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Physiol. Genomics 28: 213-222, 2007. First published September 19, 2006; doi:10.1152/physiolgenomics.00155.2006
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Received 18 July 2006; accepted in final form 13 September 2006.
Physiological Genomics 28:213-222 (2007)
1094-8341/07 $8.00 © 2007 American Physiological Society

Differential brain transcriptome of ß4 nAChR subunit-deficient mice: is it the effect of the null mutation or the background strain?

Merav Kedmi and Avi Orr-Urtreger

Genetic Institute, Tel Aviv Sourasky Medical Center, and Sackler Faculty of Medicine, Tel Aviv University, Tel Aviv, Israel


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Studies using mice with ß4 nicotinic acetylcholine receptor (nAChR) subunit deficiency (ß4–/– mice) helped reveal the roles of this subunit in bradycardiac response to vagal stimulation, nicotine-induced seizure activity and anxiety. To identify genes that might be related to ß4-containing nAChRs activity, we compared the mRNA expression profiles of brains from ß4–/– and wild-type mice using Affymetrix U74Av2 microarray. Seventy-seven genes significantly differentiated between these two experimental groups. Of them, the two most downregulated were spastic paraplegia 21 (human) homolog (Spg21) and 6-pyruvoyl-tetrahydropterin synthase (Pts) genes. Since the targeted mutagenesis of the ß4 nAChR subunit was done by using two mouse strains, 129SvEv and C57BL/6J, it is possible that the genes closely linked to the mutated ß4 gene represent the 129SvEv allele and not the control C57BL/6J-driven allele. We examined this possibility by using public database and quantitative RT-PCR. The expression levels of Spg21 and Pts genes that, like the ß4 gene, are localized on mouse chromosome 9, as well as the expression levels of other genes located on this chromosome, were dependent on the mouse background strain. The 67 differentially expressed genes that are not located on chromosome 9 were further analyzed for overrepresented functional annotations and transcription regulatory elements compared with the entire microarray. Genes encoding for proteins involved in tyrosine phosphatase activity, calcium ion binding, cell growth and/or maintenance, and chromosome organization were overrepresented. Our data enhance the understanding of the molecular interactions involved in the ß4 nAChR subunit function. They also emphasize the need for careful interpretation of expression microarray studies done on genetically manipulated animals.

nicotinic acetylcholine receptor; ß4 subunit; knockout mice; microarray; gene expression; background mouse strain


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
THE NICOTINIC ACETYLCHOLINE RECEPTORS (nAChRs) are allosteric membrane proteins that belong to a large family of ligand-gated ion channels and are expressed throughout both the central and peripheral nervous systems and in nonneuronal cells (19, 25). These receptors mediate the effects of the endogenous neurotransmitter, acetylcholine, and the tobacco alkaloid, nicotine, and are involved in a wide range of neuronal processes, including cognition, memory (28, 30, 34), and autonomic nervous system functions (53, 55). Furthermore, the nAChRs have been implicated in complex diseases affecting the nervous system, including epilepsy, schizophrenia, as well as the neurodegenerative Parkinson and Alzheimer diseases (2, 39).

The nAChRs are composed of {alpha}- and ß-subunits, which can assemble as hetero- or alpha-only homo-pentamers (19). To date, 12 distinct genes encoding neuronal nAChR subunits have been identified ({alpha}2–10, ß2–4), generating an abundance of structurally and functionally distinct receptors. The combinations of {alpha}- and ß-subunits determine channel properties such as desensitization, specificity to agonists and antagonists and permeability to ions (3, 17, 18, 27, 36).

The study of knockout mice, deficient for specific nAChR subunits, has advanced our understanding of the function of distinct subunits and has further revealed roles for endogenous receptors normally assembled with the deficient subunit (6, 33). Studies of mice lacking the ß4 subunit have helped to define the roles of both central and peripheral ß4-containing nAChRs. The ß4 subunit is widely expressed in the peripheral nervous system (1, 13, 29, 41), and studies in mice lacking this subunit revealed altered autonomic functions, including attenuated bradycardiac response to vagal stimulation, increased sensitivity to hexamethonium blockade, and significantly reduced ileal contractile responses to nicotinic agonists (52). In the central nervous system, ß4 expression has been detected in the olfactory bulb, medial habenula, ventral thalamic nucleus, interpeduncular nucleus, and the hippocampal CA1 region (11, 14, 35, 42, 54), and knockout mouse studies have supported an important role for this subunit in centrally mediated mechanisms. ß4 Null mice are highly resistant to nicotine-induced seizure activity (26, 44), exhibit altered anxiety-related behaviors (46) and decreased nicotine withdrawal symptoms (45). Additionally, ß4–/– mice were shown to have decreased sensitivity to the hypolocomotive effects of low doses of nicotine (44), lower core body temperature, and decreased nicotine-induced hypothermia (43).

To further understand the molecular basis of ß4 nAChR subunit function and to identify genes possibly related to ß4-containing nAChRs functions, we compared the global gene expression profile in brains of ß4 subunit-deficient mice to that of wild-type mice.


   METHODS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals
All mice used in this study were from congenic lines that were back-crossed onto a C57BL/6J background for seven generations after germline transmission. The mice used for microarray expression analysis were ß4 nAChR subunit-deficient [ß4–/– mice, (55)] and wild-type littermate control mice. The experimental mice were generated by breeding pairs of mice heterozygous for the ß4 nAChR subunit null mutation (ß4+/–). In each experimental group the male-to-female ratio was of ~50/50. Each experimental mouse was genotyped twice, once before and once after the experiment. The PCRreaction conditions and primer pairs used for genotyping these mice were previously described (26).

Prior to the experiments, the mice were housed in groups of 2–5 per cage under a 12:12-h light-dark cycle, with food and water ad libitum. The study was approved by the institutional animal and care committee, in accordance with the NIH Guide for the Care and Use of Laboratory Animals.

DNA Microarray Analysis
Target preprocessing and hybridization.
The experimental groups included six wild-type mice and five ß4–/– mice. All mice were 6–9 wk old. Microarray experiments were designed to comply with Minimum Information About a Microarray Experiment guidelines (4). Total RNA was isolated from whole brains using TRI reagent (Sigma-Aldrich, St. Louis, MO) according to the manufacturer's instructions and further purified on a Qiagen RNeasy mini kit (Qiagen Sciences, Germantown, MD). Ten micrograms of total RNA were used to synthesize double-stranded cDNA with the Superscript Choice System (Invitrogen, Carlsbad, CA) and 100 pmol of oligo-dT primer attached to a T7 promoter sequence (Genset, Paris, France). The double-stranded cDNAs were phenol-chloroform extracted with Phase Lock Gels tubes (Eppendorf, Hamburg, Germany). In vitro transcription of the double-stranded cDNA products was performed with an Enzo BioArray High Yield Transcript labeling kit in the presence of biotin-labeled nucleotides (Affymetrix, Santa Clara, CA). Following purification by an RNeasy mini kit (Qiagen Sciences), the biotin-labeled cRNA products were subjected to alkaline lysis (200 mM Tris-acetate pH 8.1, 500 mM potassium acetate, 150 mM magnesium acetate), and the fragmented cRNAs were hybridized overnight at 45°C to Affymetrix Gene Chip Murine Genome U74Av2 arrays (Affymetrix) that contain ~12,000 sequences. Following posthybridization washes, the arrays were stained with streptavidin-phycoerythrin (Molecular Probes, Eugene, OR) and scanned with an Affymetrix Gene-Array Scanner (Affymetrix).

Data analysis.
The statistical algorithm, implanted in Affymetrix Suite version 5.0 software (MAS5, Affymetrix) generated a signal value (a relative measure of the abundance of the transcript), a detection call (present, absent, or marginal) and a P value (which indicates the reliability of the transcript's detection call) for each transcript on the array. The detection calls were calculated, based on the detection P value, as follows: probe sets with P value of >0.06 were designated as absent, P value of >0.04 and <0.06 as marginal and P value of <0.04 as present. For interarray comparisons, data from each array were scaled using MAS5 software, and the mean intensity for each array was adjusted by a scaling factor to a set target intensity of 150. Raw data have been deposited to the National Center for Biotechnology Information Gene Expression Omnibus (GEO; http://www.ncbi.nlm.nih.gov/geo) and are accessible through GEO Series accession number GSE5320.

We further analyzed the microarray data using the GeneSpring version 7 software (Silicon Genetics, Redwood City, CA). The signal values were normalized both per each gene and per the entire array by dividing each gene's signal by its median value, and by the median of the array, respectively. The normalized data were then subjected to filtering, leaving only probe sets that had present or marginal calls in at least five out of the eleven tested arrays. Only transcripts that passed the filtration procedure were used for the following statistical analysis. The statistical analysis was applied on all 11 arrays. Nonparametric t-test was conducted with Benjamini and Hochberg false discovery rate (FDR) correction for multiple comparisons with a P value cutoff of 0.05 to detect genes that significantly discriminate between brains of wild-type and ß4 nAChR subunit-deficient mice.

Expression Analysis of Chromosome 9-located Genes
The ß4 nAChR subunit gene (Chrnb4) is localized on mouse chromosome 9. To assess the "background flanking genes" effect on the expression of the significantly changed genes in brains of ß4–/– mice, genes localized to chromosome 9 were analyzed. Their expression was downloaded from a published microarray database [(56), www.barlow-lockhartbrainmapnimhgrant.org]. This database contains microarray analysis obtained by Affymetrix Gene Chip Murine Genome U74Av2 arrays of 24 neural tissues from 8 wk mice from five different mouse strains, including 129SvEv and C57BL/6J mouse strains, which were used to generate the ß4–/– mice (55). We used 2–3 replicates for each brain area. For the analysis of the expression levels of the significantly changed chromosome 9-located genes, we calculated the replicates average and then the ratios between 129SvEv and C57BL/6J mice in each brain area. Finally, the average ± SD of the ratios of all brain areas was used as a value representing the ratio of individual gene expression in 129SvEv/C57BL/6J. We then compared this expression ratio to that of ß4–/– and wild-type mice in our experiment.

Gene Ontology Annotation Analysis
Genes that significantly differentiate between brains of wild-type and ß4 null mice, and are not localized on chromosome 9, were categorized into gene ontologies (GO) according to their molecular functions, associated biological processes and/or cellular components in a species-independent manner, using EASE software online [(10, 23), http://david.abcc.ncifcrf.gov/]. Fisher exact probability test with a P value threshold of 0.05 was applied to filter the significantly overrepresented GO annotations compared with all genes on the U74Av2 microarray.

Transcriptional Regulatory Network Analysis
We used the PAINT v3.3 program [(49), http://www.dbi.tju.edu/dbi/tools/paint/] to analyze the promoters of the genes that significantly discriminated between brains of ß4–/– and control mice and are not linked to chromosome 9. This program contains a database of sequences located upstream to the transcription start site (TSS) for all annotated (known and putative) genes in the Ensembl genome database. The 5000 bp sequences upstream to the TSS, which is conventionally considered to contain the potential promoters, were retrieved for each gene. Then, PAINT searched in these upstream sequences for transcription regulatory elements (TREs) of known transcription factors. The TREs were retrieved from the TRANSFAC Public database (http://www.gene-regulation.com/cgi-bin/pub/databases/transfac/search.cgi). The representation of these TREs among the genes that significantly differentiated between brains of ß4–/– and wild-type mice was compared with their representation in all the genes on the U74Av2 microarray. Significant over- or underrepresentation of TREs was defined by a threshold of P < 0.1, calculated by PAINT software that use the hypergeometric distribution as previously described (49).

Quantitative Real-time PCR Assay
Real-time PCR analyses using LightCycler (Roche Applied Science, Mannheim, Germany) were performed to determine the expression levels of the two spastic paraplegia 21 (human) homolog (Spg21) gene transcripts (long and short Spg21) and the 6-pyruvoyl-tetrahydropterin synthase (Pts) gene in whole brain RNA. The cDNA synthesis was performed using 100 units of Superscript III reverse transcriptase (Invitrogen), 125 µM each dNTP (Pharmacia), and 75ng/µl random primers (Invitrogen) in a total volume of 10 µl. Quantitative RT-PCRs were performed using LightCycler FastStart DNA Master SYBR Green I (Roche Applied Science). The PCR reactions were performed in a total volume of 10 µl, with 3 mM of MgCl2 and 0.5 µM of each primer (except for Gapdh where primer concentrations of 0.2 µM were used). All primer pair sequences (Sigma-Genosys, Rehovot, Israel) and their product sizes are detailed in Table 1. The expression value of each gene was normalized using Gapdh expression levels. The quantification procedure was done as previously described (26). Amplified products were verified by electrophoresis on 2% agarose gel stained with ethidium bromide (Sigma-Aldrich) and confirmed by sequencing using BigDye Terminator v1.1 on ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Foster City, CA).


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  		Table 1. Primer pairs for real-time quantitative RT-PCR analyses

 
Additional Statistical Analyses
Results are expressed as means ± SD. Statistical significance was determined by Student's t-test or by 1-way ANOVA followed by Bonferroni post hoc (SPSS, Chicago, IL). Graphs were constructed with Prism software (Prism; GraphPad Software, San Diego, CA).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Identification of Genes With Expression Levels That Significantly Discriminate Between Brains of Wild-type and ß4 nAChR Subunit-deficient Mice
The global expression profiles of whole brains from six wild-type and five ß4–/– mice were compared. After filtration of the microarray data, leaving only transcripts that were either present or marginal in at least five out of the eleven experimental arrays, a list of 6,167 probe sets was generated. These 6,167 transcripts were used for statistical analysis to find genes with expression levels that significantly changed in brains of ß4–/– mice. Nonparametric t-test with Benjamini and Hochberg FDR correction and P value cutoff of 0.05 identified 77 genes that significantly differentiated between brains of ß4–/– and control mice. These genes are listed in the supplemental data table with their expression levels and functional annotations (the online version of this article contains supplemental material). Among these genes, 57 were downregulated and 20 genes were upregulated in brains of ß4–/– mice. Hierarchical clustering of these 77 differentially expressed genes (Supplemental data table), using GeneSpring version 7 software (Silicon Genetics), resulted in a graphical representation (Fig. 1) and demonstrated that all samples were clearly clustered into two distinct groups according to their wild-type or ß4–/– genotypes. Among the 77 significantly changed genes, the most downregulated gene was the Spg21 gene, and the most upregulated gene was the cell division cycle 2 homolog (S. pombe)-like 1 (Cdc2l1) (0.17- and 2.14-fold change in brains of ß4–/– mice compared with wild-type controls, respectively).


Figure 1
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  		Fig. 1. Hierarchical clustering of the 77 genes that significantly distinguished between brains of ß4–/– and wild-type (WT) mice. Lower expression levels are represented in black and high expression in white.

 
One of the Two Spg21 Transcripts Expressed in Mouse Brain is Downregulated in ß4–/– Mice
The Spg21 gene was the most downregulated of the 77 significantly changed genes in brains of ß4–/– mice. According to Affymetrix GeneChip results, its transcript level was 5.9-fold lower in brains of ß4–/– mice (P < 0.0005, Student's t-test). However, quantitative RT-PCR analysis using primers that encompass exons 3–5 of this gene failed to validate this result. The relative expression levels of exon 3–5 transcripts were 2.31 ± 0.24 and 2.63 ± 0.22 in brains of wild-type and ß4–/– mice, respectively (n = 5 brains in each group, values represent the ratio between relative expression levels of Spg21 and Gapdh genes).

We further analyzed the Spg21 expression level by quantitative RT-PCR using a primer pair designed for the target sequence of the Affymetrix's Spg21 probe set, which is located in the 3'-untranslated region (UTR) of this gene (Table 1). The expression level of the 3'-Spg21 region was 6.2 times lower in brains of ß4-deficient mice compared with wild-type mice (n = 5 in each group, P < 0.05, Student's t-test), confirming the significantly decreased expression observed in brains of ß4–/– mice with the Affymetrix GeneChip. Using basic local alignment search tool (http://www.ncbi.nlm.nih.gov/BLAST) and MapViewer (http://www.ncbi.nlm.nih.gov/mapview), we detected two alternatively spliced transcripts with different 3'-ends of the Spg21 gene, referred to here as long Spg21 and short Spg21. The long Spg21 transcript (GenBank accession number: NM_138584) includes all 9 exons, and the short variant (GenBank accession number: AK090017) includes only 8 exons (Fig. 2).


Figure 2
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  		Fig. 2. Schematic representation of the long and short transcripts of the spastic paraplegia 21 (human) homolog (Spg21) gene expressed in the mouse brain. Schematic structure of the long (NM_138584) and the short (AK090017) transcripts of the mouse Spg21 gene. The exons depicted as boxes, the coding sequences are in light gray and the untranslated regions (UTRs) in dark gray. The upper bold number in each box represents the exon number, and the lower number represents the exon's expected size in base pair. The ratio values of ß4/WT demonstrates the expression fold change between brains of ß4–/– and WT mice analyzed by quantitative RT-PCR.

 
Different sets of primer pairs were used for each transcript (Table 1 and Fig. 2): unique reverse primers for specific amplification of either the long Spg21 transcript from its 3'-UTR region (nucleotides 1573–1598) or for the short Spg21 transcript from its exon 8 (nucleotides 1066–1084) were used. The forward primers for these two specific PCR reactions were located on sequences that are common to both transcripts.

The expression levels were determined using LightCycler quantitative RT-PCR. Standard curves of copy numbers were generated for each of the transcripts and for Gapdh. The expression level values for the long and the short Spg21 transcripts are represented as the number of mRNA copies of either the long or the short transcript/Gapdh copies number x 103. Using known copy numbers as a standard curve also allowed us to compare between the abundance of these two transcripts. We found that the long Spg21 transcript is 85.4 times more abundantly expressed in whole brains of wild-type mice than the short Spg21 transcript.

The expression level of the long Spg21 transcript was significantly downregulated in brains of ß4–/– compared with wild-type control mice (P < 0.0005, Student's t-test). The expression levels of the long Spg21 transcript were 3.55 ± 1.12 and 26.94 ± 8.79 in brains of ß4–/– and wild-type mice, respectively, representing a 0.13-fold decrease (7.6 times lower) in the expression levels of this transcript in brains of ß4–/– mice. Conversely, the expression levels of the short Spg21 transcript were significantly higher in brains of ß4–/– compared with wild-type control mice (Student's t-test P < 0.005; 0.69 ± 0.22 and 0.32 ± 0.13 in brains of ß4–/– and wild-type mice, respectively), representing a 2.6-fold increase in the expression levels of the short Spg21 transcript in brains of ß4–/– mice.

Background Flanking Genes
The genetically manipulated mice used in this study were derived from 129SvEv mouse strain embryonic stem (ES) cells and blastocysts from C57BL/6J mouse strain. The chimeric mice were further bred with C57BL/6J mice to achieve first germline transmission of the mutation, and then congenic line (55). As a consequence, even after backcrossing with C57BL/6J mice for seven generations, the genes closely linked to the mutated Chrnb4 gene can potentially represent the 129SvEv allele, while the wild-type control allele is a C57BL/6J-driven allele. Since mice from different genetic background strains exhibit changes in behavior as well as in gene expression (16, 47), we further analyzed the possibility that the differences between 129SvEv and C57BL/6J mouse strains influenced the expression of genes in control and ß4–/– mice. To assess this "background flanking genes" effect, the following approach was taken: We used the published microarray database that compared the brain expression profile of five different mouse strains, including 129SvEv and C57BL/6J mouse strains [(56), www.barlow-lockhartbrainmapnimhgrant.org, Table 2]. Since the Chrnb4 gene is localized to mouse chromosome 9, all chromosome 9-localized genes with significantly altered expression were examined; we found 10 out of the 77 significantly changed genes in the brains of ß4–/– mice localized to chromosome 9. Among them were the Spg21 and the Pts, which were the two most downregulated genes among the 77 significantly changed genes. On the basis of the Barlow-Lockhart public database, their expression levels were lower in the brain areas of 129SvEv mice compared with C57BL/6J mice (Table 2), suggesting that the expression of the Spg21 and Pts genes is affected by the background mouse strains. In contrast, we identified genes on chromosome 9 with no altered expression between brains of 129SvEv and C57BL/6J mice in the Barlow-Lockhart database that were significantly changed in brains of ß4–/– mice. For example, the fold changes of Acaa1 gene that encodes the acetyl-CoA acyltransferase 1 enzyme, which is involved in peroxisomal ß-oxidation and in the metabolism of very long chain fatty acids (7, 50), were 0.99 between 129SvEv and C57BL/6J mice and 0.65 between ß4–/– and wild-type mice (Table 2).


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  		Table 2. Comparison between brain expression level of chromosome 9-linked genes in 129SvEv and C57BL/6J mouse strains

 
For the differentially changed genes on chromosome 9 ("flanking genes"), a relationship between the physical distance from the null allele and the probability of strain related differences is expected. Such a tendency is demonstrated in Table 2. Genes that are located closer to the Chrnb4 gene, like Adpgk and Dlat (4.29 and 4.46 Mbp away from the Chrnb4, respectively), are affected by the background mouse strains. Conversely, genes located farther away from the Chrnb4 gene, such as Acaa1 and Deb1 (64.2 and 66.6 Mbp from the Chrnb4, respectively), are likely not affected by the background strains.

Downregulation of Spg21 and Pts Genes in Brains of ß4–/– Mice is the Result of the Background Flanking Gene Effect
In addition to our analysis of the published data, the expression levels of the long and short Spg21 transcripts and the Pts gene were analyzed in brains of 129SvEv and C57BL/6J wild-type mice and in brains of ß4 nAChR subunit-deficient mice. The expression of the two most significantly downregulated genes, the long Spg21 and Pts, in brains of 129SvEv wild-type mice was significantly lower than in brains of C57BL/6J wild-type mice and similar to their expression levels in brains of ß4–/– mice (P < 0.0005, one-way ANOVA followed by Bonferroni post hoc, Fig. 3). The expression of the short Spg21 transcript was significantly higher in brains of 129SvEv wild-type mice and ß4–/– mice compared with its expression in brains of C57BL/6J wild-type mice (P < 0.001, one-way ANOVA followed by Bonferroni post hoc, Fig. 3). These results support the results obtained using the Barlow-Lockhart public database, suggesting that the expression levels of Spg21 and Pts genes are determined by the background mouse strains.


Figure 3
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  		Fig. 3. Expression levels of the long and short Spg21 transcripts and the 6-pyruvoyl-tetrahydropterin synthase (Pts) gene in whole brains of C57BL/6J and 129SvEv WT mice and in brains of ß4–/– mice. The expression levels of Spg21 transcripts and Pts gene were determined relative to a dilution series of cDNA and normalized to Gapdh gene expression. The expression of Spg21 long transcript and Pts gene were significantly reduced in brains of ß4–/– mice and 129SvEv WT mice compared with brains of C57BL/6J WT mice. The expression of the short Spg21 transcript were significantly higher in brains of ß4–/– mice and 129SvEv WT mice compared with brains of C57BL/6J WT mice. Bars represent means ± SD. *P < 0.0005, **P < 0.001, ANOVA followed by Bonferroni post hoc.

 
Genes Localized to Mouse Chromosome 9 are Significantly Overrepresented Among the Significantly Changed Genes in Brains of ß4–/– Mice
We determined the chromosomal assignment of the significantly changed genes and of all the U74Av2 array genes. The representations of each chromosome in the significantly changed genes and in all the U74Av2 array genes were analyzed for over- or underrepresentation. This analysis was performed by comparing the proportion of number of genes located on each chromosome, out of the 77 significantly altered genes in brains of ß4–/– mice, to the proportion of genes on each chromosome out of the entire U74Av2 array. Of the 77 changed genes, 10 are localized to chromosome 9 (12.99%), while only 4.90% of the genes on the array are on chromosome 9 (P < 0.001, {chi}2, Fig. 4). None of the other chromosomes showed significant over- or underrepresentation when we compared the 77 changed genes to the entire array (Fig. 4).


Figure 4
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  		Fig. 4. Chromosome 9-located genes are significantly overrepresented among the significantly changed genes in brains of ß4–/– mice. A comparison of the chromosomal representations of the 77 significantly changed genes and the genes on the entire U74Av2 array. Bars represent the percentage of genes localized to each chromosome among the 77 significantly changed genes (light gray) and among all the genes on the array (dark gray). Chromosome 9 is overrepresented among the 77 significantly changed genes. NA, not assigned. *P < 0.001, {chi}2.

 
Assessment of the Background Mouse Strain Effect on Genes That are not Localized on Chromosome 9
The expression levels of the remaining 67 genes, which were significantly changed in brains of ß4 nAChR subunit-deficient mice and that are not linked to chromosome 9, are likely influenced by the mutation. However, it is also possible that the strain-affected chromosome 9-linked genes modify their expression. To assess this possibility, we randomly selected one gene from each chromosome out of the 67 significantly changed genes, and compared their expression levels in brains of 129SvEv and C57BL/6J mouse strains using the Barlow-Lockhart public database [(56), www.barlow-lockhartbrainmapnimhgrant.org, Table 3]. The expression levels of two genes, Hist1h2bc and Anxa3, were lower in brains of 129SvEv mice compared with C57BL/6J mice, as well as in brains of ß4–/– mice compared with wild-type controls (Table 3). One gene, Gpr178, was 1.53-fold upregulated in brains of 129SvEv mice compared with C57BL/6J mice but was 0.45-fold downregulated in brains of ß4-deficient mice. The expression levels of all other 16 genes analyzed were not changed between the brains of 129SvEv and C57BL/6J mice.


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  		Table 3. Comparison of the expression levels of genes that are not linked to chromosome 9 between brains of 129SvEv and C57BL/6J mouse strains

 
Genes That Encode Proteins With Tyrosine Phosphatase Activity are Upregulated and Genes Involved in Cell Growth are Downregulated in Brains of ß4–/– Mice
Of the 77 differentially expressed genes (Supplemental data table), the 67 genes that are not localized on chromosome 9 were further classified into GO annotation categories. The annotations that were significantly overrepresented in these genes are shown in Fig. 5, A (upregulated genes) and B (downregulated genes). Among the upregulated genes, the two most significantly overrepresented annotation categories were those of proteins with protein metabolism and tyrosine phosphatase activity (53.3 and 12.5% of the upregulated genes compared with 20.9 and 0.8% of the genes on the array, P = 0.0055 and 0.0071, respectively, Fig. 5A). The most significant overrepresented biological process and molecular function annotation categories among the downregulated genes included proteins involved in calcium ion binding and transferring alkyl or aryl groups activity and cell growth and/or maintenance (18.5, 7.4, and 54.2% of the downregulated genes compared with 4.7, 0.59, and 31.3% of the genes on the array, P = 0.0076, 0.011, and 0.016, respectively; Fig. 5B). Of note is that the annotation category "chromosome organization and biogenesis" included two downregulated genes and one upregulated gene. Interestingly, 23 of the 67 differentially expressed genes (6 upregulated and 17 downregulated genes) encode for membrane proteins.


Figure 5
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  		Fig. 5. Functional annotation analysis of the genes that significantly differentiate between brains of WT and ß4–/– mice and are not located on mouse chromosome 9. Annotation analysis was done using EASE online software and categories with P < 0.05 (Fisher exact probability) and a threshold of 2 genes were chosen. The gene ontology (GO) annotation trees demonstrate the categories that were overrepresented among the upregulated (A) or the downregulated (B) genes. In brackets, the 1st number represents the number of significantly changed genes that belong to the specific main GO annotation category (i.e., biological process, molecular function or cellular component), and the 2nd is the number of genes on the entire U74Av2 array that belong to this main category. In parentheses, the 1st number indicates the number of significantly altered genes found in this annotation category; the 2nd number is the percent of significantly changed genes that belong to this category (out of the significantly changed genes in the main category); the 3rd number represents the percent of genes in the entire array belonging to this category (out of all array genes in the main category); and the 4th number is the calculated P value.

 
Transcriptional Regulatory Network Analysis of the Genes That Discriminate Between Brains of Wild-type and ß4–/– Mice
Using the PAINT v3.3 program, we identified TREs that were overrepresented among the 67 genes that differentiated between wild-type and ß4–/– mice and are not linked to chromosome 9, compared with all genes on the U74Av2 microarray (Table 4). The most significantly overrepresented TRE in the promoters of the 19 upregulated genes in brains of ß4–/– mice was a promoter element that binds the Evi-1 transcription factor. Overrepresentation of two elements of NF-{kappa}B and of Gata1 transcription factors was detected among the group of the upregulated genes. Among the promoters of the 48 downregulated genes, the most significantly overrepresented TRE was for the Zfp482 transcription factor. Overrepresentation of the Foxj2 and Areb6 TREs was also significant among the promoters of the downregulated genes (Table 4).


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  		Table 4. Significantly enriched TREs in the 5' upstream region of the 67 genes that discriminate between brains of wild-type and ß4-deficient mice and are not located on mouse chromosome 9

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
We hereby report the first global microarray expression analysis of brains of adult mice with ß4 nAChR subunit deficiency. The expression level of 77 genes significantly distinguished between brains of control and mutant mice. Among them, the two most downregulated were the Spg21 and the Pts genes. Both Spg21 and Pts genes are located on mouse chromosome 9, as is the nAChR ß4 subunit (Chrnb4) gene. Our combined analysis using quantitative RT-PCR and a public expression database demonstrated that their expression levels were determined by the mouse background strain and not by the ß4 nAChR subunit deletion. Like most null mutant mice, the ß4 nAChR subunit-deficient mice were generated using ES cells derived from 129SvEv mouse strain and blastocysts from C57BL/6J (55). As a result, null mutants are of mixed strain, and even in the congenic lines, which were obtained by seven generations of backcrossing with C57BL/6J mice, the region flanking the mutant ß4 gene represents the 129SvEv allele, while the wild-type control allele is a C57BL/6J-driven allele (16). Mice from different genetic background strains exhibit changes in behaviors, such as anxiety-related behaviors (24), prepulse inhibition of acoustic and tactile startle responses (38), voluntary activity on a running wheel (5), fear conditioning (15), and hippocampus-dependent learning and memory (9). In addition, alteration in genes expression levels were observed in mRNA obtained from mice of different background strains (5, 12, 24, 37, 47). Thus, the phenotypic and transcriptional characteristics seen in knockout or transgenic mice might be influenced by the contribution of the parental alleles.

Ten of the 77 genes that significantly changed in brains of ß4–/– mice are located on chromosome 9. Zapala et al. (56) published a microarray database (www.barlow-lockhartbrainmapnimhgrant.org) that included a comparison of C57BL/6J and 129SvEv mRNA expression in >20 different brain areas. Our analysis of this database demonstrated that of the 10 differentially expressed genes located on chromosome 9, the expression levels of seven differed between these two mouse strains and are therefore strain-dependent. In contrast, the expression of three chromosome 9-located genes, Neo1, Acaa1, and Ptplad1, which significantly changed in ß4-deficient mice did not change between C57BL/6J and 129SvEv mice. The expression of these genes could be therefore modified by the ß4 subunit deficiency and not by the mouse background strain. Using this public database, we were able to assess the effect of the mouse background strain on the expression of genes flanking Chrnb4 on chromosome 9 and to detect genes whose expression is likely modified by the mutation and not by the background strain.

Using the Barlow-Lockhart database (56), we also assessed the possibility that the expression levels of differentially changed genes that are not localized on chromosome 9 might also be modified by the strain effect. The expression of most genes was not changed between the mouse strains (Table 3, 16/19, 84%), suggesting that the expression changes were due to the null mutation. In contrast, the expression of two genes, Hist1h2bc and Anxa3, was downregulated in brains of 129SvEv mice compared with C57BL/6J mice, as well as in brains of ß4–/– mice compared with wild-type controls, and the expression of another gene, Gpr178, was upregulated in brains of 129SvEv (compared to C57BL/6J) but was downregulated in brains of ß4-deficient mice. These results may suggest, but not necessarily prove, the possibility that chromosome-9 strain-affected genes can disrupt extra-chromosome 9 genes in a trans way. However, more experiments are needed to determine whether these patterns of gene expressions are related or are independent events.

The expression of Spg21 gene was further analyzed. Recently, it was shown that a frame-shift mutation (601insA) in the human SPG21 gene causes Mast syndrome, an autosomal recessive disease detected among the Old Order Amish. This is a slowly progressive disease, with onset in childhood, leading to spastic paraplegia and dementia (48). Two alternative spliced variants of the Spg21 gene were detected in mouse brains, which differ at their 3'-end. The expression level of the long Spg21 was ~85-fold higher than that of the short splice variant, suggesting a more significant role for the long Spg21 splice variant in mouse brains. In addition, upregulation of the short Spg21 transcript alone cannot explain the normal mRNA levels of exons 3–5 of the Spg21 gene that were found in brains of ß4-deficient mice. It is therefore possible that other, yet unidentified transcripts of Spg21 gene are expressed in the mouse brain. The analysis of the long Spg21 transcript expression pattern demonstrated lower expression levels in brains of both ß4 subunit-deficient mice and 129SvEv wild-type mice compared with C57BL/6J wild-type mice, while the short Spg21 transcript demonstrated an opposite pattern of expression. Our data therefore suggest that splicing of Spg21 is different between mouse strains. Differential expression of alternative spliced transcripts between mouse strains was previously detected. For example, differential alternative splicing of H2-Bl gene, a member of the MHC class Ib, was identified in the gut of inbred, outbred, and wild-derived mouse strains (20). Another example is the strain-specific alternative splicing of Pas1c1 gene, which might be the result of a polymorphism in the 3'-acceptor splice site of the alternatively spliced exon (51).

Since the expression of the genes located on chromosome 9 is likely affected by the mouse strain, while the expression of genes on other chromosomes is possibly influenced by the ß4 nAChR subunit deficiency, we further analyzed 67 differentially expressed genes in brains of ß4–/– mice that are not located on mouse chromosome 9. Several common overrepresented functional motifs were identified among this group of genes. Among them were genes encoding for membrane proteins, for proteins involved in cell growth, chromosome organization, and calcium ion binding and for proteins with GTPase and protein-tyrosine phosphatase activities, suggesting that ß4-containing nAChRs may be involved in these cellular processes. Interestingly, the most significant annotation group of upregulated genes in brains of ß4-deficient mice encodes for proteins involved in protein metabolism and, more specifically, for proteins with tyrosine phosphatase activity. Members of this phosphatase family are known to be involved in the regulation of cellular processes including cell growth, differentiation, mitotic cycle, and oncogenic transformation. It has also been shown that phosphorylation and dephosphorylation are important regulatory mechanisms involved in the activation, localization, and desensitization of ligand-gated ion channels (reviewed in Ref. 22), and that functional {alpha}7 nAChRs are indirectly regulated by tyrosine phosphorylation (8). Additionally, Marszalec et al. (32) have recently demonstrated that inhibition of phosphatase activity accelerates the desensitization recovery rate of {alpha}-bungarotoxin-insensitive (non-{alpha}7) nAChRs. Therefore, our results suggest a possible feedback loop between nAChRs and protein tyrosine phosphatases whereby the deficiency of a specific nAChR subunit, such as ß4, influences the expression of several tyrosine phosphatase genes.

Our analysis of the promoter regions of the genes that were significantly altered in brains of ß4-deficient mice, and are not located on chromosome 9, demonstrated that they share several specific TREs. These results suggest that gene expression changes observed in brains of ß4 nAChR subunit-deficient mice may be mediated by several distinct transcription factors as a part of a nAChR-associated gene regulatory network. For example, two significantly overrepresented TREs, for the Nkx2–5 and Zfhx1a transcription factors, which were previously shown to compete for an overlapping DNA binding site (40), were detected in the promoters of the downregulated genes in brains of ß4–/– mice. Another example of a more complex interaction is that the null mutation modulates the expression of genes that in turn influence the activation of transcription factors, which then regulate the transcription of other genes. Of note, the G protein G{alpha}11, which was downregulated in brains of ß4–/– mice, can activate the Srf transcription factor (31). Srf TRE was overrepresented in the promoters of the downregulated genes. Another example for such interaction is the GEFT (D10Ertd610e), which was upregulated in brains of ß4-deficient mice, and can stimulate the transcriptional activities of the Elk1 transcription factor (21). Elk1 TRE was overrepresented in the promoters of the upregulated genes. Taken together, data from the promoter analysis demonstrate the possible complex interaction between ß4-containing nAChRs and gene expression.

In summary, this is to our knowledge the first study of the brain transcriptome of mice with null mutation in an nAChR subunit. We demonstrated that the expression levels of genes in brains of ß4–/– mice can be modified by either the mouse background strain or by the ß4 null mutation or possibly by a combination of both. This work highlights the pitfalls of studies using genetically manipulated animals and emphasizes the importance of cautious interpretation of both behavioral and molecular results from these studies.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by M. K. Humanitarian Fund and by the Wolfson Foundation.


   ACKNOWLEDGMENTS
 
This work was performed in partial fulfillment of the requirements for a PhD degree of M. Kedmi, Sackler Faculty of Medicine, Tel Aviv University, Israel.


   FOOTNOTES
 
Address for reprint requests and other correspondence: A. Orr-Urtreger, The Genetic Inst., Tel-Aviv Sourasky Medical Ctr., 6 Weizmann St., Tel Aviv, 64239, Israel (e-mail: aviorr{at}tasmc.health.gov.il).

Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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M. Kedmi and A. Orr-Urtreger
Expression changes in mouse brains following nicotine-induced seizures: the modulation of transcription factor networks
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