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. 2017 Sep 1;31(17):1770-1783.
doi: 10.1101/gad.305482.117.

ZNF281 Enhances Cardiac Reprogramming by Modulating Cardiac and Inflammatory Gene Expression

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Free PMC article

ZNF281 Enhances Cardiac Reprogramming by Modulating Cardiac and Inflammatory Gene Expression

Huanyu Zhou et al. Genes Dev. .
Free PMC article

Abstract

Direct reprogramming of fibroblasts to cardiomyocytes represents a potential means of restoring cardiac function following myocardial injury. AKT1 in the presence of four cardiogenic transcription factors, GATA4, HAND2, MEF2C, and TBX5 (AGHMT), efficiently induces the cardiac gene program in mouse embryonic fibroblasts but not adult fibroblasts. To identify additional regulators of adult cardiac reprogramming, we performed an unbiased screen of transcription factors and cytokines for those that might enhance or suppress the cardiogenic activity of AGHMT in adult mouse fibroblasts. Among a collection of inducers and repressors of cardiac reprogramming, we discovered that the zinc finger transcription factor 281 (ZNF281) potently stimulates cardiac reprogramming by genome-wide association with GATA4 on cardiac enhancers. Concomitantly, ZNF281 suppresses expression of genes associated with inflammatory signaling, suggesting the antagonistic convergence of cardiac and inflammatory transcriptional programs. Consistent with an inhibitory influence of inflammatory pathways on cardiac reprogramming, blockade of these pathways with anti-inflammatory drugs or components of the nucleosome remodeling deacetylase (NuRD) complex, which associate with ZNF281, stimulates cardiac gene expression. We conclude that ZNF281 acts at a nexus of cardiac and inflammatory gene programs, which exert opposing influences on fibroblast to cardiac reprogramming.

Keywords: ZFP281; anti-inflammation; cardiac gene activation; cardiomyocytes; direct cellular reprogramming; heart regeneration.

Figures

Figure 1.
Figure 1.
Identification of activators and inhibitors of 5F-mediated cardiac reprogramming from a human ORF cDNA screen. (A) Schematic diagram of the human ORF cDNA library screen strategy for cardiac reprogramming in adult TTFs. (B) Venn diagram showing the number of activators identified from the screen. Genes with Z-scores of αMHC-GFP or cTnT expression ≥2 were defined as activators. Twenty-five genes induced αMHC-GFP expression only, 35 genes induced cTnT expression only, and 11 genes induced expression of both markers. (C) Venn diagram showing the number of inhibitors identified from the screen. Genes with Z-scores of αMHC-GFP or cTnT expression −2 or lower were defined as inhibitors. One-hundred-twenty-one genes repressed αMHC expression only, 41 genes repressed cTnT expression only, and 33 genes repressed both αMHC-GFP and cTnT expression. (D) Representative immunocytochemistry images of TTFs from adult αMHC-GFP transgenic mice treated with 5F and either empty virus or viruses encoding activators (ZNF281 or PHF7) and inhibitors (FOXA3 or SOX9). Cells were fixed and stained for αMHC-GFP (green), cTnT (red), and Hoechst (blue) 9 d after infection. Bars, 2 mm. (E) Pathways enriched in activators (n = 49) and inhibitors (n = 129), respectively, by DAVID pathway analysis.
Figure 2.
Figure 2.
ZNF281 enhances cardiac reprogramming of adult fibroblasts. (A) Immunocytochemistry images of adult αMHC-GFP transgenic TTFs 7 d after infection with empty, ZNF281, 5F, or 6F retroviruses show that ZNF281 enhances expression of cardiac markers with 5F. (Green) αMHC-GFP; (red) cTnT; (blue) Hoechst. Bars, 500 µm. (B,C) Representative flow cytometry plot (B) and analyses (C) of αMHC-GFP+ and cTnT+ TTFs 7 d after infection with empty, 5F, or 6F. (D) TTFs were infected with empty, 5F plus empty, and ZNF281 for 7 d. Transcript levels of cardiac marker genes (Myh6 and Actc1) and fibroblast marker genes (Col1a2 and Sox9) were determined by qPCR. (*) P < 0.05.
Figure 3.
Figure 3.
RNA-seq analysis shows that ZNF281 enhances cardiac genes and represses inflammatory genes. (A) Heat map of 1500 differentially expressed genes in 5F-treated versus 6F-treated TTFs identified by RNA-seq. (Red) Up-regulation; (blue) down-regulation. RNA-seq samples were prepared from adult TTFs reprogrammed for 7 d. (B,C) GO analysis showing biological processes associated with genes up-regulated (B) and down-regulated (C) by ZNF281. (D,E) Gene expression changes between 6F and 5F for selected cardiac markers (D) or inflammatory markers (E) as determined by RNA-seq. (F–H) Enrichment plots of the indicated gene sets and their nominal P-value of genes up-regulated by ZNF281. (I,J) Enrichment plots of the indicated gene sets and their nominal P-value of genes down-regulated by ZNF281.
Figure 4.
Figure 4.
ZNF281 interacts with GATA4 to synergistically activate cardiac genes. (A) Coimmunoprecipitation assays were performed using HEK293 cells transfected with equal amounts of plasmid DNA encoding Myc-tagged GATA4, HAND2, MEF2C, or TBX5 and/or Flag-tagged ZNF281. (IP) Immunoprecipitation; (IB) immunoblot. (B) Heat maps showing ChIP-seq (chromatin immunoprecipitation [ChIP] followed by high-throughput sequencing) data for H3K27ac in adult mouse hearts and ZNF281 and GATA4 binding in reprogrammed TTFs at ±5 kb around the peak center. ChIP-seq experiments were performed using TTFs infected with 6F for 2 d. (C) ZNF281-binding motif enriched within ZNF281-binding peaks. (D) GATA4-binding motif enriched within GATA4-binding peaks. (E) Venn diagram showing the number of overlapping peaks between heart H3K27ac, ZNF281, and GATA4. (F) Total number of peaks identified by ChIP-seq.
Figure 5.
Figure 5.
GATA4 recruits ZNF281 to cardiac enhancers. (A,B) Heat map for ZNF281 or GATA4 genomic binding at ±2 kb around the peak center in each cluster. ChIP-seq experiments were performed using adult TTFs reprogrammed for 2 d with 6F, 6F − G, or 6F − Z. (C) Integrative Genomics Viewer (IGV) browser tracks at chromosome 1 (137,694,960–137,749,970; mm9) show an example of peaks that belong to each indicated cluster. (D,E) GOs (D) and pathways (E) enriched in genes that associate with each indicated cluster identified by GO enrichment analysis and pathway analysis.
Figure 6.
Figure 6.
ZNF281 represses the inflammatory response through the NuRD complex. (A) TTFs were infected with empty, 5F plus empty, ZNF281, or each individual NuRD complex subunit retrovirus for 7 d. Transcript levels of inflammatory (IL6 and Ccl2) and cardiac (Myh6 and Actc1) marker genes were determined by qPCR. (B) Representative flow cytometry plot of αMHC-GFP+ and cTnT+ TTFs 7 d after infection with empty, 5F plus empty, ZNF281, or each individual NuRD complex subunit retroviruses. The dashed line indicates control. (*) P < 0.05.
Figure 7.
Figure 7.
Anti-inflammatory drugs promote cardiac reprogramming. (A) 5F reprogrammed TTFs treated with either DMSO or the indicated anti-inflammatory drugs for 7 d after infection. Expression of inflammatory genes (IL6, Ccl2, and Ptgs1), and cardiac genes (Myh6, Actc1, and Nppa) was determined by qPCR. (Dex) 10 µM Dex; (Nab) 10 µM Nab. (B) Immunocytochemistry images of 5F reprogrammed adult αMHC-GFP transgenic TTFs treated with DMSO or the indicated anti-inflammatory drugs for 7 d. (Green) αMHC-GFP; (red) cTnT; (blue) Hoechst. Bars, 500 µm. (C,D) Representative flow cytometry plot (C) and analyses (D) of αMHC-GFP+ and cTnT+ cells in 5F reprogrammed adult αMHC-GFP transgenic TTFs treated with DMSO or the indicated anti-inflammatory drugs for 7 d. (*) P < 0.05. (E) Model showing the mechanism of action of ZNF281 on 5F-mediated direct cardiac reprogramming. ZNF281 is a cardiac transcription coactivator recruited by GATA4 to cardiac enhancers to activate cardiac gene expression. ZNF281 also represses the inflammatory response, which acts as a barrier pathway to cardiac reprogramming.

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