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. 2005 Feb 15;14(4):483-92.
doi: 10.1093/hmg/ddi045. Epub 2004 Dec 22.

Epigenetic overlap in autism-spectrum neurodevelopmental disorders: MECP2 deficiency causes reduced expression of UBE3A and GABRB3

Rodney C Samaco  1 , Amber Hogart, Janine M LaSalle
Affiliations

Epigenetic overlap in autism-spectrum neurodevelopmental disorders: MECP2 deficiency causes reduced expression of UBE3A and GABRB3

Rodney C Samaco et al. Hum Mol Genet. .

Abstract

Autism is a common neurodevelopmental disorder of complex genetic etiology. Rett syndrome, an X-linked dominant disorder caused by MECP2 mutations, and Angelman syndrome, an imprinted disorder caused by maternal 15q11-q13 or UBE3A deficiency, have phenotypic and genetic overlap with autism. MECP2 encodes methyl-CpG-binding protein 2 that acts as a transcriptional repressor for methylated gene constructs but is surprisingly not required for maintaining imprinted gene expression. Here, we test the hypothesis that MECP2 deficiency may affect the level of expression of UBE3A and neighboring autism candidate gene GABRB3 without necessarily affecting imprinted expression. Multiple quantitative methods were used including automated quantitation of immunofluorescence and in situ hybridization by laser scanning cytometry on tissue microarrays, immunoblot and TaqMan PCR. The results demonstrated significant defects in UBE3A/E6AP expression in two different Mecp2 deficient mouse strains and human Rett, Angelman and autism brains compared with controls. Although no difference was observed in the allelic expression of several imprinted transcripts in Mecp2-null brain, Ube3a sense expression was significantly reduced, consistent with the decrease in protein. A non-imprinted gene from 15q11-q13, GABRB3, encoding the beta3 subunit of the GABAA receptor, also showed significantly reduced expression in multiple Rett, Angelman and autism brain samples, and Mecp2 deficient mice by quantitative immunoblot. These results suggest an overlapping pathway of gene dysregulation within 15q11-q13 in Rett, Angelman and autism and implicate MeCP2 in the regulation of UBE3A and GABRB3 expressions in the postnatal mammalian brain.

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Figures

Figure 1
Figure 1
UBE3A expression in Mecp2-null mouse brain. (A) A multiple tissue microarray containing wt and knock-out mouse cerebral cortex samples (16) was stained for immunofluorescence using a C-terminal reactive anti-MeCP2 and an anti-UBE3A antibody. The MeCP2-negative cells (Mecp2 mt-expressing, blue histograms, percentage shown) were separately gated from MeCP2-positive cells (Mecp2 wt-expressing, red histograms) and the total population (black histograms) and compared with age-matched wt control female or male samples (orange histograms). (B) The graph shows combined LSC results (mean ± SEM) from four replicate slides using two different anti-UBE3A antibodies. (C) Protein extracts from whole adult mouse brain were probed on an immunoblot with anti-UBE3A or anti-GAPDH. A representative image shows lower expression in brain of UBE3A in both hemizygous male (−/y) and heterozygous female (−/+) brain compared with wild-type (wt, +/y and +/+) littermate controls for both Mecp2tm1.1Bird and Mecp2tm1.1Jae strains. The graph demonstrates combined GAPDH-normalized results (mean ± SEM, quantitation as previously described (32), example ratios shown below each lane) from eight different −/+ and six different +/+ brain samples from Mecp2tm1.1Bird and Mecp2tm1.1Jae strains. *P < 0.05, ****P < 0.0001 by t-test.
Figure 2
Figure 2
Imprinting and transcriptional analyses in mouse brain. (A) Chromatin from adult mouse cerebrum samples [C57B6, PWK or (B6 × PWK)F1] was isolated for ChIP. Anti-MeCP2 (C-terminal) was used to immunoprecipitate DNA fragments from ‘Input’ control. Ube3a and Gabrb3 promoters were not detected in the anti-MeCP2 precipitated chromatin, in contrast to the Snrpn promoter sequences that showed association with MeCP2. U2af1-rs1 was a positive control for a promoter previously demonstrated to bind MeCP2 in brain (34). (B) Mecp2tm1.1Bird/+ (B6) females were crossed with wt PWK males to obtain F1 mice heterozygous for single nucleotide polymorphisms in the coding regions of several imprinted genes. RT–PCR followed by restriction enzyme digestion (+) was performed on RNA from adult brain samples of all Mecp2 genotypes as well as parental B6 and PWK samples (P, paternal; M, maternal). As reported previously for wt (B6 × PWK)F1 brain (67), Ube3a sense exhibited preferential maternal expression, Ube3a antisense and Snrpn were exclusively paternal and Gabrb3 was biallelic, with no significant effect of Mecp2 genotype on these imprints. In addition, Rasgrf1 showed preferential paternal expression (68), whereas H19 showed exclusive maternal expression (70) in all samples. Results are representative of adult and neonatal F1 brain samples. (C) TaqMan PCR was used to quantitatively determine expression levels of Ube3a, Snrpn and Gabrb3 in 10-week-old Mecp2−/y and Mecp2+/y brain RNA. Primers spanned intron/exon boundaries and are specific to the Ube3a sense transcript. Results shown are for the average ± SEM of four experimental replicates of two mice per genotype. Although Ube3a and Gabrb3 showed consistently lower expression in Mecp2-deficient brain compared with controls, Snrpn was not significantly changed. (D) Sense and antisense transcripts of Ube3a were detected by fluorescence in situ hybridization using single stranded riboprobes and quantitated by LSC as described previously (16). Results (mean ± SEM of 3 wt and 10 Mecp2−/+ samples) were normalized to control β-actin probe. *P < 0.05, ***P < 0.001 by t-test.
Figure 3
Figure 3
Quantitation of UBE3A expression in Rett, Angelman and autism brain samples. (A) The human tissue microarray previously described (16) containing frontal cortex layers III–V from Brodman area (BA9) was analyzed for UBE3A expression by LSC. Histograms show lower expression of patient samples (black) from Rett (RTT), Angelman (AS) and autism (AUT) compared with age-matched controls (gray). (B) UBE3A immunofluorescence values were normalized to those of control histone H1 and the results of four replicate slides using two different anti-UBE3A antibodies (Affinity Bioreagents and BD Biosciences) were averaged (mean ± SEM) for each brain sample shown in (A) (n, number of different samples). (C) Immunoblot analysis of UBE3A expression (anti-UBE3A, Affinity, recognizing all known isoforms) confirmed decreased expression in AS, RTT and AUT compared with controls (UBE3A/GAPDH ratios are shown). *P < 0.05, **P < 0.01 by t-test.
Figure 4
Figure 4
Analysis of GABRB3 expression in mouse and human brain. (A) Immunoblot analysis of GABRB3 expression (anti-GABRB3, Affinity Bioreagents, recognizing all known isoforms) shows lower expression in Mecp2−/+ and Mecp2−/y adult mouse brain compared with controls. Combined GAPDH-normalized results from eight different Mecp2−/+ and six different Mecp2+/+ adult brain samples from Mecp2tm1.1Bird and Mecp2tm1.1Jae strains were significantly lower than wild-type controls (P < 0.01). (B) Representative immunoblot analyses of GABRB3 and GAPDH in AS, RTT, autism and control (NS) cerebral samples with GABRB3/GAPDH ratios shown. Replicate immunoblot results for all samples and significance tests are shown in Table 1. Additional information about postmortem brain samples used in these studies is provided online (Supplementary Material, Table S1).
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