The regulation of the histone code during cardiac hypertrophy

Abstract
Our goal is to investigate the impact of diet and pressure overload on the histone code, and how this influences
changes in gene expression in the healthy and hypertrophied/failing hearts and, in turn, how it impacts
progression of the disease. Deciphering the histone code and how diet can modify it, provides us an educated
means to exploit it to our advantage, especially during pathological conditions. Acetylation and methylation of
histone lysine (K) residues were the first histone modifications discovered and are, therefore, the most widely
studied and understood. However, to-date, there are 11 confirmed modifiers of histone lysine residues, including
the acyl groups butyryl (Bu), crotonyl (Cr), and b-hydroxybutyrate (bHB) 1, whose source, genomic distribution,
and functional relevance, remain largely unknown in the heart, and are the focus of our study. Our recent findings
uniquely show that dietary fat is a major regulator of histone butyrylation, including H3K9-butyryl (H3K9Bu).
Using genome-wide chromatin immunoprecipitation-sequencing (ChIP-Seq), we show that H3K9Bu is abundant
at all transcriptionally active promoters. Both a high-fat diet and stress accelerated the conversion of butyryl-CoA
to crotonyl-CoA via acyl-CoA dehydrogenase short chain (ACADS), resulting in a substantial reduction in global
promoter-H3K9Bu. A deletion of ACADS both in the mouse heart and in human cells reversed this effect and
increased promoter and gene-body H3K9Bu. Paradoxically, though, a fat-free diet had the highest levels of
H3K9Bu. Deletion of fatty acid synthetase (FASN), abolished H3K9Bu in cells maintained in a glucose-rich, fatty
acid-free, but not in a fatty acid-rich, medium, proving that fatty acid synthesis from carbohydrates substitutes
for dietary fat as a source butyryl-CoA. In contrast to H3K9Bu, there were minimal dietary-induced changes in
H3K9-acetyl (H3K9ac) levels. Importantly, RNA-sequencing (RNA-Seq) revealed that diet-induced changes in
H3K9Bu abundance in the mouse heart was associated with differential changes in gene expression, but only
when stressed by pressure overload. Moreover, promoter-H3K9Bu levels inversely correlated with the extent of
changes in gene expression levels, as evidenced by the more robust changes seen in the hearts of mice on a,
short-term, high-fat vs a fat-free diet, as well as, after deletion of the ACADS. Interestingly, H3K9Bu abundance
inversely correlated with H3K9-crotonyl (H3K9Cr) and Cdk9. In sum, our data uniquely show that H3K9Bu is
enriched at active promoters, is negatively regulated by high-fat and stress in an ACADS-dependent fashion,
and its abundance inversely correlates with stress-induced changes in gene expression. We are proposing that
histone H3K9Bu, H3K9Cr, and H3K9-b-hydroxybutyryl (H3K9bHB), are products of the b-oxidation
intermediates, butyryl-CoA, crotonyl-CoA, and b-hydroxybutyryl-CoA, or the ketone body, b-hydroxybutyrate,
which serve as substrates for histones modifications. These marks are labile and differentially influence pressure
overload-induced gene expression, but not baseline expression. Specifically, as H3K9Bu decreases it is replaced
by H3K9Cr during a high-fat diet. This exchange exaggerates gene expression and worsens the outcome of
cardiac failure. Conversely, H3K9bHB that increases during a ketogenic diet has the opposite effect, as it is
reported to have beneficial effects on health and aging. This differential influence of the histone marks on gene
expression is mediated by regulating the recruitment of Cdk9 to gene promoters. We hypothesize that 1) A
high-fat diet (60 Kcal% fat, 20 Kcal% carb), or pressure overload, accelerates the conversion of nuclear butyryl-
CoA to crotonyl-CoA in an ACADS-dependent manner, thus, reducing H3K9Bu and increasing H3K9Cr, which
is responsible for exaggerating stress-induced gene expression and worsening the outcome of heart failure (HF).
In contrast, a ketogenic diet (84 Kcal% fat, 0% carb) will produce high levels of b-hydroxybutyryl that will increase
H3K9bHB, which curbs changes in stress-induced gene expression in a b-hydroxybutyrate dehydrogenase
(BDH1)-dependent fashion, improving the outcome of HF. Supplementing a diet with b-hydroxybutyrate will also
increase H3K9bHB, with similar beneficial effects. 2) Therefore, knockdown of ACADS reduces the conversion
of butyryl-CoA to crotonyl-CoA, increasing H3K9Bu and improving the outcome of heart failure during a high-fat
diet. Conversely, deletion or inhibition of BDH1 reduces H3K9bHB and worsens conditions. 3) H3K9Cr enhances
the dynamics of cyclin-dependent kinase 9 (Cdk9) recruitment to promoters during stress, whereas, H3K9Bu
and H3K9bHB temper it, thus, reducing the extent of changes in gene expression and improving disease
outcome. The specific aims are: 1) Examine the effects of high-fat, ketogenic, and b-hydroxybutyrate-enriched
diets on the genome-wide distribution and changes in H3K9Bu, H3K9Cr and H3K9bHB, changes in ge