Yosuke Okuno

PKA-dependent regulation of the histone lysine demethylase complex PHF2–ARID5B

Reversible histone methylation and demethylation are highly regulated processes that are crucial for chromatin reorganization and regulation of gene transcription in response to extracellular conditions. However, the mechanisms that regulate histone-modifying enzymes are largely unknown. Here, we characterized a protein kinase A (PKA)-dependent histone lysine demethylase complex, PHF2–ARID5B. PHF2, a jmjC demethylase, is enzymatically inactive by itself, but becomes an active H3K9Me2 demethylase through PKA-mediated phosphorylation. We found that phosphorylated PHF2 then associates with ARID5B, a DNA-binding protein, and induce demethylation of methylated ARID5B. This modification leads to targeting of the PHF2–ARID5B complex to its target promoters, where it removes the repressive H3K9Me2 mark. These findings suggest that the PHF2–ARID5B complex is a signal-sensing modulator of histone methylation and gene transcription, in which phosphorylation of PHF2 enables subsequent formation of a competent and specific histone demethylase complex.

Epigenetic Regulation of Adipogenesis by PHF2 Histone Demethylase

PHF2 is a JmjC family histone demethylase that removes the methyl group from H3K9me2 and works as a coactivator for several metabolism-related transcription factors. In this study, we examined the in vivo role of PHF2 in mice. We generated Phf2 floxed mice, systemic Phf2 null mice by crossing Phf2 floxed mice with CMV-Cre transgenic mice, and tamoxifen-inducible Phf2 knockout mice by crossing Phf2 floxed mice with Cre-ERT2 transgenic mice. Systemic Phf2 null mice had partial neonatal death and growth retardation and exhibited less adipose tissue and reduced adipocyte numbers compared with control littermates. Tamoxifen-induced conditional knockout of PHF2 resulted in impaired adipogenesis in stromal vascular cells from the adipose tissue of tamoxifen-inducible Phf2 knockout mice as well as of Phf2 knocked-down 3T3-L1 cells. PHF2 interacts with CEBPA and demethylates H3K9me2 in the promoters of CEBPA-regulated adipogenic genes. These findings suggest that PHF2 histone demethylase potentiates adipogenesis through interaction with CEBPA in vivo. Taken together, PHF2 may be a novel therapeutic target in the treatment of obesity and the metabolic syndrome.

KIAA1718 is a histone demethylase that erases repressive histone methyl marks

The methylation states of histone lysine residues are regarded as significant epigenetic marks governing transcriptional regulation. A number of histone demethylases containing a jumonji C (JmjC) domain have been recognized; however, their properties remain to be investigated. Here, we show that KIAA1718, a PHF2/PHF8 subfamily member, possesses histone demethylase activity specific for H3K9 and H3K27, transcriptionally repressive histone marks. Biochemical purification of the KIAA1718 interactants reveals that KIAA1718 forms complexes with several factors including KAP1, a transcriptional co‐activator. Consistent with these findings, KIAA1718 shows a transcriptional activation function in the chromatin context. Thus, our study identifies KIAA1718 as a histone demethylase for repressive methyl marks and shows that it is involved in transcriptional activation.

Competitive binding of Musclin to natriuretic peptide receptor 3 with atrial natriuretic peptide.

Musclin is a novel skeletal muscle-derived secretory factor that was isolated by our group. Musclin contains a region homologous to natriuretic peptides (NPs). This study investigated the interaction between musclin and NP receptors (NPRs). Musclin specifically bound to NPR3, but not to NPR1 or NPR2. Musclin and atrial natriuretic peptide (ANP) competed for binding to NPR3. We conducted binding assays using various synthetic musclin peptides and mutant musclin proteins. The first NP-homologous region in musclin ((88)LDRL(91)) and the second homologous region ((117)MDRI(120)) were responsible cooperatively for high-affinity binding to NPR3. The first NP-homologous region was more importantly associated with binding to NPR3, than the second homologous region. The competitive nature of musclin with ANP for the natriuretic clearance receptor NPR3 was also confirmed in vivo. We conclude that musclin binds to NPR3 competitively with ANP and may affect ANP concentrations in a local or systemic manner.

Novel insights into histone modifiers in adipogenesis

Recently, it has been progressively recognized that gene expression is regulated by histone methylation status, which is dynamically modulated by histone methyltransferases (HMTs) and histone demethylases (HDMs). In the past decade, many HMTs and HDMs were identified and their biological and biochemical functions have been characterized. As with other cells, several HMTs and HDMs are known to be indispensable for appropriate differentiation of adipocytes from mesenchymal stem cells. Phf2 is a recently identified dimethylated histone H3 lysine 9 (H3K9me2) demethylase that has a significant function in hepatocytes and macrophages in vitro; however, the in vivo significance of Phf2 remains unclear. To determine the physiological role of Phf2, we recently generated Phf2 knockout mice. Our analyses of these mice revealed that Phf2 has a positive role in adipogenesis by coactivating CEBPA, one of the master regulators of adipogenesis, through its demethylation activity toward H3K9me2. In this commentary, we discuss several remaining questions that underlie phenotypic abnormalities seen in our investigations of Phf2 knockout mice. These studies are related to novel functions of histone modifiers and may help identify new therapeutic targets for metabolic syndrome.

Oxidative Stress Inhibits Healthy Adipose Expansion Through Suppression of SREBF1-Mediated Lipogenic Pathway

Recent studies have emphasized the association of adipose oxidative stress (Fat reactive oxygen species [ROS]) with the pathogenesis of metabolic disorders in obesity. However, the causal roles of Fat ROS in metabolic disturbances in vivo remain unclear because no mouse model has been available in which oxidative stress is manipulated by targeting adipocytes. In this research, we generated two models of Fat ROS–manipulated mice and evaluated the metabolic features in diet-induced obesity. Fat ROS–eliminated mice, in which Cat and Sod1 were overexpressed in adipocytes, exhibited adipose expansion with decreased ectopic lipid accumulation and improved insulin sensitivity. Conversely, Fat ROS–augmented mice, in which glutathione was depleted specifically in adipocytes, exhibited restricted adipose expansion associated with increased ectopic lipid accumulation and deteriorated insulin sensitivity. In the white adipose tissues of these mice, macrophage polarization, tissue fibrosis, and de novo lipogenesis were significantly changed. In vitro approaches identified KDM1A-mediated attenuation of sterol-regulatory element-binding transcription factor 1 (SREBF1) transcriptional activities as the underlying mechanism for the suppression of de novo lipogenesis by oxidative stress. Thus, our study uncovered the novel roles of Fat ROS in healthy adipose expansion, ectopic lipid accumulation, and insulin resistance, providing the possibility for the adipocyte-targeting antioxidant therapy.

Metabolomic and microarray analyses of adipose tissue of dapagliflozin-treated mice, and effects of 3-hydroxybutyrate on induction of adiponectin in adipocytes

Sodium/glucose cotransporter 2 (SGLT2) inhibitor improves systemic glucose metabolism. To clarify the effect of dapagliflozin, we performed gene expression microarray and metabolomic analyses of murine adipose tissue. Three groups of mice were used; non-diabetic control KK mice (KK), diabetic KKAy mice (KKAy), and KKAy mice treated with dapagliflozin (KKAy + Dapa). Plasma glucose levels were significantly reduced in KKAy + Dapa compared with KKAy. Food consumption was larger in KKAy + Dapa than KKAy, and there were no significant differences in body and adipose tissue weight among the groups. Metabolomic analysis showed higher levels of many intermediate metabolites of the glycolytic pathway and TCA cycle in KKAy than KK, albeit insignificantly. Dapagliflozin partially improved accumulation of glycolytic intermediate metabolites, but not intermediate metabolites of the TCA cycle, compared with KKAy. Interestingly, dapagliflozin increased plasma and adipose 3-hydroxybutyric acid (3-HBA) levels. Microarray analysis showed that adipocytokines were downregulated in KKAy compared with KK mice, and upregulated by dapagliflozin. In vitro, 3-HBA induced β-hydroxybutyrylation of histone H3 at lysine 9 and upregulation of adiponectin in 3T3-L1 adipocytes independent of their acetylation or methylation. Our results suggest that 3-HBA seems to provide protection through epigenetic modifications of adiponectin gene in adipocytes.