Transcription Factor hDREF Is a Novel SUMO E3 Ligase of Mi2α.

The human transcription factor DNA replication-related element-binding factor (hDREF) is essential for the transcription of a number of housekeeping genes. The mechanisms underlying constitutively active transcription by hDREF were unclear. Here, we provide evidence that hDREF possesses small ubiquitin-like modifier (SUMO) ligase activity and can specifically SUMOylate Mi2α, an ATP-dependent DNA helicase in the nucleosome remodeling and deacetylation complex. Moreover, immunofluorescent staining and biochemical analyses showed that coexpression of hDREF and SUMO-1 resulted in dissociation of Mi2α from chromatin, whereas a SUMOylation-defective Mi2α mutant remained tightly bound to chromatin. Chromatin immunoprecipitation and quantitative RT-PCR analysis demonstrated that Mi2α expression diminished transcription of the ribosomal protein genes, which are positively regulated by hDREF. In contrast, coexpression of hDREF and SUMO-1 suppressed the transcriptional repression by Mi2α. These data indicate that hDREF might incite transcriptional activation by SUMOylating Mi2α, resulting in the dissociation of Mi2α from the gene loci. We propose a novel mechanism for maintaining constitutively active states of a number of hDREF target genes through SUMOylation.

Lamin A reassembly at the end of mitosis is regulated by its SUMO-interacting motif.

Modification of proteins with small ubiquitin-related modifier (SUMO; SUMOylation) is involved in the regulation of various biological processes. Recent studies have demonstrated that noncovalent associations between SUMOylated proteins and co-operative proteins containing SUMO-interacting motifs (SIMs) are important for the spatiotemporal organization of many protein complexes. In this study, we demonstrate that interactions between lamin A, a major component of the nuclear lamina, and SUMO isoforms are dependent on one of the four SIMs (SIM3) resided in lamin A polypeptide in vitro. Live cell imaging and immunofluorescence staining showed that SIM3 is required for accumulation of lamin A on the chromosomes during telophase, and subsequent evaluation of a panel of deletion mutants determined that a 156-amino acid region spanning the carboxyl-terminal Ig-fold domain of lamin A is sufficient for this accumulation. Notably, mutation of SIM3 abrogated the dephosphorylation of mitosis-specific phosphorylation at Ser-22 of lamin A, which normally occurs during telophase, and the subsequent nuclear lamina reorganization. Furthermore, expression of a conjugation-defective SUMO2 mutant, which was previously shown to inhibit endogenous SUMOylation in a dominant-negative manner, also impaired the accumulation of wild type lamin A on telophase chromosomes. These findings suggest that interactions between SIM3 of lamin A and a putative SUMO2-modified protein plays an important role in the reorganization of the nuclear lamina at the end of mitosis.

Long-term expression of the lamin A mutant associated with dilated cardiomyopathy induces senescence.

Mutation of the lamin A gene (LMNA) causes a diverse range of diseases referred to as laminopathies. Because most laminopathies have a dominant inheritance pattern and progress gradually, cultured cells stably expressing mutant lamin A at the same level as endogenous wild-type cells are required for chronological analysis. In this study, we showed that an expression system involving a lentiviral vector that carries the human metallothionein gene basal promoter ensures stable and basal-level expression of proteins and is thus suitable for investigating the properties of lamin A mutants. The small ubiquitin-related modifier (SUMO) modification (SUMOylation)-defective E203G mutant that is associated with familial dilated cardiomyopathy exhibited abnormal subnuclear distribution and inhibited normal localization of WT lamin A in a dominant-negative manner. Low-level and long-term expression of the E203G mutant resulted in multinucleated giant cells, aberrant lipid droplet accumulation in the cytoplasm and premature senescence. Expression of another SUMOylation-defective mutant (K201R) did not induce any phenotypes observed in cells expressing E203G. These results indicate that the E203G mutant may inhibit the normal functions of wild-type lamin A in a dominant-negative manner, but a defect in SUMOylation itself may not be involved in disease pathogenesis.

Active involvement of micro-lipid droplets and lipid-droplet-associated proteins in hormone-stimulated lipolysis in adipocytes.

The regulation of lipolysis in adipocytes involves coordinated actions of many lipid droplet (LD)-associated proteins such as perilipin, hormone sensitive lipase (HSL), adipose triglyceride lipase (ATGL), and its activator protein, CGI-58. Here, we describe the cellular origin and physiological significance of micro LDs (mLDs) that emerge in the cytoplasm during active lipolysis, as well as the roles of key lipolytic proteins on mLDs in differentiated 3T3-L1 adipocytes. Multiplex coherent anti-Stokes Raman scattering (CARS) microscopy demonstrated that mLDs receive the fatty acid (FA) moiety of triglyceride from pre-existing LDs during lipolysis. However, when FA re-esterification was blocked, mLDs did not emerge. Time-lapse imaging of GFP-tagged LD-associated proteins and immunocytochemical analyses showed that particulate structures carrying LD-associated proteins emerged throughout the cells upon lipolytic stimulation, but not when FA re-esterification was blocked. Overall lipolysis, as estimated by glycerol release, was significantly lowered by blocking re-esterification, whereas release of free FAs was enhanced. ATGL was co-immunoprecipitated with CGI-58 from the homogenates of lipolytically stimulated cells. Following CGI-58 knockdown or ATGL inhibition with bromoenol lactone, release of both glycerol and FA was significantly lowered. AICAR, an activator of AMP-activated protein kinase, significantly increased FA release, in accordance with increased expression of ATGL, even in the absence of CGI-58. These results suggest that, besides on the surface of pre-existing central LDs, LD-associated proteins are actively involved in lipolysis on mLDs that are formed by FA re-esterification. Regulation of mLDs and LD-associated proteins may be an attractive therapeutic target against lipid-associated metabolic diseases.

Single-cell cloning and expansion of human induced pluripotent stem cells by a microfluidic culture device.

The microenvironment of cells, which includes basement proteins, shear stress, and extracellular stimuli, should be taken into consideration when examining physiological cell behavior. Although microfluidic devices allow cellular responses to be analyzed with ease at the single-cell level, few have been designed to recover cells. We herein demonstrated that a newly developed microfluidic device helped to improve culture conditions and establish a clonality-validated human pluripotent stem cell line after tracing its growth at the single-cell level. The device will be a helpful tool for capturing various cell types in the human body that have not yet been established in vitro.

Perilipin 5, a lipid droplet-binding protein, protects heart from oxidative burden by sequestering fatty acid from excessive oxidation.

Lipid droplets (LDs) are ubiquitous organelles storing neutral lipids, including triacylglycerol (TAG) and cholesterol ester. The properties of LDs vary greatly among tissues, and LD-binding proteins, the perilipin family in particular, play critical roles in determining such diversity. Overaccumulation of TAG in LDs of non-adipose tissues may cause lipotoxicity, leading to diseases such as diabetes and cardiomyopathy. However, the physiological significance of non-adipose LDs in a normal state is poorly understood. To address this issue, we generated and characterized mice deficient in perilipin 5 (Plin5), a member of the perilipin family particularly abundant in the heart. The mutant mice lacked detectable LDs, containing significantly less TAG in the heart. Particulate structures containing another LD-binding protein, Plin2, but negative for lipid staining, remained in mutant mice hearts. LDs were recovered by perfusing the heart with an inhibitor of lipase. Cultured cardiomyocytes from Plin5-null mice more actively oxidized fatty acid than those of wild-type mice. Production of reactive oxygen species was increased in the mutant mice hearts, leading to a greater decline in heart function with age. This was, however, reduced by the administration of N-acetylcysteine, a precursor of an antioxidant, glutathione. Thus, we conclude that Plin5 is essential for maintaining LDs at detectable sizes in the heart, by antagonizing lipase(s). LDs in turn prevent excess reactive oxygen species production by sequestering fatty acid from oxidation and hence suppress oxidative burden to the heart.

FSP27 contributes to efficient energy storage in murine white adipocytes by promoting the formation of unilocular lipid droplets.

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 PPARgamma 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.

Chanarin-Dorfman syndrome: deficiency in CGI-58, a lipid droplet-bound coactivator of lipase.

Chanarin-Dorfman syndrome (CDS) is a rare autosomal recessive disease of lipid metabolism; it is associated with congenital ichthyosis typed as non-bullous congenital ichthyosiform erythroderma (NCIE). CDS is characterized by the presence of an abnormally large number of cytosolic lipid droplets containing triacylglycerol (TG) in various tissues such as the skin, liver, and leukocytes. Mutations in the CGI-58 (also called ABHD5) gene encoding a 39-kDa protein of the alpha/beta hydrolase domain subfamily have been shown to be responsible for this disorder. In adipocytes, CGI-58 is involved in TG degradation on lipid droplets; in doing so, it coordinates with several lipolytic factors including perilipin, a member of the PAT protein family, and ATGL, a putative rate-limiting lipase in adipocytes. In quiescent adipocytes, CGI-58 interacts with perilipin on the surfaces of lipid droplets. Upon hormonal stimulation, CGI-58 facilitates massive lipolysis by activating ATGL. Some CGI-58 mutations found in CDS patients cancel the ability to interact with perilipin or activate ATGL, indicating that the loss of these interactions is physiologically important. However, based on the tissue distributions of these lipolytic factors, there are likely multiple molecular targets of CGI-58 actions. This in turn gives rise to the multiple phenotypes of CDS, such as ichthyosis, liver steatosis, or neurosensory diseases.

hDREF regulates cell proliferation and expression of ribosomal protein genes.

Although ribosomal proteins (RPs) are essential cellular constituents in all living organisms, mechanisms underlying regulation of their gene expression in mammals remain unclear. We have established that 22 out of 79 human RP genes contain sequences similar to the human DREF (DNA replication-related element-binding factor; hDREF) binding sequence (hDRE) within 200-bp regions upstream of their transcriptional start sites. Electrophoretic gel mobility shift assays and chromatin immunoprecipitation analysis indicated that hDREF binds to hDRE-like sequences in the RP genes both in vitro and in vivo. In addition, transient luciferase assays revealed that hDRE-like sequences act as positive elements for RP gene transcription and cotransfection of an hDREF-expressing plasmid was found to stimulate RP gene promoter activity. Like that of hDREF, expression of RP genes is increased during the late G(1) to S phases, and depletion of hDREF using short hairpin RNA-mediated knockdown decreased RP gene expression and cell proliferation in normal human fibroblasts. Knockdown of the RPS6 gene also resulted in impairment of cell proliferation. These data suggest that hDREF is an important transcription factor for cell proliferation which plays roles in cell cycle-dependent regulation of a number of RP genes.

Heme-binding to the nuclear receptor retinoid X receptor alpha (RXRalpha) leads to the inhibition of the transcriptional activity.

Heme acts as a ligand for transcription factors and regulates the expression of several genes. The nuclear receptor retinoid X receptor alpha (RXRalpha) plays important roles in various nuclear receptor-dependent signaling pathways. We here show that heme binds to RXRalpha and impairs its DNA-binding activity. Deletion and mutation studies of RXRalpha revealed that the binding region of hemin corresponded to the ligand binding domain of mouse RXRalpha and cysteine 374 was involved in the binding. The DNA-binding activity using the DR-1 consensus sequence of RXRalpha in electrophoretic mobility shift assays was inhibited by heme. The reporter assay also showed a decrease of RXRalpha-dependent transcriptional activity. It was reported that hemin enhanced the adipocyte differentiation of mouse 3T3-L1 cells, where the functions of several nuclear receptors including RXRalpha and peroxisome proliferator-activated receptor-gamma (PPAR-gamma) are activated. However, the inductions of adipogenic factor mRNAs including PPAR-gamma, fatty acid binding protein-4 and glucose transporter-4 were markedly repressed by heme during adipocyte differentiation. These results suggest that heme causes the impairment of RXRalpha-dependent signal pathways and inhibits the adipocyte differentiation of 3T3-L1 cells.

Perilipin, a critical regulator of fat storage and breakdown, is a target gene of estrogen receptor-related receptor alpha.

Perilipin is a protein localized on lipid droplet surfaces in adipocytes and steroidogenic cells, playing a central role in regulated lipolysis. Expression of the perilipin gene is markedly induced during adipogenesis. We found that transcription from the perilipin gene promoter is activated by an orphan nuclear receptor, estrogen receptor-related receptor (ERR)alpha. A response element to this receptor was identified in the promoter region by a gene reporter assay, the electrophoretic-gel mobility-shift assay and the chromatin immunoprecipitation assay. Peroxisome proliferator-activated receptor gamma coactivator (PGC)-1alpha enhanced, whereas small heterodimer partner (SHP) repressed, the transactivating function of ERRalpha on the promoter. Thus, the perilipin gene expression is regulated by a transcriptional network controlling energy metabolism, substantiating the functional importance of perilipin in the maintenance of body energy balance.

Target specificities of estrogen receptor-related receptors: analysis of binding sequences and identification of Rb1-inducible coiled-coil 1 (Rb1cc1) as a target gene.

Estrogen receptor-related receptors (ERRs) are orphan members of the nuclear receptor superfamily. A single AGGTCA sequence element preceded by three conserved nucleotides has been identified as a specific recognition motif of ERRs. Here we performed systematic analyses of target sequences on all three ERR subtypes, alpha, beta and gamma. In electrophoretic gel-mobility shift assay and transcriptional reporter assays, they exhibited similar patterns of recognition specificities, showing extremely broad ranges of target sequences. We searched a mouse promoter database for a gene carrying possible ERR-binding sequences. The Rb-1 inducible coiled-coil 1 (Rb1cc1) gene was found to contain two putative ERR binding elements, named response element (RE)-1 and RE-2, in the promoter region. In gene reporter assays, RE-2, but not RE-1, functioned as an effective cis-regulatory element for transactivation by ERRalpha in the presence of a coactivator, peroxisome proliferator-activated receptor gamma coactivator-1alpha. Mutational analyses suggested that RE-2 is recognized by ERRalpha partly as a monovalent element, but also as a direct repeat motif separated by four spacer nucleotides. In vivo binding of ERRalpha to the Rb1cc1 promoter region was confirmed by the chromatin immunoprecipitation assay. Thus, Rb1cc1 is a target gene of ERRalpha, driven by a novel type of recognition sequence.

Metabolic pathway that produces essential fatty acids from polymethylene-interrupted polyunsaturated fatty acids in animal cells.

Sciadonic acid (20:3 Delta-5,11,14) and juniperonic acid (20:4 Delta-5,11,14,17) are polyunsaturated fatty acids (PUFAs) that lack the Delta-8 double bond of arachidonic acid (20:4 Delta-5,8,11,14) and eicosapentaenoic acid (20:5 Delta-5,8,11,14,17), respectively. Here, we demonstrate that these conifer oil-derived PUFAs are metabolized to essential fatty acids in animal cells. When Swiss 3T3 cells were cultured with sciadonic acid, linoleic acid (18:2 Delta-9,12) accumulated in the cells to an extent dependent on the concentration of sciadonic acid. At the same time, a small amount of 16:2 Delta-7,10 appeared in the cellular lipids. Both 16:2 Delta-7,10 and linoleic acid accumulated in sciadonic acid-supplemented CHO cells, but not in peroxisome-deficient CHO cells. We confirmed that 16:2 Delta-7,10 was effectively elongated to linoleic acid in rat liver microsomes. These results indicate that sciadonic acid was partially degraded to 16:2 Delta-7,10 by two cycles of beta-oxidation in peroxisomes, then elongated to linoleic acid in microsomes. Supplementation of Swiss 3T3 cells with juniperonic acid, an n-3 analogue of sciadonic acid, induced accumulation of alpha-linolenic acid (18:3 Delta-9,12,15) in cellular lipids, suggesting that juniperonic acid was metabolized in a similar manner to sciadonic acid. This PUFA remodeling is thought to be a process that converts unsuitable fatty acids into essential fatty acids required by animals.

Orphan nuclear receptor Nur77 accelerates the initial phase of adipocyte differentiation in 3T3-L1 cells by promoting mitotic clonal expansion.

Nur77 is an orphan member of the nuclear receptor superfamily that is expressed in various types of cells and mediates diverse biological processes. Although Nur77 mRNA is induced in the early stage of adipogenesis of 3T3-L1 cells, its roles are not known. To address this issue, we closely inspected the expression of Nur77 mRNA and protein during differentiation of 3T3-L1 cells. Nur77 was induced rapidly and transiently at both mRNA and protein levels only in the initial phase of differentiation induction, and localized almost exclusively in the nuclei. Isobutylmethylxanthine was essential for the induction of Nur77 protein, acting by at least in part protecting the protein from rapid degradation by proteasome. Nur77 siRNA resulted in delayed adipogenesis in 3T3-L1, accompanied by retarded mitotic clonal expansion. These effects were mediated at least partly by decreased expression of cyclins D and E. Constitutive expression of Nur77 inhibited adipogenesis of 3T3-L1, accompanied by enhanced expression of cyclin D1 and prolonged mitotic clonal expansion. Moreover, constitutive expression of Nur77 inhibited, but transient induction of Nur77 promoted, adipogenesis in NIH-3T3 cells. These results suggest that Nur77 accelerates adipocyte differentiation by regulating cell cycle progression and the rapid and transient induction is crucial for its action.

Tissue-selective, bidirectional regulation of PEX11 alpha and perilipin genes through a common peroxisome proliferator response element.

Most cis-acting regulatory elements have generally been assumed to activate a single nearby gene. However, many genes are clustered together, raising the possibility that they are regulated through a common element. We show here that a single peroxisome proliferator response element (PPRE), located between the mouse PEX11 alpha and perilipin genes, confers on both genes activation by peroxisome proliferator-activated receptor alpha (PPAR alpha) and PPAR gamma. A functional PPRE 8.4 kb downstream of the promoter of PEX11 alpha, a PPAR alpha target gene, was identified by a gene transfection study. This PPRE was positioned 1.9 kb upstream of the perilipin gene and also functioned with the perilipin promoter. In addition, this PPRE, when combined with the natural promoters of the PEX11 alpha and perilipin genes, conferred subtype-selective activation by PPAR alpha and PPAR gamma 2. The PPRE sequence specifically bound to the heterodimer of RXR alpha and PPAR alpha or PPAR gamma 2, as assessed by electrophoretic gel mobility shift assays. Furthermore, tissue-selective binding of PPAR alpha and PPAR gamma to the PPRE was demonstrated in hepatocytes and adipocytes, respectively, by chromatin immunoprecipitation assay. Hence, the expression of these genes is induced through the same PPRE in the liver and adipose tissue, where the two PPAR subtypes are specifically expressed.

Peroxisomes are formed from complex membrane structures in PEX6-deficient CHO cells upon genetic complementation.

Pex6p belongs to the AAA family of ATPases. Its CHO mutant, ZP92, lacks normal peroxisomes but contains peroxisomal membrane remnants, so called peroxisomal ghosts, which are detected with anti-70-kDa peroxisomal membrane protein (PMP70) antibody. No peroxisomal matrix proteins were detected inside the ghosts, but exogenously expressed green fluorescent protein (GFP) fused to peroxisome targeting signal-1 (PTS-1) accumulated in the areas adjacent to the ghosts. Electron microscopic examination revealed that PMP70-positive ghosts in ZP92 were complex membrane structures, rather than peroxisomes with reduced matrix protein import ability. In a typical case, a set of one central spherical body and two layers of double-membraned loops were observed, with endoplasmic reticulum present alongside the outer loop. In the early stage of complementation by PEX6 cDNA, catalase and acyl-CoA oxidase accumulated in the lumen of the double-membraned loops. Biochemical analysis revealed that almost all the peroxisomal ghosts were converted into peroxisomes upon complementation. Our results indicate that 1) Peroxisomal ghosts are complex membrane structures; and 2) The complex membrane structures become import competent and are converted into peroxisomes upon complementation with PEX6.

Peroxisome proliferator-activated receptor subtypes differentially cooperate with other transcription factors in selective transactivation of the perilipin/PEX11 alpha gene pair.

Perilipin is an adipocyte-specific protein associated with lipid droplets that is crucial for the regulation of storage and mobilization of lipids. We earlier reported that the mouse perilipin gene is regulated by peroxisome proliferator-activated receptor (PPAR) gamma through a peroxisome proliferator-response element (PPRE) positioned upstream of the perilipin promoter. Moreover, we showed that this PPRE also controls expression of the PEX11alpha gene, which is located further upstream. We show here that three elements, A, B, and C, in close proximity downstream of the PPRE, are essential for transactivation of the perilipin gene by PPARgamma. Electrophoretic gel-mobility shift assays demonstrated that nuclear factor (NF)-1 subtypes bind specifically to element B. Furthermore, chromatin immunoprecipitation using 3T3-L1 cells revealed that NF-1A and NF-1B bind to element B in a differentiation-dependent fashion, whereas binding is constitutive with NF-1C and NF-1X. Element C is likely to be a binding motif for nuclear receptors. With PPARalpha, elements A-C do not appear to be required for transactivation of the PEX11alpha gene, so that cooperation with other transcription factors may be differentially involved in selective transactivation of the PEX11alpha and perilipin genes by different PPAR subtypes.

Aberrant peroxisome morphology in peroxisomal beta-oxidation enzyme deficiencies.

Peroxisomes are ubiquitous organelles in eukaryotic cells and surrounded by a single membrane, and undergo considerable changes in size, shape and number. Peroxisomal disorders are classified into two categories: peroxisome biogenesis disorders (PBDs) and single-enzyme deficiencies (SEDs). Morphologically aberrant peroxisomes called 'peroxisomal ghosts' in PBDs are well known, however, a morphological approach to the study of peroxisomes in SEDs has been rarely reported. Here, we investigated the morphology of peroxisomes in cultured fibroblasts from patients lacking peroxisomal beta-oxidation enzymes, including acyl-CoA oxidase (AOX) or D-3-hydroxyacyl-CoA dehydratase/D-3-hydroxyacyl-CoA dehydrogenase bifunctional protein (D-BP). Morphological analysis by immunofluorescence examination using an antibody against catalase revealed a smaller number of large peroxisomes in fibroblasts from these patients. Moreover, immunoelectron microscopy using an antibody against the 70-kDa peroxisomal membrane protein (PMP70) showed large peroxisomes with various horseshoe-shaped membrane structures. These results give an important clue to elucidating the division of peroxisomes and how peroxisomes change in size, shape, number and position within cells, which are subjects for future study.

Identification of a new complementation group of the peroxisome biogenesis disorders and PEX14 as the mutated gene.

Peroxisome biogenesis disorders (PBD) are lethal hereditary diseases caused by abnormalities in the biogenesis of peroxisomes. At present, 12 different complementation groups have been identified and to date, all genes responsible for each of these complementation groups have been identified. The peroxisomal membrane protein PEX14 is a key component of the peroxisomal import machinery and may be the initial docking site for the two import receptors PEX5 and PEX7. Although PEX14 mutants have been identified in yeasts and CHO-cells, human PEX14 deficiency has apparently not been documented. We now report the identification of a new complementation group of the peroxisome biogenesis disorders with PEX14 as the defective gene. Indeed, human PEX14 rescues the import of a PTS1-dependent as well as a PTS2-dependent protein into the peroxisomes in fibroblasts from a patient with Zellweger syndrome belonging to the new complementation group. This patient was homozygous for a nonsense mutation in a putative coiled-coil region of PEX14, c.553C>T (p.Q185X). Furthermore, we showed that the patient's fibroblasts lacked PEX14 as determined by immunocytochemical analysis. These findings indicate that there are 13 genotypes in PBD and that the role of PEX14 is also essential in humans.