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, 29 (7), 2001-2015.e5

Chromatin Environment and Cellular Context Specify Compensatory Activity of Paralogous MEF2 Transcription Factors

Affiliations

Chromatin Environment and Cellular Context Specify Compensatory Activity of Paralogous MEF2 Transcription Factors

Shahriyar P Majidi et al. Cell Rep.

Abstract

Compensation among paralogous transcription factors (TFs) confers genetic robustness of cellular processes, but how TFs dynamically respond to paralog depletion on a genome-wide scale in vivo remains incompletely understood. Using single and double conditional knockout of myocyte enhancer factor 2 (MEF2) family TFs in granule neurons of the mouse cerebellum, we find that MEF2A and MEF2D play functionally redundant roles in cerebellar-dependent motor learning. Although both TFs are highly expressed in granule neurons, transcriptomic analyses show MEF2D is the predominant genomic regulator of gene expression in vivo. Strikingly, genome-wide occupancy analyses reveal upon depletion of MEF2D, MEF2A occupancy robustly increases at a subset of sites normally bound to MEF2D. Importantly, sites experiencing compensatory MEF2A occupancy are concentrated within open chromatin and undergo functional compensation for genomic activation and gene expression. Finally, motor activity induces a switch from non-compensatory to compensatory MEF2-dependent gene regulation. These studies uncover genome-wide functional interdependency between paralogous TFs in the brain.

Keywords: MEF2; cerebellum; chromatin; chromatin accessibility; compensation; gene expression; interdependency; learning; neuron; paralog; transcription factor.

Conflict of interest statement

DECLARATION OF INTERESTS

The authors declare no conflicts of interest.

Figures

Figure 1.
Figure 1.. MEF2A and MEF2D Regulate Cerebellar-Dependent Motor Learning in a Compensatory Manner
(A) Schematic depiction of single- and double-conditional knockouts of MEF2A and MEF2D in cerebellar granule neurons in mice. Transgenic mice expressing the recombinase Cre downstream of the granule-neuron-enriched GABA(A)a6-receptor (G6R) gene promoter are crossed to mice harboring conditional alleles for Mef2a, Mef2d, or both Mef2a and Mef2d to generate Mef2afl/fl;G6RCre+/− (AcKO), Mef2dfl/fl;G6RCre+/− (DcKO), and Mef2a/dfl/fl;G6RCre+/− (ADcKO), respectively. (B) Immunoblotting of MEF2A (top left) and MEF2D (bottom left) in lysates of cerebellum harvested from postnatal day 3 through 56 (P3–P56) AcKO (top) and DcKO (bottom) mice, respectively. Immunoblotting also performed for MEF2D in P22 AcKO mice (top right) and MEF2A in P22 DcKO mice (bottom right). (C) Sagittal sections of P22 cerebellum from different MEF2 conditional knockout mouse lines were subjected to immunohistochemistry by using antibodies recognizing calbindin (first column), and brain-enriched MEF2 family members (second column), as well as the DNA dye bisbenzimide (Hoechst) (third column). Immunohistochemical analyses of MEF2A performed on control (top row) and AcKO (second row) mouse cerebellum, of MEF2D on control (third row) and DcKO (fourth row) mouse cerebellum, and of MEF2C on control (fifth row) mouse cerebellum. IGL, internal granule layer; PCL, Purkinje cell layer; ML, molecular layer. Scale bar: 100 μm, 20× magnification. (D) Cerebellar-dependent eyeblink conditioning learning paradigm was performed on AcKO and control (n = 6 per genotype), DcKO and control (n = 7 and n = 8, respectively), and ADcKO and control (n = 11 and n = 9, respectively) mice. Percent conditioned response (CR) is shown as mean ± SEM for each session day. ****p < 10−4, **p < 10−2 repeated-measures ANOVA, Sidak’s multiple comparison test. See also Figure S1.
Figure 2.
Figure 2.. MEF2A and MEF2D Exhibit Complex Patterns of Gene Regulation in Cerebellum
(A) Hierarchical clustering of gene expression of AcKO, DcKO, ADcKO, and respective control (Ctrl) P22 mouse cerebellum for genes detected as significantly dysregulated (false discovery rate [FDR], <0.05) in analysis of RNA-seq from Ctrl and ADcKO cerebellum (n = 4 biological replicates per genotype). Four clusters are indicated on the left side of the heatmap. Heat represents Z score of log2 cpm (counts per million) for a given gene. (B) Box-whisker plots representing median and distribution of the Z score of log2 cpm for control, AcKO, DcKO, and ADcKO mice show distinct trends in gene expression for each of the four clusters (C1–C4) of genes identified in (A). ****p < 10−4, ***p < 10−3, one-way ANOVA, Tukey’s multiple comparison test; n.s., not significant. (C) WashU Epigenome browser view of RNA-seq coverage from AcKO, DcKO, ADcKO, and respective control (Ctrl) mice, illustrating changes in gene expression for each of the four clusters (C1–C4). See also Figure S2.
Figure 3.
Figure 3.. 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.
Figure 4.
Figure 4.. 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.
Figure 5.
Figure 5.. 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.

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