Human DNA samples were obtained in full compliance with Yale and NIH Human Investigation Committee regulations.
All cell lines were obtained from American Type Culture Collection (ATCC; Manassas, Vermont, USA). Mouse embryonic carcinoma cells (P19) and human embryonic kidney 293 cells (HEK-293) were cultured in Dulbecco’s modified Eagle medium (GIBCO invitrogen cell culture, Carlsbad, California, USA). Media were supplemented with 10% fetal bovine serum, 2 U/ml penicillin, 2 μg/ml streptomycin, and 2mmol/l L-glutamine (GIBCO invitrogen cell culture). Human neuroblastoma cells [SK-NBE( 2)] were cultured in a 1: 1 mixture of Eagle’s minimum essential medium and F-12K media (ATCC) supplemented with 10% fetal bovine serum, 2 U/ml penicillin, and 2 μg/ml streptomycin. All cells were grown in a humidified incubator at 37°C and 5% CO2.
Electromobility shift assay
Nuclear protein extracts from P19, SK-N-BE(2), and HEK-293 were prepared using the NE-PER Nuclear and Cytoplasmic Extraction kit (PIERCE, Rockford, Illinois, USA) and quantified by BCA protein assay kit (PIERCE). Two sets of double-stranded DNA probes were constructed coding for one copy of GAT1-21. One set of probes was labeled with LI-COR IRDye 700-red, the other with LI-COR IRDye 800-green phosphoramidite (LI-COR Bioscience, Lincoln, Nebraska, USA). The IRDye 800 competitor probe was used in a manner analogous to a nonradioactive competitor probe in radioisotope- based electromobility shift assay (EMSA) assays. For the EMSA binding reactions, a simple competition of the two probes was performed where the ratio of probe to competitor was varied from 1: 1, 1: 2, 1: 5, and 1: 10. Probes were mixed with 1x binding buffer [2.5mmol/l dithiothreitol (DTT)/0.25% Tween-20], 1 μg of Poly (dI-dC) (LI-COR bioscience) and mixed with nuclear lysates. Binding reactions were carried out for 30 min at room temperature with end-over-end mixing and loaded on 4% native acrylamide gels containing 0.38 mol/l of glycine. Gel imaging was carried out using an Odyssey Imaging System (LI-COR Bioscience) at 700 and 800nm wavelengths.
Construction of reporter plasmids
Genomic DNA from samples that were determined to be homozygous for the SLC6A1 insertion or noninsertion alleles with otherwise identical sequences were identified by DNA sequencing. Genomic DNA was amplified by nested PCR. The first PCR used the primers 5′-CTGGGCTGGAGAGAAGGAATCTTTT-3′ and 5′-ATGCAACTCTCGCCTCTGTTCCAG-3′ to yield a DNA fragment of 1.52 kb containing the 5′ UTR and exon 1 of the SLC6A1 gene. PCR reactions were carried out in 50 μl volumes containing 50 ng genomic DNA, 10mmol/l of each dNTP (New England BioLabs, Beverly, Massachusetts, USA), 2 μmol/l of primer, 1x reaction buffer, 1 μl of PfuUltra II Fusion HS DNA Polymerase (Stratagene, La Jolla, California, USA) and 10% sulfolane. PCR products were gel purified and amplified in a second PCR reaction using primers 5′-CTGAGTTCCTGGGGACCCCAGAGGGAAGG-3′ and 5′-CGAGCGGCGCCTCTGCTCCTTCATGTGG-3′. Each 50 μl of reaction contained 1 μl of purified PCR product, 10 mmol/l of each dNTP (New England BioLabs), 2 μmol/l of each primer, 1x reaction buffer, 1 μl of PfuUltra II Fusion HS DNA Polymerase (Stratagene), and 10% dimethyl sulfoxide. PCR products were gel purified and subcloned into the pSTBlue-1 vector (EMD Biosciences, Darmstadt, Germany). Recombinant plasmid clones were sequenced in both directions for verification. Desired clones with and without the 21 bp insertion with otherwise identical sequences and sequences identical to the Genbank reference sequence were selected, digested with NheI and MluI (New England BioLabs), and subcloned into the pGL3-basic reporter vector (Promega, Madison, Wisconsin, USA) to create pGL3-SLC6A1-Ins (two copies of GAT1-21) and pGL3-SLC6A1-Del (one copy of GAT1-21).
Transcription factor constructs
The transcription factors SP1, MAZ1, and ZNF148 were amplified from mouse cDNA using Pfu Ultra II polymerase (Stratagene), cloned in to pSTBlue-1 (Novagen), and subjected to DNA sequencing. SP1 primers included 5′-CGAATTCGCCACCATGAGCGACCAAGATCACTCCATGGA-3′ and 5′-CAAGCTTTCACTTGTCGTCATCGTCTTTGTAGTCGAAGCCATTGCCACTGATATTAATGGACT-3′. ZNF148 primers included 5′-CTCTAGAGCCACCATGAACATTGACGACAAACTGGAAGGATTG-3′ and 5′-CAAGCTTTCACTTGTCGTCATCGTCTTTGTAGTCGCCAAAAGTCTGGCCAGTTGTGGC-3′. MAZ1 primers included 5′-CGAATTCGCCACCATGTTCCCGGTGTTTCCTTGCACGC-3′ and 5′-CAAGCTTTCACTTGTCGTCATCGTCTTTGTAGTCCCAGGGTTGGGAGGGAAGTGGC-3′. Inserts were then subcloned into the pcDNA3.1-Zeo vector (Invitrogen) for overexpression studies. Data were analyzed using SYSTAT version 12 by one-way analysis of variance (ANOVA).
Transient transfection and dual-luciferase assay
P19 and HEK-293 cells were transiently transfected with pGL3-SLC6A1-Ins, pGL3- SLC6A1-Del, or the promoterless pGL3-basic using Fugene 6 transfection reagent (Roche, Basel, Switzerland). SK-N-BE(2) cells were transfected by calcium phosphate. Cells were treated with 125 mmol/l CaCl2 in 2 × (4-(2-hydroxyethyl)- 1-piperazineethanesulfonic acid)-buffered saline buffer (500mmol/l (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), 100 mmol/l Na2HPO4, 1 mol/l NaCl, pH=7.10) for 6 h after 15% glycerol shock for 40 min. Transfections were optimized to yield greater than 60% efficiency as determined by lacZ plasmid transfection and X-gal histochemistry. pRL-SV40 plasmid (Promega) coding for Renilla luciferase was included in all transfection reactions to allow normalization of luciferase activity for differences in transfection efficiencies. Forty-eight hours after transfection cells were harvested and luciferase activity was measured by the Dual-Luciferase Reporter Assay System (Promega) on a DTX 880 Series Multimode Detector (Beckman Coulter, Fullerton, California, USA). Firefly luciferase luminescence values were divided by Renilla luciferase luminescence values from the same transfection to control for differences in transfection efficiency. Of note, one-way ANOVA revealed no significant differences in Renilla luminescence for these experiments. Band intensities were determined using NIH ImageJ software and analyzed using SYSTAT version 12 by one-way ANOVA.
Human SK-N-BE(2) cells (ATCC) were plated into 24-well polystyrene plates (BD Falcon) coated with polyethyleneimine (800 kD; Sigma) at a density of 2 × 105 cells per well. The next day cells were transfected with 200 ng per well of cDNA coding for ZNF148 in the CMV-driven expression vector pCMV-MCS (Stratagene), or with pCMV-MCS as a control. After 48 h cells were harvested and total RNA was prepared using the RNeasy kit (Qiagen). For each well, 0.5 μg of RNA was reverse transcribed using the Superscript II kit and dT primer (Invitrogen) and subsequently treated with 0.5 μl of RNase H (Invitrogen) for 30 min. cDNA was subjected to real-time quantitative PCR (qPCR). TATA-binding protein (TBP) was utilized for the purpose of normalization and was detected with the primers TBPF 5′-TCAGCAGTCAACGTCCCAGCAGGCA-3′ and TBPR 5′-GCTGCGGTACAATCCCAGAACTCTCCGA-3′. Human SLC6A1 was detected with the primers S1F 5′-TGCCGTGGAAACAGTGCGACAACCC-3′ and S1R 5′-TGCATGTTGCGCTCCCAGAACTCCACC-3′ by cycling for 10 min at 95°C followed by 40 cycles of 95°C for 20 s, 72°C for 20 s, and 80°C for 10 s. Fluorescence was acquired at the 80°C step. Amplification efficiencies were determined for each primer set using the dilution series method. Data were analyzed by calculating ratios of SLC6A1 expression relative to TBP expression as [(Efficiency SLC6A1)−CT(SLC6A1)/(Efficiency TBP)−CT(TBP)] where the efficiencies (ranging from 1 to 2) were calculated by the dilution series method and CT(i) refers to the observed threshold crossing for gene i. Data were analyzed using SYSTAT version 12 using one-way ANOVA.
DNA affinity pull-down assay
DNA templates that contained zero, one, or two copies of GAT1-21 were designed for pull-down assays. The noninsertion (one copy) and insertion (two copy) templates were generated by PCR from pGL3-SLC6A1
- Del and pGL3-SLC6A1
-Ins, respectively. The zero-copy template was generated by deletion mutagenesis using overlap extension PCR [22
] using four primers, 5′-TGTAAAACGACGGCCAGTGGCAGACAGGCTGGTGACCCAGGATGA-3′, 5′-TCTCTTCCTCCCTCCCTCGCCTGCCCCGCCGT-3′, 5′-GCAGGCGAGGGAGGGAGGAAGAGA-3′, and 5′-CTTCTTTCCTCTCGCATTC-3′. PCR products were labeled at the 5′ ends by amplification with a dual biotin containing M13 oligonucleotide sequence [23
]. Biotinylated DNA was incubated with 1x-binding buffer (10mmol/l Tris, 50 mmol/l KCl, 1 mmol/l DTT pH=7.5) and nuclear protein lysates at room temperature for 30min with end-over-end mixing. DNA–protein complexes were mixed with Dynabeads MyOne Streptavidin beads (Dynal Biotech ASA, Smestad Oslo, Norway) and incubated for 30 min with continuous mixing. Complexes were purified using a microcentrifuge tube magnet (Dynal). Supernatants containing unbound material were removed using aspiration and beads were washed three times with buffer containing 20mmol/l Tris, 100 mmol/l KCl, 2mmol/l DTT, pH=7.5. Complexes were eluted by adding 6 × SDS sample buffer and heating at 95°C for 5 min. One-dimensional electrophoresis was carried out using 10% SDS–polyacrylamide gel electrophoresis gels run at 10 mA/gel at room temperature. Gels were stained by coomassie blue (GelCode Blue Stain reagent, PIERCE) and destained overnight with ultra pure water, or were stained using silver staining (SilverSNAP Stain kit II, PIERCE). Bands were cut and were subjected to liquid chromatography followed by mass spectroscopy (Yale/NIDA Proteomic Center, Yale University, New Haven, Connecticut, USA). Gel fragments were trypsinized and proteins were analyzed on a Waters Q-Tof ABI mass spectrometer. All MS/MS spectra were searched using the automated Mascot algorithm against the NCBI nr database. Criteria for positive protein identification were: two or more MS/MS spectra matched the same protein entry in the database and the matched peptides were derived from the type of enzymatic digestion performed on the protein. We report only proteins that fulfilled these criteria and where at least 10 peptides matched the protein. Proteins were annotated using STRING (Search Tool for the Retrieval of Interacting Proteins; http://string.embl.de/
) and Bioinformatics Harvester (http://harvester.embl.de/
). Liquid chromatography/tandem mass spectrometry was conducted by the Yale Proteomics Center.
The GAT1-21 sequence (GGGTGGGGAGAGGGAGGGAGG) was cloned upstream of the SV40 promoter in the pGL3-Promoter vector (Promega) in the forward direction in one, two, or four tandem copies. As a control, two tandem copies of a randomly generated sequence (TTATGACGTTATTCTACTTTG) with no homology to any REFSEQ promoter sequence was inserted upstream of the same promoter. Constructs were verified by DNA sequencing. For these experiments we noted that the resulting constructs led to increased Renilla activity. This is a well-known difficulty with the dual luciferase method, and is thought to be because of the ability of very strong promoters to affect other promoters in trans. To prevent any spurious results from regulation of Renilla expression, we normalized our data for these experiments within but not between constructs, with mean Renilla values normalized to control (pGL3 promoter) values. To verify that this procedure did not bias our results, we compared our results to non-normalized values (i.e. absolute firefly luciferase luminescence values) and observed precisely the same pattern that we report here. Two independent sets of experiments were conducted. Data were analyzed using SYSTAT version 12 using one-way or two-way ANOVA with Tukey honestly significant difference (HSD) post-hoc comparisons.
Human postmortem expression analysis
Postmortem brains were collected at the Clinical Brain Disorders Branch, National Institute of Mental Health with informed consent from the legal next of kin under National Institute of Mental Health protocol 90-M-0142 and processed as described previously [24
]. In brief, hippocampus and dorsolateral prefrontal cortex (DLPFC) were dissected. Diagnoses were determined by independent reviews of clinical records and family interviews by two psychiatrists using Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition criteria. Toxicological analysis was conducted on every patient. Normal controls had no known history of psychiatric symptoms or substance abuse, and negative toxicological results. Positive toxicology was not an exclusion criterion for cases with schizophrenia.
For this study, postmortem brain tissue exclusively from African–American individuals between the ages of 18 and 74 years of age (mean=43.21 years) was used because the SLC6A1 insertion polymorphism is found only in individuals of African descent. The sample included 43 males and 30 females and 69 individuals were successfully genotyped. Of these 73 patients, 49 were healthy controls and 24 had a diagnosis of schizophrenia (14 males and 10 females). RNA from hippocampus and DLPFC was reverse transcribed and subjected to real-time qPCR.
We quantified mRNA expression levels of SLC6A1 transcripts by real-time qPCR, using an ABI Prism 7900 sequence detection system (Applied Biosystems). The assay Hs01104469_m1, which contained the primers and a probe, was purchased from Applied Biosystems. Each 10 μl of reaction contained 500 nl of the assay, 5 μl of Taqman Universal PCR Mastermix (Applied Biosystems) containing Hot Goldstar DNA Polymerase, dNTPs with dUTP, uracil-N-glycosylase, passive reference, and 100 ng of cDNA template. PCR cycle parameters were 50°C for 2min, 95°C for 10 min, 40 cycles of 95°C for 15s, and 60°C for 1min. PCR data were acquired from the Sequence Detector Software (SDS version 2.0, Applied Biosystems) and quantified by a standard curve method with serial dilutions of pooled cDNA derived from commercially available human brain reference RNA. In each experiment, the R2 value of the curve was more than 0.99, and controls comprising no-template cDNA resulted in no detectable signal. All samples were measured in a single plate for each transcript, and cycle number at threshold values were in the linear range of the standard curve. All measurements were performed in triplicate, and the gene expression level was calculated as an average of the triplicates. Expression was normalized to the geometric mean of β-actin, β-2- microglobulin, and β-glucuronidase. SLC6A1 genotypes were determined as detailed above except that PCR products were sized on an Agilent Bioanalyzer and PCR products were also subjected to direct sequencing to confirm the genotypes. Genotypes were scored as 0 (only noninsertion alleles present) or 1 (at least one insertion allele present). This coding scheme was used in part because only one patient was homozygous for the insertion allele in our sample. Data were analyzed using a multiple regression model with SAS PROC GLM version 9.1.3. This model included diagnosis, age at death, SLC6A1 genotype, sex, and their interactions, as well as an index of RNA quality (Agilent RIN index). Interaction effects were interpreted by calculating a categorical age at death variable as a median split of the age at death variable. Means and standard errors for graphical analysis were calculated with SAS GLM using model-based least squares means and standard errors.