Diet-induced obesity leads to metabolic dysregulation in offspring via endoplasmic reticulum stress in a sex-specific manner.

BACKGROUND/OBJECTIVES: Exposure to metabolic stress has been suggested to influence the susceptibility to metabolic disorders in offspring according to epidemiological and animal studies. Nevertheless, molecular mechanisms remain unclear. We investigated impacts of diet-induced paternal obesity on metabolic phenotypes in offspring and its underlying molecular mechanism. SUBJECTS/METHODS: Male founder mice (F0), fed with control diet (CD) or high-fat diet (HFD), were mated with CD-fed females. F1 progenies were mated with outbred mice to generate F2 mice. All offspring were maintained on CD. Metabolic phenotypes, metabolism-related gene expression and endoplasmic reticulum (ER) stress markers were measured in serum or relevant tissues of F2 mice. DNA methylation in sperm and testis of the founder and in the liver of F2 mice was investigated. RESULTS: Male founder obesity, instigated by HFD, led to glucose dysregulation transmitted down to F2. We found that F2 males to HFD founders were overweight and had a high fasting glucose relative to F2 to CD founders. F2 females to HFD founders, in contrast, had a reduced bodyweight relative to F2 to CD founders and exhibited an early onset of impaired glucose homeostasis. The sex-specific difference was associated with distinct transcriptional patterns in metabolism-related organs, showing altered hepatic glycolysis and decreased adipose Glucose transporter 4 (Glut4) in males and increased gluconeogenesis and lipid synthesis in females. Furthermore, the changes in females were linked to hepatic ER stress, leading to suppressed insulin signaling and non-obese hyperglycemic phenotypes. DNA methylation analysis revealed that the Nr1h3 locus was sensitive to HFD at founder germ cells and the alteration was also detected in the liver of F2 female. CONCLUSION: Our findings demonstrate that male founder obesity influences impaired glucose regulation in F2 progeny possibly via ER stress in a sex-specific manner and it is, in part, contributed by altered DNA methylation at the Nr1h3 locus.

TET-mediated hydroxymethylcytosine at the Pparγ locus is required for initiation of adipogenic differentiation.

BACKGROUND/OBJECTIVES: Adipose tissue is one of the main organs regulating energy homeostasis via energy storage as well as endocrine function. The adipocyte cell number is largely determined by adipogenesis. While the molecular mechanism of adipogenesis has been extensively studied, its role in dynamic DNA methylation plasticity remains unclear. Recently, it has been shown that Tet methylcytosine dioxygenase (TET) is catalytically capable of oxidizing DNA 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC) toward a complete removal of the methylated cytosine. We investigate whether expression of the Tet genes and production of hydroxymethylcytosine are required for preadipocyte differentiation. SUBJECTS/METHODS: Murine 3T3-L1 preadipocytes were used to evaluate the role of Tet1 and Tet2 genes during adipogenesis. Changes in adipogenic ability and in epigenetic status were analyzed, with and without interfering Tet1 and Tet2 expression using small interfering RNA (siRNA). The adipogenesis was evaluated by Oil-Red-O staining and induced expression of adipogenic genes using quantitative polymerase chain reaction (qPCR). Levels of 5-hmC and 5-mC were measured by MassARRAY, immunoprecipitation and GC mass spectrometry at specific loci as well as globally. RESULTS: Both Tet1 and Tet2 genes were upregulated in a time-dependent manner, accompanied by increased expression of hallmark adipogenic genes such as Pparγ and Fabp4 (P<0.05). The TET upregulation led to reduced DNA methylation and elevated hydroxymethylcytosine, both globally and specifically at the Pparγ locus (P<0.05 and P<0.01, respectively). Knockdown of Tet1 and Tet2 blocked adipogenesis (P<0.01) by repression of Pparγ expression (P<0.05). In particular, Tet2 knockdown repressed conversion of 5-mC to 5-hmC at the Pparγ locus (P<0.01). Moreover, vitamin C treatment enhanced adipogenesis (P<0.05), while fumarate treatment inhibited it (P<0.01) by modulating TET activities. CONCLUSIONS: TET proteins, particularly TET2, were required for adipogenesis by modulating DNA methylation at the Pparγ locus, subsequently by inducing Pparγ gene expression.

Requirement of CHROMOMETHYLASE3 for maintenance of CpXpG methylation.

Epigenetic silenced alleles of the Arabidopsis SUPERMAN locus (the clark kent alleles) are associated with dense hypermethylation at noncanonical cytosines (CpXpG and asymmetric sites, where X = A, T, C, or G). A genetic screen for suppressors of a hypermethylated clark kent mutant identified nine loss-of-function alleles of CHROMOMETHYLASE3 (CMT3), a novel cytosine methyltransferase homolog. These cmt3 mutants display a wild-type morphology but exhibit decreased CpXpG methylation of the SUP gene and of other sequences throughout the genome. They also show reactivated expression of endogenous retrotransposon sequences. These results show that a non-CpG DNA methyltransferase is responsible for maintaining epigenetic gene silencing.

Knock-down of oncohistone H3F3A-G34W counteracts the neoplastic phenotype of giant cell tumor of bone derived stromal cells.

Giant cell tumors of bone (GCTB) are semi-malignant tumors associated with extensive osteolytic defects and massive bone destructions. They display a locally aggressive behavior and a very high recurrence rate. Recently, a single mutation has been identified in GCTB affecting the H3F3A gene coding for the histone variant H3.3 (H3.3-G34W). The aim of this study was to investigate whether H3.3-G34W is sufficient to drive tumorigenesis in GCTB. Initially, we confirmed the high frequency of this mutation in 94% of 84 analyzed tissue samples. Using a siRNA based approach we could selectively knockdown H3.3-G34W in primary neoplastic stromal cells isolated from tumor tissue (GCTSC). H3.3-G34W knockdown caused a significant inhibition of cell proliferation, migration and colony formation capacity in vitro. Xenotransplantation of GCTSCs onto the chorioallantoic membrane of fertilized chicken eggs further demonstrated a significant impact of H3.3-G34W knockdown on tumor engraftment and growth in vivo. Our data indicate that H3.3-G34W is sufficient to drive tumorigenesis in GCTB. Apart from the application of H3.3-G34W screening as diagnostic tool, our data suggest that H3.3-G4W represents a promising target for the development of new GCTB therapies.

Two S-adenosylmethionine synthetase-encoding genes differentially expressed during adventitious root development in Pinus contorta.

Two S-adenosylmethionine synthetase (SAMS) cDNAs, PcSAMS1 and PcSAMS2, have been identified in Pinus contorta. We found that the two genes are differentially expressed during root development. Thus, PcSAMS1 is preferentially expressed in roots and exhibits a specific expression pattern in the meristem at the onset of adventitious root development, whereas PcSAMS2 is expressed in roots as well as in shoots and is down-regulated during adventitious root formation. The expression of the two SAMS genes is different from the SAMS activity levels during adventitious root formation. We conclude that other SAMS genes that remain to be characterized may contribute to the observed SAMS activity, or that the activities of PcSAMS1 and PcSAMS2 are affected by post-transcriptional regulation. The deduced amino acid sequences of PcSAMS1 and PcSAMS2 are highly divergent, suggesting different functional roles. However, both carry the two perfectly conserved motifs that are common to all plant SAMS. At the protein level, PcSAMS2 shares about 90% identity to other isolated eukaryotic SAMS, while PcSAMS1 shares less than 50% identity with other plant SAMS. In a phylogenetic comparison, PcSAMS1 seems to have diverged significantly from all other SAMS genes. Nevertheless, PcSAMS1 was able to complement a Saccharomyces cerevisiae sam1 sam2 double mutant, indicating that it encodes a functional SAMS enzyme.