Fig 1.
MeCP2 interacts with multiple splicing regulators.
(a) Gene ontology (GO) analysis of proteins identified from anti-Flag co-immunoprecipitation and mass spectrometry (Co-IP/MS) analysis in Mecp2-Flag knockin mouse brain. Top five GOTERM_BP_FAT terms were shown and –log10 Benjamini P-value is plotted. (b) Western blot validation of interactions between MeCP2 and splicing regulators identified in Co-IP/MS. Anti-Flag immunoprecipitate in nuclear extract from WT and Mecp2-Flag knockin mouse brain is resolved in SDS-PAGE and probed with antibodies against indicated proteins. (c) Western blot analysis of MeCP2 in anti-TDP-43, anti-LEDGF, anti-DHX9, anti-FUS and anti-hnRNP F+H immunoprecipitate. Rabbit (Rb) IgG is negative control for rabbit antibody (anti-TDP-43, LEDGF, DHX9 and FUS) and mouse (Ms) IgG is negative control for mouse antibody (anti-hnRNP F+H).
Fig 2.
RTT mutations disrupt MeCP2 interaction with LEDGF and DHX9.
(a) Schematic diagram of HA-MeCP2 constructs used in this study. The methyl-CpG-binding domain (MBD) domain is shown in yellow and the transcription repression domain (TRD) in blue. (b) Western blot analysis of Myc-LEDGF/p52 in anti-HA immunoprecipitate (top panel) from cells transfected with different combinations of plasmids as labeled on top. HA-MeCP2 (middle panel) and Myc-LEDGF/p52 (bottom panel) in input were also analyzed by western blot. (c) Western blot analysis of Myc-DHX9 in anti-HA immunoprecipitate (top panel) from cells transfected with different combinations of plasmids as labeled on top. HA-MeCP2 (middle panel) and Myc-DHX9 (bottom panel) in input were also analyzed by Western blot. (d) Representative western blot analysis of Myc-LEDGF/p52 in HA immunoprecipitate (top panel) from cells transfected with different combinations of plasmids as labeled on top. HA-MeCP2 (middle panel) and Myc-LEDGF/p52 (bottom panel) in input were also analyzed by Western blot. (e) Quantification of Myc-LEDGF/p52 IP over input signal intensity of three independent experiments. Error bar represents S.E.M, **P < 0.01, *P < 0.05; two-tailed t-test. (f) Representative Western blot analysis of Myc-DHX9 in HA immunoprecipitate (top panel) from cells transfected with different combinations of plasmids as labeled on top. HA-MeCP2 (middle panel) and Myc-DHX9 (bottom panel) in input were also analyzed by Western blot. (g) Quantification of Myc-DHX9 IP over input signal intensity of four independent experiments. Error bar represents S.E.M, **P < 0.01, *P < 0.05; two-tailed t-test.
Fig 3.
Global splicing changes in Mecp2 KO mouse cortex and enrichment of MeCP2 around the exon/intron boundary.
(a) Number of misregulated alternative splicing (AS) events by category in Mecp2 KO cortex. (b) Relative fraction of each AS events type positively or negatively affected by loss of MeCP2. (c) Histogram of total gene expression changes in Mecp2 KO cortex for those 232 genes associated with splicing changes in Mecp2 KO cortex. FC: fold change. (d) Functional enrichment analysis of genes with MeCP2 peak(s) identified in ChIP-Seq data from WT mouse cortex. Top six terms are shown and –log10 Benjamini P-value is plotted for each GO term. (e) Read counts per millions of mapped reads (ChIP minus input) across regions spanning from 500bp upstream and 500bp downstream of all exons. Coverage for ChIP and input data are calculated for each biological replicate and normalized to be equal length across all the mm9 exons by sampling at equal intervals. Displayed is the average ChIP minus input profile of two biological replicates. The graph is generated using ngs.plot[41].
Fig 4.
Loss of MeCP2 affects flip/flop splicing of AMPAR genes.
(a) Reads distribution surrounding the flip and flop exons of Gria2 locus from RNA-Seq data of two pairs of WT and Mecp2 KO mouse cortex. Percentage of flip isoform (% flip) for each sample is estimated by Mixture of Isoform pipeline (MISO) and plotted on the right. (b) Quantification of flip/flop ratio of Gria2 gene in the cortex of WT and Mecp2 KO mice. Mean ± S.E.M is plotted. n = 8 per genotype. *** P < 0.001; two-tailed t-test. (c) Quantification of Gria2 total mRNA level in the cortex of WT and Mecp2 KO mice. Mean ± S.E.M is plotted. n = 8 per genotype. (d) Quantification of the flip/flop ratio of Gria1, Gria3 and Gria4 in the cortex of WT and Mecp2 KO mice. Mean ± S.E.M is plotted. n = 8 per genotype. *** P < 0.001; two-tailed t-test. (e) Quantification of Gria1, Gria3 and Gria4 total mRNA level in the cortex of WT and Mecp2 KO mice. Mean ± S.E.M is plotted. n = 8 per genotype. (f) Quantification of the flip/flop ratio of Gria1, Gria2, Gria3 and Gria4 in the cortex of WT and Mecp2-/+ female mice. Mean ± S.E.M is plotted. n = 4 per genotype. * P < 0.05, ** P < 0.01; two-tailed t-test. (g) Quantification of Gria1, Gria3 and Gria4 total mRNA level in the cortex of WT and Mecp2-/+ female mice. Mean ± S.E.M is plotted. n = 4 per genotype.
Fig 5.
LEDGF is involved in regulation of Gria2 flip/flop splicing.
(a) ChIP-qPCR analysis of MeCP2 occupancy on Gapdh promoter, Gria2 flip and flop exon in Mecp2-Flag mouse cortex. pr: promoter. Mean ± S.E.M is plotted. n = 4 per genotype. * P < 0.05; one-way ANOVA followed with Holm-Sidak’s multiple comparisons test. (b) ChIP-qPCR analysis of PolII occupancy on Gria2 flip and flop exon in WT and Mecp2 KO cortex. Mean ± S.E.M is plotted. n = 4 per genotype. (c) ChIP-qPCR analysis of LEDGF occupancy on Gria2 flip and flop exon in WT mouse cortex. Mean ± S.E.M; n = 5; *** P < 0.001; one-way ANOVA followed with Holm-Sidak's multiple comparisons test. (d) ChIP-qPCR analysis of H3K36me3 occupancy on Gria2 flip and flop exons in WT and Mecp2 KO cortex. n = 4 per genotype. (e-g) Representative Western blot analysis of MeCP2 (e), quantification of MeCP2 protein level (f), and flip/flop ratio (g) in Neuro-2A cells co-transfected with Mecp2 shRNA plasmid, MeCP2 expression plasmid and Gria2 minigene plasmid. Mean ± S.E.M of three independent experiments, * P < 0.05, *** P < 0.001; two-tailed t-test. (h-j) Representative Western blot analysis of LEDGF (h), quantification of LEDGF protein level (i), and flip/flop ratio (j) in Neuro-2A cells co-transfected with Ledgf shRNA plasmid and Gria2 minigene plasmid. Mean ± S.E.M of three (i) or four (j) independent experiments, ** P < 0.01; two-tailed t-test.
Fig 6.
Altered AMPA receptor decay kinetics and synaptic transmission in Mecp2 KO cortical neurons can be reversed by ESF.
(a) Representative sample trace of glutamate-evoked current in WT and Mecp2 KO cortical neurons. Scale bar = 15ms. (b) Quantification of decay time constant τ of glutamate-evoked current in WT and Mecp2 KO cortical neurons. Mean ± S.E.M is plotted. n = 37 for WT, n = 33 for KO. ** P < 0.01; two tailed t-test. (c) Representative mEPSC sample trace in WT and Mecp2 KO cortical neurons with or without CTZ treatment. Scale bar = 10ms. (d) Quantification of decay time constant τ of mEPSC. Mean ± S.E.M is plotted. n = 42 for WT, n = 46 for KO, n = 38 for KO + CTZ. ** P < 0.01; one-way ANOVA with Tukey’s multiple comparisons test. (e-f) qRT-PCR analysis the flip/flop ratio (e) and total Gria2 minigene (f) expression in HEK 293 cells co-transfected with indicated ESF and the Gria2 minigene. Mean ± S.E.M is plotted. n = 4 for each group. * P < 0.05, *** P < 0.001; two tailed t-test. (g) Quantification of decay time constant τ of glutamate-evoked current in WT, Mecp2 KO (KO+Ctrl) and Mecp2 KO cortical neurons infected with ESF-flop-Gly lentivirus (KO+ESF). Recording was done on acute slice from mouse infected with lentivirus two weeks after stereotaxic injection. Mean ± S.E.M is plotted. n = 50 for WT, n = 26 for KO+Ctrl, n = 27 for KO+ESF. ** P < 0.01, *** P < 0.001; one-way ANOVA with Tukey’s multiple comparisons test. (h) Representative sample trace of AMPAR-mediated current in response to repetitive stimulations in WT and Mecp2 KO neurons infected with control or ESF-flop-Gly lentivirus (KO+Ctrl or KO+ESF). Scale bar, 100ms. (i) Relative current amplitude of the xth stimulation to first stimulation in WT and Mecp2 KO neurons infected with either control or ESF-flop-Gly lentivirus (KO+Ctrl or KO+ESF). Mean ± S.E.M is plotted. n = 36 for WT, n = 18 for KO+Ctrl, n = 18 for KO+ESF. Asterisk denotes P-value for comparison between WT and KO+Ctrl, *** P < 0.001. Pound sign denotes P-value for comparison between KO+ESF and KO+Ctrl, # P < 0.05, ## P < 0.01; repeated measures two-way ANOVA with Tukey’s multiple comparisons test. (j) Quantification of decay time constant τ of glutamate-evoked current in neurons infected with shCtrl or shLedgf lentivirus. Mean ± S.E.M is plotted. n = 13 for shCtrl, n = 12 for shLedgf, * P < 0.05; two-tailed t-test. (k) Representative sample trace of AMPAR-mediated current in response to repetitive stimulations in neurons infected with shCtrl or shLedgf lentivirus. Scale bar, 100ms. (l) Relative current amplitude of xth stimulation to first stimulation in neurons infected with shCtrl or shLedgf. Mean ± S.E.M is plotted. n = 12 for each group, *** P < 0.001; repeated measures two-way ANOVA with Tukey's multiple comparisons test.