Interplay between the Chd4/NuRD Complex and the Transcription Factor Znf219 Controls Cardiac Cell Identity

Figures

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  Figure 1. Chd4 and Znf219 physically interact in the mouse heart. (a) Bubble plot representation of the enrichment analysis for transcription factor (TF) binding sites in the set of upregulated genes in adult Chd4corin hearts (TRANSFAC version 7.4). (b) Bubble plot representation of similar enrichment analysis for TF binding sites present in the set of upregulated genes in ED10.5 Chd4nkx hearts. (c) The Chd4/NuRD complex composition in heart tissue; components were identified from peptides (pept.) recovered by mass spectrometry after Chd4 immunoprecipitation (IP) from 4-week-old WT mouse hearts (Chd4F/F; Corinwt/w). The table represents the number of distinct peptides (IP: pept.) derived from the proteins associated with Chd4 (Mi-2β) in WT hearts, and in Chd4corin (Chd4F/F; Corincre/cre) cKO hearts. (d) The specificity of the association of Znf219 with WT Chd4 was demonstrated by analyzing the extracted ion chromatography traces of the peptides from the proteins indicated in each graph. M* means oxidated methionine. (e) Western blot of WT and Chd4corin cardiac nuclear extracts (Ext.) and Chd4 (α-Chd4) immunoprecipitated (IP), showing an association between Chd4 (arrow) and Znf219 (*)) only…

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  Figure 2. Znf219 represses the skeletal-muscle sarcomeric program in vitro. (a) Relative mRNA expression of Znf219 in different mouse organs (n = 4). Sk-muscle = skeletal muscle (tibialis anterior). (b) Western blot of different mouse organs, showing predominant Znf219 protein expression in the heart. (c) Relative mRNA expression of Znf219 during mouse cardiac development (n = 3). (d) Western blot of skeletal and cardiac cell lines, showing Znf219 protein expression in myocytic cell line C2C12 and the cardiomyocytic cell line HL-1. DM = differentiation media; GM = growth media. (e) qPCR analysis of representative skeletal muscle genes in HL-1 cells silenced for Znf219 or Chd4 with corresponding shRNAs. Cells infected with scrambled shRNA were used as a control (n = 4); two-way ANOVA (Bonferroni); * adj-p-value < 0.05, **** p-value < 0.0001. (f) Western blot showing the extent of knockdown of Znf219 (lane 2, middle) and Chd4 (lane 3, up) with the corresponding shRNAs. Lane 1 corresponds to HL-1 cells infected with scrambled shRNA (Scramble).

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  Figure 3. Znf219 regulates heart homeostasis in vivo. (a) Schematic representation of the experimental design after subcutaneous AAV9 injection into CD1 PD1-2 pups. (b) Gross morphological appearance of whole hearts of 8-week-old infected mice (upper panels) and fluorescence microscopy showing GFP expression in both atria and ventricles (lower panels). (c) Heart weight/tibia length ratios in infected mice. Paired t-test (two-tailed), ** adj. p-value < 0.01. (d) H&E (HE), Sirius Red (SR), and GFP staining on transverse heart sections from 8-week-old infected mice. Hearts were infected with 2 × 1011 particles of AAV9-shRNA-Scramble (left) or 2 × 1011 AAV9-shRNA-Znf219 (right). Mice infected with shRNA-Znf219 show interstitial fibrosis (middle right). ShRNA-Scramble (n = 9); shRNAZnf219 (n = 13).

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  Figure 4. Znf219 represses the skeletal-muscle sarcomeric program in vivo. (a) qPCR analysis showing downregulation of Znf219 but no change in Chd4 in mice infected with ShZnf219; paired t-test (two-tailed) **** p-value < 0.0001). (b) Western blot showing Znf219 protein expression in ShScramble- and ShZnf219-infected mice. (c) qPCR analysis of selected genes encoding sarcomeric skeletal-muscle proteins (two-way ANOVA: ns = not significant; ** p-value ≤ 0.01, **** p-value ≤ 0.0001); ns = non-significant. (d) Western blot showing α-troponin I and T fast skeletal protein expression. (e) IHC analysis of Atp2a1 in longitudinal heart sections of 8-week-old infected mice. ShZnf219 mice (right) show ectopic atrial expression of Atp2a1.

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  Figure 5. Znf219 knockdown induces cardiac arrhythmias. (a) Electrocardiography (ECG) traces of 4-week-old and (b) 8-week-old mice infected with shRNA-Scramble or shRNA-Znf219, the latter showing arrhythmias (c). PR and QRS intervals in 4-week-old mice infected with shRNA-Scramble (ShScramble) or shRNA-Znf219 (ShZnf219)., ** p ≤ 0.01, (mean + S.E.M.), ns = non-significant.

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  Figure 6. Model of the function of Znf219 in the mammalian heart. Proposed model of Znf219-Chd4/NuRD interplay in cardiac regulation. (a) Znf219/Chd4 complexes silence the expression of the skeletal-muscle gene program in cardiac tissue. (b) In the absence of Znf219 or Chd4, the skeletal-muscle gene program is activated ectopically in the heart, leading to fibrosis, conformational arrhythmias, and cardiac remodeling. This figure was created with biorender.com.