Figure 4. Integrative gene expression and methylation analysis shows mCH and mCG loss contribute to Dnmt3a cKO DEGs, and reveals a strong mCH contribution to RTT.

(A) Gene-body mCH levels in different categories of DEGs in WT mice are plotted, highlighting that misregulated genes have higher mCH than genes that are unchanged and the significant differences between mCH levels on up- and down-regulated genes. (B) Gene-body mCG levels in different categories of DEGs in WT mice are plotted, demonstrating that misregulated genes (with the exception of MeCP2 down-regulated genes) have higher mCG than genes that are unchanged and the significant differences between mCG levels on up- and down-regulated genes. (C) Running average plot of log2fold change in gene expression for genes significantly misregulated only in the Dnmt3a cKO model versus the change in mCH methylation observed in the Dnmt3a cKO model (‘Dnmt3a dependent mCH methylation’) (left). Running average plot of log2fold change in gene expression for these same genes versus the change in mCG methylation observed in the Dnmt3a cKO (‘Dnmt3a dependent mCG methylation’) (right). (D) Running average plots of log2fold change in gene expression in the Dnmt3a cKO model for genes commonly misregulated in both cKO models versus Dnmt3a dependent mCH (right) and mCG (left). (E) Running average plots of log2fold change in gene expression in the MeCP2 cKO for genes commonly misregulated in both cKO models versus Dnmt3a dependent mCH (right) and mCG (left). (F) Running average plots of log2fold change in gene expression for genes that are only significantly misregulated in the MeCP2 cKO model. (G–J) R² values from analysis in panels C-F shown as blue, orange or green dots, respectively, plotted over 1000 random repetitions of the analysis with each repetition containing the same number of non-DEGs (padj >0.01). The results of random repetitions are shown as gray dots. All plots were made with DEGs padj <0.01. n = 2 mice per genotype for methylation data. n = 4 mice per genotype (RNA-seq). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 4—figure supplement 1. Methylation levels on categories of DEG genes in WT, Dnmt3a cKO, and Mecp2 cKO mice with statistical comparisons.

(A) Averaged gene-body mCH levels (average of two replicates) in different categories of DEGs in WT mice, highlighting the direction of difference in mCH levels between up- and down-regulated DEGs. The box plots within each segment highlights the median of the distribution, while the red point represents the mean of the distribution. (B) Averaged gene-body mCG levels (average of two replicates) in different categories of DEGs in WT mice, highlighting the direction of difference in mCG levels between up- and down-regulated DEGs. The box plots within each segment highlights the median of the distribution, while the red point represents the mean of the distribution. (C) Gene-body mCH levels in different categories of DEGs in WT, Dnmt3a cKO and Mecp2 cKO mice. (D) Gene-body mCG levels in different categories of DEGs in WT, Dnmt3a cKO and Mecp2 cKO mice. (E) P-value matrix for comparison of mCH levels of different categories of genes. The boxes are colored such that more significant p-values are darker shades of red. (F) P-value matrix for comparison of mCG levels of different categories of genes. As in C, the boxes are colored such that more significant p-values are darker shades of red.

Figure 4—figure supplement 2. Integrative gene expression and methylation analysis excluding interneuron specific DEGs determined in PSEA analysis shows the same trends and statistical significance as in Figure 4.

(A–H) Running average and validation plots from Figure 4 re-plotted after removing DEGs that may represent changes in gene expression in the minor population of co-purified inhibitory interneurons. All plots were made with DEGs padj <0.01. n = 2 mice per genotype for methylation data. n = 4 mice per genotype (RNA-seq). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.