Masashi Okamura

The Peroxisome Proliferator-Activated Receptor γ/Retinoid X Receptor α Heterodimer Targets the Histone Modification Enzyme PR-Set7/Setd8 Gene and Regulates Adipogenesis through a Positive Feedback Loop

ABSTRACT Control of cell differentiation occurs through transcriptional mechanisms and through epigenetic modification. Using a chromatin immunoprecipitation-on-chip approach, we performed a genome-wide search for target genes of peroxisome proliferator-activated receptor γ (PPARγ) and its partner protein retinoid X receptor α during adipogenesis. We show that these two receptors target several genes that encode histone lysine methyltransferase SET domain proteins. The histone H4 Lys 20 (H4K20) monomethyltransferase PR-Set7/Setd8 gene is upregulated by PPARγ during adipogenesis, and the knockdown of PR-Set7/Setd8 suppressed adipogenesis. Intriguingly, monomethylated H4K20 (H4K20me1) levels are robustly increased toward the end of differentiation. PR-Set7/Setd8 positively regulates the expression of PPARγ and its targets through H4K20 monomethylation. Furthermore, the activation of PPARγ transcriptional activity leads to the induction of H4K20me1 modification of PPARγ and its targets and thereby promotes adipogenesis. We also show that PPARγ targets PPARγ2 and promotes its gene expression through H4K20 monomethylation. Our results connect transcriptional regulation and epigenetic chromatin modulation through H4K20 monomethylation during adipogenesis through a feedback loop.

COUP-TFII acts downstream of Wnt/β-catenin signal to silence PPARγ gene expression and repress adipogenesis

Wnt signaling through β-catenin and TCF maintains preadipocytes in an un-differentiated proliferative state; however, the molecular pathway has not been completely defined. By integrating gene expression microarray, chromatin immunoprecipitation-chip, and cell-based experimental approaches, we show that Wnt/β-catenin signaling activates the expression of COUP-TFII which recruits the SMRT corepressor complex to the first introns located downstream from the first exons of both PPARγ1 and γ2 mRNAs. This maintains the local chromatin in a hypoacetylated state and represses PPARγ gene expression to inhibit adipogenesis. Our experiments define the COUP-TFII/SMRT complex as a previously unappreciated component of the linear pathway that directly links Wnt/β-catenin signaling to repression of PPARγ gene expression and the inhibition of adipogenesis.

Obesity and metabolic syndrome in histone demethylase JHDM2a-deficient mice.

Histone H3 lysine 9 (H3K9) methylation is a crucial epigenetic mark of heterochromatin formation and transcriptional silencing. Recent studies demonstrated that most covalent histone lysine modifications are reversible and the jumonji C (JmjC)‐domain‐containing proteins have been shown to possess such demethylase activities. However, there is little information available on the biological roles of histone lysine demethylation in intact animal model systems. JHDM2A (JmjC‐domain‐containing histone demethylase 2A, also known as JMJD1A) catalyses removal of H3K9 mono‐ and dimethylation through iron and α‐ketoglutarate dependent oxidative reactions. Here, we demonstrate that JHDM2a also regulates metabolic genes related to energy homeostasis including anti‐adipogenesis, regulation of fat storage, glucose transport and type 2 diabetes. Mice deficient in JHDM2a (JHDM2a−/−) develop adult onset obesity, hypertriglyceridemia, hypercholesterolemia, hyperinsulinemia and hyperleptinemia, which are hallmarks of metabolic syndrome. JHDM2a−/− mice furthermore exhibit fasted induced hypothermia indicating reduced energy expenditure and also have a higher respiratory quotient indicating less fat utilization for energy production. These observations may explain the obesity phenotype in these mice. Thus, H3K9 demethylase JHDM2a is a crucial regulator of genes involved in energy expenditure and fat storage, which suggests it is a previously unrecognized key regulator of obesity and metabolic syndrome.

SOX6 suppresses cyclin D1 promoter activity by interacting with β-catenin and histone deacetylase 1 and its down-regulation induces pancreatic β-cell proliferation

Sex-determining region Y-box (SOX) 6 negatively regulates glucose-stimulated insulin secretion from β-cells and is a down-regulated transcription factor in the pancreatic islet cells of hyperinsulinemic obese mice. To determine the contribution of SOX6 to insulin resistance, we analyzed the effects of SOX6 on cell proliferation. Small interfering RNA-mediated attenuation of SOX6 expression stimulated the proliferation of insulinoma INS-1E and NIH-3T3 cells, whereas retroviral overexpression resulted in inhibition of cell growth. Quantitative real time-PCR analysis revealed that the levels of cyclin D1 transcripts were markedly decreased by SOX6 overexpression. Luciferase-reporter assay with β-catenin showed that SOX6 suppresses cyclin D1 promoter activities. In vitro binding experiments showed that the LZ/Q domain of SOX6 physically interacts with armadillo repeats 1-4 of β-catenin. Furthermore, chromatin immunoprecipitation assay revealed that increased SOX6 expression significantly reduced the levels of acetylated histones H3 and H4 at the cyclin D1 promoter. By using a histone deacetylase (HDAC) inhibitor and co-immunoprecipitation analysis, we showed that SOX6 suppressed cyclin D1 activities by interacting withβ-catenin and HDAC1. The data presented suggest that SOX6 may be an important factor in obesity-related insulin resistance.

Cooperative Interaction between Hepatocyte Nuclear Factor 4α and GATA Transcription Factors Regulates ATP-Binding Cassette Sterol Transporters ABCG5 and ABCG8

ABSTRACT Cholesterol homeostasis is maintained by coordinate regulation of cholesterol synthesis and its conversion to bile acids in the liver. The excretion of cholesterol from liver and intestine is regulated by ATP-binding cassette half-transporters ABCG5 and ABCG8. The genes for these two proteins are closely linked and divergently transcribed from a common intergenic promoter region. Here, we identified a binding site for hepatocyte nuclear factor 4α (HNF4α) in the ABCG5/ABCG8 intergenic promoter, through which HNF4α strongly activated the expression of a reporter gene in both directions. The HNF4α-responsive element is flanked by two conserved GATA boxes that were also required for stimulation by HNF4α. GATA4 and GATA6 bind to the GATA boxes, coexpression of GATA4 and HNF4α leads to a striking synergistic activation of both the ABCG5 and the ABCG8 promoters, and binding sites for HNF4α and GATA were essential for maximal synergism. We also show that HNF4α, GATA4, and GATA6 colocalize in the nuclei of HepG2 cells and that a physical interaction between HNF4α and GATA4 is critical for the synergistic response. This is the first demonstration that HNF4α acts synergistically with GATA factors to activate gene expression in a bidirectional fashion.

Role of histone methylation and demethylation in adipogenesis and obesity

Adipocyte differentiation is a complex developmental process that involves the coordinated interplay of numerous transcription factors. PPARγ has emerged as a master regulator of adipogenesis and recent global target gene analysis demonstrated that PPARγ targets many genes encoding chromatin modification enzymes as well as genes of lipid metabolism and storage. Among such modification enzymes are histone lysine methyltransferases, which play important roles in transcriptional regulation. Histone methyltransferases are involved in PPARγ gene expression and subsequent adipogenesis. In addition, recent studies revealed that demethylation of histone H3 at lys9 is associated with resistance to obesity. We here review the role of histone methylation and demethylation in adipogenesis, metabolism, and obesity.

Fasting-Induced Hypothermia and Reduced Energy Production in Mice Lacking Acetyl-CoA Synthetase 2

Acetate is activated to acetyl-CoA by acetyl-CoA synthetase 2 (AceCS2), a mitochondrial enzyme. Here, we report that the activation of acetate by AceCS2 has a specific and unique role in thermogenesis during fasting. In the skeletal muscle of fasted AceCS2(-/-) mice, ATP levels were reduced by 50% compared to AceCS2(+/+) mice. Fasted AceCS2(-/-) mice were significantly hypothermic and had reduced exercise capacity. Furthermore, when fed a low-carbohydrate diet, 4-week-old weaned AceCS2(-/-) mice also exhibited hypothermia accompanied by sustained hypoglycemia that led to a 50% mortality. Therefore, AceCS2 plays a significant role in acetate oxidation needed to generate ATP and heat. Furthermore, AceCS2(-/-) mice exhibited increased oxygen consumption and reduced weight gain on a low-carbohydrate diet. Our findings demonstrate that activation of acetate by AceCS2 plays a pivotal role in thermogenesis, especially under low-glucose or ketogenic conditions, and is crucially required for survival.

Methylglyoxal Is a Predictor in Type 2 Diabetic Patients of Intima-Media Thickening and Elevation of Blood Pressure

We test whether plasma level of methylglyoxal (MG) is an independent risk factor predicting the progression of diabetic macroangiopathy or microangiopathy in type 2 diabetic patients. We measured in 50 type 2 diabetic patients plasma levels of MG and 3-deoxyglucosone (DG) using an electrospray ionization-liquid chromatography-mass spectrometry. We assessed the correlations between baseline levels of MG or DG and the percentage changes after 5 years of clinical parameters linked to diabetic macroangiopathy or microangiopathy, that is, intima-media thickness (IMT), systolic blood pressure (SBP), the amount of urinary albumin excretion (ACR), pulse wave velocity (PWV), and estimated glomerular filtration rate (eGFR). Multiple regression analysis was performed using the percentage changes in IMT, SBP, ACR, PWV, and eGFR over the 5-year period as the independent or objective variables and the values of MG, DG, glycohemoglobin A1c, body mass index, triglyceride, and diabetic duration at the baseline as the dependent variables. The values of IMT, PWV, SBP, and ACR all increase, but eGFR reduces with time during the 5-year period. Baseline level of MG correlates significantly with the percentage changes of IMT, SBP, ACR, PWV, and eGFR, whereas that of DG does only with ACR. A multiple regression analysis reveals that MG is an independent risk factor for the percentage changes of IMT, PWV, and SBP but not for those of ACR and eGFR. DG is an independent risk factor for the percentage change of ACR. MG is a predictor in type 2 diabetic patients of intima-media thickening, of increase of PWV, and of elevation of SBP.

SOX6 attenuates glucose stimulated insulin secretion by repressing PDX1 transcriptional activity and is down-regulated in hyperinsulinemic obese mice

In obesity-related insulin resistance, pancreatic islets compensate for insulin resistance by increasing secretory capacity. Here, we report the identification of sex-determining region Y-box 6 (SOX6), a member of the high mobility group box superfamily of transcription factors, as a co-repressor for pancreatic-duodenal homeobox factor-1 (PDX1). SOX6 mRNA levels were profoundly reduced by both a long term high fat feeding protocol in normal mice and in genetically obese ob/ob mice on a normal chow diet. Interestingly, we show that SOX6 is expressed in adult pancreatic insulin-producing β-cells and that overexpression of SOX6 decreased glucose-stimulated insulin secretion, which was accompanied by decreased ATP/ADP ratio, Ca2+ mobilization, proinsulin content, and insulin gene expression. In a complementary fashion, depletion of SOX6 by small interfering RNAs augmented glucose-stimulated insulin secretion in insulinoma mouse MIN6 and rat INS-1E cells. These effects can be explained by our mechanistic studies that show SOX6 acts to suppress PDX1 stimulation of the insulin II promoter through a direct protein/protein interaction. Furthermore, SOX6 retroviral expression decreased acetylation of histones H3 and H4 in chromatin from the promoter for the insulin II gene, suggesting that SOX6 may decrease PDX1 stimulation through changes in chromatin structure at specific promoters. These results suggest that perturbations in transcriptional regulation that are coordinated through SOX6 and PDX1 in β-cells may contribute to the β-cell adaptation in obesity-related insulin resistance.

Hepatocyte Nuclear Factor 4α Contributes to Thyroid Hormone Homeostasis by Cooperatively Regulating the Type 1 Iodothyronine Deiodinase Gene with GATA4 and Krüppel-Like Transcription Factor 9

ABSTRACT Type 1 iodothyronine deiodinase (Dio1), a selenoenzyme catalyzing the bioactivation of thyroid hormone, is highly expressed in the liver. Dio1 mRNA and enzyme activity levels are markedly reduced in the livers of hepatocyte nuclear factor 4α (HNF4α)-null mice, thus accounting for its liver-specific expression. Consistent with this deficiency, serum T4 and rT3 concentrations are elevated in these mice compared with those in HNF4α-floxed control littermates; however, serum T3 levels are unchanged. Promoter analysis of the mouse Dio1 gene demonstrated that HNF4α plays a key role in the transactivation of the mouse Dio1 gene. Deletion and substitution mutation analyses demonstrated that a proximal HNF4α site (direct repeat 1 [TGGACAAAGGTGC]; HNF4α-RE) is crucial for transactivation of the mouse Dio1 gene by HNF4α. Mouse Dio1 is also stimulated by thyroid hormone signaling, but a direct role for thyroid hormone receptor action has not been reported. We also showed that thyroid hormone-inducible Krüppel-like factor 9 (KLF9) stimulates the mouse Dio1 promoter very efficiently through two CACCC sequences that are located on either side of HNF4α-RE. Furthermore, KLF9 functions together with HNF4α and GATA4 to synergistically activate the mouse Dio1 promoter, suggesting that Dio1 is regulated by thyroid hormone in the mouse through an indirect mechanism requiring prior KLF9 induction. In addition, we showed that physical interactions between the C-terminal zinc finger domain (Cf) of GATA4 and activation function 2 of HNF4α and between the basic domain adjacent to Cf of GATA4 and a C-terminal domain of KLF9 are both required for this synergistic response. Taken together, these results suggest that HNF4α regulates thyroid hormone homeostasis through transcriptional regulation of the mouse Dio1 gene with GATA4 and KLF9.