Leveraging dominant-negative histone H3 K-to-M mutations to study chromatin during differentiation and development.

Histone modifications are associated with regulation of gene expression that controls a vast array of biological processes. Often, these associations are drawn by correlating the genomic location of a particular histone modification with gene expression or phenotype; however, establishing a causal relationship between histone marks and biological processes remains challenging. Consequently, there is a strong need for experimental approaches to directly manipulate histone modifications. A class of mutations on the N-terminal tail of histone H3, lysine-to-methionine (K-to-M) mutations, was identified as dominant-negative inhibitors of histone methylation at their respective and specific residues. The dominant-negative nature of K-to-M mutants makes them a valuable tool for studying the function of specific methylation marks on histone H3. Here, we review recent applications of K-to-M mutations to understand the role of histone methylation during development and homeostasis. We highlight important advantages and limitations that require consideration when using K-to-M mutants, particularly in a developmental context.

Mechanical memory stored through epigenetic remodeling reduces cell therapeutic potential.

Understanding how cells remember previous mechanical environments to influence their fate, or mechanical memory, informs the design of biomaterials and therapies in medicine. Current regeneration therapies, such as cartilage regeneration procedures, require 2D cell expansion processes to achieve large cell populations critical for the repair of damaged tissues. However, the limit of mechanical priming for cartilage regeneration procedures before inducing long-term mechanical memory following expansion processes is unknown, and mechanisms defining how physical environments influence the therapeutic potential of cells remain poorly understood. Here, we identify a threshold to mechanical priming separating reversible and irreversible effects of mechanical memory. After 16 population doublings in 2D culture, expression levels of tissue-identifying genes in primary cartilage cells (chondrocytes) are not recovered when transferred to 3D hydrogels, while expression levels of these genes were recovered for cells only expanded for eight population doublings. Additionally, we show that the loss and recovery of the chondrocyte phenotype correlates with a change in chromatin architecture, as shown by structural remodeling of the trimethylation of H3K9. Efforts to disrupt the chromatin architecture by suppressing or increasing levels of H3K9me3 reveal that only with increased levels of H3K9me3 did the chromatin architecture of the native chondrocyte phenotype partially return, along with increased levels of chondrogenic gene expression. These results further support the connection between the chondrocyte phenotype and chromatin architecture, and also reveal the therapeutic potential of inhibitors of epigenetic modifiers as disruptors of mechanical memory when large numbers of phenotypically suitable cells are required for regeneration procedures.

Regression from pathological hypertrophy in mice is sexually dimorphic and stimulus specific.

Pathological cardiac hypertrophy is associated with increased morbidity and mortality. Understanding the mechanisms whereby pathological cardiac growth can be reversed could be of therapeutic value. Here, we show that pathways leading to regression of pathological cardiac hypertrophy are strongly dependent on the hypertrophic trigger and are significantly modified by sex. Two pathological stimuli causing hypertrophy via distinct pathways were administered to male and female mice: angiotensin II (ANG II) or isoproterenol (Iso). Stimuli were removed after 7 days of treatment, and left ventricles (LVs) were studied at 1, 4, and 7 days. ANG II-treated females did not show regression after stimulus removal. Iso-treated males showed rapid LV hypertrophy regression. Somewhat surprisingly, RNAseq analysis at day 1 after removal of triggers revealed only 45 differentially regulated genes in common among all the groups, demonstrating distinct responses. Ingenuity pathway analysis predicted strong downregulation of the TGFß1 pathway in all groups except for ANG II-treated females. Consistently, we found significant downregulation of Smad signaling after stimulus removal including in ANG II-treated females. In addition, the ERK1/2 pathway was significantly reduced in the groups showing regression. Finally, protein degradation pathways were significantly activated only in Iso-treated males 1 day after stimulus removal. Our data indicate that TGFß1 downregulation may play a role in the regression of pathological cardiac hypertrophy via downregulation of the ERK1/2 pathway and activation of autophagy and proteasome activity in Iso-treated males. This work highlights that the reversal of pathological hypertrophy does not use universal signaling pathways and that sex potently modifies this process.NEW & NOTEWORTHY Pathological cardiac hypertrophy is a major risk factor for mortality and is thought to be largely irreversible in many individuals. Although cardiac hypertrophy itself has been studied extensively, very little is understood about its regression. It is important that we have a better understanding of mechanisms leading to regression, why this process is not reversible in some individuals and that sex differences need to be considered when contemplating therapies.

Nuclear deformation guides chromatin reorganization in cardiac development and disease.

In cardiovascular tissues, changes in the mechanical properties of the extracellular matrix are associated with cellular de-differentiation and with subsequent functional declines. However, the underlying mechanoreceptive mechanisms are largely unclear. Here, by generating high-resolution, full-field strain maps of cardiomyocyte nuclei during contraction in vitro, complemented with evidence from tissues from patients with cardiomyopathy and from mice with reduced cardiac performance, we show that cardiomyocytes establish a distinct nuclear organization during maturation, characterized by the reorganization of H3K9me3-marked chromatin towards the nuclear border. Specifically, we show that intranuclear tension is spatially correlated with H3K9me3-marked chromatin, that reductions in nuclear deformation (through environmental stiffening or through the disruption of complexes of the linker of nucleoskeleton and cytoskeleton) abrogate chromatin reorganization and lead to the dissociation of H3K9me3-marked chromatin from the nuclear periphery, and that the suppression of H3K9 methylation induces chromatin reorganization and reduces the expression of cardiac developmental genes. Overall, our findings indicate that, by integrating environmental mechanical cues, the nuclei of cardiomyocytes guide and stabilize the fate of cells through the reorganization of epigenetically marked chromatin.

Myocardial Lipin 1 knockout in mice approximates cardiac effects of human LPIN1 mutations.

Lipin 1 is a bifunctional protein that is a transcriptional regulator and has phosphatidic acid (PA) phosphohydrolase activity, which dephosphorylates PA to generate diacylglycerol. Human lipin 1 mutations lead to episodic rhabdomyolysis, and some affected patients exhibit cardiac abnormalities, including exercise-induced cardiac dysfunction and cardiac triglyceride accumulation. Furthermore, lipin 1 expression is deactivated in failing heart, but the effects of lipin 1 deactivation in myocardium are incompletely understood. We generated mice with cardiac-specific lipin 1 KO (cs-Lpin1-/-) to examine the intrinsic effects of lipin 1 in the myocardium. Cs-Lpin1-/- mice had normal systolic cardiac function but mild cardiac hypertrophy. Compared with littermate control mice, PA content was higher in cs-Lpin1-/- hearts, which also had an unexpected increase in diacylglycerol and triglyceride content. Cs-Lpin1-/- mice exhibited diminished cardiac cardiolipin content and impaired mitochondrial respiration rates when provided with pyruvate or succinate as metabolic substrates. After transverse aortic constriction-induced pressure overload, loss of lipin 1 did not exacerbate cardiac hypertrophy or dysfunction. However, loss of lipin 1 dampened the cardiac ionotropic response to dobutamine and exercise endurance in association with reduced protein kinase A signaling. These data suggest that loss of lipin 1 impairs cardiac functional reserve, likely due to effects on glycerolipid homeostasis, mitochondrial function, and protein kinase A signaling.

High-sucrose-induced maternal obesity disrupts ovarian function and decreases fertility in Drosophila melanogaster.

As the obesity epidemic worsens, the prevalence of maternal obesity is expected to rise. Both high-fat and high-sucrose diets are known to promote maternal obesity and several studies have elucidated the molecular influence of high-fat feeding on female reproduction. However, to date, the molecular impact of a high-sucrose diet on maternal obesity remains to be investigated. Using our previously reported Drosophila high-sucrose maternal obesity model, we sought to determine how excess dietary sucrose impacted the ovary. High-sucrose diet (HSD) fed adult females developed systemic insulin resistance and exhibited an ovarian phenotype characterized by excess accumulation of lipids and cholesterol in the ovary, decreased ovary size, and impaired egg maturation. We also observed decreased expression of antioxidant genes and increased protein carbonylation in the ovaries of HSD females. HSD females laid fewer eggs; however, the overall survival of offspring was unchanged relative to lean control females. Ovaries of HSD females had increased mitochondrial DNA copy number and decreased expression of key mitochondrial regulators, suggestive of an ineffective compensatory response to mitochondrial dysfunction. Mitochondrial alterations were also observed in male offspring of obese females. This study demonstrates that high-sucrose-induced maternal obesity promotes insulin resistance, while disrupting ovarian metabolism and function.