Affinity chromatography reveals direct binding of the GATA4-NKX2-5 interaction inhibitor (3i-1000) with GATA4.

Heart failure is a serious medical condition with a poor prognosis. Current treatments can only help manage the symptoms and slow the progression of heart failure. However, there is currently no cure to prevent and reverse cardiac remodeling. Transcription factors are in a central role in various cellular processes, and in the heart, GATA4 and NKX2-5 transcription factors mediate hypertrophic responses and remodeling. We have identified compounds that modulate the synergistic interaction of GATA4 and NKX2-5 and shown that the most promising compound (1, 3i-1000) is cardioprotective in vitro and in vivo. However, direct evidence of its binding site and mechanism of action has not been available. Due to the disordered nature of transcription factors, classical target engagement approaches cannot be utilized. Here, we synthesized a small-molecule ligand-binding pulldown probe of compound 1 to utilize affinity chromatography alongside CETSA, AlphaScreen, and molecular modeling to study ligand binding. These results provide the first evidence of direct physical binding of compound 1 selectively to GATA4. While developing drugs that target transcription factors presents challenges, advances in technologies and knowledge of intrinsically disordered proteins enable the identification of small molecules that can selectively target transcription factors.

Enhanced GATA4 expression in senescent systemic lupus erythematosus monocytes promotes high levels of IFNα production.

Enhanced interferon α (IFNα) production has been implicated in the pathogenesis of systemic lupus erythematosus (SLE). We previously reported IFNα production by monocytes upon activation of the stimulator of IFN genes (STING) pathway was enhanced in patients with SLE. We investigated the mechanism of enhanced IFNα production in SLE monocytes. Monocytes enriched from the peripheral blood of SLE patients and healthy controls (HC) were stimulated with 2'3'-cyclic GAMP (2'3'-cGAMP), a ligand of STING. IFNα positive/negative cells were FACS-sorted for RNA-sequencing analysis. Gene expression in untreated and 2'3'-cGAMP-stimulated SLE and HC monocytes was quantified by real-time PCR. The effect of GATA binding protein 4 (GATA4) on IFNα production was investigated by overexpressing GATA4 in monocytic U937 cells by vector transfection. Chromatin immunoprecipitation was performed to identify GATA4 binding target genes in U937 cells stimulated with 2'3'-cGAMP. Differentially expressed gene analysis of cGAS-STING stimulated SLE and HC monocytes revealed the enrichment of gene sets related to cellular senescence in SLE. CDKN2A, a marker gene of cellular senescence, was upregulated in SLE monocytes at steady state, and its expression was further enhanced upon STING stimulation. GATA4 expression was upregulated in IFNα-positive SLE monocytes. Overexpression of GATA4 enhanced IFNα production in U937 cells. GATA4 bound to the enhancer region of IFIT family genes and promoted the expressions of IFIT1, IFIT2, and IFIT3, which promote type I IFN induction. SLE monocytes with accelerated cellular senescence produced high levels of IFNα related to GATA4 expression upon activation of the cGAS-STING pathway.

GATA4 regulates the transcription of MMP9 to suppress the invasion and migration of breast cancer cells via HDAC1-mediated p65 deacetylation.

GATA-binding protein 4 (GATA4) is recognized for its significant roles in embryogenesis and various cancers. Through bioinformatics and clinical data, it appears that GATA4 plays a role in breast cancer development. Yet, the specific roles and mechanisms of GATA4 in breast cancer progression remain elusive. In this study, we identify GATA4 as a tumor suppressor in the invasion and migration of breast cancer. Functionally, GATA4 significantly reduces the transcription of MMP9. On a mechanistic level, GATA4 diminishes MMP9 transcription by interacting with p65 at the NF-κB binding site on the MMP9 promoter. Additionally, GATA4 promotes the recruitment of HDAC1, amplifying the bond between p65 and HDAC1. This leads to decreased acetylation of p65, thus inhibiting p65's transcriptional activity on the MMP9 promoter. Moreover, GATA4 hampers the metastasis of breast cancer in vivo mouse model. In summary, our research unveils a novel mechanism wherein GATA4 curtails breast cancer cell metastasis by downregulating MMP9 expression, suggesting a potential therapeutic avenue for breast cancer metastasis.

Switching of hypertrophic signalling towards enhanced cardiomyocyte identity and maturity by a GATA4-targeted compound.

BACKGROUND: The prevalence of heart failure is constantly increasing, and the prognosis of patients remains poor. New treatment strategies to preserve cardiac function and limit cardiac hypertrophy are therefore urgently needed. Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are increasingly used as an experimental platform for cardiac in vitro studies. However, in contrast to adult cardiomyocytes, hiPSC-CMs display immature morphology, contractility, gene expression and metabolism and hence express a naive phenotype that resembles more of a foetal cardiomyocyte. METHODS: A library of 14 novel compounds was synthesized in-house and screened for GATA4-NKX2-5 reporter activity and cellular toxicity. The most potent compound, 3i-1262, along with previously reported GATA4-acting compounds, were selected to investigate their effects on hypertrophy induced by endothelin-1 or mechanical stretch. Morphological changes and protein expression were characterized using immunofluorescence staining and high-content analysis. Changes in gene expression were studied using qPCR and RNA sequencing. RESULTS: The prototype compound 3i-1262 inhibited GATA4-NKX2-5 synergy in a luciferase reporter assay. Additionally, the isoxazole compound 3i-1262 inhibited the hypertrophy biomarker B-type natriuretic peptide (BNP) by reducing BNP promoter activity and proBNP expression in neonatal rat ventricular myocytes and hiPSC-CMs, respectively. Treatment with 3i-1262 increased metabolic activity and cardiac troponin T expression in hiPSC-CMs without affecting GATA4 protein levels. RNA sequencing analysis revealed that 3i-1262 induces gene expression related to metabolic activity and cell cycle exit, indicating a change in the identity and maturity status of hiPSC-CMs. The biological processes that were enriched in upregulated genes in response to 3i-1262 were downregulated in response to mechanical stretch, and conversely, the downregulated processes in response to 3i-1262 were upregulated in response to mechanical stretch. CONCLUSIONS: There is currently a lack of systematic understanding of the molecular modulation and control of hiPSC-CM maturation. In this study, we demonstrated that the GATA4-interfering compound 3i-1262 reorganizes the cardiac transcription factor network and converts hypertrophic signalling towards enhanced cardiomyocyte identity and maturity. This conceptually unique approach provides a novel structural scaffold for further development as a modality to promote cardiomyocyte specification and maturity.

GATA4 inhibits odontoblastic differentiation of dental pulp stem cells through targeting IGFBP3.

OBJECTIVE: The odontogenic differentiation of human dental pulp stem cells (HDPSCs) is associated with reparative dentinogenesis. Transcription factor GATA binding protein 4 (GATA4) is proved to be essential for osteoblast differentiation and bone remodeling. This study clarified the function of GATA4 in HDPSCs odontoblast differentiation. METHODS: The change in GATA4 expression during reparative dentin formation was detected by immunohistochemistry staining. The expression of GATA4 during HDPSCs odontoblastic differentiation was detected by western blot and quantitative polymerase chain reaction. The effect of GATA4 on odontoblast differentiation was investigated following overexpression lentivirus transfection. RNA sequencing, dual luciferase assay and chromatin immunoprecipitation (CHIP) were conducted to verify downstream targets of GATA4. GATA4 overexpression lentivirus and small interference RNA targeting IGFBP3 were co-transfected to investigate the regulatory mechanism of GATA4. RESULTS: Upregulated GATA4 was observed during reparative dentin formation in vivo and the odontoblastic differentiation of HDPSCs in vitro. GATA4 overexpression suppressed the odontoblastic potential of HDPSCs, demonstrated by decreased alkaline phosphatase activity (p < 0.0001), mineralized nodules formation (p < 0.01), and odonto/osteogenic differentiation markers levels (p < 0.05). RNA sequencing revealed IGFBP3 was a potential target of GATA4. CHIP and dual luciferase assays identified GATA4 could activate IGFBP3 transcription. Additionally, IGFBP3 knockdown recovered the odontoblastic differentiation defect caused by GATA4 overexpression (p < 0.05). CONCLUSIONS: GATA4 inhibited odontoblastic differentiation of HDPSCs via activating the transcriptional activity of IGFBP3, identifying its promising role in regulating HDPSCs odontoblast differentiation and reparative dentinogenesis.

Induction of Senescence by Loss of Gata4 in Cardiac Fibroblasts.

Cardiac fibroblasts are a major source of cardiac fibrosis during heart repair processes in various heart diseases. Although it has been shown that cardiac fibroblasts become senescent in response to heart injury, it is unknown how the senescence of cardiac fibroblasts is regulated in vivo. Gata4, a cardiogenic transcription factor essential for heart development, is also expressed in cardiac fibroblasts. However, it remains elusive about the role of Gata4 in cardiac fibroblasts. To define the role of Gata4 in cardiac fibroblasts, we generated cardiac fibroblast-specific Gata4 knockout mice by cross-breeding Tcf21-MerCreMer mice with Gata4fl/fl mice. Using this mouse model, we could genetically ablate Gata4 in Tcf21 positive cardiac fibroblasts in an inducible manner upon tamoxifen administration. We found that cardiac fibroblast-specific deletion of Gata4 spontaneously induces senescence in cardiac fibroblasts in vivo and in vitro. We also found that Gata4 expression in both cardiomyocytes and non-myocytes significantly decreases in the aged heart. Interestingly, when αMHC-MerCreMer mice were bred with Gata4fl/fl mice to generate cardiomyocyte-specific Gata4 knockout mice, no senescent cells were detected in the hearts. Taken together, our results demonstrate that Gata4 deficiency in cardiac fibroblasts activates a program of cellular senescence, suggesting a novel molecular mechanism of cardiac fibroblast senescence.

Determination of Complex Formation between Drosophila Nrf2 and GATA4 Factors at Selective Chromatin Loci Demonstrates Transcription Coactivation.

Nrf2 is the dominant cellular stress response factor that protects cells through transcriptional responses to xenobiotic and oxidative stimuli. Nrf2 malfunction is highly correlated with many human diseases, but the underlying molecular mechanisms remain to be fully uncovered. GATA4 is a conserved GATA family transcription factor that is essential for cardiac and dorsal epidermal development. Here, we describe a novel interaction between Drosophila Nrf2 and GATA4 proteins, i.e., cap'n'collar C (CncC) and Pannier (Pnr), respectively. Using the bimolecular fluorescence complementation (BiFC) assay-a unique imaging tool for probing protein complexes in living cells-we detected CncC-Pnr complexes in the nuclei of Drosophila embryonic and salivary gland cells. Visualization of CncC-Pnr BiFC signals on the polytene chromosome revealed that CncC and Pnr tend to form complexes in euchromatic regions, with a preference for loci that are not highly occupied by CncC or Pnr alone. Most genes within these loci are activated by the CncC-Pnr BiFC, but not by individually expressed CncC or Pnr fusion proteins, indicating a novel mechanism whereby CncC and Pnr interact at specific genomic loci and coactivate genes at these loci. Finally, CncC-induced early lethality can be rescued by Pnr depletion, suggesting that CncC and Pnr function in the same genetic pathway during the early development of Drosophila. Taken together, these results elucidate a novel crosstalk between the Nrf2 xenobiotic/oxidative response factor and GATA factors in the transcriptional regulation of development. This study also demonstrates that the polytene chromosome BiFC assay is a valuable tool for mapping genes that are targeted by specific transcription factor complexes.

Oncogenic SIRT7 inhibits GATA4 transcriptional activity and activates the Wnt signaling pathway in ovarian cancer.

OBJECTIVE: Sirtuin-7 (SIRT7) is a class III histone deacetylase that plays an important role in cancer development and frequently overexpressed in carcinomas. In this study, the tumor-supporting role and underlying mechanisms of SIRT7 were characterized in ovarian cancer (OC) aggressiveness. METHODS: SIRT7 expression was examined in OC tissues and cells. Interactions among SIRT7, GATA4, Wnt signaling pathway were explored by bioinformatics tools and experimental validations. The effect of SIRT7 and GATA4 on malignant phenotypes of OC cells were examined with gain- and loss-of-function experiments. A nude mouse model of OC was developed to verify the in vitro findings. RESULTS: It was noted that SIRT7 was highly expressed in OC tissues and cells. Cell lines with higher SIRT7 expression (OVCAR-3 and OVCAR-8) were used for subsequent in vitro experiments. The experimental data indicated that silencing of SIRT7 suppressed the OC cell proliferation, colony formation, migration, and invasion, and promoted cell senescence, which could be abolished by GATA4 knockdown. Mechanistically, SIRT7 promoted deacetylation of GATA4 and consequently inhibited the transcriptional activity of GATA4. In addition, GATA4 induced OC cell senescence by inhibiting Wnt signaling pathway. Further in vivo experiments substantiated that SIRT7 knockdown or overexpressed GATA4 could effectively inhibit tumor growth of nude mice. CONCLUSION: Taken together, our findings indicated that SIRT7 enhanced development of OC by suppressing GATA4 and activating Wnt signaling pathway, suggesting the potential of SIRT7/GATA4/Wnt axis as a therapeutic target for OC.

GATA-binding protein 4 promotes neuroinflammation and cognitive impairment in Aß1-42 fibril-infused rats through small nucleolar RNA host gene 1/miR-361-3p axis.

Aging with dysregulated metabolic and immune homeostasis stimulates pyroptosis, neuroinflammation, and cellular senescence, thus contributing to etiopathogenesis of Alzheimer's disease. GATA-binding protein 4 (GATA4) functions as a transcriptional factor in response to DNA damage, and is associated with neuroinflammation and cellular senescence. The role of GATA4 in Alzheimer's disease was investigated. GATA4 was elevated in hippocampus of Aß1-42 fibril-infused rats. Injection with shRNA targeting GATA4 reduced escape latency with increase of time in target quadrant and number of platform crossings in Aß1-42 fibril-infused rats. Moreover, knockdown of GATA4 ameliorated morphological changes of hippocampus and reduced amyloid plaque deposition in Aß1-42 fibril-infused rats. Silence of GATA4 repressed neuroinflammation and apoptosis in Aß1-42 fibril-infused rats. Loss of GATA4 in Aß1-42 fibril-infused rats reduced the expression of specificity protein 1 (Sp1) to downregulate long noncoding RNA small nucleolar RNA host gene 1 (SNHG1) and upregulated miR-361-3p. Loss of SNHG1 ameliorated learning and memory impairments in Aß1-42 fibril-infused rats. Overexpression of Sp1 attenuated GATA4 silence-induced decrease of escape latency, increase of time in target quadrant, and number of platform crossings in Aß1-42 fibril-infused rats. In conclusion, silence of GATA4 ameliorated cognitive dysfunction and inhibited hippocampal inflammation and cell apoptosis through regulation of Sp1/SNHG1/miR-361-3p.

Bmi-1 Overexpression Improves Sarcopenia Induced by 1,25(OH)2 D3 Deficiency and Downregulates GATA4-Dependent Rela Transcription.

Sarcopenia increases with age, and an underlying mechanism needs to be determined to help with designing more effective treatments. This study aimed to determine whether 1,25(OH)2 D3 deficiency could cause cellular senescence and a senescence-associated secretory phenotype (SASP) in skeletal muscle cells to induce sarcopenia, whether GATA4 could be upregulated by 1,25(OH)2 D3 deficiency to promote SASP, and whether Bmi-1 reduces the expression of GATA4 and GATA4-dependent SASP induced by 1,25(OH)2 D3 deficiency in skeletal muscle cells. Bioinformatics analyses with RNA sequencing data in skeletal muscle from physiologically aged and young mice were conducted. Skeletal muscles from 2-month-old young and 2-year-old physiologically aged wild-type (WT) mice and 8-week-old WT, Bmi-1 mesenchymal transgene (Bmi-1Tg ), Cyp27b1 homozygous (Cyp27b1-/- ), and Bmi-1Tg Cyp27b1-/- mice were observed for grip strength, cell senescence, DNA damage, and NF-κB-mediated SASP signaling of skeletal muscle. We found that muscle-derived Bmi-1 and vitamin D receptor (VDR) decreased with physiological aging, and DNA damage and GATA4-dependent SASP activation led to sarcopenia. Furthermore, 1,25(OH)2 D3 deficiency promoted DNA damage-induced GATA4 accumulation in muscles. GATA4 upregulated Rela at the region from -1448 to -1412 bp at the transcriptional level to cause NF-κB-dependent SASP for aggravating cell senescence and muscular dysfunction and sarcopenia. Bmi-1 overexpression promoted the ubiquitination and degradation of GATA4 by binding RING1B, which prevented cell senescence, SASP, and dysfunctional muscle, and improved sarcopenia induced by 1,25(OH)2 D3 deficiency. Thus, Bmi-1 overexpression improves sarcopenia induced by 1,25(OH)2 D3 deficiency, downregulates GATA4-dependent Rela transcription, and sequentially inhibits GATA4-dependent SASP in muscle cells. Therefore, Bmi-1 overexpression could be used for translational gene therapy for the ubiquitination of GATA4 and prevention of sarcopenia. © 2023 American Society for Bone and Mineral Research (ASBMR).

GATA4 controls regionalization of tissue immunity and commensal-driven immunopathology.

There is growing recognition that regionalization of bacterial colonization and immunity along the intestinal tract has an important role in health and disease. Yet, the mechanisms underlying intestinal regionalization and its dysregulation in disease are not well understood. This study found that regional epithelial expression of the transcription factor GATA4 controls bacterial colonization and inflammatory tissue immunity in the proximal small intestine by regulating retinol metabolism and luminal IgA. Furthermore, in mice without jejunal GATA4 expression, the commensal segmented filamentous bacteria promoted pathogenic inflammatory immune responses that disrupted barrier function and increased mortality upon Citrobacter rodentium infection. In celiac disease patients, low GATA4 expression was associated with metabolic alterations, mucosal Actinobacillus, and increased IL-17 immunity. Taken together, these results reveal broad impacts of GATA4-regulated intestinal regionalization on bacterial colonization and tissue immunity, highlighting an elaborate interdependence of intestinal metabolism, immunity, and microbiota in homeostasis and disease.

GATA4 and GATA6 loss-of-expression is associated with extinction of the classical programme and poor outcome in pancreatic ductal adenocarcinoma.

OBJECTIVE: GATA6 is a key regulator of the classical phenotype in pancreatic ductal adenocarcinoma (PDAC). Low GATA6 expression associates with poor patient outcome. GATA4 is the second most expressed GATA factor in the pancreas. We assessed whether, and how, GATA4 contributes to PDAC phenotype and analysed the association of expression with outcome and response to chemotherapy. DESIGN: We analysed PDAC transcriptomic data, stratifying cases according to GATA4 and GATA6 expression and identified differentially expressed genes and pathways. The genome-wide distribution of GATA4 was assessed, as well as the effects of GATA4 knockdown. A multicentre tissue microarray study to assess GATA4 and GATA6 expression in samples (n=745) from patients with resectable was performed. GATA4 and GATA6 levels were dichotomised into high/low categorical variables; association with outcome was assessed using univariable and multivariable Cox regression models. RESULTS: GATA4 messenger RNA is enriched in classical, compared with basal-like tumours. We classified samples in 4 groups as high/low for GATA4 and GATA6. Reduced expression of GATA4 had a minor transcriptional impact but low expression of GATA4 enhanced the effects of GATA6 low expression. GATA4 and GATA6 display a partially overlapping genome-wide distribution, mainly at promoters. Reduced expression of both proteins in tumours was associated with the worst patient survival. GATA4 and GATA6 expression significantly decreased in metastases and negatively correlated with basal markers. CONCLUSIONS: GATA4 and GATA6 cooperate to maintain the classical phenotype. Our findings provide compelling rationale to assess their expression as biomarkers of poor prognosis and therapeutic response.

Insulin-like growth factor 1 enhances follicle-stimulating hormone-induced phosphorylation of GATA4 in rat granulosa cells.

Preovulatory granulosa cell (GC) differentiation is essential for the maturation and release of oocytes from the ovary. We have previously demonstrated that follicle-stimulating hormone (FSH) and insulin-like growth factors (IGFs) closely interact to control GC function. Similarly, we showed that GATA4 mediates FSH actions and it is required for preovulatory follicle formation. This report aimed to determine in vivo the effect of FSH on GATA4 phosphorylation and to investigate whether FSH and IGF1 interact to regulate GATA4 activity. In rat ovaries, treatment with equine chorionic gonadotropin (eCG) increased the phosphorylation of GATA4, which was confined to the nucleus of GCs. Using primary rat GCs, we observed that GATA4 phosphorylation at serine 105 increases the transcriptional activity of this transcription factor. Like FSH, IGF1 stimulated GATA4 phosphorylation at serine 105. Interestingly, GATA4 phosphorylation was significantly higher in cells cotreated with FSH and IGF1 when compared to FSH or IGF1 alone, suggesting that IGF1 augments the effects of FSH on GATA4. It was also found that the enhancing effect of IGF1 requires AKT activity and is mimicked by the inhibition of glycogen synthase kinase-3 ß (GSK3ß), suggesting that AKT inhibition of GSK3ß may play a role in the regulation of GATA4 phosphorylation. The data support an important role of the IGF1/AKT/GSK3ß signaling pathway in the regulation of GATA4 transcriptional activity and provide new insights into the mechanisms by which FSH and IGF1 regulate GC differentiation. Our findings suggest that GATA4 transcriptional activation may, at least partially, mediate AKT actions in GCs.

Bicuspid Aortic Valve-Associated Regulatory Regions Reveal GATA4 Regulation and Function During Human-Induced Pluripotent Stem Cell-Based Endothelial-Mesenchymal Transition-Brief Report.

BACKGROUND: The endothelial-mesenchymal transition (EndoMT) is a fundamental process for heart valve formation and defects in EndoMT cause aortic valve abnormalities. Our previous genome-wide association study identified multiple variants in a large chromosome 8 segment as significantly associated with bicuspid aortic valve (BAV). The objective of this study is to determine the biological effects of this large noncoding segment in human induced pluripotent stem cell (hiPSC)-based EndoMT. METHODS: A large genomic segment enriched for BAV-associated variants was deleted in hiPSCs using 2-step CRISPR/Cas9 editing. To address the effects of the variants on GATA4 expression, we generated CRISPR repression hiPSC lines (CRISPRi) as well as hiPSCs from BAV patients. The resulting hiPSCs were differentiated to mesenchymal/myofibroblast-like cells through cardiovascular-lineage endothelial cells for molecular and cellular analysis. Single-cell RNA sequencing was also performed at different stages of EndoMT induction. RESULTS: The large deletion impaired hiPSC-based EndoMT in multiple biallelic clones compared with their isogenic control. It also reduced GATA4 transcript and protein levels during EndoMT, sparing the other genes nearby the deletion segment. Single-cell trajectory analysis revealed the molecular reprogramming during EndoMT. Putative GATA-binding protein targets during EndoMT were uncovered, including genes implicated in endocardial cushion formation and EndoMT process. Differentiation of cells derived from BAV patients carrying the rs117430032 variant as well as CRISPRi repression of the rs117430032 locus resulted in lower GATA4 expression in a stage-specific manner. TWIST1 was identified as a potential regulator of GATA4 expression, showing specificity to the locus tagged by rs117430032. CONCLUSIONS: BAV-associated distal regions regulate GATA4 expression during hiPSC-based EndoMT, which in turn promotes EndoMT progression, implicating its contribution to heart valve development.

The flavonoid GL-V9 alleviates liver fibrosis by triggering senescence by regulating the transcription factor GATA4 in activated hepatic stellate cells.

BACKGROUND AND PURPOSE: Liver fibrosis is a critical risk factor for the progression from chronic liver injury to hepatocellular carcinoma. Clinically, there is a lack of therapeutic drugs for liver fibrosis. Previous studies have confirmed that GL-V9, a newly synthesized flavonoid derivative, exhibits anti-inflammatory activity, but whether it has anti-hepatic fibrosis actions remains unclear. This study aimed to investigate the anti-fibrotic activities and potential mechanisms of GL-V9. EXPERIMENTAL APPROACH: Bile duct ligation (BDL) and carbon tetrachloride (CCl4 ) challenges were used to assess the anti-fibrotic effects of GL-V9 in vivo. Mouse primary hepatic stellate cells (pHSCs) and the human HSC line LX2 also served as a liver fibrosis model in vitro. Cellular functions and molecular mechanism were analysed using senescence-associated beta-galactosidase staining, real-time PCR, western blotting, immunofluorescence, and co-immunoprecipitation. KEY RESULTS: GL-V9 attenuated hepatic histopathological injury and collagen accumulation, as well as decreasing the expression of fibrotic genes in vivo. GL-V9 promoted senescence and inhibited the expression of fibrogenic genes in HSCs in vitro. Mechanistic studies revealed that GL-V9 induced senescence by upregulating GATA4 expression in HSCs. Further studies confirmed that GL-V9 stabilized GATA4 by promoting autophagic degradation of P62. CONCLUSION AND IMPLICATIONS: GL-V9 exerted potent anti-fibrotic effects both in vivo and in vitro by stabilizing GATA4, thereby promoting the senescence of HSCs, and by avoiding its activation and ultimately inhibiting liver fibrosis. This action indicated that the flavonoid GL-V9 is a potential therapeutic candidate for the treatment of liver fibrosis.

Deleting Gata4 in hepatocytes promoted the progression of NAFLD via increasing steatosis and apoptosis, and desensitizing insulin signaling.

Gata4 is a member of the zinc finger GATA transcription factor family and is required for liver development during the embryonic stage. Gata4 expression is repressed during NAFLD progression, however how it functions in this situation remains unclear. Here, Gata4 was deleted specifically in hepatocytes via Cre recombinase driven by the Alb promoter region. Under a high-fat diet (HFD) or methionine and choline deficient diet (MCD), Gata4 knockout (KO) male, but not female, mice displayed more severe NAFLD or NASH, evidenced by increased steatosis, fibrosis, as well as a higher NAS score and serum ALT level. The Gata4KO male liver exposed to a HFD or MCD had a reduced ratio of pACC/ACC, similar to the Gata4KO hepatocytes treated with palmitic acid. More cell apoptosis, which is associated with activated JNK signaling and inhibited NFκB signaling, was observed in the Gata4KO male liver and isolated hepatocytes. However, the inflammatory status in the Gata4KO male liver was similar to the control liver. Importantly, lower activation of AKT signaling in the liver, which is consistent with de-sensitized insulin signaling in isolated hepatocytes, was found in the Gata4KO male. In summary, our data demonstrated that loss of Gata4 in hepatocytes promoted NAFLD progression in male mice.

Hepatic GATA4 regulates cholesterol and triglyceride homeostasis in collaboration with LXRs.

GATA4 is a transcription factor known for its crucial role in the development of many tissues, including the liver; however, its role in adult liver metabolism is unknown. Here, using high-throughput sequencing technologies, we identified GATA4 as a transcriptional regulator of metabolism in the liver. GATA4 expression is elevated in response to refeeding, and its occupancy is increased at enhancers of genes linked to fatty acid and lipoprotein metabolism. Knocking out GATA4 in the adult liver (Gata4LKO) decreased transcriptional activity at GATA4 binding sites, especially during feeding. Gata4LKO mice have reduced plasma HDL cholesterol and increased liver triglyceride levels. The expression of a panel of GATA4 binding genes involved in hepatic cholesterol export and triglyceride hydrolysis was down-regulated in Gata4LKO mice. We further demonstrate that GATA4 collaborates with LXR nuclear receptors in the liver. GATA4 and LXRs share a number of binding sites, and GATA4 was required for the full transcriptional response to LXR activation. Collectively, these results show that hepatic GATA4 contributes to the transcriptional control of hepatic and systemic lipid homeostasis.

TRIM35-mediated degradation of nuclear PKM2 destabilizes GATA4/6 and induces P53 in cardiomyocytes to promote heart failure.

Pyruvate kinase M2 (PKM2) is a glycolytic enzyme that translocates to the nucleus to regulate transcription factors in different tissues or pathologic states. Although studied extensively in cancer, its biological role in the heart remains unresolved. PKM1 is more abundant than the PKM2 isoform in cardiomyocytes, and thus, we speculated that PKM2 is not genetically redundant to PKM1 and may be critical in regulating cardiomyocyte-specific transcription factors important for cardiac survival. Here, we showed that nuclear PKM2 (S37P-PKM2) in cardiomyocytes interacts with prosurvival and proapoptotic transcription factors, including GATA4, GATA6, and P53. Cardiomyocyte-specific PKM2-deficient mice (Pkm2 Mut Cre+) developed age-dependent dilated cardiac dysfunction and had decreased amounts of GATA4 and GATA6 (GATA4/6) but increased amounts of P53 compared to Control Cre+ hearts. Nuclear PKM2 prevented caspase-1-dependent cleavage and degradation of GATA4/6 while also providing a molecular platform for MDM2-mediated reduction of P53. In a preclinical heart failure mouse model, nuclear PKM2 and GATA4/6 were decreased, whereas P53 was increased in cardiomyocytes. Loss of nuclear PKM2 was ubiquitination dependent and associated with the induction of the E3 ubiquitin ligase TRIM35. In mice, cardiomyocyte-specific TRIM35 overexpression resulted in decreased S37P-PKM2 and GATA4/6 along with increased P53 in cardiomyocytes compared to littermate controls and similar cardiac dysfunction to Pkm2 Mut Cre+ mice. In patients with dilated left ventricles, increase in TRIM35 was associated with decreased S37P-PKM2 and GATA4/6 and increased P53. This study supports a previously unrecognized role for PKM2 as a molecular platform that mediates cell signaling events essential for cardiac survival.

GATA4 Regulates Developing Endocardium Through Interaction With ETS1.

BACKGROUND: The pioneer transcription factor (TF) GATA4 (GATA Binding Protein 4) is expressed in multiple cardiovascular lineages and is essential for heart development. GATA4 lineage-specific occupancy in the developing heart underlies its lineage specific activities. Here, we characterized GATA4 chromatin occupancy in cardiomyocyte and endocardial lineages, dissected mechanisms that control lineage specific occupancy, and analyzed GATA4 regulation of endocardial gene expression. METHODS: We mapped GATA4 chromatin occupancy in cardiomyocyte and endocardial cells of embryonic day 12.5 (E12.5) mouse heart using lineage specific, Cre-activated biotinylation of GATA4. Regulation of GATA4 pioneering activity was studied in cell lines stably overexpressing GATA4. GATA4 regulation of endocardial gene expression was analyzed using single cell RNA sequencing and luciferase reporter assays. RESULTS: Cardiomyocyte-selective and endothelial-selective GATA4 occupied genomic regions had features of lineage specific enhancers. Footprints within cardiomyocyte- and endothelial-selective GATA4 regions were enriched for NKX2-5 (NK2 homeobox 5) and ETS1 (ETS Proto-Oncogene 1) motifs, respectively, and both of these TFs interacted with GATA4 in co-immunoprecipitation assays. In stable NIH3T3 cell lines expressing GATA4 with or without NKX2-5 or ETS1, the partner TFs re-directed GATA4 pioneer binding and augmented its ability to open previously inaccessible regions, with ETS1 displaying greater potency as a pioneer partner than NKX2-5. Single-cell RNA sequencing of embryonic hearts with endothelial cell-specific Gata4 inactivation identified Gata4-regulated endocardial genes, which were adjacent to GATA4-bound, endothelial regions enriched for both GATA4 and ETS1 motifs. In reporter assays, GATA4 and ETS1 cooperatively stimulated endothelial cell enhancer activity. CONCLUSIONS: Lineage selective non-pioneer TFs NKX2-5 and ETS1 guide the activity of pioneer TF GATA4 to bind and open chromatin and create active enhancers and mechanistically link ETS1 interaction to GATA4 regulation of endocardial development.

Chicken ovalbumin upstream promoter transcription factor type II interacts and functionally cooperates with GATA4 to regulate anti-Müllerian hormone receptor type 2 transcription in mouse MA-10 Leydig cells.

BACKGROUND: Leydig cells produce testosterone and insulin-like 3, two hormones essential for male sex differentiation and reproductive function. The orphan nuclear receptor, chicken ovalbumin upstream promoter transcription factor type II (COUP-TFII), and the zinc finger factor GATA4 are two transcription factors involved in Leydig cell differentiation, gene expression, and function. OBJECTIVES: Several Leydig cell gene promoters contain binding motifs for both GATA factors and nuclear receptors. The goal of the present study is to determine whether GATA4 and COUP-TFII cooperate to regulate gene expression in Leydig cells. MATERIALS AND METHODS: The transcriptomes from GATA4- and COUP-TFII-depleted MA-10 Leydig cells were analyzed using bioinformatic tools. Functional cooperation between GATA4 and COUP-TFII, and other related family members, was assessed by transient transfections in Leydig (MA-10 and MLTC-1) and fibroblast (CV-1) cell lines on several gene promoters. Recruitment of GATA4 and COUP-TFII to gene promoters was investigated by chromatin immunoprecipitation. Co-immunoprecipitation was used to determine whether GATA4 and COUP-TFII interact in MA-10 Leydig cells. RESULTS: Transcriptomic analyses of GATA4- and COUP-TFII-depleted MA-10 Leydig cells revealed 44 commonly regulated genes including the anti-Müllerian hormone receptor type (Amhr2) gene. GATA4 and COUP-TFII independently activated the Amhr2 promoter, and their combination led to a stronger activation. A GC-rich element, located in the proximal Amhr2 promoter was found to be essential for GATA4- and COUP-TFII-dependent activation as well as for the COUP-TFII/GATA4 cooperation. COUP-TFII and GATA4 directly interacted in MA-10 Leydig cell extracts. Chromatin immunoprecipitation revealed that GATA4 and COUP-TFII are recruited to the proximal Amhr2 promoter, which contains binding sites for both factors in addition to the GC-rich element. Cooperation between COUP-TFII and GATA6, but not GATA1 and GATA3, was also observed. DISCUSSION AND CONCLUSION: Our results establish the importance of physical and functional cooperation between COUP-TFII/GATA4 in the regulation of gene expression in MA-10 Leydig cells, and more specifically the Amhr2 gene.