Fig 2: MEF2A Displays Functionally Compensatory Binding Activity at a Subset of MEF2D-Bound Genomic Sites(A) Aggregate plot and heatmap of ChIP-seq signal for MEF2A (red, n = 203) and MEF2D (green, n = 1388) genomic binding sites in Ctrl, AcKO, and DcKO P22 mouse cerebellum (n = 3–4 biological replicates per ChIP condition). ChIP-seq signal for H3K27ac and H3K4me3 (purple) from P22 mouse cerebellum centered on MEF2 genomic binding sites.(B) Significantly enriched de novo binding motifs at MEF2A and MEF2D peaks. Below each position, weighted matrix is the E-value followed by the most significant match to a TF motif.(C) Pie charts displaying regulatory element distribution of MEF2A (top) and MEF2D (bottom) peaks. Pro, promoter; Enh, enhancer. For regulatory element distribution of genomic background, refer to Figure S3D.(D) MEF2D sites experiencing increased MEF2A occupancy in DcKO mouse cerebellum are termed compensatory. Aggregate plots are shown for MEF2A and MEF2D ChIP-seq performed in different control (Ctrl) or cKO mice. A schematic depicts outcome based on the ChIP-seq peak signal detected in each condition.(E) Non-compensatory sites, defined as control MEF2D sites without increased MEF2A binding in DcKO mouse cerebellum, are shown. Aggregate plots are shown for MEF2A and MEF2D ChIP-seq performed in different control (Ctrl) or cKO mice. A schematic depicts outcome based on the ChIP-seq peak signal detected in each condition.(F) Heatmap of ChIP-seq signal for MEF2A (left) and MEF2D (right) at compensatory sites, sorted in descending order based on MEF2A ChIP-seq signal.(G) Scatterplot of MEF2A and MEF2D ChIP-seq signal (RPKM) at compensatory sites. Coefficient of determination using Pearson correlation.(H) For compensatory genomic binding sites, box-whisker plots for log2-transformed fold change of H3K27ac in AcKO, DcKO, and ADcKO over respective control mice. ****p < 10-4, ANOVA followed by Tukey’s multiple comparison test. n.s., not significant.(I) WashU Epigenome Browser view of a compensatory site (highlighted), showing MEF2A, MEF2D, and H3K27ac ChIP-seq coverage from AcKO, DcKO, AdcKO, and respective control (Ctrl) mice.(J) Same format as (I) for non-compensatory sites.See also Figure S3.

Fig 3: Compensatory Binding by MEF2A at a Subset of MEF2-Activated Target Genes Confers Robustness to MEF2D Depletion on Gene Expression(A) Pie chart representing proportion of MEF2-repressed (top) and MEF2-activated (bottom) genes associated with MEF2A and/or MEF2D genomic binding sites.(B) Table represents significance of overlap of RNA-seq clusters with MEF2A and MEF2D peaks identified in various ChIP-seq conditions. Columns from left to right: MEF2A ChIP in control mice; MEF2A ChIP in DcKO mice (compensatory sites); MEF2D ChIP in control mice; and MEF2D ChIP in AcKO mice. Rows from top to bottom represent RNA-seq clusters 1, 2, 3, and 4 (C1, C2, C3, and C4), as depicted by a schematic of the trends in gene expression (schematization of Figure 2B): control (black circle), AcKO (red circle), DcKO (green circle), and ADcKO (blue circle) mice. Heat represents -log-10 p value significance of overlap determined by hypergeometric test, with significant values displayed.(C) For compensatory direct target genes (defined as genes from compensatory C4 associated with MEF2-bound sites): box-whisker plots show median and distribution of log2-transformed fold change of H3K27ac in different conditional knockout mice over respective control mice. ***p < 10-3, ANOVA followed by Tukey’s multiple comparison test.(D) WashU Epigenome Browser view of an intragenic enhancer site (highlighted) of a compensatory direct target gene, showing MEF2A, MEF2D, and H3K27ac ChIP-seq and RNA-seq coverage from AcKO, DcKO, AdcKO, and respective control (Ctrl) mice.(E) Same format as (D) for intragenic enhancer site (highlighted) at non-compensatory direct target gene.See also Figure S4.

Fig 4: Chromatin Accessibility and Cellular State Specify Compensatory Action of MEF2A(A) Box-whisker plot of MRE degeneracy scores relative to a consensus MRE for compensatory and non-compensatory sites. n.s., not significant.(B) Box-whisker plot of chromatin accessibility for compensatory and non-compensatory sites. Two-sided unpaired t test, ****p < 10-4.(C) Plots represent bins of MEF2 sites sorted by increasing MEF2A peak read density in DcKO mice. Relative distribution of compensatory (light gray) and non-compensatory (black) MEF2 sites are shown as percentage (%) of regions comprising each bin.(D) Box-whisker plots of DNaseI sequencing read density for bins of increasing MEF2A density in DcKO mice (classified in C). ANOVA followed by Tukey’s multiple comparison test. **p < 10-2; n.s., not significant.(E) (Top) Relative motif enrichment for compensatory sites relative to non-compensatory sites. (Bottom) Relative motif enrichment at MEF2A ChIP peaks (some of which are overlap MEF2C) relative to MEF2C-only ChIP peaks in cortical neurons (Telese et al., 2015). Predicted binding factor followed in parentheses by number of significant motif occurrences and the top q-value representing significance of relative enrichment.(F) ChIP-seq signal of MEF2A (red, left) and MEF2D (green, right) at stimulus responsive genes listed in the “Response to Stimulus” Gene Ontology term (GO 0050896).(G) Schematic depicting accelerating rotarod paradigm (Yang et al., 2016; Yamada et al., 2019).(H) Rotarod paradigm performed for control and ADcKO mice followed by qRT-PCR on cerebellar RNA for select rotarod-activated genes experiencing compensatory MEF2A binding. ****p < 10-4, ***p < 10-3, **p < 10-2, *p < 10-4, two-way repeated-measures ANOVA, Sidak’s multiple comparison test.(I) Total RNA-seq analysis of rotarod-activated gene expression in cerebellum of AcKO, DcKO, ADcKO, and respective control mice subjected to the rotarod paradigm (n = 4 biological replicates per condition). Box-whisker plots of log2-transformed fold change (FC) of gene expression in AcKO, DcKO, and ADcKO over respective control mice for MEF2-occupied rotarod-activated genes at baseline (left) and after rotarod stimulation (middle), as well as unoccupied rotarod-activated genes after rotarod stimulation (right). ****p < 10-4, ANOVA followed by Tukey’s multiple comparison test. n.s., not significant.See also Figure S5.