White adipocytes are unique in that they contain large unilocular lipid droplets that occupy most of the cytoplasm. To identify genes involved in the maintenance of mature adipocytes, we expressed dominant-negative PPARγ in 3T3-L1 cells and performed a microarray screen. The fat-specific protein of 27 kDa (FSP27) was strongly downregulated in this context. FSP27 expression correlated with induction of differentiation in cultured preadipocytes, and the protein localized to lipid droplets in murine white adipocytes in vivo. Ablation of FSP27 in mice resulted in the formation of multilocular lipid droplets in these cells. Furthermore, FSP27-deficient mice were protected from diet-induced obesity and insulin resistance and displayed an increased metabolic rate due to increased mitochondrial biogenesis in white adipose tissue (WAT). Depletion of FSP27 by siRNA in murine cultured white adipocytes resulted in the formation of numerous small lipid droplets, increased lipolysis, and decreased triacylglycerol storage, while expression of FSP27 in COS cells promoted the formation of large lipid droplets. Our results suggest that FSP27 contributes to efficient energy storage in WAT by promoting the formation of unilocular lipid droplets, thereby restricting lipolysis. In addition, we found that the nature of lipid accumulation in WAT appears to be associated with maintenance of energy balance and insulin sensitivity.

In animals and plants, the H3K9me3 and H3K27me3 chromatin silencing marks are deposited by different protein machineries. H3K9me3 is catalyzed by the SET-domain SU(VAR)3–9 enzymes, while H3K27me3 is catalyzed by the SET-domain Enhancer-of-zeste enzymes, which are the catalytic subunits of Polycomb Repressive Complex 2 (PRC2). Here, we show that the Enhancer-of-zeste-like protein Ezl1 from the unicellular eukaryote Paramecium tetraurelia , which exhibits significant sequence and structural similarities with human EZH2, catalyzes methylation of histone H3 in vitro and in vivo with an apparent specificity toward K9 and K27. We find that H3K9me3 and H3K27me3 co-occur at multiple families of transposable elements in an Ezl1-dependent manner. We demonstrate that loss of these histone marks results in global transcriptional hyperactivation of transposable elements with modest effects on protein-coding gene expression. Our study suggests that although often considered functionally distinct, H3K9me3 and H3K27me3 may share a common evolutionary history as well as a common ancestral role in silencing transposable elements.

Photoirradiation of a solution of BH(3).NHR(2) (1a: R = Me, 1b: R = 1/2C(4)H(8), 1c: R = 1/2C(5)H(10), 1f: R = Et) containing a catalytic amount of a group-6 metal carbonyl complex, [M(CO)(6)] (M = Cr, Mo, W), led to dehydrogenative B-N covalent bond formation to produce aminoborane dimers, [BH(2)NR(2)](2) (2a-c, f), in high yield. During these reactions a borane sigma complex, [M(CO)(5)(eta(1)-BH(3).NHR(2))] (3), was detected by NMR spectroscopy. Similar catalytic dehydrogenation of bulkier amineboranes, BH(3).NH(i)Pr(2) (1d) and BH(3).NHCy(2) (1e, Cy = cyclo-C(6)H(11)), afforded monomeric products BH(2) horizontal lineNR(2) (4d, e). The reaction mechanism of the dehydrocoupling was investigated by DFT calculations. On the basis of the computational study, we propose that the catalytic dehydrogenation reactions proceed via an intramolecular pathway and that the active catalyst is [Cr(CO)(4)]. The reaction follows a stepwise mechanism involving NH and BH activation. Dehydrocoupling of borane-primary amine adducts BH(3).NH(2)R (1g: R = Me, 1h: R = Et, 1i: R = (t)Bu) gave borazine derivatives [BHNR](3) (5g-i).

SUV39H is the major histone H3 lysine 9 (H3K9)-specific methyltransferase that targets pericentric regions and is crucial for assembling silent heterochromatin. SUV39H recognizes trimethylated H3K9 (H3K9me3) via its chromodomain (CD), and enriched H3K9me3 allows SUV39H to target specific chromosomal regions. However, the detailed targeting mechanisms, especially for naïve chromatin without preexisting H3K9me3, are poorly understood. Here we show that Suv39h1’s CD (Suv39h1-CD) binds nucleic acids, and this binding is important for its function in heterochromatin assembly. Suv39h1-CD had higher binding affinity for RNA than DNA, and its ability to bind nucleic acids was independent of its H3K9me3 recognition. Suv39h1 bound major satellite RNAs in vivo, and knockdown of major satellite RNAs lowered Suv39h1 retention on pericentromere. Suv39h1 mutational studies indicated that both the nucleic acid–binding and H3K9me–binding activities of Suv39h1-CD were crucial for its pericentric heterochromatin assembly. These results suggest that chromatin-bound RNAs contribute to creating SUV39H’s target specificity.DOI: http://dx.doi.org/10.7554/eLife.25317.001

Chronic inflammation in adipose tissue is thought to be important for the development of insulin resistance in obesity. Furthermore, the level of monocyte chemoattractant protein-1 (MCP-1) is increased not only in adipose tissue but also in the circulation in association with obesity. However, it has remained unclear to what extent the increased circulating level of MCP-1 contributes to insulin resistance. We have now examined the relevance of circulating MCP-1 to the development of insulin resistance in mice. The plasma concentration of MCP-1 was increased chronically or acutely in mice to the level observed in obese animals by chronic subcutaneous infusion of recombinant MCP-1 with an osmotic pump or by acute intravenous infusion of MCP-1 with an infusion pump, respectively. Whole-body metabolic parameters as well as inflammatory changes in adipose tissue were examined. A chronic increase in the circulating level of MCP-1 induced insulin resistance, macrophage infiltration into adipose tissue, and an increase in hepatic triacylglycerol content. An acute increase in the circulating MCP-1 concentration also induced insulin resistance but not macrophage infiltration into adipose tissue. In addition, inhibition of signaling by MCP-1 and its receptor CCR2 by administration of a novel CCR2 antagonist ameliorated insulin resistance in mice fed a high-fat diet without affecting macrophage infiltration into adipose tissue. These data indicate that an increase in the concentration of MCP-1 in the circulation is sufficient to induce systemic insulin resistance irrespective of adipose tissue inflammation.

No abstract

SNARE proteins (VAMP2, syntaxin4, and SNAP23) have been thought to play a key role in GLUT4 trafficking by mediating the tethering, docking and subsequent fusion of GLUT4-containing vesicles with the plasma membrane. The precise functions of these proteins have remained elusive, however. We have now shown that depletion of the vesicle SNARE (v-SNARE) VAMP2 by RNA interference in 3T3-L1 adipocytes inhibited the fusion of GLUT4 vesicles with the plasma membrane but did not affect tethering of the vesicles to the membrane. In contrast, depletion of the target SNAREs (t-SNAREs) syntaxin4 or SNAP23 resulted in impairment of GLUT4 vesicle tethering to the plasma membrane. Our results indicate that the t-SNAREs syntaxin4 and SNAP23 are indispensable for the tethering of GLUT4 vesicles to the plasma membrane, whereas the v-SNARE VAMP2 is not required for this step but is essential for the subsequent fusion event.

Chromobox 2 (CBX2), a component of polycomb repressive complex 1 (PRC1), binds lysine 27-methylated histone H3 (H3K27me3) via its chromodomain (CD) and plays a critical role in repressing developmentally regulated genes. The phosphorylation of CBX2 has been described in several studies, but the biological implications of this modification remain largely elusive. Here, we show that CBX2's phosphorylation plays an important role in its nucleosome binding. CBX2 is stably phosphorylated in vivo, and domain analysis showed that residues in CBX2's serine-rich (SR) region are the predominant phosphorylation sites. The serine residues in an SR region followed by an acidic-residue (AR) cluster coincide with the consensus target of casein kinase II (CK2), and CK2 efficiently phosphorylated the SR region in vitro. A nucleosome pull-down assay revealed that CK2-phosphorylated CBX2 had a high specificity for H3K27me3-modified nucleosomes. An electrophoretic mobility-shift assay showed that CK2-mediated phosphorylation diminished CBX2's AT-hook-associated DNA-binding activity. Mutant CBX2 lacking the SR region or its neighboring AR cluster failed to repress the transcription of p21, a gene targeted by PRC1. These results suggest that CBX2's phosphorylation is critical for its transcriptional repression of target genes.