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1.  Dynamin 2 homozygous mutation in humans with a lethal congenital syndrome 
Heterozygous mutations in dynamin 2 (DNM2) have been linked to dominant Charcot-Marie-Tooth neuropathy and centronuclear myopathy. We report the first homozygous mutation in the DNM2 protein p.Phe379Val, in three consanguineous patients with a lethal congenital syndrome associating akinesia, joint contractures, hypotonia, skeletal abnormalities, and brain and retinal hemorrhages. In vitro membrane tubulation, trafficking and GTPase assays are consistent with an impact of the DNM2p.Phe379Val mutation on endocytosis. Although DNM2 has been previously implicated in axonal and muscle maintenance, the clinical manifestation in our patients taken together with our expression analysis profile during mouse embryogenesis and knockdown approaches in zebrafish resulting in defects in muscle organization and angiogenesis support a pleiotropic role for DNM2 during fetal development in vertebrates and humans.
PMCID: PMC3658203  PMID: 23092955
DNM2; dynamin; centronuclear myopathy; Charcot-Marie-Tooth; fetal akinesia deformation sequence; endocytosis
2.  Transcriptome analysis for Notch3 target genes identifies Grip2 as a novel regulator of myogenic response in the cerebrovasculature 
Notch3 is critically important for the structure and myogenic response of distal arteries, particularly of cerebral arteries. However, signaling pathways acting downstream of Notch3 remain largely unknown.
Methods and results
Transcriptome analysis using tail arteries of Notch3-null mice identified a core set of 17 novel Notch3-regulated genes confirmed in tail or brain arteries. Post-natal deletion of RBP-Jκ in smooth muscle cells recapitulated the structural, functional and molecular defects of brain arteries induced by Notch3 deficiency. Transient in vivo blockade of the Notch pathway with γ-secretase inhibitors uncovered, in addition to Notch3, six immediate responders including the voltage-gated potassium channel Kv1.5, which opposes to myogenic constriction of brain arteries, and the glutamate receptor interacting protein 2 (GRIP-2), with no previously established role in the cerebrovasculature. We identified a vascular smooth muscle cell isoform of Grip2. We showed that Notch3-RBP-Jκ specifically regulates this isoform. Finally, we found that cerebral arteries of Grip2 mutant mice, which express a N-terminally truncated GRIP-2 protein, exhibited selective attenuation of pressure-induced contraction.
Our data provide insight into how Notch3 signals in the brain arteries, establishing the postnatal requirement of smooth muscle RBP-Jκ in this context. Notch3-regulated transcriptome provides potential for modulating myogenic response in the cerebrovasculature.
PMCID: PMC3524357  PMID: 23117660
Notch3; cerebrovasculature; myogenic response; smooth muscle cell; transcriptome
3.  Profiling target genes of FGF18 in the postnatal mouse lung: possible relevance for alveolar development 
Physiological Genomics  2011;43(21):1226-1240.
Better understanding alveolarization mechanisms could help improve prevention and treatment of diseases characterized by reduced alveolar number. Although signaling through fibroblast growth factor (FGF) receptors is essential for alveolarization, involved ligands are unidentified. FGF18, the expression of which peaks coincidentally with alveolar septation, is likely to be involved. Herein, a mouse model with inducible, lung-targeted FGF18-transgene was used to advance the onset of FGF18 expression peak, and genome-wide expression changes were determined by comparison with littermate controls. Quantitative RT-PCR was used to confirm expression changes of selected up and down regulated genes, and to determine their expression profiles in the course of lung postnatal development. This allowed identifying so far unknown target genes of the factor, among which a number are known to be involved in alveolarization. The major target was adrenomedullin, a promoter of lung angiogenesis and alveolar development, whose transcript was increased 6.9-fold. Other genes involved in angiogenesis presented marked expression increases, including Wnt2 and cullin2. Although it appeared to favor cell migration notably through enhanced expression of Snai1/2, FGF18 also induced various changes consistent with prevention of epithelial-mesenchymal transition. Together with anti-fibrotic effects driven by induction of E prostanoid receptor 2 and repression of numerous myofibroblast markers, this could prevent alveolar septation-driving mechanisms from becoming excessive and deleterious. Last, FGF18 up or down regulated genes of ECM components and epithelial-cell markers previously shown to be up or down regulated during alveolarization. These findings therefore argue for an involvement of FGF18 in the control of various developmental events during the alveolar stage.
PMCID: PMC3622615  PMID: 21878612
Animals; Animals, Newborn; Down-Regulation; drug effects; genetics; Doxycycline; pharmacology; Fibroblast Growth Factors; metabolism; Gene Expression Profiling; Gene Expression Regulation, Developmental; drug effects; Mice; Oligonucleotide Array Sequence Analysis; Pulmonary Alveoli; cytology; drug effects; growth & development; metabolism; Reverse Transcriptase Polymerase Chain Reaction; Transgenes; genetics; Up-Regulation; drug effects; genetics; transgenic mice; alveolarization; angiogenesis; fibrosis; epithelial-mesenchymal transition.
4.  SOX2 Is an Oncogene Activated by Recurrent 3q26.3 Amplifications in Human Lung Squamous Cell Carcinomas 
PLoS ONE  2010;5(1):e8960.
Squamous cell carcinoma (SCC) of the lung is a frequent and aggressive cancer type. Gene amplifications, a known activating mechanism of oncogenes, target the 3q26-qter region as one of the most frequently gained/amplified genomic sites in SCC of various types. Here, we used array comparative genomic hybridization to delineate the consensus region of 3q26.3 amplifications in lung SCC. Recurrent amplifications occur in 20% of lung SCC (136 tumors in total) and map to a core region of 2 Mb (Megabases) that encompasses SOX2, a transcription factor gene. Intense SOX2 immunostaining is frequent in nuclei of lung SCC, indicating potential active transcriptional regulation by SOX2. Analyses of the transcriptome of lung SCC, SOX2-overexpressing lung epithelial cells and embryonic stem cells (ESCs) reveal that SOX2 contributes to activate ESC-like phenotypes and provide clues pertaining to the deregulated genes involved in the malignant phenotype. In cell culture experiments, overexpression of SOX2 stimulates cellular migration and anchorage-independent growth while SOX2 knockdown impairs cell growth. Finally, SOX2 over-expression in non-tumorigenic human lung bronchial epithelial cells is tumorigenic in immunocompromised mice. These results indicate that the SOX2 transcription factor, a major regulator of stem cell function, is also an oncogene and a driver gene for the recurrent 3q26.33 amplifications in lung SCC.
PMCID: PMC2813300  PMID: 20126410
5.  Transcriptome analysis identifies genes with enriched expression in the mouse central Extended Amygdala 
Neuroscience  2008;156(4):950-965.
The central Extended Amygdala (EAc) is an ensemble of highly interconnected limbic structures of the anterior brain, and forms a cellular continuum including the Bed Nucleus of the Stria Terminalis (BNST), the central nucleus of the Amygdala (CeA) and the Nucleus Accumbens shell (AcbSh). This neural network is a key site for interactions between brain reward and stress systems, and has been implicated in several aspects of drug abuse. In order to increase our understanding of EAc function at the molecular level, we undertook a genome-wide screen (Affymetrix) to identify genes whose expression is enriched in the EAc. We focused on the less-well known BNST-CeA areas of the EAc, and identified 121 genes that exhibit more than 2-fold higher expression level in the EAc compared to whole brain. Among these, forty-three genes have never been described to be expressed in the EAc. We mapped these genes throughout the brain, using non-radioactive in situ hybridization, and identified eight genes with a unique and distinct rostro-caudal expression pattern along AcbSh, BNST and CeA. Q-PCR analysis performed in brain and peripheral organ tissues indicated that, with the exception of one (Spata13), all these genes are predominantly expressed in brain. These genes encode signaling proteins (Adora2, GPR88, Arpp21 and Rem2), a transcription factor (Limh6) or proteins of unknown function (Rik130, Spata13 and Wfs1). The identification of genes with enriched expression expands our knowledge of EAc at a molecular level, and provides useful information to towards genetic manipulations within the EAc.
PMCID: PMC2629946  PMID: 18786617
adult mouse; Affymetrix microarray; in-situ hybridization; central Extended Amygdala; gene; marker
6.  Transcriptomic Analysis of the Sulfate Starvation Response of Pseudomonas aeruginosa▿ †  
Journal of Bacteriology  2007;189(19):6743-6750.
Pseudomonas aeruginosa is an opportunistic pathogen that causes a number of infections in humans, but is best known for its association with cystic fibrosis. It is able to use a wide range of sulfur compounds as sources of sulfur for growth. Gene expression in response to changes in sulfur supply was studied in P. aeruginosa E601, a cystic fibrosis isolate that displays mucin sulfatase activity, and in P. aeruginosa PAO1. A large family of genes was found to be upregulated by sulfate limitation in both isolates, encoding sulfatases and sulfonatases, transport systems, oxidative stress proteins, and a sulfate-regulated TonB/ExbBD complex. These genes were localized in five distinct islands on the genome and encoded proteins with a significantly reduced content of cysteine and methionine. Growth of P. aeruginosa E601 with mucin as the sulfur source led not only to a sulfate starvation response but also to induction of genes involved with type III secretion systems.
PMCID: PMC2045191  PMID: 17675390
7.  Novel insights into the relationships between dendritic cell subsets in human and mouse revealed by genome-wide expression profiling 
Genome Biology  2008;9(1):R17.
Genome-wide expression profiling of mouse and human leukocytes reveal conserved transcriptional programs of plasmacytoid or conventional dendritic cell subsets.
Dendritic cells (DCs) are a complex group of cells that play a critical role in vertebrate immunity. Lymph-node resident DCs (LN-DCs) are subdivided into conventional DC (cDC) subsets (CD11b and CD8α in mouse; BDCA1 and BDCA3 in human) and plasmacytoid DCs (pDCs). It is currently unclear if these various DC populations belong to a unique hematopoietic lineage and if the subsets identified in the mouse and human systems are evolutionary homologs. To gain novel insights into these questions, we sought conserved genetic signatures for LN-DCs and in vitro derived granulocyte-macrophage colony stimulating factor (GM-CSF) DCs through the analysis of a compendium of genome-wide expression profiles of mouse or human leukocytes.
We show through clustering analysis that all LN-DC subsets form a distinct branch within the leukocyte family tree, and reveal a transcriptomal signature evolutionarily conserved in all LN-DC subsets. Moreover, we identify a large gene expression program shared between mouse and human pDCs, and smaller conserved profiles shared between mouse and human LN-cDC subsets. Importantly, most of these genes have not been previously associated with DC function and many have unknown functions. Finally, we use compendium analysis to re-evaluate the classification of interferon-producing killer DCs, lin-CD16+HLA-DR+ cells and in vitro derived GM-CSF DCs, and show that these cells are more closely linked to natural killer and myeloid cells, respectively.
Our study provides a unique database resource for future investigation of the evolutionarily conserved molecular pathways governing the ontogeny and functions of leukocyte subsets, especially DCs.
PMCID: PMC2395256  PMID: 18218067
8.  Notch Activation Is an Early and Critical Event during T-Cell Leukemogenesis in Ikaros-Deficient Mice 
Molecular and Cellular Biology  2006;26(1):209-220.
The Ikaros transcription factor is both a key regulator of lymphocyte differentiation and a tumor suppressor in T lymphocytes. Mice carrying a hypomorphic mutation (IkL/L) in the Ikaros gene all develop thymic lymphomas. IkL/L tumors always exhibit strong activation of the Notch pathway, which is required for tumor cell proliferation in vitro. Notch activation occurs early in tumorigenesis and may precede transformation, as ectopic expression of the Notch targets Hes-1 and Deltex-1 is detected in thymocytes from young IkL/L mice with no overt signs of transformation. Notch activation is further amplified by secondary mutations that lead to C-terminal truncations of Notch 1. Strikingly, restoration of Ikaros activity in tumor cells leads to a rapid and specific downregulation of Notch target gene expression and proliferation arrest. Furthermore, Ikaros binds to the Notch-responsive element in the Hes-1 promoter and represses Notch-dependent transcription from this promoter. Thus, Ikaros-mediated repression of Notch target gene expression may play a critical role in defining the tumor suppressor function of this factor.
PMCID: PMC1317628  PMID: 16354692
9.  Distinct Mutations in Yeast TAFII25 Differentially Affect the Composition of TFIID and SAGA Complexes as Well as Global Gene Expression Patterns 
Molecular and Cellular Biology  2002;22(9):3178-3193.
The RNA polymerase II transcription factor TFIID, composed of the TATA-binding protein (TBP) and TBP-associated factors (TAFIIs), nucleates preinitiation complex formation at protein-coding gene promoters. SAGA, a second TAFII-containing multiprotein complex, is involved in transcription regulation in Saccharomyces cerevisiae. One of the essential protein components common to SAGA and TFIID is yTAFII25. We define a minimal evolutionarily conserved 91-amino-acid region of TAFII25 containing a histone fold domain that is necessary and sufficient for growth in vivo. Different temperature-sensitive mutations of yTAFII25 or chimeras with the human homologue TAFII30 arrested cell growth at either the G1 or G2/M cell cycle phase and displayed distinct phenotypic changes and gene expression patterns. Immunoprecipitation studies revealed that TAFII25 mutation-dependent gene expression and phenotypic changes correlated at least partially with the integrity of SAGA and TFIID. Genome-wide expression analysis revealed that the five TAFII25 temperature-sensitive mutant alleles individually affect the expression of between 18 and 33% of genes, whereas taken together they affect 64% of all class II genes. Thus, different yTAFII25 mutations induce distinct phenotypes and affect the regulation of different subsets of genes, demonstrating that no individual TAFII mutant allele reflects the full range of its normal functions.
PMCID: PMC133751  PMID: 11940675

Results 1-9 (9)