AIMS:Heart failure is marked by suppression of fatty acid oxidation (FAO) and mitochondrial ATP production gene expression. While transcriptional downregulation via PGC-1 and PPARα/ERRα has been well documented, the involvement of the general transcriptional machinery remains insufficiently understood. Distinct from its salutary role in many cardiac conditions, endogenous Sirt1 negatively affects cardiac function during pressure overload (PO). This study investigates how Sirt1 modulates preinitiation complex (PIC) assembly and RNA polymerase II (Pol II) recruitment during pathological PO. METHODS AND RESULTS:Cardiac-specific Sirt1 knock-out (Sirt1 cKO) mice were subjected to PO. Interaction between Sirt1 and Sub1, a protein recruiting GTFs to the gene promoter, was assessed with co-immunoprecipitation, protein pull-down and molecular docking. Pol II recruitment was evaluated with ChIP-sequencing and -qPCR analyses. The binding affinity of Sirt1 and GTF to the Sub1 GTF binding domain was assessed by immunoprecipitation and protein pull-down assays. Unbiased ChIP-sequencing and -qPCR analyses showed that Pol II binding to metabolic gene promoters was downregulated during PO, which was reversed in Sirt1 cKO mice. PO upregulated Sirt1 in the heart and increased its binding to Sub1, which interacts PPARα and ERRα, but not NF-kB. The Sirt1 binding to Sub1 competitively displaced the interaction between Sub1 and GTFs, thereby inhibiting the PIC formation at the Sub1-PPARα/ERRα complex. CONCLUSIONS:Sirt1 impairs metabolic gene transcription during PO by competitively inhibiting Sub1-mediated recruitment of GTFs and PIC formation. This repression of general transcriptional machinery contributes to the metabolic disturbance and may represent a maladaptive component of the heart failure phenotype.

Summary Our prior work demonstrated that lowering dietary branched-chain amino acids (BCAAs) improves cardiac outcomes during pressure overload-induced stress. Here, we identify isoleucine restriction (IleR) as the key driver of this effect. Dietary isoleucine restriction induces hypophagia and weight loss, recapitulating the effects of caloric restriction (CR). Although it does not prevent the initial development of left ventricular hypertrophy, it halts its progression and the decline in ejection fraction compared with controls. This is associated with preservation of electron transport chain (ETC) gene expression, cristae structure, NAD + /NADH levels, and mitochondrial respiratory capacity in cardiomyocytes, which is recapitulated by CR. Mechanistically, both IleR and CR diets increase Foxo3 expression, thereby blocking the decline in expression of its target ETC and mitochondrial genome-encoded genes. Consequently, this improves mitochondrial respiratory capacity and reduces cardiac fibrosis. We conclude that restricting dietary isoleucine improves cardiac health by increasing Foxo3 expression and mitochondrial function via a cell-autonomous mechanism and by reducing caloric intake.

Klf9 is a cardiac-enriched transcription factor of the Krüppel-like factor (Klf) family. Klf9 levels decrease during cardiac hypertrophy; however, no studies have examined its transcriptional targets or role in the progression of hypertrophy. Here, we report genome-wide differential Klf9 occupancy during cardiac hypertrophy, with a predominant enrichment at the metabolic gene promoters. Further, using conditional Klf9 knock-in mice subjected to pressure overload for 1 or 2 weeks, we show that restoring Klf9 expression initially inhibits hypertrophy but later leads to early-onset heart failure. We conclude that a decrease in Klf9 is required for metabolic adaptations that support the development of compensatory hypertrophy.

Adaptation of gene expression is the earliest response during work overload to maintain cardiac homeostasis and function. We reported a novel function of Krüppel-like factor (Klf) 9 in mediating metabolic adaptations in response to Dexamethasone in cardiomyocytes. Klf9 expression decreases in hearts undergoing cardiac hypertrophy and failure, suggesting that differential regulation of Klf9 could be contributing to the pathogenesis. Here, we present the characterization of a conditional (αMHC-Cre) Klf9 knock-in (Klf9KI) mouse. Constitutive expression of exogenous Klf9 results in spontaneous cardiac dysfunction and the onset of failure by 8 weeks of age, and an early mortality by 12-14 weeks, suggesting that Klf9 dysregulation is sufficient for maladaptation. Transcriptome data from 1-week-old Klf9KI hearts show dysregulation of genes involved in lipid, carbohydrate, and glutathione metabolism, and transcripts of transport, transcription, and motor proteins. At 4 weeks and 8 weeks, we observe a differential expression of genes involved in innate immunity and extracellular matrix, along with metabolic and contractile signaling. These data correlate with the untargeted metabolome analysis, showing dysregulation in metabolites of lipid and fatty acids, glutathione, purine and pyrimidines, and sucrose metabolic pathways. Functional data in Klf9KI hearts show a decrease in mitochondrial ETC complex activity and ATP production, and an increase in ROS. Structural mitochondrial defects include distortion and sparse cristae in Klf9KI vs. Wt-Cre hearts. In conclusion, we show that Klf9 is critical for metabolic adaptations in postnatal heart development. Constitutive expression of Klf9 results in metabolic maladaptation, which precipitates dysfunction, early onset of heart failure, and death.

We reported that Krüppel-like factor 9 (Klf9) regulates metabolic adaptations in neonatal cardiomyocytes. In this study, we characterize conditional Klf9 knock-in (Klf9KI) mice with 3-4-fold increase in Klf9 expression when crossed with αMHC-Cre. Our data shows that Klf9KI mice develop spontaneous hypertrophy by 4wks, followed by cardiac dysfunction and failure by 8wks (EF=24 ±1%; FS=11±0.5%), and early mortality by 12-14 wks. Transcriptome analysis in 1wk, 4wk and 8wks old hearts shows that the number of differentially expressed genes increases over time, with significant changes observed in 108 genes at 1 wk, and further dysregulation of 949 genes and 1247 genes at 4wks and 8wks, respectively. Consistent with Klf9's predicted role as a transcriptional repressor, 64.81% of differentially expressed genes are downregulated at 1wk. Functional annotation reveals a dysregulation of mostly metabolic genes including those involved in lipid metabolism (Bdh1), glutathione pathways (Gstk1), and carbohydrate metabolism (Fbp2). By 4wks and 8wks, along with dysregulation of metabolic pathways, we observe upregulation of genes involved in apoptosis (Bcl2), innate immunity (Tlr genes), and extracellular matrix remodeling (Col8a1). Untargeted metabolomics at 4wks identifies significant alterations in 125 tissue metabolites [VIP >1.5, ILog2FCI ≥0.5, p<0.05]). Pathway enrichment analysis shows Arachidonic acid, Lipid and Fatty acids metabolism and biosynthesis, Sucrose and Glutathione metabolism as top enriched metabolite sets. These findings correlate with dysregulated genes identified in 4wks old Klf9KI mice (RNA-seq). To assess mitochondrial function, we measured mitochondrial electron transport chain complex activities in Klf9KI and Wt-Cre mice at 2 and 4wks. Basal mitochondrial respiration decreases at both time points, while complex II (succinate) and IV (ascorbate +TMPD) activities decline significantly by 4wks. A 3.5-fold increase in ROS production is observed in these hearts, consistent with a decline in mitochondrial function. In conclusion, constitutive expression of Klf9 disrupts transcriptional and metabolic homeostasis, which precipitates spontaneous progressive hypertrophy, dysfunction, and early onset of heart failure.

Krüppel-like factors (Klfs) regulate cellular processes, including metabolism, differentiation, and proliferation, which have implications in development and diseases of the heart. We reported Klf9’s role in transcriptional control of metabolic genes and the GR-Klf9 axis in metabolic adaptation in response to Dexamethasone in neonatal myocytes. Here, we examine the role of Klf9 in adult hearts undergoing pressure overload-induced hypertrophy. Our RNA polymerase II (pol II) ChIPSeq data from mouse hearts undergoing transverse aortic constriction (TAC) induced hypertrophy show reduced pol II occupancy across the Klf9 gene compared to sham hearts, suggesting reduced transcription. Consistently, we observe reduced Klf9 transcript and protein levels in TAC hearts compared to sham, indicating decreased Klf9 expression. Klf9-ChIPSeq identified 5238 genes with Klf9 binding, with 2250 with MaxTag ≥50. 51% of the genes showed decreased Klf9 genomic binding in TAC vs Sham hearts. KEGG pathway showed metabolic pathways on top of the list with 131 genes, including those involved in carbon metabolism, insulin signaling, fatty acid degradation, glycolysis/gluconeogenesis, amino acid, and nucleotide metabolism. Conversely, 22% of genes showed increased Klf9 binding, mostly those involved in innate immunity, apoptosis, mitochondrial organization, and autophagy. Further, we subjected inducible conditional Klf9 knock-in (Klf9KI) mice to sham/TAC operations for 2 weeks, followed by functional and molecular analysis. Contrary to our hypothesis, restoring Klf9 levels with TAC showed systolic dysfunction in Klf9KI-TAC (%EF-38.65±1.9,%FS-18.34±0.77) compared to Wt-TAC (%EF-53.88±3.33,%FS-27.03±1.96) hearts, with no significant change in LV mass within the TAC groups (75.66±13.73 vs. 78.7±8.71). Interestingly, our preliminary data examining gene expression showed a significant increase in hypertrophy markers, Mhy7 (bMHC) and NPPB (BNP), and a decrease in Hmgcs2 (Klf9 target from ChIPSeq) in Klf9KI mice within sham and TAC groups. We conclude that Klf9 expression is critical for metabolic homeostasis, and dysregulated Klf9 levels are sufficient for transcriptional changes and with stress can precipitate early cardiac dysfunction and failure.

Background: Neurofibromin 2 (NF2) is a tumor suppressor that can engage signaling pathways to modulate cell proliferation and survival. We previously demonstrated that NF2 mediates cardiomyocyte apoptosis and cardiac injury caused by acute myocardial infarction. Research Aim: The role of NF2 in heart failure remains uncharacterized. This study sought to determine whether NF2 modulates heart failure due to chronic stress. Approach: We generated cardiomyocyte-specific NF2 knockout (cKO) mice and used transverse aortic constriction (TAC) to generate chronic pressure overload (PO) stress, which elicits cardiac remodeling and failure. Complementary cell-based experiments were performed in neonatal rat ventricular myocytes (NRVMs). We analyzed cardiac function by echocardiographic and hemodynamic analysis. We used RNAseq and validated the novel findings using promoter pulldown, Seahorse metabolic profiling, and biochemical analyses to investigate underlying mechanisms. Results: We found that NF2 is transiently upregulated in wild-type mouse myocardium in response to early phase of PO, but is downregulated during heart failure. Following TAC, NF2 cKO hearts unexpectedly showed significantly worsened cardiac function, compared to controls. RNAseq analysis followed by qPCR indicated downregulation of several metabolic pathways and impaired Estrogen Related Receptor (ERR) signaling in NF2 cKO hearts. Luciferase experiments employing NRVMs confirmed that NF2 promoted expression of ERR isoforms (a, g), and DNA pulldown assays demonstrated NF2 association with ERR proximal promoters. Analysis of NRVM bioenergetics demonstrated that NF2 depletion impaired mitochondrial respiration, which was reversed by concomitant expression of ERRg. Moreover, AAV9-mediated restoration of ERRg in NF2 cKO mice normalized cardiac function in response to PO. As NF2 does not directly bind DNA, leveraging a proteomics-based approach we identified the transcription factor linking NF2 to ERR isoform expression and further validated its regulation using NRVMs based experiments. Conclusion: Based on these findings, we conclude that NF2 is essential and therefore transiently upregulated during PO stress to compensate and regulate metabolic demand.

Cardiac malformations and ventricular remodeling due to heart diseases result in compromised cardiac function, eventually leading to heart failure. In this study, we examine the role of cardiac Wolf-Hirschhorn Syndrome candidate 2 (Whsc2), also known as Negative elongation factor A (NELFA), one of the genes encoded in the WHS critical region. The Wolf-Hirschhorn Syndrome is a contiguous genetic disorder due to microdeletions in the critical region, with clinical manifestations of neurological defects frequently associated with congenital malformations, including cardiac defects. NelfA has been implicated in RNA polymerase II (pol II) pausing, suggesting a role in pol II-dependent gene transcription. We previously reported an early onset of heart failure with the acute knockdown of NelfA in hearts undergoing pressure overload-induced cardiac hypertrophy. Here, we characterize a mouse model with cardiomyocyte-specific loss of NelfA function, in which these mice develop spontaneous cardiomyopathy at 2 months of age and exhibit early mortality by 3 months, suggesting a critical role for postnatal NelfA in the heart. Interactome data show that chromatin-bound NelfA interacts with proteins involved in chromatin remodeling (Trim28) and pre-mRNA processing (Adrph1l), along with expected binding partners like RNA pol II, Supt5, and other Nelf proteins. Examination of genomic occupancy of these NelfA-associated proteins in the NelfA knockout (KO) hearts reveals a disassembly of the NelfA nucleated complex at promoters of cardiac-enriched genes, including cytoskeletal and metabolic genes. This deconstruction of the NelfA-dependent complex results in the inhibited expression of these essential genes during postnatal cardiac development, leading to a cardiac contractile and metabolic crisis that precipitates dilated cardiomyopathy.

Background: Neurofibromin 2 (NF2) is a tumor suppressor that can engage multiple signaling pathways to modulate cell proliferation and survival. We previously demonstrated that NF2 mediates cardiomyocyte apoptosis and cardiac injury caused by acute myocardial infarction. Research Aim: The role of NF2 in heart failure remains uncharacterized. This study sought to determine whether NF2 modulates heart failure due to chronic stress. Approach: We generated cardiomyocyte-specific NF2 knockout (cKO) mice, and used transverse aortic constriction (TAC) to generate chronic pressure overload (PO) stress, which elicits cardiac remodeling and failure. Complementary cell-based experiments were performed in neonatal rat ventricular myocytes (NRVMs). We analyzed cardiac function by echocardiographic and hemodynamic analysis. We used RNAseq, ChIP, Seahorse metabolic profiling, and biochemical analyses to investigate underlying mechanisms. Results: We found that NF2 is transiently upregulated in wild-type mouse myocardium in response to early phase of PO, but is downregulated during heart failure. Following TAC, NF2 cKO hearts unexpectedly showed significantly worsened cardiac function, compared to controls. RNAseq analysis indicated downregulation of several metabolic pathways and impaired ERR activity in NF2 cKO hearts. qPCR confirmed significantly reduced ERRβ and ERRγ transcripts and decreased metabolic gene expression. Experiments employing NRMVs confirmed that NF2 promoted expression of ERRβ and ERRγ, and DNA pulldown assays demonstrated NF2 association with ERRβ and ERRγ proximal promoters. Analysis of NRVM bioenergetics demonstrated that NF2 depletion impaired mitochondrial respiration, which was reversed by concomitant expression of ERRγ. Moreover, AAV9-mediated restoration of ERRγ in NF2 cKO mice normalized cardiac function in response to PO. Because NF2 does not directly bind DNA, ongoing studies are leveraging a proteomics-based approach to identify the mediator linking NF2 to ERR isoform expression. Conclusion: Based on these findings, we conclude that transient upregulation of myocardial NF2 expression during PO stress is compensatory and regulates metabolic gene expression according to energy demand.

Department of Cell Biology and Molecular Medicine, New Jersey Medical School, Rutgers University, Newark, NJ 07103.Neurofibromin 2 (NF2) is a tumor suppressor that can engage the Hippo signaling pathway and modulate cell proliferation and survival. We previously demonstrated that NF2 mediates cardiomyocyte apoptosis and injury caused by acute myocardial infarction. However, the function of NF2 in the heart remains largely uncharacterized. Our current study sought to determine whether NF2 modulates heart failure due to chronic stress. We used a transverse aortic constriction (TAC) model in WT C57BL/6J mice to generate chronic pressure overload (PO) stress, which elicits cardiac remodeling and failure. We found that NF2 is transiently upregulated in mouse myocardium in response to early phase of PO, and is downregulated during late phase PO. We generated cardiomyocyte-specific NF2 knockout (cKO) mice, which had normal cardiac morphology and function at baseline. Following TAC, NF2 cKO hearts unexpectedly showed worsened cardiac function, assessed by echocardiography and hemodynamic analysis, compared to controls. RNAseq analysis indicated downregulation of several metabolic pathways including oxidative phosphorylation and fatty acid oxidation in NF2 cKO hearts at baseline. Additionally, we observed reduced ATP content in baseline NF2 cKO hearts compared to controls. Fractionation experiments indicated that nuclear NF2 is enriched following PO stress. RNAseq revealed the downregulation of ERRβ and ERRγ in NF2 cKO hearts, which was confirmed by qPCR. Experiments employing neonatal rat ventricular myocytes (NRMVs) confirmed that NF2 regulated expression of ERRβ and ERRγ, and DNA pulldown assays demonstrated NF2 association with ERRβ and ERRγ proximal promoters. Luciferase reporter assays demonstrated that NF2 modulates ERR function in NRVMs. Based on these findings, we propose that transient upregulation of myocardial NF2 expression during PO stress is compensatory and regulates metabolic gene expression according to energy demand.

Glucocorticoids (GCs) through activation of the Glucocorticoid receptor (GR) plays an essential role in cellular homeostasis during physiological variations and in response to stress. GC-GR signaling is involved in regulating several cellular processes including metabolism, circadian rhythm and inflammation for diurnal adaptations. Our genomic GR binding (ChIP) and transcriptome (RNAseq) data from Dexamethasone (Dex) treatment in cardiomyocytes show an early (1hr) differential regulation of mostly transcription factors, followed by sequential change in downstream signaling pathways (6-12hr). Here, we examine the role of an early direct target of GR in cardiomyocytes, Krüppel-like factor 9 (Klf9) in metabolic homeostasis. Our Klf9-ChIPseq identified 4100 genes with change in promoter Klf9 binding in response to Dex. Functional annotation of these genes lists metabolic pathway on the top of KEGG pathway, along with genes regulating transcription and survival. Interestingly, integration of GR and Klf9 data show overlapping targets (1181), suggesting that Klf9 could be serving as feedback regulator for these genes. Further, our transcriptome analysis of Dex treated cardiomyocytes with knockdown of Klf9 reveal differential regulation of 1777 genes (Dex+siKLf9 vs Dex+siLUC), of which a reversal in expression is seen in 1640 (92%) genes with siKlf9 vs. Dex. Conversely, only 137 (8%) genes show further dysregulation in expression with siKLf9 as Dex. Gene ontology of these 1640 genes show metabolic pathway on the top, including genes involved in glycolysis and oxidative phosphorylation. Conversely, mostly immuno-regulatory genes are among those 137 genes. Expectedly, knockdown of Klf9 in cardiomyocytes inhibits Dex induced increase in glycolysis and glycolytic capacity by 24%, and spare respiratory capacity by 50%, as measured by ECAR and OCR with glycolysis and mito stress tests, respectively. Thus, we conclude that cyclic, diurnal GC mediated GR activation, through Klf9 -dependent feedforward signaling plays a central role in maintaining cellular metabolic homeostasis. With hypertrophic stress a decrease in cardiac GR and dysregulated Klf9 signaling might result in metabolic crisis, contributing to mitochondrial and cardiac dysfunction

RNA polymerase II (pol II) pausing, a wide-spread genomic phenomenon regulates pol II dependent transcription and plays a critical role in gene expression during development and disease. Negative elongation factor (Nelf) complex consisting of five subunits, namely NelfA, NelfB, NelfC/D and NelfE, in coordination with Drb sensitive inducing factor (DISF) complex has been implicated in mediating pol II pausing. Structural studies show NelfA is indispensable for nucleation of paused complex. We have reported widespread promoter NelfA occupancy on cardiac genome, and showed that it is required for compensatory gene expression in hearts during cardiac hypertrophy. Here, we examine paused complex, with respect to its interactome, influence on gene expression and cardiac function. Immunoprecipitation of chromatin bound NelfA, followed by mass spectrometry in sham and TAC induced hypertrophied hearts identified Nelf proteins, pol II and Supt5, along with novel partners like Trim28 (chromatin remodeler), Adprhl1 (cardiac restricted, nuclear enriched, involved in DNA repair) and Numa1 (nuclear structural protein). Conditional NelfA knockout mice developed progressive dilated cardiomyopathy (LVID’d: 4.72 ±0.1, LVID;s: 3.96±0.13, %EF: 33.7±2.12, %FS: 16.15±1.12, LV vol;d: 104.5±21.9, LV vol;s: 70.26±5.5) compared to Wt-Cre (LVID’d: 4.16 ±0.09, LVID;s: 3.14±0.12, %EF: 49.1±2.3, %FS: 24.6±1.3, LV vol;d: 77.5±13.9, LV vol;s: 40.9±3.9), with sudden death by 3mths of age, in males and females KO mice compared to Wt-Cre. Interestingly, absence of NelfA resulted in decrease in cardiomyocyte size and increase in apoptosis. Single nuclei RNAseq on these hearts show decrease in cardiac-enriched sarcomeric genes, along with increase in genes involved in fibrosis and endothelial dysfunction. Further, ChIP-seq showed reduced Trim28 and NelfE promoter occupancy on essential gene, while below threshold or absent on cardiac-enriched and selected inducible promoters suggesting disruption of paused complex. Thus, we conclude that NelfA induced paused complex plays an essential role in efficient pol II dependent gene transcription and expression in heart, and deconstruction of the paused complex precipitates cardiac dysfunction and dilated cardiomyopathy

Identification of branched-chain amino acid (BCAA) oxidation enzymes in the nucleus led us to predict that they are a source of the propionyl-CoA that is utilized for histone propionylation and, thereby, regulate gene expression. To investigate the effects of BCAAs on the development of cardiac hypertrophy and failure, we applied pressure overload on the heart in mice maintained on a diet with standard levels of BCAAs (BCAA control) versus a BCAA-free diet. The former was associated with an increase in histone H3K23-propionyl (H3K23Pr) at the promoters of upregulated genes (e.g., cell signaling and extracellular matrix genes) and a decrease at the promoters of downregulated genes (e.g., electron transfer complex [ETC I-V] and metabolic genes). Intriguingly, the BCAA-free diet tempered the increases in promoter H3K23Pr, thus reducing collagen gene expression and fibrosis during cardiac hypertrophy. Conversely, the BCAA-free diet inhibited the reductions in promoter H3K23Pr and abolished the downregulation of ETC I-V subunits, enhanced mitochondrial respiration, and curbed the progression of cardiac hypertrophy. Thus, lowering the intake of BCAAs reduced pressure overload-induced changes in histone propionylation-dependent gene expression in the heart, which retarded the development of cardiomyopathy.

The heart utilizes multiple adaptive mechanisms to maintain pump function. Compensatory cardiac hypertrophy reduces wall stress and oxygen consumption, thereby protecting the heart against acute blood pressure elevation. The nuclear effector of the Hippo pathway, Yes-associated protein 1 (YAP), is activated and mediates compensatory cardiac hypertrophy in response to acute pressure overload (PO). In this study, YAP promoted glycolysis by upregulating glucose transporter 1 (GLUT1), which in turn caused accumulation of intermediates and metabolites of the glycolytic, auxiliary, and anaplerotic pathways during acute PO. Cardiac hypertrophy was inhibited and heart failure was exacerbated in mice with YAP haploinsufficiency in the presence of acute PO. However, normalization of GLUT1 rescued the detrimental phenotype. PO induced the accumulation of glycolytic metabolites, including l-serine, l-aspartate, and malate, in a YAP-dependent manner, thereby promoting cardiac hypertrophy. YAP upregulated the GLUT1 gene through interaction with TEA domain family member 1 (TEAD1) and HIF-1α in cardiomyocytes. Thus, YAP induces compensatory cardiac hypertrophy through activation of the Warburg effect.

Objective: We previously reported that 13-oxidation enzymes are present in the nucleus in close proximity to transcriptionally active promoters. Thus, we hypothesized that the fatty acid intermediate, butyryl-CoA, is the substrate for histone butyrylation and its abundance is regulated by acyl-CoA dehydrogenase short chain (ACADS). The objective of this study was to determine the genomic distribution of H3K9-butyryl (H3K9Bu) and its regulation by dietary fat, stress, and ACADS and its impact on gene expression. Methods and results: Using genome-wide chromatin immunoprecipitation-sequencing (ChIPeSeq), we show that H3K9Bu is abundant at all transcriptionally active promoters, where, paradoxically, it is most enriched in mice fed a fat-free vs high-fat diet. Deletion of fatty acid synthetase (FASN) abolished H3K9Bu in cells maintained in a glucose-rich but not fatty acid-rich medium, signifying that fatty acid synthesis from carbohydrates substitutes for dietary fat as a source of butyryl-CoA. A high-fat diet induced an increase in ACADS expression that accompanied the decrease in H3K9Bu. Conversely, the deletion of ACADS increased H3K9Bu in human cells and mouse hearts and reversed high -fat-and stress induced reduction in promoter-H3K9Bu, whose abundance coincided with diminished stress-regulated gene expression as revealed by RNA sequencing. In contrast, H3K9-acetyl (H3K9Ac) abundance was minimally impacted by diet. Conclusion: Promoter H3K9 butyrylation is a major histone modification that is negatively regulated by high fat and stress in an ACADSdependent fashion and moderates stress-regulated gene expression. (c) 2021 The Author(s). Published by Elsevier GmbH. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).