Tissue-Specific Overexpression of the HSA21 Gene Gabpa: Implications for DS

Biochimica et Biophysica Acta 1739 (2004) 81–87 http://www.elsevier.com/locate/bba

Tissue-specific overexpression of the HSA21 gene GABPa: implications for DS

Debra A. O’Leary, Melanie A. Pritchard, Dakang Xu, Ismail Kola1, Paul J. Hertzog, Sika Ristevski*

Centre for Functional Genomics and Human Disease, Monash Institute of Reproduction and Development, Monash University, Clayton, Victoria 3168, Australia

Received 8 January 2004; received in revised form 7 September 2004; accepted 8 September 2004 Available online 19 September 2004

Abstract

The ETS transcription factor GABPa is encoded by a gene on HSA21 and interacts with an ankyrin repeat-containing h subunit to form the GABP complex. GABP regulates expression of genes involved in mitochondrial respiration and neuromuscular signalling. When GABPa mRNA is overexpressed in human DS fibroblast cell lines, or by tranfection in NIH3T3 cells, no increase in protein level is detected. However, increased Gabpa gene dosage in the Ts65Dn segmental trisomy mouse model of DS (DS) results in elevated Gabpa protein levels in brain and skeletal muscle only. These findings suggest that GABPa protein levels are tightly regulated in a tissue-specific manner, and consequently GABP may play a role in DS pathologies in tissues where GABPa protein levels are elevated. D 2004 Elsevier B.V. All rights reserved.

Keywords: ETS; GA-binding protein; HSA21; Tissue-specific regulation; Down syndrome

1. Introduction encoding E26 Transformation Specific (ETS) transcription factors, GA-binding protein (GABPa), Erythroblastosis Down syndrome (DS), a result of trisomy human virus E26 oncogene Related Gene (ERG) and E26 Trans- chromosome 21 (HSA21), occurs at a frequency of ~1/ formation Specific gene 2 (ETS-2). The most commonly 700 live births [1] and results in a number of distinct studied segmental trisomy 16 mice, Ts65Dn, encompass pathologies with varying expressivity. DS individuals dis- trisomy of genes from Gabpa to Tmprss2 (Transmembrane play distinct craniofacial and skeletal abnormalities and Protease, Serine 2) [5] and model many of the craniofacial commonly present with premature aging, Alzheimer’s [6], neurodegenerative [7,8], behavioural [9], motor [7] and disease-like pathology, mental retardation and muscular learning impairments [10] associated with DS. Therefore, hypotonia [2]. Genes localized on HSA21 are implicated in genes located within the Ts65Dn trisomic region are key the pathophysiological features of DS in a dosage-sensitive regulators of DS pathology. manner [2]. GABPa is an ETS protein which, together with an The long arm of mouse chromosome 16 (MMU16) unrelated GABPb subunit, forms the functional GABP shares synteny with the long arm of HSA21, from Stch transcription factor complex (also known as Nuclear (Stress 70 protein chaperone) to marker D16Xrf306 [3], Respiratory Factor 2 (NRF-2) or Adenovirus E4 Gene encompassing more than 100 genes [4], including three Transcription Factor (E4TF-1)). Gabpa is localized to HSA21q21–22.1 [11], is trisomic in Ts65Dn mice, and therefore a prime candidate for contributing to the patho- * Corresponding author. Tel.: +61 3 9594 7217; fax: +61 3 9594 7211. E-mail address: [email protected] (S. Ristevski). physiological features of DS. The GABP complex has been 1 Current Address: Merck Research Laboratories, Merck and Co., Inc., shown to regulate expression of many genes necessary for Rahway, NJ, USA. cellular respiration in mitochondria. These genes include

Cytochrome Oxidase subunits COXIV and Vb [12], VIA1 2.3. Western blot analysis [13], VIIAL [14], VIIC [15], XVII [16] and Mitochondrial Transcription Factor MTFA (the principal transcription Protein extracts were prepared by homogenisation in a factor in mitochondrial gene expression) [17,18]. GABP is buffer of 10 mM Tris, 0.1 M EDTA (pH 7.4) (1 Al per redox-regulated, with oxidation of cysteine residues within mg of tissue), containing Complete Mini EDTA Free GABPa resulting in impairment of DNA binding and Protease Inhibitor Cocktail (Roche). Protein concentration reduced GABP complex activity by preventing heterodime- was determined by Bradford assay, using the Bio-Rad rization with GABPb [19]. Thus, in DS, or models thereof, Protein Assay Reagent, and samples were boiled in SDS- where cells are sensitized to oxidative stress [20], GABPa PAGE buffer (0.5 M Tris–HCl, 0.4% SDS, 20% glycerol, function may be further compromised. 1.4 M h-mercaptoethanol, 0.3 mg bromophenol blue) for In addition, GABP is a regulator of neuromuscular 10 min. Reducing SDS-PAGE (8%) was performed with junction gene expression, up-regulating the Delta [21] and 50 Ag of protein per sample using duplicate gels. The Epsilon [22] subunits of the Nicotinic Acid Acetylcholine proteins were then electroblotted to Immobilon membrane Receptor (AChRd and e), Acetylcholine Esterase [23] and (Millipore) using the semi-dry transfer method. After Utrophin [24]. Acetylcholine is one of the major neuro- blocking the membranes with 10% fat-free milk powder transmitters affected in Alzheimer’s disease and DS [25]. 1Â TBST (Tris, HCl) for at least 2 h at room temper- These disorders and their respective mouse models present ature, primary antibodies (optimised concentration of anti- with loss of cholinergic neurons [26]. Notably, DS patients Gabpa polyclonal rabbit antisera as determined by serial also suffer from muscular hypotonia (floppy muscles) during dilution [29] or 0.05 Ag/ml anti-h-tubulin mouse mono- the early years of life [6,27]. To address the role of GABPa in clonal antibody (Chemicon)) were added to each of the DS, we have examined the effect of increased gene dosage on duplicate membranes and incubated overnight at 4 8C. GABPa protein levels in human DS cell lines, transfected Membranes were washed three times by 20 min each with NIH3T3 fibroblasts and Ts65Dn mouse tissues, and show 1Â TBST followed by incubation for 1 h at room that GABPa protein levels are subject to tight regulation in temperature with 0.15 Ag/ml goat anti-rabbit IgG HRP most tissues, except brain and skeletal muscle. This implies (DAKO) or 0.3 Ag/ml rabbit anti-mouse IgG HRP that GABP may contribute to DS pathologies in a tissue- (DAKO), followed by three washes. HRP was detected specific manner, namely brain and skeletal muscle pathology. by Super-Signal (Pierce) chemiluminescence and exposure to BioMax autoradiographic film (Kodak). A gradient strip was used to obtain exposure times that gave signal 2. Materials and methods intensities within the linear range of the film. The relative quantities of Gabpa protein to h-tubulin were quantified 2.1. Cell lines and culture conditions by using MacBas v2.5 software. Results represent an average of three independent Western blotting experiments NIH3T3 (CRL-1658, ATCC), DS fibroblast cell lines and carried out per animal. It was determined that multiple their age- and sex-matched fibroblast controls (a kind gift Western blots with equal protein loading would be from A. Choo, Murdoch Children’s Research Institute, performed for each individual protein sample rather than Melbourne, Australia) were maintained in DMEM contain- stripping and re-probing the same membrane. Gabpa ing 10% FCS, 5 Ag/ml penicillin and 5 Ag/ml streptomycin. levels for individual tissue samples were determined as a The DS fibroblasts and their matched controls were early ratio to h-tubulin levels in those tissues. In each case, the passage (between passages 12 and 16) primary cells. wild-type tissue was given a value of 1 and the equivalent Ts65Dn tissue or DS cell line was represented 2.2. Northern blot analysis relative to this wild-type value.

GABPa mRNA levels were determined by Northern blot 2.4. Generation of Gabpa stably transfected NIH3T3 cells analysis and hybridization with a Gabpa cDNA probe spanning 413–1884 bp (representing the full length ORF The full-length 1.5-kb cDNA sequence of Gabpa (RT- of sequence GI:193382). Expression levels of the house- PCR product spanning 413–1884 bp of GI:193382) was keeping mRNA Glyceraldehyde-3-phosphate dehydrogen- inserted into the XbaI site of the pEF-BOS-puromycin ase (GAPDH) were also determined to allow for vector [30] (under control of the human polypeptide chain quantification, using a probe spanning nucleotides 585– elongation factor 1a (EF-1a) promoter) in both sense and 985 of sequence GI:193423. Poly-A+ RNA was prepared and antisense orientations. Ten micrograms of SalI linearized filters hybridized as previously described [28]. RNA species sense and mock (vector alone) DNA was each added to were viewed and quantified using a FujiFilm FLA-2000 2Â106 cells and subjected to 350 V/960 AF for 10 s. v1.21 Phosphorimager, with Image Reader and Image Gauge Cells were then grown in Dulbeccos Modified Eagles (v.3.46) software. Values were adjusted such that the GABPa Medium (DMEM) supplemented with 10% FCS (v/v). to GAPDH ratio of wild-type cells was equal to 1. After 2-day recovery, 5 Ag/ml puromycin was added for 3 D.A. O’Leary et al. / Biochimica et Biophysica Acta 1739 (2004) 81–87 83 days and surviving clones were individually isolated and of resulting cDNAwas used as template for real-time RT-PCR passaged. reactions of 45 cycles, using a Light Cycler (Roche) (v3.5 software). Fast start SYBR Green Master Mix (Roche) was 2.5. RT-PCR analysis of Gabpa-transfected NIH3T3 clones used, with 10 pmol of each primer, in 10-Al reactions. Primers spanning Gabpa exon 9 to exon 10, from 1441–1707 bp Single-stranded cDNA was generated using 500-ng poly- of GI:193382, were used to generate a 266-bp PCR A+ RNA as template and AMV reverse transcriptase product. Gabpa expression levels were expressed relative (Promega), as per manufacturer’s instructions. Expression to Gapdh, by amplification of a 400-bp product spanning of the exogenous Gabpa cDNA was confirmed in 3T3 585–985 bp of GI:193423. To create a standard curve, five clones by RT-PCR with a forward 5Vprimer within Gabpa 10-fold dilutions of plasmid DNA were used as template exon 9 (1441–1460 bp of GI:193382) and a reverse primer (10À5–10À9 ng/Al). Results represent an average of three in the 3VUTR of pEF-BOS (5V-GTCTCCCACGTGG- independent experiments. GAGACCT-3V). Products of 496 bp resulted from cDNA of clones expressing exogenous Gabpa, not mock controls. 2.7. Mice The integrity of cDNA samples was confirmed by RT-PCR amplification of a 400 bp Gapdh product, spanning 585– Ts65Dn mice (strain name: B6EiC3Sn a/A-Ts(1716)65Dn, 985 bp of GI:193423. stock number: 001924) were obtained from the Jackson Laboratories, Bar Harbor, ME, USA and maintained in a 2.6. Real-time RT-PCR analysis of Gabpa-transfected specific pathogen facility at Monash University. Ts65Dn NIH3T3 clones mice were bred with (C57BL/6JEixC3H/HeSnJ)F1 hybrid mice and progeny were genotyped by quantitative PCR as Single-stranded cDNA was generated using 500-ng poly- described by Liu et al. [31]. A+ RNA as template and AMV reverse transcriptase All experimental procedures involving mice were (Promega), as per manufacturer’s instructions. A 1/5 dilution approved by the appropriate ethics committee within

Fig. 1. Effect of increased GABPa gene dosage in DS fibroblasts. (a) Northern blot analysis of GABPa mRNA levels in two DS (DS) and two wild-type (WT) fibroblast cell lines. GAPDH was used as a loading control. (b) Northern blots quantified for GABPa mRNA levels relative to GAPDH in DS and wild-type fibroblast cell lines, where wild-type GABPa levels were given a value of 1.0 and DS levels were expressed relative to this. Error bars represent S.D., where n=2. * represents statistical significance, as determined by a two-tailed t-test, where P=b0.05. (c) Western blot analysis of GABPa protein levels in wild-type and DS fibroblast cell lines. The h-tubulin housekeeping protein was used as a loading control. (d) Western blots quantified for GABPa protein levels relative to h-tubulin in wild-type and DS fibroblast cell lines. Wild-type GABPa levels were given a value of 1.0 and DS levels were expressed relative to this. Error bars represent S.E., where n=9. 84 D.A. O’Leary et al. / Biochimica et Biophysica Acta 1739 (2004) 81–87

Monash University and performed according to the Uni- lines). This is as expected from the increased gene dosage of versity’s guide for the care and use of laboratory animals. trisomy 21. However, this increase in GABPa mRNA is not reflected in increased GABPa protein levels, which remain unchanged relative to diploid fibroblasts (Fig. 1c and d). 3. Results 3.2. Overexpression of Gabpa cDNA in NIH3T3 cells 3.1. Expression analysis of GABPa in DS fibroblast cell lines To examine the effect of Gabpa overexpression, we stably transfected mouse NIH3T3 fibroblast cells with We examined the effect of increased gene dosage on Gabpa cDNA under regulatory control of the human GABPa protein levels in fibroblast cell lines isolated from polypeptide chain elongation factor 1a (EF-1a) promoter DS individuals. As shown in Fig. 1a and b, the average (see Fig. 2a). Expression of the exogenous Gabpa cDNA expression level of GABPa mRNA (detected by Northern was detected by RT-PCR analysis (Fig. 2b) of cDNA from blot analysis) is increased approximately 1.7-fold in DS Gabpa transfected NIH3T3 clones, but not from mock samples (relative to matched human diploid fibroblast cell controls. Real-time RT-PCR analysis showed a three- to

Fig. 2. Overexpression of Gabpa in fibroblasts. (a) Gabpa-pEF-BOS-puromycin overexpression construct containing the human Elongation Factor 1a promoter and a Puromycin resistance cassette. Restriction endonuclease sites used for cloning are indicated: SalI (S), XbaI (X), NotI (N), NcoI (Nc) and EcoRI (E). (b) RT-PCR analysis of cDNA made from 3T3 clones transfected with mock (M) or Gabpa (G) constructs, using a 5V primer within Gabpa and 3V primer within the 3VUTR of the vector backbone to generate a 436-bp product in clones expressing exogenous Gabpa. Integrity of cDNA was confirmed by amplification of a 400-bp Gapdh housekeeping gene product. (c) Real-time RT-PCR analysis of total Gabpa mRNA levels in mock and Gabpa transfected 3T3 clones, using primers spanning Gabpa exon 9 to exon 10, expressed as a ratio to Gapdh. Error bars represent S.D., where n=3. (d) Western blot analysis of protein lysates of 3T3 mock or Gabpa overexpressing clones. The h-tubulin housekeeping protein was used as a loading control. (e) Western blots quantified for Gabpa protein levels in Gabpa and mock transfected 3T3 clones. Error bars represent S.D., where n=17 for mock, n=21 for Gabpa clones. D.A. O’Leary et al. / Biochimica et Biophysica Acta 1739 (2004) 81–87 85 eightfold increase in total Gabpa mRNA levels in Gabpa elevated levels of Gabpa protein, these tissues may be clones compared to mock controls (Fig. 2c). However, relatively more sensitive to increased GABPa gene dosage Western blotting showed no corresponding increase in (as occurs in DS), leading to increased Gabpa protein and Gabpa protein levels relative to h-tubulin in Gabpa clones possible contribution to DS pathology. (Fig. 2d and e), suggesting tight regulation of Gabpa protein levels in fibroblast cells. Accordingly there was no significant difference in the morphological or growth 4. Discussion characteristics of the Gabpa transfected cell lines (data not shown). Gabpa and h are co-expressed throughout embryogenesis, consistent with the function of these genes in a 3.3. Expression analysis of Gabpa in Ts65Dn mice complex (O’Leary et al. 2004, manuscript in preparation). This indicates co-ordinate regulation of the two genes, Ts65Dn mice are trisomic for genes between Gabpa although localized to different chromosomes. It is likely and Tmprss2 [5]. Therefore, the effect of increased that translation of these genes is co-ordinately regulated to Gabpa gene dosage upon resultant protein expression preserve effective Gabp complex function although the levels was examined in various tissues. Tissue extracts mechanism for co-ordinate regulation of Gabpa and b from three Ts65Dn mice and three wild-type littermates at remains to be elucidated. It is possible that altered gene 6–8 weeks of age were examined for Gabpa protein by dosage of GABPa in DS may impair GABP function due Western blot analysis (see Fig. 3a for a representative to an imbalance in stoichiometry of the a/h complex, image) and quantified relative to h-tubulin protein levels. although it is also possible that translation mechanisms Gabpa protein is expressed in all tissues examined, yet is may be in place to maintain the stoichiometric balance of elevated in a tissue-specific manner in Ts65Dn animals. the proteins. An average increase of 2.2-fold was observed in brain Elevated GABPa gene dosage in DS fibroblasts and and 1.4-fold in skeletal muscle (Fig. 3b). Given the overexpression of Gabpa mRNA in NIH3T3 fibroblasts do not lead to increased protein levels, despite elevated mRNA levels. The Ts65Dn mouse model, which has three alleles of Gabpa, shows increased Gabpa protein levels in brain and skeletal muscle. Interestingly, we did not detect elevated Gabpa mRNA levels in brains of three Ts65 individuals relative to their control littermates (data not shown). This discordance between elevated gene dosage, unaltered mRNA levels and elevated protein levels may be explained by the operation of both transcription and translation control mechanisms. It is possible that mRNA turnover is altered in the brain such that increased transcription from the third Gabpa allele is not detected. We have identified alternative 5V and 3V UTRs for Gabpa that can result in 12 alternative transcripts. These variations display tissue restricted expres- sionandarepredictedtovaryintheirstabilityand translation efficiency (O’Leary et al. 2004, manuscript in preparation). Furthermore, the brain and skeletal muscle may be sensitive to increased gene dosage, whereas increased Gabpa dosage and/or expression in other tissues (and up to eightfold in NIH3T3 cells) has no impact on Gabpa protein levels and therefore is unlikely to contribute to whole body pathologies in DS. The high mitochondrial load Fig. 3. Effect of increased Gabpa gene dosage in Ts65Dn mice. (a) of brain and skeletal muscle makes these tissues highly Representative image of Western blot analysis of Gabpa protein levels in tissues from adult Ts65Dn (Ts) and wild-type (wt) mice. dependent upon mitochondrial respiration and therefore The h-tubulin protein was used as a loading control. (b) Western blots Gabp regulation of target genes functioning within mito- quantified for Gabpa protein levels relative to h-tubulin in Ts65Dn chondria. It is possible that although Gabpa levels are tissues. Wild-type Gabpa levels were given a value of 1.0 and Ts65Dn generally tightly regulated, in tissues such as brain and levels were expressed relative to this. Error bars represent S.D., where skeletal muscle the demand for Gabp function may result in n=3 for wild-type tissues and Ts65Dn brain, heart, liver and skeletal muscle, and n=6 for Ts65Dn lung, thymus, spleen and kidney. less stringent mechanisms for regulation. Furthermore, * represents statistical significance, as determined by a two-tailed t-test, given the pro-oxidant cellular conditions associated with where P=b0.05. DS and the redox sensitivity of Gabp [19], Gabpa protein 86 D.A. O’Leary et al. / Biochimica et Biophysica Acta 1739 (2004) 81–87 levels may be increased in order to compensate for reduced impaired hippocampal function, Behavioural Brain Research 118 Gabp activity in these high energy tissues. (2001) 53–60. [11] M. 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