated at 65 °C for 10-20 hours. Following the removal of the hybridization solution the membrane filters were first washed in a 2xSSC (0.3M NaCl, 0.03M Na-citrate), 1 % SDS solution two times for 5 minutes at room temperature. In the next step, the membrane filters were incubated at 60°C two times for 30 minutes each in the same solution. In a third step, the filters were washed two times at room temperature in 0. lxSSC. Finally, the membrane filters were placed on a sheet of filter paper with the DNA face up, and allowed to dry. The dried membrane filters were then exposed to X-ray films and autoradiographed. Of the approximately 2x10^ individual plaques analyzed, four hybridizing plaques were detected and isolated. These four isolates were designated #GR1, #GR12, #GR13 and #GR41, respectively. After several rescreening experiments, the recombinant phage DNA was purified using Qiagen Lambda Midi Kit (Qiagen GmbH, Germany). The purified DNA was digested with Sail and the fragments representing the inserts were isolated by agarose electrophoresis. The 16kb insert of isolate #GR13 was cloned into Sail digested linearized pUC19, resulting in the plasmid pAM364. The insert was analyzed by PCR, restriction mapping and hybridization to 3 p_ι a 5 e ι e d DNA fragments representing various regions of the GABA B receptor cDNA. The cloned fragment in the plasmid pAM364 was characterized by restriction enzyme mapping, using EcoRI. Hindiπ, Pstl, and BamHI. The approximate positions of the exons and the approximate size of the introns were analyzed and determined by PCR-based exon- exon linking and agarose gel electrophoresis. In order to facilitate nucleotide sequence analysis. 7 restriction sub-fragments derived from pAM364 were isolated and cloned individually into pUC19. The following strategy was emploved, by combining PCR primers located within the pUC19 sequence either upstream or downstream of the cloning site, with a PCR primer with defined orientation and specific for the GABA B receptor derived subcloned fragment allowed the sequence determination The inserts were subjected to nucleotide sequence analysis The nucleotide sequences for all subclones were determined using a Thermo Sequenase dye terminator cycle sequencing pre-mix kit (Amersham Pharmacia Biotech, Uppsala, Sweden) As primers for sequencing reactions specific o gonucleotides complementary to pUC19 or primers complementary to the human GABA B receptor cDNA were used The sequence of the human GABA B receptor gene fragment cloned in the plasmid pAM364 has previously been revealed (see PCT/SE98/01947) This genomic fragment was shown to contain the complete exons 1-11 and the complete introns 1-10 of the human GABAβ receptor gene as well as > 3kb sequence upstream of exon 1 The elucidation of the gene organization revealed that the human GABA B Rla and GABA B Rlb are splice variants encoded by a single gene In order to localize the putative human GABA B receptor promoter, we investigated the genomic sequence for the presence of consensus sequences of known regulatory promoter elements To our surprise, we found that promoter elements, clustered in two regions one region upstream of exon 1, and the other region in intron 5, just upstream of exon 6 We concluded that the human GABA B receptor may be regulated by two independent promoters, and not by one single promoter as expected The first putative promoter, denoted Pla (SEQ ED NO 1) and described in detail below, may regulate transcription of GAB A B R la-type splice variants and the second putative promoter, denoted Plb (SEQ ID NO 2) and described in detail below, may regulate transcription of GAB A B R lb-type splice variants As indicated in the schematic representation of Pla and Plb (Figure 2), both putative promoters lack a TATA box However Plb has an initiator (Inr) element in position 4375- 4381 which is located 24-30 bp upstream of the position corresponding to the 5' end of the longest known cDNA isolated ith "rapid amplification ot cDNA ends " ( RACE) PCR amplification The Inr element may therefore direct the start ot transcription from Plb Pl a contains neither a TATA or an Inr element and the transcription from R l a may therefore initiate from different start sites w hich is often the case in promoters lacking TATA boxes or Inr elements Both Pla and Plb contain multiple GC rich regions at pos 3009-3016, 3037-3044 and 31 16-3123 in R 1 a and pos 4080-4087. 4196-4204, 4241 -4249 and 4272- 4279 in Rib, which are potential binding site for the SP1 family of transcription factors SP1 binding sites are often found in TATA lacking promoters where thev often substantially contribute to transcription In addition, to the indicated GC sequences in Figure 2 there are also other GC motifs that may function as SP1 binding sites. Pla further contains an activator protein- 1 (AP- 1 ) site at position 1497- 1503 AP- 1 sites are recognized by AP- 1 transcription factors which consists of homodimers ot members of the Jun family or Fos/Jun heterodimeπc complexes. AP-1 complexes also interact, by protein-protein interactions, with members of the steroid receptor family and are therefore also targets for steroid receptor signaling. In addition to the GC motifs Plb also contain an activator proteιn-2 (AP-2) site at position 3844-3851 and a cAMP responsive element (CRE) at position 4308-4315. Especially the finding of a consensus CRE (TGACGTCA) is interesting as this promoter element is found in many genes regulated by cAMP which are bound and regulated by members of the ATF/CREB gene family This sequence may therefore be an important target for cAMP mediated signaling via G-protein coupled receptors, including GABA B receptors We conclude that transcription of the human GABA B receptor gene may be regulated by two putative promoters, Pl a and Plb, that may independently regulate expression of human GABA B receptor la and lb splice isoforms, respectively Example 2 Determination of GAB AR R 1 promoter P 1 a and P 1 b activity To experimentally determine if Pla and Plb indeed act as promoters, we fused these regions to a cDNA encoding firefly luciferase to be used as a reporter of promoter activity in transfected cells Reporter constructs containing Rla and Rib promoter fragments were generated by PCR using plasmid pAM 364, containing genomic sequence covering the promoter regions, as template PCR reaction was performed by standard procedure (Perkin Elmer) Briefly, an initial denaturation at 95°C for 4 min was followed by 35 cycles of denaturation at 95°C for 1 min, annealing at 60°C for 1 rrun, elongation at 72°C for 1 mm and finally a 7 min elongation at 72°C In the sequence for primers used to generate promoter fragments (for details see Table 1 below), a Nhe I and Hind EH endonuclease restriction enzyme site was introduced, in 5 'and 3 ' primers respectively, to enable sub-cloning into Nhe I/Hind HE digested pGL3-Basιc luciferase reporter vector (Promega) Hence complete promoter- reporter constructs (pAM440, pAM438 and pAM436) contain Rla and Rib promoter fragments (indicated size see table) fused to the luciferase reporter gene Plasmid DNA were purified using Qiagen tip- 100 columns according to suppliers instruction Correct fragment insertion was verified by DNA sequencing Table 1 Nucleotide sequence of primers used to generate promoter fragments To determine putative promoter activity the reporter gene constructs were introduced into mammalian cells by transfection In this study the cell line ND7/23 (ECACC Ref No 92090903) was used ND7/23 is a hybrid cell line originating from a mouse neuroblastoma (N18tg2) fused by PEG to a rat dorsal root ganglion neuron cell line This cell line was chosen since it express functional GABA B receptors as evidenced by radio gand binding studies Cells were cultured in a 1 1 mixture of Dulbecco s modified medium (DMEM) and Ham's F12 medium supplemented with 10% (v/v) fetal bovine serum (FBS) Cells were grown at 37°C in an atmosphere of 5% CO 2 ND7/23 cells (4x 10 ^ ) were transfected using the DMREE-C reagent according to manufacturer s protocol (Gibco) Briefly, cells were seeded in 6 well tissue culture plates (Nunc) the day before transfection Next day. 2μg of promoter- reporter construct and lμg of transfection control construct (pSV-β- Galactosidase, Promega) was mixed with 0 5 ml Optimem media (Gibco) DNA containing media was then mixed with an equal volume (0 5ml) of Optimem containing 4μl of DMREE-C reagent and the combined mixture was then incubated for 45 min After incubation, the transfection mixture was added to the cells, which were washed with Optimem media just prior to addition of transfection mixture Following 5h incubation at 37°C, an equal volume of 1 1 mixture of Dulbecco ' s modified medium (DMEM) and Ham's F12 medium supplemented with 20% (v/v) fetal bovine serum (FBS) was added to the cells Cells were incubated for 24h at 37°C with or without cAMP enhancing supplement as indicated in Figure 3 Before cell harvest, cells were washed in PBS (7 6 mM Na 2 HPO 4 /NaH 2 PO 4 pH 7 4 and 120 mM NaCl), then cell extracts were prepared by addition of 250μl reporter lysis buffer (Promega) to cells, follo ed by transfer of cell suspension to 1 5 ml tubes Cells were further lysed by one round of freeze-thawmg and 15s of vortex Cell debris were removed by 2 minutes of centπfugation at 12 000 g Luciferase activity in cell extracts was measured in a Luciferase Assay System (Promega). where 40μl of cell extracts was added to a 96-well plate (Maxisorp Nunc) and mixed with 50μl of luciferase substrate Luciferase activity was then measured in a LUMIstar (BMG Lab technologies) As internal control for transfection efficiency, β-Galactosidase (β-Gal ) activity was measured in 96-well plate (Maxisorp Nunc) using a β-Galactosidase Enzyme Assay (Promega) according to suppliers protocol As seen in Figure 3 transient transfection of ND7/23 cells shows that both Pl a and Plb has promoter activity as pAM438 pAM436 and pAM440 (Figure 3 ) result in reporter expression while the pAM442 (vector control) has very low activity In addition, this experiment demonstrates that especially reporter expression originating from pAM440 (Rib) can be induced by the cAMP activating agent forskolm Moreover, forskolin induced expression may also be further enhanced in the presence of the phosphodiesterase inhibitor l-methyl-3- lsobutylxanthine (IB MX) although this experiment does not show a significant difference In conclusion, this experiment demonstrate that Pla and Plb both have promoter activity and that the degree of activity can be modulated using the cAMP activating agent forsko ne Example 3 Screening for substances modulating Pla and Plb activity Modulating GABAβRl expression in a controlled way is a means to regulate signaling through GABA B receptors which could be of significant therapeutic value for a variety of conditions Particularly, the ability to specifically regulate expression of either la- or lb- type GABA B receptor splice isoforms could be of medical value if these isoforms could be attributed to specific conditions The experiment presented in Example 2 demonstrates that the use of Pla and Plb promoter/reporter constructions can be used to monitor GABA B Rl expression in screens for therapeutic agents that can modify the expression of GABA B receptor 1 isoforms A screen for Pla and Plb modulating substances could be performed in ND7/23 cells as described in Example 2 A screen for Pla and Plb modulating substances could in addition be performed in any cell type with endogenous expression of GABA B receptor 1 and 2 isoforms. in cells expressing recombinant GABA B receptor 1 and 2 isoforms and in intact cells and in extract or fractions of cells expressing endogenous and recombinant proteins modulating GABA B receptor function Such screens could furthermore be done in tissues and in living organisms Example 4 Functional analysis of GABAR-R 1 promoter fragments and modified forms In order to identify functionally active promoter fragments of Pla and Plb. a deletion analysis of DNA fragments containing promoter fragments can be performed in two steps It is anticipated that Pla and Plb comprise active fragments that can mediate increased expression by binding of transcription factors that are activators as well as active fragments which can mediate decreased expression by binding of transcription factors that are repressors In the first step, promoter fragments mediating expression of a reporter gene when used in reporter constructs can be stepwise deleted or truncated to identify important regions Briefly, truncated or deleted promoter fragments are created by PCR using specific primers and the already identified promoter sequences as template Reporter constructs (as exemplified in this application) comprising the deleted or truncated promoter fragments are then created These reporter constructs can then be used in transfection experiments, as described above, to identify important regions of the promoters manifested in altered expression from constructs lacking active fragments compared to none-deleted constructs In the second step, the exact location and sequence of transcription factor binding sites within active fragments can be determined by PCR technique using specific primers harbouring desired mutations Such promoter fragments, with specifically mutated promoter regions, can be used in transfection experiments, similar to those described above, to determine the exact sequence of functionally important nuclear factor binding sites within active promoter fragments, manifested in altered expression from constructs with mutated DNA sequence compared to none-mutated control constructs with equal size The above mentioned strategy can also be used to identify the specific active promoter fragments which are important for the effect on promoter activity of active substances identified when screening for therapeutic agents regulating GABA B -R1 expression Example 5 Activity of GAB A 3 R 1 promoter P 1 a and P 1 b fragments Reporter constructs containing Pla and Plb promoter fragments were generated by PCR as described in Example 2 and fused to the firefly luciferase reporter gene The generated constructs are visualised in Figure 4 Putative transcription initiation sites were identified at position 3207 m SEQ ED NO 1 for Pla and at position 4405 in SEQ D NO 2 for Plb The positions in the promoter region shown in Figure 4 were calculated setting the transcription initiation sites as position +1 The generated promoter reporter constructs (see Figure 4) were used to transfect ND7/23 cells. As shown in Figure 4, deletion of Pla promoter region in between position -2549 bp and -361 caused an increase in expression, indicating putative repressor regions between position -2549 and -361 Moreover, when 175 bp of the 5 ' untranslated region were removed (compare the third and fourth Pla constructs from the top) there was no detected difference in promoter activity and constructs with the shorter Pla 5 ' -untranslated region were therefore used for mutational analysis throughout the rest of the study Additional deletions of the Pla promoter from position -361 to -46 caused a stepwise decrease of the expression that seem to correlate with removal of promoter regions containing GC elements (I- HE) This indicate that 361 bp of the Pla promoter can confer optimal expression and that promoter elements essential for R la-type expression, including GC elements I-EQ are located between position -361 and -46 Similar to the Pla promoter, deletion of Plb promoter region from position -3238 bp to -390 caused an increase in expression, indicating putative repressor regions between -1084 and -390 Also, additional deletions of the Plb promoter from position -390 to -88 caused a stepwise decrease of the expression that correlate with removal of promoter region GC elements (EV-VEI) as well as the consensus CRE Although there was still some promoter activity left when the Plb promoter region was deleted down to position -88. the major promoter elements essential for Rib-type expression seem to be located between position -390 and -88. Comparison of Pla and Plb expression also indicates that, Plb mediated expression was higher (approximately 3-4 fold) than for Pla. Example 6. Mutational analysis of Pla and Plb promoter element function As shown in Figure. 4, reporter constructs -361 in Pl a (short 5TJTR) and -390 in Plb conferred optimal expression of the respective promoters. In order to determine the importance of the promoter elements that was found within these regions, Pla (-361 ) and Plb (-390) constructs were used as templates to obtain reporter constructs with promoter regions mutated at specific sites. For the mutated promoter constructs, a 3 bp substitution was introduced by "quick change" site directed mutagenesis. Site-directed mutagenesis: Mutant constructs were prepared with a QuickChange site- directed mutagenesis kit (Strategene, La Jolla, CA) according to the manufacturer's instructions. The mutagenic oligonucleotide primers are listed in Table 2. All constructs were verified by DNA sequencing. Table 2. Oligonucleotide primers used in the mutagenesis experiments Bases that are labelled bold correspond to designed mutations Obtained promoter constructs were then used to transfect ND7/23 cells. As shown in Figure 5, single point mutations of GC elements I, IE and HE reduced Pla expression to approximately 65-75% compared to wild-type, while double-mutations (I/O, I EH and Et EQ, respectively) resulted in a further decrease to approximately 55-60% of wild-type expression levels. When all three GC elements (I-EEI) were mutated, the reporter expression was reduced to approximately one third (33%) of wild-type expression. Together, these results suggest that the three GC-elements found in Pla, all contribute to Pla mediated expression in a substantial and additive manner. However, the fact that one third of the Pla promoter activity still remains suggests that there are additional promoter elements within Pla contributing to Pla mediated expression. In contrast to Pla single mutations of the tour Plb GC elements (IV. \ VI and VII) only caused small reductions of expression to approximately 75-90% compared to wild-type The relatively modest contribution by the four Plb GC elements was also demonstrated by a promoter construct were all four GC elements (IV-VII) had been mutated This construct retained 63% of Plb expression compared to the wild-type construct However, when the Plb CRE consensus site was mutated (CRE), a dramatic reduction of Plb mediated expression (approximately 50%) w as obtained Moreover, when the CRE mutation was combined with mutations of each GC element respectively, a further reduction was observed Most notably, the promoter construct containing a double mutation of the CRE and GC V (CREN) promoter elements resulted in a substantial reduction of Plb expression to approximately 26%. similar to the construct where all fiv e Plb promoter elements were mutated (CRE/IV-VH) where 24% of the expression still remained compared to wild-type Together these data demonstrates the absolute importance of the consensus CRE site for Plb expression, alone or in combination with the GC elements (EV, V, VI and VU), of which GC element V seems to contribute most The fact that one fourth of Plb promoter activity still remains suggests that, as for Pla. there are additional promoter elements within Plb that may contribute to Plb mediated expression Example 7 Identification of factors interacting with the Plb CRE site Electrophoretic mobility shift assays (EMSA) Electrophoretic mobility shift assays (EMSA) can be used to identify nuclear factors that interact with Pla and Plb promoter elements In an attempt to identify factors interacting with the Plb CRE site, we performed gelshift analysis with super-shift antibodies that recognise members of the CREB/ATF family of transcription factors The DNA-binding reactions (12 μl) were done as follows, 2-3 fmol " P-labeled double-stranded oligonucleotide corresponding to the Plb CRE site was mixed with 5 μg crude nuclear extracts from ND7/23 cells and 1 μg poly (dl-dC). 25 mM Hepes (pH 7,9), 150 mM KC1. 5 mM dithiothreitol and 10% glycerol (Schneider et al 1986, Nucleic Acids Research 14 1303-17) In the supershift lane (Figure 6) the ATF- 1 p35/CREB- l P 43/CREM- 1 reactive antibody (sc-270 from Santa Cruz Biotechnology Santa Cruz, CA) was pre- mcubated at room temperature for 20 mm before addition of '"P-labeled probe After incubation at room temperature for 15 min, proteιn-D\A complexes were resolved on precasted DNA-retard gels (Novex ) containing 6% polyacrvlamide prepared with 0.5x TBE as gel buffer . Following electrophoresis, gels were dried and visualised by autoradiography As shown in Figure 6. addition of a monoclonal CREB/ATF supershift antibody (reactive with members of the ATF/CREB family such as ATF-1 p35 CREB-1 p43 and CREM-1 ot mouse, rat and human origin) into the EMSA reaction mixture results in a distinct shift of the nuclear factor(s) that interacts with the Plb CRE site while no super-shift was observed when the same antibody was added to other EMSA reactions containing other promoter elements (data not shown) This data suggests that the complex formed with the Plb CRE site contains a member of the CREB/ATF family Similar further studies can be performed in order to determine which factor(s) that interact with the various promoter elements in Pla and Plb Example 8 Use of recombinant transcription factors in the study of Pl a and Plb promoter activities Cells with stably integrated or with transiently transfected Pla/Plb reporter constructs can be transfected with cDNA encoding specific transcription factors to make reporter cells suitable for investigations of Pla and Plb activities mediated by said transcription factors Reporter cells can alternatively be generated by delivery of transcription factors into similar cells by various means such as e g microinjection and pofection Reporter cells can be utilised for screening of compounds, which are modulators of GABA B receptor 1 transcription BREEF DESCRIPTION OF DRAWINGS Figure 1. The human GABA R receptor gene The figure shows the organization of the human GABA B receptor gene. Exons, represented by vertical squares/bars, are numbered ( 1-23). Translational start and stop sites are indicated by arrows. Location of human GABA B receptor 1 promoters Pla (SEQ ED NO: 1 ) and Plb (SEQ ED NO: 2) are indicated. The extent of human GABA B receptor genomic sequence cloned in plasmid pAM 364 is indicated by a horizontal bar. Figure 2. Schematic representation of the Pl a and Plb promoters DNA fragments used to generate reporter constructs, corresponding to the positions in the Pla and Plb promoter sequence, are shown below each promoter. Putative promoter elements in each promoter are indicated. Arrows indicate putative positions for start of transcription. Figure 3. Determination of GABAB receptor 1 promoter Pla and Plb activity ND7/23 cells (4 l0 5 ) were transfected with promoter-luciferase constructs as described above. After transfection, cells were cultured in media without supplement (basal) or in the presence of Forskolin (lOμM) or Forskolin (lOμM) + l-methyl-3-isobutylxanthine (0.125mM) for 24h. After incubation, cells were harvested and luciferase activity measured. Luciferase activity, minus background, is shown as arbitrary light units measured in 40 μl of cell extracts. Relative values represent the mean+SEM of two individual experiments. Figure 4: Deletion analysis of the GBR 1 promoters in ND7/23 cells ND7/23 cells (4 1 O^) were transfected with Pla and Plb promoter-luciferase constructs as outlined above. After transfection, cells were cultured for 24h. harvested and luciferase activity was measured. Luciferase activity, minus background, is shown as arbitrary light units measured in 10 μl of cell extracts. Relative values represent the mean+SEM of duplicate samples from at least three individual experiments. Figure 5. Effect of Pla and Pl b promoter element mutations on promoter activities. ND7/23 cells (4xl0 5 ) were transfected with wild-type and mutated Pla and Plb promoter- luciferase constructs as described above. The point mutations in the promoter constructs are outlined to the left. After transfection, the cells were cultured for 24h. After incubation, cells were harvested and luciferase activity measured. Luciferase activity, minus background, is shown as arbitrary light units measured in 10 μl of cell extracts. Relative values represent the mean+SEM of duplicate samples from at least three individual experiments. Relative expression level of mutated constructs are indicated to the left with the Pla and Plb "wild-type" expression level set as 100%. Figure 6. Identification of nuclear factors binding to the Plb consensus CRE site using CREB/ATF super-shift antibodies. Nuclear extracts (5 μg) from ND7/23 cells were incubated with double-stranded 32 P- labeled oligonucleotides containing the Plb consensus CRE site (sense: 5'- CGCCGCCCGTGACGTCAGAGCCCCCT-3'). In lane 1 , no antibody was added. In lane 2, a mouse monoclonal antibody (sc-270 Santa Cruz Biotechnology. Santa Cruz, CA) reactive with members of the ATF/CREB family such asATF-1 p35, CREB-1 p43 and CREM- 1 was pre-incubated at room temperature for 20 min before addition of J "P-labeled probe. The specific complex between nuclear factors and the CRE is indicated by a star and the super-shifted complex is indicated by two stars.

Claims: CLALMS 1. A nucleic acid molecule constituting a human GABA B receptor 1 promoter Pla. or a functionally equivalent modified form thereof, or an active fragment thereof. 2. A nucleic acid molecule according to claim 1 selected from: (a) a nucleic acid molecule comprising a nucleotide sequence set forth as SEQ ED NO: 1 ; (b) a nucleic acid molecule comprising a nucleotide sequence capable of hybridizing, under stringent conditions, to a nucleotide sequence complementary to the polypeptide coding region of a DNA molecule as defined in (a). 3. A nucleic acid molecule constituting a human GABA B receptor 1 promoter Plb, or a functionally equivalent modified form thereof, or an active fragment thereof. 4. A nucleic acid molecule according to claim 1 selected from: (a) a nucleic acid molecule comprising a nucleotide sequence set forth as SEQ ED NO: 2; (b) a nucleic acid molecule comprising a nucleotide sequence capable of hybridizing, under stringent conditions, to a nucleotide sequence complementary to the polypeptide coding region of a DNA molecule as defined in (a). 5. A nucleic acid molecule comprising a nucleic acid molecule according to claim 1, in combination with a nucleic acid molecule according to claim 3. 6. A nucleic acid molecule comprising a nucleic acid molecule according to claim 2, in combination with a nucleic acid molecule according to claim 4. 7. A vector transformed with a nucleic acid molecule according to any one of claims 1 to 6. 8 A cultured cell host harboring a vector according to claim 7 9 An expression system comprising a nucleic acid molecule constituting a human GABA B receptor 1 promoter Pl a. or a functionally equivalent modified form thereof, or an active fragment thereof 10 An expression system according to claim 9. comprising a nucleic acid molecule selected from (a) a nucleic acid molecule comprising a nucleotide sequence set forth as SEQ ED NO 1 , (b) a nucleic acid molecule comprising a nucleotide sequence capable of hybridizing, under stringent conditions, to a nucleotide sequence complementary to the polypeptide coding region of a DNA molecule as defined in (a) 11. An expression system comprising a nucleic acid molecule constituting a human GABAβ receptor 1 promoter Plb, or a functionally equivalent modified form thereof, or an active fragment thereof 12 An expression system according to claim 1 1, comprising a nucleic acid molecule selected from (a) a nucleic acid molecule comprising a nucleotide sequence set forth as SEQ ED NO: 2; (b) a nucleic acid molecule comprising a nucleotide sequence capable of hybridizing, under stringent conditions, to a nucleotide sequence complementary to the polypeptide coding region of a DNA molecule as defined in (a) 13. An expression system comprising a nucleic acid molecule according to claim 1. in combination with a nucleic acid molecule according to claim 3 14 An expression system comprising a nucleic acid molecule according to claim 2, in combination with a nucleic acid molecule according to claim 4 15. An expression system according to any one of claims 9 to 14. which, in addition, comprises a reporter gene. 16. An expression system according to claim 15, wherein the reporter gene is selected from: (a) the firefly luciferase gene, (b) the bacterial amphenicol acetyl transferase (CAT) gene, (c) the β-galactosidase (β-GAL) gene, and (d) the green fluorescent (GFP) gene. 17. An expression system according to claim 15 or claim 16, wherein the promoter and the reporter gene are positioned so that the expression system of the reporter gene is regulated by the GABA B receptor 1 promoter. 18. An expression system according to any one of claims 9 to 17, wherein the said nucleic acid molecule is transformed in a vector. 19. An expression system according to claim 18, wherein said vector comprises an origin of replication and/or a dominant selection marker. 20. A host cell transfected with an expression system according to any one of claims 9 to 19. 21. A method of assay for GAB A B receptor promoter activity, comprising the use of a host cell according to claim 20. 22. A method for screening compounds which are modulators of GABA B receptor 1 transcription, comprising the steps of: (a) transfecting a cell host with a suitable expression system comprising a nucleic acid molecule constituting a human GABA B receptor 1 promoter Pla, and/or a human GABA B receptor 1 promoter Plb, or functionally equivalent modified forms, or active fragments thereof coupled to a reporter gene; (b) contacting a test compound with the cell: and (c) determining whether the test compound modulates the level of expression of the reporter gene. 23. A method according to claim 22. wherein the said expression system is an expression system according to any one of claims 9 to 19. 24. A method according to claim 22, wherein the said the reporter gene is selected from: (a) the firefly luciferase gene, (b) the bacterial amphenicol acetyl transferase (CAT) gene, (c) the β-galactosidase (β-GAL) gene, and (d) the green fluorescent (GFP) gene. 25. The method according to claim 22, wherein the host cell endogenously expresses GABA B receptor 1. 26. The method according to claim 22, wherein the host cell is further transfected with a suitable expression system comprising a nucleic acid molecule encoding one or more specific transcription factors. 27. The method according to claim 26, where the transcription factor is selected from the group: CREB-1, CREB-2, CREM-1, ATF-1, ATF-2, ATF-3, ATF-4, Spl , Sp2, Sp3, Sp4, AP-1, and AP-2. 28. A transgenic non-human animal whose genome comprises an expression system comprising nucleic acid molecules constituting GABA B receptor promoters Pla and/or Plb, or functionally equivalent modified forms thereof, or active fragments thereof, coupled to a reporter gene. 29. A transgenic non-human animal whose genome comprises an expression system according to any one of claims 9 to 19. 30. A method for the screening of compounds which are modulators of GABA B receptor 1 transcription, comprising the use of a transgenic non-human animal according to claim 28 or claim 29. 31. A method for the screening of compounds which are modulators of GABA B receptor 1 transcription, comprising the use of tissues or cells isolated from a transgenic non- human animal according to claim 28 or claim 29.

Inventors: EDLUND ANDERS, EKSTRAND JONAS, JOHANSSON THORE, LEONARDSSON GOERAN, EDLUND, ANDERS, EKSTRAND, JONAS, JOHANSSON, THORE, LEONARDSSON, GOERAN
Applicants: ASTRAZENECA AB, EDLUND ANDERS, EKSTRAND JONAS, JOHANSSON THORE, LEONARDSSON GOERAN, EDLUND, ANDERS, EKSTRAND, JONAS, JOHANSSON, THORE, LEONARDSSON, GOERAN
Application year: 2000
Main classification: C07K14
IPC codes: A01K67, C12N1, C12N5, C12N15, C12Q1, G01N33