Genome-wide analysis reveals methyl-CpG-binding protein 2-dependent regulation of microRNAs in a mouse model of Rett syndrome

Abstract

MicroRNAs (miRNAs) are a class of small, noncoding RNAs that function as posttranscriptional regulators of gene expression. Many miRNAs are expressed in the developing brain and regulate multiple aspects of neural development, including neurogenesis, dendritogenesis, and synapse formation. Rett syndrome (RTT) is a progressive neurodevelopmental disorder caused by mutations in the gene encoding methyl-CpG-binding protein 2 (MECP2). Although Mecp2 is known to act as a global transcriptional regulator, miRNAs that are directly regulated by Mecp2 in the brain are not known. Using massively parallel sequencing methods, we have identified miRNAs whose expression is altered in cerebella of Mecp2-null mice before and after the onset of severe neurological symptoms. In vivo genome-wide analyses indicate that promoter regions of a significant fraction of dysregulated miRNA transcripts, including a large polycistronic cluster of brain-specific miRNAs, are DNA-methylated and are bound directly by Mecp2. Functional analysis demonstrates that the 3' UTR of messenger RNA encoding Brain-derived neurotrophic factor (Bdnf) can be targeted by multiple miRNAs aberrantly up-regulated in the absence of Mecp2. Taken together, these results suggest that dysregulation of miRNAs may contribute to RTT pathoetiology and also may provide a valuable resource for further investigations of the role of miRNAs in RTT.

Fig. 1.

Systematic identification of dysregulated miRNAs in WT and KO cerebella. (A) Venn diagram of the number of up- or down-regulated miRNAs detected in Mecp2-null cerebella. Note that a significant portion of aberrantly up-regulated mature miRNAs is detected consistently in Mecp2-null mice at both the pre-/early-symptomatic stage (6 wk postnatal) and the symptomatic stage (8 wk postnatal). (B) Heat map representation of 18 mature miRNAs that are dysregulated consistently in Mecp2-null mice at both the pre-/early-symptomatic stage (four pairs of 6-wk-postnatal WT and KO littermates sequenced as pooled samples) and the symptomatic stage (four pairs of >8-wk-postnatal WT and KO samples sequenced individually). Color intensity represents the degree of differential expression of miRNAs between WT and KO cerebella. (C) Genomic representation (NCBI build 36) of aggregated sequencing reads mapped to dysregulated miRNAs at the pre-/early-symptomatic (6-wk-postnatal) stage. Sequencing read counts for a given mature miRNA sequence are indicated also.

Fig. 2.

MeCP2 binds directly to gene-regulatory regions of a cohort of miRNAs. (A) Number of Mecp2 directly bound miRNA targets that are up-regulated, down-regulated, and unchanged (Other) in 6-wk-postnatal cerebella. MiRNA transcripts without Mecp2-binding sites within 1-kb (proximal) or 5-kb (distal) regions flanking their TSSs are considered nontargets. Note that only up-regulated miRNAs are significantly enriched in both proximal (P = 0.0054, hypergeometric test) and distal (P = 0.0066) Mecp2 miRNA targets. (B) MeCP2 occupancy and DNA methylation (−log₁₀ P value) are shown for two targets (miR-137 and miR-34b/34c) and one nontarget (miR-10a) in WT and KO cerebella. Genomic positions for nearby known protein-coding genes and CpG islands are shown also. Selected regions that are validated by ChIP/MeDIP-qPCR for Mecp2 occupancy and DNA methylation are highlighted in gray. The DNA methylation level within two regions (indicated by blue bars) flanking the miR-137 gene is validated further by bisulphite conversion followed by mass spectrometry (Sequenom). (C) ChIP of endogenous Mecp2 occupancy (Upper) and MeDIP-based DNA methylation analyses (Lower) for two miRNA targets and one nontarget in WT and KO cerebella. Error bars represent SEM. *P < 0.01.

Fig. 3.

Mecp2 binds to specific genomic regions within the Dlk1-Gtl2 imprinting domain. Mecp2 occupancy, DNA methylation, and histone H3/4 acetylation (H3/4ac) in WT and/or KO cerebella (6 wk postnatal) within a 300-kb region of the Dlk1-Gtl2 imprinting domain are shown. Statistically enriched regions (−log₁₀ P > 2) are denoted as vertical bars. Mecp2 enrichment at the putative transcription start site for the polycistronic miRNA transcript is indicated by arrows.

Fig. 4.

Essential roles of Mecp2-regulated miRNAs in regulation of Bdnf expression. (A) Schematic diagram of the pISO luciferase reporter containing the first 800 bp of the Bdnf 3′ UTR (pISO-Bdnf). Also shown are aberrantly up-regulated miRNAs (>1.5-fold) in KO cerebella at the early-symptomatic stage (6 wk postnatal) that are predicted to target the Bdnf 3′ UTR by at least two independent target-prediction algorithms (TargetScan, miRanda, or PicTar/DIANA microT). (B) Luciferase assays in 293T cells transfected with miRNA mimics duplexes (100 nM). Error bars represent SEM. *P < 0.05; n ≥ 4. Note that other miRNAs [cel-miR-67 (specifically expressed in C. elegans) and miR-137] were used as negative controls for pISO-Bdnf. (C) Luciferase assays in WT cerebellar neurons transfected with control (100 nM) or miR-381/495 (100 nM) 2’-O-Methyl oligonucleotide inhibitors. Error bars represent SEM. *P < 0.05; n ≥ 5. (D) Immunoblotting of Mecp2 in postnatal cortical neurons infected with lentiviruses expressing a control shRNA or a shRNA specific for Mecp2. (E) Bdnf ELISAs in postnatal cortical neurons. Error bars represent SEM. *P < 0.05; n = 4.