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) Mecp2^tm1.1Bird/+ (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.