BRD4 (Bromodomain-Containing Protein 4) Interacts with GATA4 (GATA Binding Protein 4) to Govern Mitochondrial Homeostasis in Adult Cardiomyocytes

Abstract

Background: Gene regulatory networks control tissue homeostasis and disease progression in a cell type-specific manner. Ubiquitously expressed chromatin regulators modulate these networks, yet the mechanisms governing how tissue specificity of their function is achieved are poorly understood. BRD4 (bromodomain-containing protein 4), a member of the BET (bromo- and extraterminal domain) family of ubiquitously expressed acetyl-lysine reader proteins, plays a pivotal role as a coactivator of enhancer signaling across diverse tissue types in both health and disease and has been implicated as a pharmacological target in heart failure. However, the cell-specific role of BRD4 in adult cardiomyocytes remains unknown.

Methods: We combined conditional mouse genetics, unbiased transcriptomic and epigenomic analyses, and classic molecular biology and biochemical approaches to understand the mechanism by which BRD4 in adult cardiomyocyte homeostasis.

Results: Here, we show that cardiomyocyte-specific deletion of Brd4 in adult mice leads to acute deterioration of cardiac contractile function with mutant animals demonstrating a transcriptomic signature characterized by decreased expression of genes critical for mitochondrial energy production. Genome-wide occupancy data show that BRD4 enriches at many downregulated genes (including the master coactivators Ppargc1a, Ppargc1b, and their downstream targets) and preferentially colocalizes with GATA4 (GATA binding protein 4), a lineage-determining cardiac transcription factor not previously implicated in regulation of adult cardiac metabolism. BRD4 and GATA4 form an endogenous complex in cardiomyocytes and interact in a bromodomain-independent manner, revealing a new functional interaction partner for BRD4 that can direct its locus and tissue specificity.

Conclusions: These results highlight a novel role for a BRD4-GATA4 module in cooperative regulation of a cardiomyocyte-specific gene program governing bioenergetic homeostasis in the adult heart.

Keywords: epigenomics; mitochondria; myocytes, cardiac.

Figure 1:. Adult cardiomyocyte-specific Brd4 deletion results in acute and persistent contractile dysfunction and lethality.

(A) Immunoblot of isolated Myh6-MCM; Brd4^flox/flox cardiomyocyte lysates from mice treated with vehicle (VEH) or tamoxifen (TAM) for 5 days using BRD4 or vinculin (loading control) antibodies. (B) Kaplan-Meier curve demonstrating survival of indicated mice treated with tamoxifen (TAM; 75 μg/g/day). p-values were calculated using a Mantel–Cox test. (C-F) H&E images of Myh6-MCM; Brd4^flox/flox mice treated with vehicle (VEH) or tamoxifen (TAM) at low and high magnification. (G) Ejection fraction and (H) left ventricular end systolic volume (LVESV) of indicated mice treated with tamoxifen (TAM) or vehicle (VEH) at indicated days after injection. Individual points and mean ± SD shown. One-way ANOVA analysis coupled with a Tukey test was used to assess significance. For B, G, and H, **** represents p<0.0001 for indicated comparison. Scale bars = 100 μm (D, F) and 500 μm (C, E)

Figure 2:. BRD4 regulates mitochondrial metabolic pathways in adult cardiomyocytes.

(A, B) Volcano plots showing Log2 fold change and adj. p-value of individual genes 2 days (A) or 5 days (B) after Brd4 deletion; genes differentially expressed between Cre-control and Control samples have been excluded. Selected categories identified from gene ontology analysis from up or down regulated genes are shown below the volcano plot. Genes are assigned with specific colors following DE analysis: grey (not significant), green (Log2 FC<−1 or >+1), blue (adj. p<0.05) or red (Log2 FC<−1 or >+1 and adj. p<0.05). (C) Track view of Ppargc1a and Ppargc1b genes showing sequencing reads mapping from RNA-seq signature at day 2 post-tamoxifen (TAM) treatment for Control and Brd4-KO samples. (D) Heatmap of expression of PPARGC1A known targets in Control and Brd4-KO samples at day 5 post-TAM treatment. (E, F) Electron micrographs of Brd4-KO and Control animals at day 5 highlighting the loss of normal mitochondrial morphology. (G) FPKMs of indicated genes related to cardiac stress and homeostasis in Control (n=3) or Brd4-KO (n=3) cardiomyocytes at day 5. Error bars represent standard deviation (SD). (H) Correlation analysis of difference in gene expression in day 5 Brd4-KO cardiomyocytes compared to sham-operated animals administered JQ1 (normalized to their respective controls). Genes highlighted in red are those which are downregulated upon BRD4 loss but not significantly changed by JQ1. (I) Gene ontology analysis of genes that were downregulated upon BRD4 loss but not affected by JQ1 treatment. For G, * represents p<0.05; ** represents p<0.01 for indicated comparison. Scale bars = 2 μm (E, F)

Figure 3:. Constitutive cardiomyocyte-specific Brd4 deletion is embryonic lethal and reveals BRD4 as a regulator of mitochondrial metabolic pathways in embryonic cardiomyocytes.

(A) Genotyping data from parental cross of Tnnt2-Cre; Brd4^flox/+ and Brd4^flox/flox animals demonstrating embryonic lethality in Tnnt2-Cre; Brd4^flox/flox offspring at postnatal days 0 to 5 (P0–5). (B-G) H&E images of cross sections from Brd4^flox/flox, Tnnt2-Cre; Brd4^flox/+, and Tnnt2-Cre; Brd4^flox/flox embryonic hearts at E14.0 at low and high magnification. (H) Volcano plots showing Log2 fold change and adj. p-value of individual genes at E14.0 between Tnnt2-Cre; Brd4^flox/+ and Tnnt2-Cre; Brd4^flox/flox microdissected embryonic hearts. Selected categories identified from gene ontology analysis from up or down regulated genes are shown below the volcano plot. Genes are assigned with specific colors following DE analysis: grey (not significant), green (Log2 FC<−1 or >+1), blue (adj. p<0.05) or red (Log2 FC<−1 or >+1 and adj. p<0.05). (I) Heatmap of expression of PPARGC1A known targets in Tnnt2-Cre; Brd4^flox/+ and Tnnt2-Cre; Brd4^flox/flox in embryonic hearts at E14.0. Scale bars = 250 μm (B-D) and 100 μm (E-G)

Figure 4:. BRD4 and GATA4 co-occupy and regulate genes controlling mitochondrial homeostasis.

(A) Venn diagram showing number of unique and shared accessible chromatin regions between Control and Brd4-KO samples. (B) Top selected categories identified from gene ontology analysis from Control and Brd4-KO specific ATAC regions. (C) Motif enrichment analysis for cardiac TFs in unique and shared accessible chromatin regions between Control and Brd4-KO samples. (D) Number of differentially expressed genes between Control and Brd4-KO at day 2 and day 5 occupied by cardiac TFs^, at their promoters (±1 TSS). (E) Heatmaps showing enrichment of BRD4 and GATA4 ChIP signals from adult mouse hearts under basal homeostatic conditions at gene promoters (±1 TSS, 55,386 mm10 annotated transcripts) ordered by BRD4 intensity identifies a cluster of strongly bound transcripts (Cluster 1, n=8080). (F) Gene ontology analysis of Cluster 1 genes identified enriched terms for biological processes, cellular components, and SNP-phenotype associations. (G-H) Track view of Ppargc1a and Ppargc1b genes showing sequencing reads mapping from BRD4 and GATA4 ChIP-seq as well as Control and Brd4-KO ATAC-seq at day 5 post-tamoxifen treatment. The promoter region and a putative regulatory element for each gene is highlighted in red.

Figure 5.. BRD4 and GATA4 interact in a bromodomain-independent manner to control the master regulator of mitochondrial homeostasis Ppargc1a.

(A) Gene reporter assay showing activation of indicated luciferase reporters upon addition of plasmids encoding indicated proteins. Statistical significance is shown between control and GATA4 + BRD4 vs. all other individual conditions (n=4). (B) Relative expression by RT-qPCR in neonatal rat ventricular myocytes. Statistical significance is indicated between siControl and siGata4 + siBrd4 vs. all other individual conditions (n=3). One-way ANOVA analysis coupled with a Tukey test was used to assess significance. (C) Immunoprecipitation (IP) of endogenous protein from human cardiac progenitor cells using anti-GATA4 or anti-IgG antibody and immunoblotting (IB) with anti-BRD4 or anti-GATA4 antibody demonstrates endogenous BRD4 co-immunoprecipitates with GATA4 but not IgG. (D) Immunoprecipitation of FLAG-BRD4 overexpressed in HEK293 cells followed by immunoblotting with anti-GFP or anti-FLAG antibodies demonstrates GFP-GATA4 still co-IPs with BRD4 even in the presence of increasing doses of JQ1 or DMSO as control. (E) Immunoprecipitation of FLAG-BRD4, FLAG-BRD4^N140A/N433A, and FLAG-BRD4^Y97A/Y390A in HEK293 cells followed by immunoblotting with anti-GFP or anti-FLAG antibodies indicates co-immunoprecipitation of BRD4 mutants with GFP-GATA4. For A and B, * represents p<0.05, ** represents p<0.01, *** represents p<0.001 and **** represents p<0.0001 for indicated comparison.