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1.
Proc Natl Acad Sci U S A ; 117(51): 32739-32749, 2020 12 22.
Article in English | MEDLINE | ID: mdl-33273113

ABSTRACT

In photosynthetic eukaryotes, thousands of proteins are translated in the cytosol and imported into the chloroplast through the concerted action of two translocons-termed TOC and TIC-located in the outer and inner membranes of the chloroplast envelope, respectively. The degree to which the molecular composition of the TOC and TIC complexes is conserved over phylogenetic distances has remained controversial. Here, we combine transcriptomic, biochemical, and genetic tools in the green alga Chlamydomonas (Chlamydomonas reinhardtii) to demonstrate that, despite a lack of evident sequence conservation for some of its components, the algal TIC complex mirrors the molecular composition of a TIC complex from Arabidopsis thaliana. The Chlamydomonas TIC complex contains three nuclear-encoded subunits, Tic20, Tic56, and Tic100, and one chloroplast-encoded subunit, Tic214, and interacts with the TOC complex, as well as with several uncharacterized proteins to form a stable supercomplex (TIC-TOC), indicating that protein import across both envelope membranes is mechanistically coupled. Expression of the nuclear and chloroplast genes encoding both known and uncharacterized TIC-TOC components is highly coordinated, suggesting that a mechanism for regulating its biogenesis across compartmental boundaries must exist. Conditional repression of Tic214, the only chloroplast-encoded subunit in the TIC-TOC complex, impairs the import of chloroplast proteins with essential roles in chloroplast ribosome biogenesis and protein folding and induces a pleiotropic stress response, including several proteins involved in the chloroplast unfolded protein response. These findings underscore the functional importance of the TIC-TOC supercomplex in maintaining chloroplast proteostasis.


Subject(s)
Chlamydomonas reinhardtii/metabolism , Chloroplasts/genetics , Multiprotein Complexes/genetics , Plant Proteins/genetics , Cell Compartmentation , Chlamydomonas reinhardtii/genetics , Chloroplasts/metabolism , Evolution, Molecular , Gene Expression Regulation, Plant , Multiprotein Complexes/metabolism , Plant Proteins/metabolism , Protein Transport , Sequence Homology, Amino Acid
2.
PLoS Pathog ; 13(4): e1006344, 2017 Apr.
Article in English | MEDLINE | ID: mdl-28414774

ABSTRACT

Autophagy is a eukaryotic catabolic process also participating in cell-autonomous defence. Infected host cells generate double-membrane autophagosomes that mature in autolysosomes to engulf, kill and digest cytoplasmic pathogens. However, several bacteria subvert autophagy and benefit from its machinery and functions. Monitoring infection stages by genetics, pharmacology and microscopy, we demonstrate that the ESX-1 secretion system of Mycobacterium marinum, a close relative to M. tuberculosis, upregulates the transcription of autophagy genes, and stimulates autophagosome formation and recruitment to the mycobacteria-containing vacuole (MCV) in the host model organism Dictyostelium. Antagonistically, ESX-1 is also essential to block the autophagic flux and deplete the MCV of proteolytic activity. Activators of the TORC1 complex localize to the MCV in an ESX-1-dependent manner, suggesting an important role in the manipulation of autophagy by mycobacteria. Our findings suggest that the infection by M. marinum activates an autophagic response that is simultaneously repressed and exploited by the bacterium to support its survival inside the MCV.


Subject(s)
Autophagy , Bacterial Proteins/metabolism , Multiprotein Complexes/metabolism , Mycobacterium Infections, Nontuberculous/metabolism , Mycobacterium Infections, Nontuberculous/physiopathology , Mycobacterium marinum/metabolism , TOR Serine-Threonine Kinases/metabolism , Bacterial Proteins/genetics , Dictyostelium/genetics , Dictyostelium/metabolism , Dictyostelium/microbiology , Host-Pathogen Interactions , Humans , Mechanistic Target of Rapamycin Complex 1 , Multiprotein Complexes/genetics , Mycobacterium Infections, Nontuberculous/genetics , Mycobacterium Infections, Nontuberculous/virology , Mycobacterium marinum/genetics , TOR Serine-Threonine Kinases/genetics , Vacuoles/microbiology
3.
Nat Commun ; 8: 14172, 2017 02 03.
Article in English | MEDLINE | ID: mdl-28155854

ABSTRACT

The cerebral cortex is organized into specialized sensory areas, whose initial territory is determined by intracortical molecular determinants. Yet, sensory cortical area size appears to be fine tuned during development to respond to functional adaptations. Here we demonstrate the existence of a prenatal sub-cortical mechanism that regulates the cortical areas size in mice. This mechanism is mediated by spontaneous thalamic calcium waves that propagate among sensory-modality thalamic nuclei up to the cortex and that provide a means of communication among sensory systems. Wave pattern alterations in one nucleus lead to changes in the pattern of the remaining ones, triggering changes in thalamic gene expression and cortical area size. Thus, silencing calcium waves in the auditory thalamus induces Rorß upregulation in a neighbouring somatosensory nucleus preluding the enlargement of the barrel-field. These findings reveal that embryonic thalamic calcium waves coordinate cortical sensory area patterning and plasticity prior to sensory information processing.


Subject(s)
Ventral Thalamic Nuclei/anatomy & histology , Ventral Thalamic Nuclei/embryology , Animals , Calcium/metabolism , Female , Gap Junctions/metabolism , Gene Expression , Humans , Mice, Inbred C57BL , Mice, Transgenic , Neuronal Plasticity , Orphan Nuclear Receptors/genetics , Pregnancy , Somatosensory Cortex/physiology , Ventral Thalamic Nuclei/metabolism , Ventral Thalamic Nuclei/physiology , Vision, Ocular
4.
EMBO Rep ; 17(5): 769-79, 2016 05.
Article in English | MEDLINE | ID: mdl-26993089

ABSTRACT

Pioneering studies within the last few years have allowed the in vitro expansion of tissue-specific adult stem cells from a variety of endoderm-derived organs, including the stomach, small intestine, and colon. Expansion of these cells requires activation of the receptor Lgr5 by its ligand R-spondin 1 and is likely facilitated by the fact that in healthy adults the stem cells in these organs are highly proliferative. In many other adult organs, such as the liver, proliferating cells are normally not abundant in adulthood. However, upon injury, the liver has a strong regenerative potential that is accompanied by the emergence of Lgr5-positive stem cells; these cells can be isolated and expanded in vitro as organoids. In an effort to isolate stem cells from non-regenerating mouse livers, we discovered that healthy gallbladders are a rich source of stem/progenitor cells that can be propagated in culture as organoids for more than a year. Growth of these organoids was stimulated by R-spondin 1 and noggin, whereas in the absence of these growth factors, the organoids differentiated partially toward the hepatocyte fate. When transplanted under the liver capsule, gallbladder-derived organoids maintained their architecture for 2 weeks. Furthermore, single cells prepared from dissociated organoids and injected into the mesenteric vein populated the liver parenchyma of carbon tetrachloride-treated mice. Human gallbladders were also a source of organoid-forming stem cells. Thus, under specific growth conditions, stem cells can be isolated from healthy gallbladders, expanded almost indefinitely in vitro, and induced to differentiate toward the hepatocyte lineage.


Subject(s)
Carrier Proteins/metabolism , Gallbladder/cytology , Stem Cells/metabolism , Thrombospondins/metabolism , Animals , Biomarkers , Carrier Proteins/genetics , Carrier Proteins/pharmacology , Cell Differentiation/genetics , Cells, Cultured , Gene Expression Profiling , Humans , Liver/cytology , Mice , Mice, Transgenic , Organoids , Protein Kinase Inhibitors/pharmacology , Receptors, Transforming Growth Factor beta/antagonists & inhibitors , Receptors, Transforming Growth Factor beta/metabolism , Stem Cells/drug effects , Thrombospondins/genetics , Thrombospondins/pharmacology , Transcriptome
5.
EMBO Rep ; 16(6): 741-52, 2015 Jun.
Article in English | MEDLINE | ID: mdl-25851648

ABSTRACT

The Wnt pathway, which controls crucial steps of the development and differentiation programs, has been proposed to influence lipid storage and homeostasis. In this paper, using an unbiased strategy based on high-content genome-wide RNAi screens that monitored lipid distribution and amounts, we find that Wnt3a regulates cellular cholesterol. We show that Wnt3a stimulates the production of lipid droplets and that this stimulation strictly depends on endocytosed, LDL-derived cholesterol and on functional early and late endosomes. We also show that Wnt signaling itself controls cholesterol endocytosis and flux along the endosomal pathway, which in turn modulates cellular lipid homeostasis. These results underscore the importance of endosome functions for LD formation and reveal a previously unknown regulatory mechanism of the cellular programs controlling lipid storage and endosome transport under the control of Wnt signaling.


Subject(s)
Cholesterol, LDL/metabolism , Lipid Droplets/metabolism , Wnt Signaling Pathway , Animals , Cell Line , Cholesterol, LDL/genetics , Endocytosis , Endosomes/metabolism , Epithelial Cells/ultrastructure , Gene Expression Profiling , HeLa Cells , High-Throughput Screening Assays , Homeostasis , Humans , L Cells , Mice , Oleic Acid/pharmacology , RNA Interference , Wnt3A Protein/metabolism
6.
Mol Biol Cell ; 25(20): 3234-46, 2014 Oct 15.
Article in English | MEDLINE | ID: mdl-25143408

ABSTRACT

The regulatory pathways required to maintain eukaryotic lipid homeostasis are largely unknown. We developed a systematic approach to uncover new players in the regulation of lipid homeostasis. Through an unbiased mass spectrometry-based lipidomic screening, we quantified hundreds of lipid species, including glycerophospholipids, sphingolipids, and sterols, from a collection of 129 mutants in protein kinase and phosphatase genes of Saccharomyces cerevisiae. Our approach successfully identified known kinases involved in lipid homeostasis and uncovered new ones. By clustering analysis, we found connections between nutrient-sensing pathways and regulation of glycerophospholipids. Deletion of members of glucose- and nitrogen-sensing pathways showed reciprocal changes in glycerophospholipid acyl chain lengths. We also found several new candidates for the regulation of sphingolipid homeostasis, including a connection between inositol pyrophosphate metabolism and complex sphingolipid homeostasis through transcriptional regulation of AUR1 and SUR1. This robust, systematic lipidomic approach constitutes a rich, new source of biological information and can be used to identify novel gene associations and function.


Subject(s)
Glycerophospholipids/metabolism , Lipid Metabolism , Phosphoric Monoester Hydrolases/genetics , Protein Kinases/genetics , Saccharomyces cerevisiae Proteins/genetics , Sphingolipids/metabolism , Sterols/metabolism , Glycerophospholipids/analysis , Homeostasis , Lipids/analysis , Mass Spectrometry , Mutation , Saccharomyces cerevisiae/genetics , Sphingolipids/analysis , Sterols/analysis
7.
Plant Cell ; 26(5): 2201-2222, 2014 May.
Article in English | MEDLINE | ID: mdl-24879428

ABSTRACT

Plastid protein homeostasis is critical during chloroplast biogenesis and responses to changes in environmental conditions. Proteases and molecular chaperones involved in plastid protein quality control are encoded by the nucleus except for the catalytic subunit of ClpP, an evolutionarily conserved serine protease. Unlike its Escherichia coli ortholog, this chloroplast protease is essential for cell viability. To study its function, we used a recently developed system of repressible chloroplast gene expression in the alga Chlamydomonas reinhardtii. Using this repressible system, we have shown that a selective gradual depletion of ClpP leads to alteration of chloroplast morphology, causes formation of vesicles, and induces extensive cytoplasmic vacuolization that is reminiscent of autophagy. Analysis of the transcriptome and proteome during ClpP depletion revealed a set of proteins that are more abundant at the protein level, but not at the RNA level. These proteins may comprise some of the ClpP substrates. Moreover, the specific increase in accumulation, both at the RNA and protein level, of small heat shock proteins, chaperones, proteases, and proteins involved in thylakoid maintenance upon perturbation of plastid protein homeostasis suggests the existence of a chloroplast-to-nucleus signaling pathway involved in organelle quality control. We suggest that this represents a chloroplast unfolded protein response that is conceptually similar to that observed in the endoplasmic reticulum and in mitochondria.

8.
PLoS One ; 9(6): e99883, 2014.
Article in English | MEDLINE | ID: mdl-24932636

ABSTRACT

The function of the nuclear receptor Rev-erbα (Nr1d1) in the brain is, apart from its role in the circadian clock mechanism, unknown. Therefore, we compared gene expression profiles in the brain between wild-type and Rev-erbα knock-out (KO) animals. We identified fatty acid binding protein 7 (Fabp7, Blbp) as a direct target of repression by REV-ERBα. Loss of Rev-erbα manifested in memory and mood related behavioral phenotypes and led to overexpression of Fabp7 in various brain areas including the subgranular zone (SGZ) of the hippocampus, where neuronal progenitor cells (NPCs) can initiate adult neurogenesis. We found increased proliferation of hippocampal neurons and loss of its diurnal pattern in Rev-erbα KO mice. In vitro, proliferation and migration of glioblastoma cells were affected by manipulating either Fabp7 expression or REV-ERBα activity. These results suggest an important role of Rev-erbα and Fabp7 in adult neurogenesis, which may open new avenues for treatment of gliomas as well as neurological diseases such as depression and Alzheimer.


Subject(s)
Aging/metabolism , Carrier Proteins/genetics , Hippocampus/growth & development , Hippocampus/metabolism , Neurogenesis , Nuclear Receptor Subfamily 1, Group D, Member 1/metabolism , Tumor Suppressor Proteins/genetics , Affect/physiology , Animals , Behavior, Animal , Carrier Proteins/metabolism , Cell Line, Tumor , Cell Movement , Cell Proliferation , Circadian Rhythm , Cognition , Dentate Gyrus/metabolism , Fatty Acid-Binding Protein 7 , Gene Expression Profiling , Gene Expression Regulation , Genome , Glioblastoma/metabolism , Glioblastoma/pathology , Humans , Immunohistochemistry , Mice, Knockout , Nuclear Receptor Subfamily 1, Group D, Member 1/deficiency , Promoter Regions, Genetic/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Tumor Suppressor Proteins/metabolism
9.
Cell Rep ; 3(3): 796-807, 2013 Mar 28.
Article in English | MEDLINE | ID: mdl-23434507

ABSTRACT

The HES proteins are known Notch effectors and have long been recognized as important in inhibiting neuronal differentiation. However, the roles that they play in the specification of neuronal fate remain largely unknown. Here, we show that in the differentiating retinal epithelium, the proneural protein ATOH7 (ATH5) is required for the activation of the transcription of the Hes5.3 gene before the penultimate mitosis of progenitor cells. We further show that the HES5.3 protein slows down the cell-cycle progression of Atoh7-expressing cells, thereby establishing conditions for Atoh7 to reach a high level of expression in S phase and induce neuronal differentiation prior to the ultimate mitosis. Our study uncovers how a proneural protein recruits a protein known to be a component of the Notch signaling pathway in order to regulate the transition between an initial phase of selection among uncommitted progenitors and a later phase committing the selected progenitors to neuronal differentiation.


Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Feedback, Physiological , Neurogenesis , Receptors, Notch/metabolism , Retina/metabolism , S Phase , Animals , Basic Helix-Loop-Helix Transcription Factors/genetics , Chick Embryo , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Epithelial Cells/cytology , Epithelial Cells/metabolism , Gene Expression Regulation, Developmental , Mitosis , Neurons/cytology , Neurons/metabolism , Retina/cytology , Retina/embryology , Signal Transduction , Transcription, Genetic
10.
PLoS Genet ; 9(1): e1003160, 2013.
Article in English | MEDLINE | ID: mdl-23300479

ABSTRACT

Mouse sex determination provides an attractive model to study how regulatory genetic networks and signaling pathways control cell specification and cell fate decisions. This study characterizes in detail the essential role played by the insulin receptor (INSR) and the IGF type I receptor (IGF1R) in adrenogenital development and primary sex determination. Constitutive ablation of insulin/IGF signaling pathway led to reduced proliferation rate of somatic progenitor cells in both XX and XY gonads prior to sex determination together with the downregulation of hundreds of genes associated with the adrenal, testicular, and ovarian genetic programs. These findings indicate that prior to sex determination somatic progenitors in Insr;Igf1r mutant gonads are not lineage primed and thus incapable of upregulating/repressing the male and female genetic programs required for cell fate restriction. In consequence, embryos lacking functional insulin/IGF signaling exhibit (i) complete agenesis of the adrenal cortex, (ii) embryonic XY gonadal sex reversal, with a delay of Sry upregulation and the subsequent failure of the testicular genetic program, and (iii) a delay in ovarian differentiation so that Insr;Igf1r mutant gonads, irrespective of genetic sex, remained in an extended undifferentiated state, before the ovarian differentiation program ultimately is initiated at around E16.5.


Subject(s)
Gonads , Insulin , Receptor, IGF Type 1 , Receptor, Insulin , Sex Determination Processes/genetics , Adrenal Cortex/growth & development , Adrenal Cortex/pathology , Adrenal Glands/growth & development , Adrenal Glands/metabolism , Animals , Cell Differentiation/genetics , Cell Lineage , Cell Proliferation , Disorders of Sex Development/genetics , Female , Gonads/growth & development , Gonads/metabolism , Gonads/pathology , Humans , Insulin/genetics , Insulin/metabolism , Male , Mice , Ovary/growth & development , Ovary/metabolism , Receptor, IGF Type 1/genetics , Receptor, IGF Type 1/metabolism , Receptor, Insulin/genetics , Receptor, Insulin/metabolism , Sex Chromosomes , Signal Transduction , Testis/growth & development , Testis/metabolism
11.
Plant Cell ; 25(1): 167-86, 2013 Jan.
Article in English | MEDLINE | ID: mdl-23292734

ABSTRACT

Although reverse genetics has been used to elucidate the function of numerous chloroplast proteins, the characterization of essential plastid genes and their role in chloroplast biogenesis and cell survival has not yet been achieved. Therefore, we developed a robust repressible chloroplast gene expression system in the unicellular alga Chlamydomonas reinhardtii based mainly on a vitamin-repressible riboswitch, and we used this system to study the role of two essential chloroplast genes: ribosomal protein S12 (rps12), encoding a plastid ribosomal protein, and rpoA, encoding the α-subunit of chloroplast bacterial-like RNA polymerase. Repression of either of these two genes leads to the arrest of cell growth, and it induces a response that involves changes in expression of nuclear genes implicated in chloroplast biogenesis, protein turnover, and stress. This response also leads to the overaccumulation of several plastid transcripts and reveals the existence of multiple negative regulatory feedback loops in the chloroplast gene circuitry.


Subject(s)
Algal Proteins/genetics , Chlamydomonas reinhardtii/genetics , Chloroplast Proteins/genetics , Chloroplasts/metabolism , Signal Transduction , Algal Proteins/immunology , Algal Proteins/metabolism , Animals , Base Sequence , Chlamydomonas reinhardtii/growth & development , Chlamydomonas reinhardtii/physiology , Chloroplast Proteins/immunology , Chloroplast Proteins/metabolism , Chloroplasts/genetics , Cluster Analysis , DNA-Directed RNA Polymerases/genetics , DNA-Directed RNA Polymerases/immunology , DNA-Directed RNA Polymerases/metabolism , Feedback, Physiological , Gene Expression , Gene Expression Regulation, Plant , Genes, Essential , Immune Sera , Molecular Sequence Data , Polyribosomes , Protein Biosynthesis , Rabbits , Ribosomal Proteins/genetics , Ribosomal Proteins/immunology , Ribosomal Proteins/metabolism , Sequence Analysis, DNA , Transcription, Genetic
12.
PLoS One ; 7(11): e49010, 2012.
Article in English | MEDLINE | ID: mdl-23152841

ABSTRACT

BACKGROUND: The diagnosis of malignant hematologic diseases has become increasingly complex during the last decade. It is based on the interpretation of results from different laboratory analyses, which range from microscopy to gene expression profiling. Recently, a method for the analysis of RNA phenotypes has been developed, the nCounter technology (Nanostring® Technologies), which allows for simultaneous quantification of hundreds of RNA molecules in biological samples. We evaluated this technique in a Swiss multi-center study on eighty-six samples from acute leukemia patients. METHODS: mRNA and protein profiles were established for normal peripheral blood and bone marrow samples. Signal intensities of the various tested antigens with surface expression were similar to those found in previously performed Affymetrix microarray analyses. Acute leukemia samples were analyzed for a set of twenty-two validated antigens and the Pearson Correlation Coefficient for nCounter and flow cytometry results was calculated. RESULTS: Highly significant values between 0.40 and 0.97 were found for the twenty-two antigens tested. A second correlation analysis performed on a per sample basis resulted in concordant results between flow cytometry and nCounter in 44-100% of the antigens tested (mean = 76%), depending on the number of blasts present in a sample, the homogeneity of the blast population, and the type of leukemia (AML or ALL). CONCLUSIONS: The nCounter technology allows for fast and easy depiction of a mRNA profile from hematologic samples. This technology has the potential to become a valuable tool for the diagnosis of acute leukemias, in addition to multi-color flow cytometry.


Subject(s)
Leukemia/genetics , Leukemia/metabolism , Proteome , Transcriptome , Antigens, CD/genetics , Antigens, CD/metabolism , Blood Cells/metabolism , Bone Marrow Cells/metabolism , Flow Cytometry/methods , Genomics/methods , Humans , Immunophenotyping , Leukemia/diagnosis , Proteomics/methods
13.
Sci Signal ; 5(215): ra21, 2012 Mar 13.
Article in English | MEDLINE | ID: mdl-22416276

ABSTRACT

Ligand binding to the epidermal growth factor receptor (EGFR) on the cell surface activates the extracellular signal-regulated kinase (ERK) cascade. Activated, ligand-bound receptors are internalized, and this process may contribute to termination of signaling or enable signaling from intracellular sites. ESCRT (endosomal sorting complex required for transport) complexes may contribute to termination of signaling by sorting receptors into intraluminal vesicles of multivesicular endosomes from which the receptors continue into lysosomes for degradation. We showed that depletion of ESCRTs, which causes the retention of the EGFR in endosomes, increased the activation of the EGFR and its downstream kinases but had little effect on the overall profile and amplitude of the EGF-induced transcriptional response. In contrast, interfering with receptor endocytosis or ubiquitination to keep the EGFR at the cell surface stimulated increases in the abundance of many EGF-induced transcripts, similar to those induced by EGFR overexpression. We also found that the complete EGF transcriptional program was rapidly activated after ligand binding to the receptor. We conclude that the transcriptional response is elicited primarily by receptor molecules at the cell surface.


Subject(s)
Endocytosis/physiology , Endosomal Sorting Complexes Required for Transport/metabolism , Epidermal Growth Factor/metabolism , ErbB Receptors/metabolism , Signal Transduction/physiology , Transcription, Genetic/physiology , Endosomal Sorting Complexes Required for Transport/genetics , Endosomes/genetics , Endosomes/metabolism , Epidermal Growth Factor/genetics , ErbB Receptors/genetics , HeLa Cells , Humans , Lysosomes/genetics , Lysosomes/metabolism , Proteolysis
14.
Toxicol Sci ; 125(1): 310-7, 2012 Jan.
Article in English | MEDLINE | ID: mdl-21998131

ABSTRACT

Several million people are exposed to dioxin and dioxin-like compounds, primarily through food consumption. Skin lesions historically called "chloracne" are the most specific sign of abnormal dioxin exposure and classically used as a key marker in humans. We followed for 5 years a man who had been exposed to the most toxic dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), at a single oral dose of 5 million-fold more than the accepted daily exposure in the general population. We adopted a molecular medicine approach, aimed at identifying appropriate therapy. Skin lesions, which progressively covered up to 40% of the body surface, were found to be hamartomas, which developed parallel to a complete and sustained involution of sebaceous glands, with concurrent transcriptomic alterations pointing to the inhibition of lipid metabolism and the involvement of bone morphogenetic proteins signaling. Hamartomas created a new compartment that concentrated TCDD up to 10-fold compared with serum and strongly expressed the TCDD-metabolizing enzyme cytochrome P450 1A1, thus representing a potentially significant source of enzymatic activity, which may add to the xenobiotic metabolism potential of the classical organs such as the liver. This historical case provides a unique set of data on the human tissue response to dioxin for the identification of new markers of exposure in human populations. The herein discovered adaptive cutaneous response to TCDD also points to the potential role of the skin in the metabolism of food xenobiotics.


Subject(s)
Hamartoma/chemically induced , Polychlorinated Dibenzodioxins/poisoning , Skin Diseases/chemically induced , Skin/drug effects , Biopsy , Gene Expression/drug effects , Gene Expression Profiling , Hamartoma/genetics , Hamartoma/pathology , Hamartoma/therapy , Humans , Male , Middle Aged , Multimodal Imaging , Polychlorinated Dibenzodioxins/pharmacokinetics , Positron-Emission Tomography , Skin/metabolism , Skin/pathology , Skin Diseases/genetics , Skin Diseases/pathology , Skin Diseases/therapy , Tomography, X-Ray Computed , Treatment Outcome
15.
Blood ; 117(17): 4490-500, 2011 Apr 28.
Article in English | MEDLINE | ID: mdl-21385848

ABSTRACT

MicroRNAs (miRNAs) are small, noncoding RNAs that regulate target mRNAs by binding to their 3' untranslated regions. There is growing evidence that microRNA-155 (miR155) modulates gene expression in various cell types of the immune system and is a prominent player in the regulation of innate and adaptive immune responses. To define the role of miR155 in dendritic cells (DCs) we performed a detailed analysis of its expression and function in human and mouse DCs. A strong increase in miR155 expression was found to be a general and evolutionarily conserved feature associated with the activation of DCs by diverse maturation stimuli in all DC subtypes tested. Analysis of miR155-deficient DCs demonstrated that miR155 induction is required for efficient DC maturation and is critical for the ability of DCs to promote antigen-specific T-cell activation. Expression-profiling studies performed with miR155(-/-) DCs and DCs overexpressing miR155, combined with functional assays, revealed that the mRNA encoding the transcription factor c-Fos is a direct target of miR155. Finally, all of the phenotypic and functional defects exhibited by miR155(-/-) DCs could be reproduced by deregulated c-Fos expression. These results indicate that silencing of c-Fos expression by miR155 is a conserved process that is required for DC maturation and function.


Subject(s)
Dendritic Cells/physiology , Gene Silencing/immunology , MicroRNAs/immunology , Proto-Oncogene Proteins c-fos/genetics , Proto-Oncogene Proteins c-fos/immunology , Animals , Cell Differentiation/genetics , Cell Differentiation/immunology , Cell Line , Dendritic Cells/cytology , Evolution, Molecular , Humans , Mice , Mice, Mutant Strains , MicroRNAs/genetics , Monocytes/cytology , RNA, Messenger/genetics , RNA, Messenger/immunology
16.
Mol Cell Proteomics ; 10(4): M900587MCP200, 2011 Apr.
Article in English | MEDLINE | ID: mdl-20467044

ABSTRACT

Sertoli cells (SCs) are the central, essential coordinators of spermatogenesis, without which germ cell development cannot occur. We previously showed that Dicer, an RNaseIII endonuclease required for microRNA (miRNA) biogenesis, is absolutely essential for Sertoli cells to mature, survive, and ultimately sustain germ cell development. Here, using isotope-coded protein labeling, a technique for protein relative quantification by mass spectrometry, we investigated the impact of Sertoli cell-Dicer and subsequent miRNA loss on the testicular proteome. We found that, a large proportion of proteins (50 out of 130) are up-regulated by more that 1.3-fold in testes lacking Sertoli cell-Dicer, yet that this protein up-regulation is mild, never exceeding a 2-fold change, and is not preceeded by alterations of the corresponding mRNAs. Of note, the expression levels of six proteins of interest were further validated using the Absolute Quantification (AQUA) peptide technology. Furthermore, through 3'UTR luciferase assays we identified one up-regulated protein, SOD-1, a Cu/Zn superoxide dismutase whose overexpression has been linked to enhanced cell death through apoptosis, as a likely direct target of three Sertoli cell-expressed miRNAs, miR-125a-3p, miR-872 and miR-24. Altogether, our study, which is one of the few in vivo analyses of miRNA effects on protein output, suggests that, at least in our system, miRNAs play a significant role in translation control.


Subject(s)
Proteome/metabolism , Ribonuclease III/deficiency , Sertoli Cells/metabolism , Testis/metabolism , 3' Untranslated Regions , Animals , Gene Expression Profiling , Gene Expression Regulation, Enzymologic , Genes, Reporter , Luciferases, Firefly/biosynthesis , Luciferases, Firefly/genetics , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , MicroRNAs/genetics , MicroRNAs/metabolism , Promoter Regions, Genetic , RNA Interference , Ribonuclease III/genetics , Sequence Deletion , Superoxide Dismutase/genetics , Superoxide Dismutase/metabolism , Superoxide Dismutase-1 , Tandem Mass Spectrometry , Testis/pathology , Transcription, Genetic , Up-Regulation
17.
Mol Cell Endocrinol ; 321(2): 152-60, 2010 Jun 10.
Article in English | MEDLINE | ID: mdl-20171261

ABSTRACT

Soy and soy-based products are widely consumed by infants and adult individuals. There has been speculation that the presence of isoflavone phytoestrogens in soybean cause adverse effects on the development and function of the male reproductive system. The purpose of this study was to examine the influence of dietary soy and phytoestrogens on testicular and reproductive functions. Male mice were fed from conception to adulthood with either a high soy-containing diet or a soy-free diet. Although adult mice fed a soy-rich diet exhibited normal male behaviour and were fertile, we observed a reduced proportion of haploid germ cells in testes correlating with a 25% decrease in epididymal sperm counts and a 21% reduction in litter size. LH and androgens levels were not affected but transcripts coding for androgen-response genes in Sertoli cells and Gapd-s, a germ cell-specific gene involved in sperm glycolysis and mobility were significantly reduced. In addition, we found that dietary soy decreased the size of the seminal vesicle but without affecting its proteolytic activity. Taken together, these studies show that long-term exposure to dietary soy and phytoestrogens may affect male reproductive function resulting in a small decrease in sperm count and fertility.


Subject(s)
Diet , Fertility/physiology , Glycine max/metabolism , Phytoestrogens/metabolism , Animals , Blotting, Western , Flow Cytometry , Hormones/blood , Isoflavones/blood , Male , Mice , Microarray Analysis , Reverse Transcriptase Polymerase Chain Reaction , Sperm Count
18.
PLoS Biol ; 7(9): e1000181, 2009 Sep.
Article in English | MEDLINE | ID: mdl-19721697

ABSTRACT

In mammals, many aspects of behavior and physiology, and in particular cellular metabolism, are coordinated by the circadian timing system. Molecular clocks are thought to rely on negative feedback loops in clock gene expression that engender oscillations in the accumulation of transcriptional regulatory proteins, such as the orphan receptor REV-ERBalpha. Circadian transcription factors then drive daily rhythms in the expression of clock-controlled output genes, for example genes encoding enzymes and regulators of cellular metabolism. To gain insight into clock output functions of REV-ERBalpha, we carried out genome-wide transcriptome profiling experiments with liver RNA from wild-type mice, Rev-erbalpha knock-out mice, or REV-ERBalpha overexpressing mice. On the basis of these genetic loss- and gain-of-function experiments, we concluded that REV-ERBalpha participates in the circadian modulation of sterol regulatory element-binding protein (SREBP) activity, and thereby in the daily expression of SREBP target genes involved in cholesterol and lipid metabolism. This control is exerted via the cyclic transcription of Insig2, encoding a trans-membrane protein that sequesters SREBP proteins to the endoplasmic reticulum membranes and thereby interferes with the proteolytic activation of SREBPs in Golgi membranes. REV-ERBalpha also participates in the cyclic expression of cholesterol-7alpha-hydroxylase (CYP7A1), the rate-limiting enzyme in converting cholesterol to bile acids. Our findings suggest that this control acts via the stimulation of LXR nuclear receptors by cyclically produced oxysterols. In conclusion, our study suggests that rhythmic cholesterol and bile acid metabolism is not just driven by alternating feeding-fasting cycles, but also by REV-ERBalpha, a component of the circadian clockwork circuitry.


Subject(s)
Bile Acids and Salts/metabolism , Circadian Rhythm/physiology , Nuclear Receptor Subfamily 1, Group D, Member 1/metabolism , Signal Transduction , Sterol Regulatory Element Binding Protein 1/metabolism , Animals , Biological Clocks/genetics , Biological Clocks/physiology , Blotting, Western , Cholesterol/metabolism , Cholesterol 7-alpha-Hydroxylase/genetics , Cholesterol 7-alpha-Hydroxylase/metabolism , Circadian Rhythm/genetics , Cluster Analysis , Endoplasmic Reticulum/metabolism , Gas Chromatography-Mass Spectrometry , Gene Expression Profiling , Golgi Apparatus/metabolism , Homeostasis , Liver/metabolism , Liver X Receptors , Membrane Proteins/genetics , Membrane Proteins/metabolism , Mice , Mice, Knockout , Mice, Transgenic , Nuclear Receptor Subfamily 1, Group D, Member 1/genetics , Oligonucleotide Array Sequence Analysis , Orphan Nuclear Receptors/genetics , Orphan Nuclear Receptors/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sterol Regulatory Element Binding Protein 1/genetics , Triglycerides/metabolism
19.
Genes Dev ; 23(11): 1313-26, 2009 Jun 01.
Article in English | MEDLINE | ID: mdl-19487572

ABSTRACT

In liver, most metabolic pathways are under circadian control, and hundreds of protein-encoding genes are thus transcribed in a cyclic fashion. Here we show that rhythmic transcription extends to the locus specifying miR-122, a highly abundant, hepatocyte-specific microRNA. Genetic loss-of-function and gain-of-function experiments have identified the orphan nuclear receptor REV-ERBalpha as the major circadian regulator of mir-122 transcription. Although due to its long half-life mature miR-122 accumulates at nearly constant rates throughout the day, this miRNA is tightly associated with control mechanisms governing circadian gene expression. Thus, the knockdown of miR-122 expression via an antisense oligonucleotide (ASO) strategy resulted in the up- and down-regulation of hundreds of mRNAs, of which a disproportionately high fraction accumulates in a circadian fashion. miR-122 has previously been linked to the regulation of cholesterol and lipid metabolism. The transcripts associated with these pathways indeed show the strongest time point-specific changes upon miR-122 depletion. The identification of Pparbeta/delta and the peroxisome proliferator-activated receptor alpha (PPARalpha) coactivator Smarcd1/Baf60a as novel miR-122 targets suggests an involvement of the circadian metabolic regulators of the PPAR family in miR-122-mediated metabolic control.


Subject(s)
Circadian Rhythm/physiology , Gene Expression Regulation , Liver/metabolism , MicroRNAs/metabolism , Animals , Circadian Rhythm/genetics , DNA-Binding Proteins/metabolism , Gene Expression Profiling , Genome/genetics , Male , Mice , Mice, Inbred C57BL , Nuclear Receptor Subfamily 1, Group D, Member 1 , Oligonucleotide Array Sequence Analysis , Peroxisome Proliferator-Activated Receptors/metabolism , RNA, Messenger/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Time Factors
20.
Mol Biol Cell ; 20(7): 2083-95, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19225153

ABSTRACT

Sterols and sphingolipids are limited to eukaryotic cells, and their interaction has been proposed to favor formation of lipid microdomains. Although there is abundant biophysical evidence demonstrating their interaction in simple systems, convincing evidence is lacking to show that they function together in cells. Using lipid analysis by mass spectrometry and a genetic approach on mutants in sterol metabolism, we show that cells adjust their membrane composition in response to mutant sterol structures preferentially by changing their sphingolipid composition. Systematic combination of mutations in sterol biosynthesis with mutants in sphingolipid hydroxylation and head group turnover give a large number of synthetic and suppression phenotypes. Our unbiased approach provides compelling evidence that sterols and sphingolipids function together in cells. We were not able to correlate any cellular phenotype we measured with plasma membrane fluidity as measured using fluorescence anisotropy. This questions whether the increase in liquid order phases that can be induced by sterol-sphingolipid interactions plays an important role in cells. Our data revealing that cells have a mechanism to sense the quality of their membrane sterol composition has led us to suggest that proteins might recognize sterol-sphingolipid complexes and to hypothesize the coevolution of sterols and sphingolipids.


Subject(s)
Cell Membrane/physiology , Saccharomyces cerevisiae/cytology , Saccharomyces cerevisiae/physiology , Sphingolipids/metabolism , Sterols/metabolism , Anisotropy , Biological Transport/drug effects , Caffeine/pharmacology , Cell Membrane/drug effects , Cluster Analysis , Gene Expression Profiling , Mutation/genetics , Phenotype , Saccharomyces cerevisiae/drug effects , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/metabolism , Sirolimus/pharmacology , Sorbic Acid/pharmacology , Sphingolipids/chemistry , Sterols/biosynthesis , Sterols/chemistry
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