Cerebellar gene expression profiles of mouse models for Rett syndrome reveal novel MeCP2 targets

<p>Abstract</p> <p>Background</p> <p>MeCP2, methyl-CpG-binding protein 2, binds to methylated cytosines at CpG dinucleotides, as well as to unmethylated DNA, and affects chromatin condensation. <it>MECP2 </it>mutations in females lead to Rett syndrome, a neurological disorder characterized by developmental stagnation and regression, loss of purposeful hand movements and speech, stereotypic hand movements, deceleration of brain growth, autonomic dysfunction and seizures. Most mutations occur <it>de novo </it>during spermatogenesis. Located at Xq28, <it>MECP2 </it>is subject to X inactivation, and affected females are mosaic. Rare hemizygous males suffer from a severe congenital encephalopathy.</p> <p>Methods</p> <p>To identify the pathways mis-regulated by MeCP2 deficiency, microarray-based global gene expression studies were carried out in cerebellum of <it>Mecp2 </it>mutant mice. We compared transcript levels in mutant/wildtype male sibs of two different MeCP2-deficient mouse models at 2, 4 and 8 weeks of age. Increased transcript levels were evaluated by real-time quantitative RT-PCR. Chromatin immunoprecipitation assays were used to document <it>in vivo </it>MeCP2 binding to promoter regions of candidate target genes.</p> <p>Results</p> <p>Of several hundred genes with altered expression levels in the mutants, twice as many were increased than decreased, and only 27 were differentially expressed at more than one time point. The number of misregulated genes was 30% lower in mice with the exon 3 deletion (<it>Mecp2</it>^tm1.1Jae) than in mice with the larger deletion (<it>Mecp2</it>^tm1.1Bird). Between the mutants, few genes overlapped at each time point. Real-time quantitative RT-PCR assays validated increased transcript levels for four genes: <it>Irak1</it>, interleukin-1 receptor-associated kinase 1; <it>Fxyd1</it>, phospholemman, associated with Na, K-ATPase;<it>Reln</it>, encoding an extracellular signaling molecule essential for neuronal lamination and synaptic plasticity; and <it>Gtl2/Meg3</it>, an imprinted maternally expressed non-translated RNA that serves as a host gene for C/D box snoRNAs and microRNAs. Chromatin immunoprecipitation assays documented <it>in vivo </it>MeCP2 binding to promoter regions of <it>Fxyd1, Reln</it>, and <it>Gtl2</it>.</p> <p>Conclusion</p> <p>Transcriptional profiling of cerebellum failed to detect significant global changes in <it>Mecp2</it>-mutant mice. Increased transcript levels of <it>Irak1, Fxyd1, Reln</it>, and <it>Gtl2 </it>may contribute to the neuronal dysfunction in MeCP2-deficient mice and individuals with Rett syndrome. Our data provide testable hypotheses for future studies of the regulatory or signaling pathways that these genes act on.</p

Cerebellar gene expression profiles of mouse models for Rett syndrome reveal novel MeCP2 targets-6

<p><b>Copyright information:</b></p><p>Taken from "Cerebellar gene expression profiles of mouse models for Rett syndrome reveal novel MeCP2 targets"</p><p>http://www.biomedcentral.com/1471-2350/8/36</p><p>BMC Medical Genetics 2007;8():36-36.</p><p>Published online 20 Jun 2007</p><p>PMCID:PMC1931432.</p><p></p>en unaffected controls (C1 – C7), two -mutant males with congenital encephalopathy (CE) and five females diagnosed with RTT, with or without identified mutations. The mutant samples are identified by their brain bank numbers. expression was normalized to , a constitutive ribosomal protein gene that is expressed at a similar level in brain. To calculate relative fold changes, control sample C4 was arbitrarily set at 1. All samples were run in triplicate on the same plate

Cerebellar gene expression profiles of mouse models for Rett syndrome reveal novel MeCP2 targets-2

<p><b>Copyright information:</b></p><p>Taken from "Cerebellar gene expression profiles of mouse models for Rett syndrome reveal novel MeCP2 targets"</p><p>http://www.biomedcentral.com/1471-2350/8/36</p><p>BMC Medical Genetics 2007;8():36-36.</p><p>Published online 20 Jun 2007</p><p>PMCID:PMC1931432.</p><p></p> J-allele mice, while B-allele mice are on a C57BL/6 background. The effects of array batch, and to a lesser extent, age, are also apparent. We, therefore, compared only microarrays from littermates, at the same age and using the same array batch, in our search for differentially expressed genes

Cerebellar gene expression profiles of mouse models for Rett syndrome reveal novel MeCP2 targets-5

<p><b>Copyright information:</b></p><p>Taken from "Cerebellar gene expression profiles of mouse models for Rett syndrome reveal novel MeCP2 targets"</p><p>http://www.biomedcentral.com/1471-2350/8/36</p><p>BMC Medical Genetics 2007;8():36-36.</p><p>Published online 20 Jun 2007</p><p>PMCID:PMC1931432.</p><p></p>e M, 100 bp ladder; lane 1, -mutant chromatin precipitated with anti-MeCP2 (negative control); lane 2, wild-type chromatin precipitated with no antibody present; lane 3, wild-type chromatin precipitated with rabbit IgG, lane 4: wild-type chromatin precipitated with anti-MeCP2, lane 5: input DNA, lane 6: no DNA control. The precipitated DNA was amplified with primers specific for the promoter regions of and , and for the promoter/DMR of . The Gtl2-B amplicon is located within the Gtl2-A amplicon. Precipitation with anti-MeCP2 shows enrichment relative to the IgG and no antibody controls, indicating that MeCP2 is associated with the designated regions . As negative controls, PCR was performed using primers covering regions that do not interact with MeCP2 for each ChIP reaction (not shown)

Cerebellar gene expression profiles of mouse models for Rett syndrome reveal novel MeCP2 targets-1

<p><b>Copyright information:</b></p><p>Taken from "Cerebellar gene expression profiles of mouse models for Rett syndrome reveal novel MeCP2 targets"</p><p>http://www.biomedcentral.com/1471-2350/8/36</p><p>BMC Medical Genetics 2007;8():36-36.</p><p>Published online 20 Jun 2007</p><p>PMCID:PMC1931432.</p><p></p>heir wt sibs rather than with mutants of other litters. Litters are identified by A – F. The array of the litter B wild-type sib did not pass quality control standards

Cerebellar gene expression profiles of mouse models for Rett syndrome reveal novel MeCP2 targets-0

<p><b>Copyright information:</b></p><p>Taken from "Cerebellar gene expression profiles of mouse models for Rett syndrome reveal novel MeCP2 targets"</p><p>http://www.biomedcentral.com/1471-2350/8/36</p><p>BMC Medical Genetics 2007;8():36-36.</p><p>Published online 20 Jun 2007</p><p>PMCID:PMC1931432.</p><p></p>tched: coding sequence. Grey: non-coding parts of transcript. Exons 1 and 2 that are alternatively spliced, and intron 2, are not drawn to scale. Horizontal bar above exon 4 denotes location of the amplicon analyzed by qRT-PCR (in Figure 1B) The (J-allele) mutant has a deletion of exon 3 that encodes the N-terminal half of the MBD. In the (B-allele) mutant, exon 3, the coding portion of exon 4 and part of the 3'UTR are deleted. This larger deletion eliminates ~ 97% of the coding sequence. Using primers within the deleted region, transcript was undetectable in mutant mice with the B-allele, when four wild-type and four mutant samples were compared at 8 wk. This proves the specificity of the primers. In mice with the J-allele, mean expression was higher in the mutants, but this difference was not statistically significant (= 0.11), when six wild-type and seven mutant samples were compared at 8 wk

Cerebellar gene expression profiles of mouse models for Rett syndrome reveal novel MeCP2 targets-7

<p><b>Copyright information:</b></p><p>Taken from "Cerebellar gene expression profiles of mouse models for Rett syndrome reveal novel MeCP2 targets"</p><p>http://www.biomedcentral.com/1471-2350/8/36</p><p>BMC Medical Genetics 2007;8():36-36.</p><p>Published online 20 Jun 2007</p><p>PMCID:PMC1931432.</p><p></p>tched: coding sequence. Grey: non-coding parts of transcript. Exons 1 and 2 that are alternatively spliced, and intron 2, are not drawn to scale. Horizontal bar above exon 4 denotes location of the amplicon analyzed by qRT-PCR (in Figure 1B) The (J-allele) mutant has a deletion of exon 3 that encodes the N-terminal half of the MBD. In the (B-allele) mutant, exon 3, the coding portion of exon 4 and part of the 3'UTR are deleted. This larger deletion eliminates ~ 97% of the coding sequence. Using primers within the deleted region, transcript was undetectable in mutant mice with the B-allele, when four wild-type and four mutant samples were compared at 8 wk. This proves the specificity of the primers. In mice with the J-allele, mean expression was higher in the mutants, but this difference was not statistically significant (= 0.11), when six wild-type and seven mutant samples were compared at 8 wk

And were selected for validation of expression changes by qRT-PCR analyses in B-allele mice

<p><b>Copyright information:</b></p><p>Taken from "Cerebellar gene expression profiles of mouse models for Rett syndrome reveal novel MeCP2 targets"</p><p>http://www.biomedcentral.com/1471-2350/8/36</p><p>BMC Medical Genetics 2007;8():36-36.</p><p>Published online 20 Jun 2007</p><p>PMCID:PMC1931432.</p><p></p> Cerebellum microarray expression data (M) are compared with qRT-PCR data from cerebellum (RC), forebrain (RF) and hippocampus (RH). For RC of and , the 8 wk samples from the microarray analysis were used. RC2 data are derived from an independent set of litters at 7.5 to 8 wk. Error bars represent SEM. One asterisk indicates < 0.05, two asterisks indicate < 0.001. expression was markedly increased in the cerebellum of 8 wk B-allele mice on microarray (2-fold, = 0.011, four mutant/wild-type pairs) and qRT-PCR (3.5 fold, < 0.001, four mutant/wild-type pairs) as well as in the forebrain of a different set of mice at 7.5 wk (1.9 fold, < 0.05, three mutant/five wild-type). expression was elevated 1.4-fold on microarrays (< 0.05, five mutant/wild-type sibs) and qRT-PCR (RC) = 0.053, four mutant/wild-type sibs). The RC2 data (five mutant/three wild-type) were more variable and yielded = 0.16. displayed an ~ 1.5 fold increase in expression on microarrays (= 0.03, four mutant/wild-type pairs) and qRT-PCR (= 0.06, five mutant/wild-type pairs) at 2 wk (RC) and was not changed in hippocampus (RH). On microarray, expression was increased at 8 wk, but by qRT-PCR we found highly variable expression in cerebellum (RC and RC2). expression was not significantly increased (= 0.29, eight mutant/eight wild-type) in the forebrain