Identification of late larval stage developmental checkpoints in Caenorhabditis elegans regulated by insulin/IGF and steroid hormone signaling pathways.

Organisms in the wild develop with varying food availability. During periods of nutritional scarcity, development may slow or arrest until conditions improve. The ability to modulate developmental programs in response to poor nutritional conditions requires a means of sensing the changing nutritional environment and limiting tissue growth. The mechanisms by which organisms accomplish this adaptation are not well understood. We sought to study this question by examining the effects of nutrient deprivation on Caenorhabditis elegans development during the late larval stages, L3 and L4, a period of extensive tissue growth and morphogenesis. By removing animals from food at different times, we show here that specific checkpoints exist in the early L3 and early L4 stages that systemically arrest the development of diverse tissues and cellular processes. These checkpoints occur once in each larval stage after molting and prior to initiation of the subsequent molting cycle. DAF-2, the insulin/insulin-like growth factor receptor, regulates passage through the L3 and L4 checkpoints in response to nutrition. The FOXO transcription factor DAF-16, a major target of insulin-like signaling, functions cell-nonautonomously in the hypodermis (skin) to arrest developmental upon nutrient removal. The effects of DAF-16 on progression through the L3 and L4 stages are mediated by DAF-9, a cytochrome P450 ortholog involved in the production of C. elegans steroid hormones. Our results identify a novel mode of C. elegans growth in which development progresses from one checkpoint to the next. At each checkpoint, nutritional conditions determine whether animals remain arrested or continue development to the next checkpoint.

Different Mechanisms Confer Gradual Control and Memory at Nutrient- and Stress-Regulated Genes in Yeast.

Cells respond to environmental stimuli by fine-tuned regulation of gene expression. Here we investigated the dose-dependent modulation of gene expression at high temporal resolution in response to nutrient and stress signals in yeast. The GAL1 activity in cell populations is modulated in a well-defined range of galactose concentrations, correlating with a dynamic change of histone remodeling and RNA polymerase II (RNAPII) association. This behavior is the result of a heterogeneous induction delay caused by decreasing inducer concentrations across the population. Chromatin remodeling appears to be the basis for the dynamic GAL1 expression, because mutants with impaired histone dynamics show severely truncated dose-response profiles. In contrast, the GRE2 promoter operates like a rapid off/on switch in response to increasing osmotic stress, with almost constant expression rates and exclusively temporal regulation of histone remodeling and RNAPII occupancy. The Gal3 inducer and the Hog1 mitogen-activated protein (MAP) kinase seem to determine the different dose-response strategies at the two promoters. Accordingly, GAL1 becomes highly sensitive and dose independent if previously stimulated because of residual Gal3 levels, whereas GRE2 expression diminishes upon repeated stimulation due to acquired stress resistance. Our analysis reveals important differences in the way dynamic signals create dose-sensitive gene expression outputs.

Functional Variants in Notch Pathway Genes NCOR2, NCSTN, and MAML2 Predict Survival of Patients with Cutaneous Melanoma.

BACKGROUND: The Notch signaling pathway is constitutively activated in human cutaneous melanoma to promote growth and aggressive metastatic potential of primary melanoma cells. Therefore, genetic variants in Notch pathway genes may affect the prognosis of cutaneous melanoma patients. METHODS: We identified 6,256 SNPs in 48 Notch genes in 858 cutaneous melanoma patients included in a previously published cutaneous melanoma genome-wide association study dataset. Multivariate and stepwise Cox proportional hazards regression and false-positive report probability corrections were performed to evaluate associations between putative functional SNPs and cutaneous melanoma disease-specific survival. Receiver operating characteristic curve was constructed, and area under the curve was used to assess the classification performance of the model. RESULTS: Four putative functional SNPs of Notch pathway genes had independent and joint predictive roles in survival of cutaneous melanoma patients. The most significant variant was NCOR2 rs2342924 T>C (adjusted HR, 2.71; 95% confidence interval, 1.73-4.23; Ptrend = 9.62 × 10(-7)), followed by NCSTN rs1124379 G>A, NCOR2 rs10846684 G>A, and MAML2 rs7953425 G>A (Ptrend = 0.005, 0.005, and 0.013, respectively). The receiver operating characteristic analysis revealed that area under the curve was significantly increased after adding the combined unfavorable genotype score to the model containing the known clinicopathologic factors. CONCLUSIONS: Our results suggest that SNPs in Notch pathway genes may be predictors of cutaneous melanoma disease-specific survival. IMPACT: Our discovery offers a translational potential for using genetic variants in Notch pathway genes as a genotype score of biomarkers for developing an improved prognostic assessment and personalized management of cutaneous melanoma patients.

A complex intronic enhancer regulates expression of the CFTR gene by direct interaction with the promoter.

Genes can maintain spatiotemporal expression patterns by long-range interactions between cis-acting elements. The cystic fibrosis transmembrane conductance regulator gene (CFTR) is expressed primarily in epithelial cells. An element located within a DNase I-hypersensitive site (DHS) 10 kb into the first intron was previously shown to augment CFTR promoter activity in a tissue-specific manner. Here, we reveal the mechanism by which this element influences CFTR transcription. We employed a high-resolution method of mapping DHS using tiled microarrays to accurately locate the intron 1 DHS. Transfection of promoter-reporter constructs demonstrated that the element displays classical tissue-specific enhancer properties and can independently recruit factors necessary for transcription initiation. In vitro DNase I footprinting analysis identified a protected region that corresponds to a conserved, predicted binding site for hepatocyte nuclear factor 1 (HNF1). We demonstrate by electromobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP) that HNF1 binds to this element both in vitro and in vivo. Moreover, using chromosome conformation capture (3C) analysis, we show that this element interacts with the CFTR promoter in CFTR-expressing cells. These data provide the first insight into the three- dimensional (3D) structure of the CFTR locus and confirm the contribution of intronic cis-acting elements to the regulation of CFTR gene expression.

Inactivation of the von Hippel-Lindau tumor suppressor leads to selective expression of a human endogenous retrovirus in kidney cancer.

A human endogenous retrovirus type E (HERV-E) was recently found to be selectively expressed in most renal cell carcinomas (RCCs). Importantly, antigens derived from this provirus are immunogenic, stimulating cytotoxic T cells that kill RCC cells in vitro and in vivo. Here, we show HERV-E expression is restricted to the clear cell subtype of RCC (ccRCC) characterized by an inactivation of the von Hippel-Lindau (VHL) tumor-suppressor gene with subsequent stabilization of hypoxia-inducible transcription factors (HIFs)-1α and -2α. HERV-E expression in ccRCC linearly correlated with HIF-2α levels and could be silenced in tumor cells by either transfection of normal VHL or small interfering RNA inhibition of HIF-2α. Using chromatin immunoprecipitation, we demonstrated that HIF-2α can serve as transcriptional factor for HERV-E by binding with HIF response element (HRE) localized in the proviral 5' long terminal repeat (LTR). Remarkably, the LTR was found to be hypomethylated only in HERV-E-expressing ccRCC while other tumors and normal tissues possessed a hypermethylated LTR preventing proviral expression. Taken altogether, these findings provide the first evidence that inactivation of a tumor suppressor gene can result in aberrant proviral expression in a human tumor and give insights needed for translational research aimed at boosting human immunity against antigenic components of this HERV-E.

Examination of Endogenous Rotund Expression and Function in Developing Drosophila Olfactory System Using CRISPR-Cas9-Mediated Protein Tagging.

The zinc-finger protein Rotund (Rn) plays a critical role in controlling the development of the fly olfactory system. However, little is known about its molecular function in vivo. Here, we added protein tags to the rn locus using CRISPR-Cas9 technology in Drosophila to investigate its subcellular localization and the genes that it regulates . We previously used a reporter construct to show that rn is expressed in a subset of olfactory receptor neuron (ORN) precursors and it is required for the diversification of ORN fates. Here, we show that tagged endogenous Rn protein is functional based on the analysis of ORN phenotypes. Using this method, we also mapped the expression pattern of the endogenous isoform-specific tags in vivo with increased precision. Comparison of the Rn expression pattern from this study with previously published results using GAL4 reporters showed that Rn is mainly present in early steps in antennal disc patterning, but not in pupal stages when ORNs are born. Finally, using chromatin immunoprecipitation, we showed a direct binding of Rotund to a previously identified regulatory element upstream of the bric-a-brac gene locus in the developing antennal disc.

Computational Inference of Genome-Wide Protein-DNA Interactions Using High-Throughput Genomic Data

Transcriptional regulation has been studied intensively in recent decades. One important aspect of this regulation is the interaction between regulatory proteins, such as transcription factors (TF) and nucleosomes, and the genome. Different high-throughput techniques have been invented to map these interactions genome-wide, including ChIP-based methods (ChIP-chip, ChIP-seq, etc.), nuclease digestion methods (DNase-seq, MNase-seq, etc.), and others. However, a single experimental technique often only provides partial and noisy information about the whole picture of protein-DNA interactions. Therefore, the overarching goal of this dissertation is to provide computational developments for jointly modeling different experimental datasets to achieve a holistic inference on the protein-DNA interaction landscape.

We first present a computational framework that can incorporate the protein binding information in MNase-seq data into a thermodynamic model of protein-DNA interaction. We use a correlation-based objective function to model the MNase-seq data and a Markov chain Monte Carlo method to maximize the function. Our results show that the inferred protein-DNA interaction landscape is concordant with the MNase-seq data and provides a mechanistic explanation for the experimentally collected MNase-seq fragments. Our framework is flexible and can easily incorporate other data sources. To demonstrate this flexibility, we use prior distributions to integrate experimentally measured protein concentrations.

We also study the ability of DNase-seq data to position nucleosomes. Traditionally, DNase-seq has only been widely used to identify DNase hypersensitive sites, which tend to be open chromatin regulatory regions devoid of nucleosomes. We reveal for the first time that DNase-seq datasets also contain substantial information about nucleosome translational positioning, and that existing DNase-seq data can be used to infer nucleosome positions with high accuracy. We develop a Bayes-factor-based nucleosome scoring method to position nucleosomes using DNase-seq data. Our approach utilizes several effective strategies to extract nucleosome positioning signals from the noisy DNase-seq data, including jointly modeling data points across the nucleosome body and explicitly modeling the quadratic and oscillatory DNase I digestion pattern on nucleosomes. We show that our DNase-seq-based nucleosome map is highly consistent with previous high-resolution maps. We also show that the oscillatory DNase I digestion pattern is useful in revealing the nucleosome rotational context around TF binding sites.

Finally, we present a state-space model (SSM) for jointly modeling different kinds of genomic data to provide an accurate view of the protein-DNA interaction landscape. We also provide an efficient expectation-maximization algorithm to learn model parameters from data. We first show in simulation studies that the SSM can effectively recover underlying true protein binding configurations. We then apply the SSM to model real genomic data (both DNase-seq and MNase-seq data). Through incrementally increasing the types of genomic data in the SSM, we show that different data types can contribute complementary information for the inference of protein binding landscape and that the most accurate inference comes from modeling all available datasets.

This dissertation provides a foundation for future research by taking a step toward the genome-wide inference of protein-DNA interaction landscape through data integration.

Wnt Protein Signaling Reduces Nuclear Acetyl-CoA Levels to Suppress Gene Expression during Osteoblast Differentiation.

Developmental signals in metazoans play critical roles in inducing cell differentiation from multipotent progenitors. The existing paradigm posits that the signals operate directly through their downstream transcription factors to activate expression of cell type-specific genes, which are the hallmark of cell identity. We have investigated the mechanism through which Wnt signaling induces osteoblast differentiation in an osteoblast-adipocyte bipotent progenitor cell line. Unexpectedly, Wnt3a acutely suppresses the expression of a large number of genes while inducing osteoblast differentiation. The suppressed genes include Pparg and Cebpa, which encode adipocyte-specifying transcription factors and suppression of which is sufficient to induce osteoblast differentiation. The large scale gene suppression induced by Wnt3a corresponds to a global decrease in histone acetylation, an epigenetic modification that is associated with gene activation. Mechanistically, Wnt3a does not alter histone acetyltransferase or deacetylase activities but, rather, decreases the level of acetyl-CoA in the nucleus. The Wnt-induced decrease in histone acetylation is independent of β-catenin signaling but, rather, correlates with suppression of glucose metabolism in the tricarboxylic acid cycle. Functionally, preventing histone deacetylation by increasing nucleocytoplasmic acetyl-CoA levels impairs Wnt3a-induced osteoblast differentiation. Thus, Wnt signaling induces osteoblast differentiation in part through histone deacetylation and epigenetic suppression of an alternative cell fate.

ERM transactivation is up-regulated by the repression of DNA binding after the PKA phosphorylation of a consensus site at the edge of the ETS domain

The final step of the transduction pathway is the activation of gene transcription, which is driven by kinase cascades leading to changes in the activity of many transcription factors. Among these latter, PEA3/E1AF, ER81/ETV1, and ERM, members of the well conserved PEA3 group from the Ets family are involved in these processes. We show here that protein kinase A (PKA) increases the transcriptional activity of human ERM and human ETV1, through a Ser residue situated at the edge of the ETS DNA-binding domain. PKA phosphorylation does not directly affect the ERM transactivation domains but does affect DNA binding activity. Unphosphorylated wild-type ERM bound DNA avidly, whereas after PKA phosphorylation it did so very weakly. Interestingly, S367A mutation significantly reduced the ERM-mediated transcription in the presence of the kinase, and the DNA binding of this mutant, although similar to that of unphosphorylated wild-type protein, was insensitive to PKA treatment. Mutations, which may mimic a phosphorylated serine, converted ERM from an efficient DNA-binding protein to a poor DNA binding one, with inefficiency of PKA phosphorylation. The present data clearly demonstrate a close correlation between the capacity of PKA to increase the transactivation of ERM and the drastic down-regulation of the binding of the ETS domain to the targeted DNA. What we thus demonstrate here is a relatively rare transcription activation mechanism through a decrease in DNA binding, probably by the shift of a non-active form of an Ets protein to a PKA-phosphorylated active one, which should be in a conformation permitting a transactivation domain to be active.

Human E2F6 is alternatively spliced to generate multiple protein isoforms

E2F6 protein belongs to the family of the E2F transcription factors. Here, we showed that the human E2F6 gene contains nine exons distributed along 20.4kbp of genomic DNA on chromosome 2 leading to the transcription of six alternatively spliced E2F6 mRNAs that encode four different E2F6 proteins. Moreover, we identified an E2F6 pseudogene localized on chromosome 22 completely spliced and devoid of exons 2, 3, and 4, and part of exons 1 and 5. Definition of the transcriptional initiation site and sequence analysis show that the gene contains a TATA less, CAAT less, GC-rich promoter with multiple transcription start sites. Regulatory elements necessary for basal transcription reside within a 134bp fragment as determined by transient transfection experiments. © 2004 Elsevier Inc. All rights reserved.

Solution structure of the N-terminal transactivation domain of ERM modified by SUMO-1.

ERM is a member of the PEA3 group of the Ets transcription factor family that plays important roles in development and tumorigenesis. The PEA3s share an N-terminal transactivation domain (TADn) whose activity is inhibited by small ubiquitin-like modifier (SUMO). However, the consequences of sumoylation and its underlying molecular mechanism remain unclear. The domain structure of ERM TADn alone or modified by SUMO-1 was analyzed using small-angle X-ray scattering (SAXS). Low resolution shapes determined ab initio from the scattering data indicated an elongated shape and an unstructured conformation of TADn in solution. Covalent attachment of SUMO-1 does not perturb the structure of TADn as indicated by the linear arrangement of the SUMO moiety with respect to TADn. Thus, ERM belongs to the growing family of proteins that contain intrinsically unstructured regions. The flexible nature of TADn may be instrumental for ERM recognition and binding to diverse molecular partners.

In vitro expansion of CD4+CD25highFOXP3+CD127low/- regulatory T cells from peripheral blood lymphocytes of healthy Mycobacterium tuberculosis-infected humans.

CD4+CD25highFOXP3+ regulatory T (Treg) cells have recently been found at elevated levels in the peripheral blood of tuberculosis patients, compared to Mycobacterium tuberculosis latently infected (LTBI) healthy individuals and non-infected controls. Here, we show that CD4+CD25highFOXP3+ T lymphocytes can be expanded in vitro from peripheral blood mononuclear cells (PBMC) of LTBI individuals, but not of uninfected controls by incubating them with BCG in the presence of TGF-beta. These expanded cells from the PBMC of LTBI subjects expressed CTLA-4, GITR and OX-40, but were CD127low/- and have therefore the phenotype of Treg cells. In addition, they inhibited in a dose-dependant manner the proliferation of freshly isolated mononuclear cells in response to polyclonal stimulation, indicating that they are functional Treg lymphocytes. In contrast, incubation of the PBMC with BCG alone preferentially induced activated CD4+ T cells, expressing CD25 and/or CD69 and secreting IFN-gamma. These results show that CD4+CD25highFOXP3+ Treg cells can be expanded or induced in the peripheral blood of LTBI individuals in conditions known to predispose to progression towards active tuberculosis and may therefore play an important role in the pathogenesis of the disease.

Regulatory T cells depress immune responses to protective antigens in active tuberculosis.

RATIONALE: Tuberculosis (TB) remains a leading cause of death, and the role of T-cell responses to control Mycobacterium tuberculosis infections is well recognized. Patients with latent TB infection develop strong IFN-gamma responses to the protective antigen heparin-binding hemagglutinin (HBHA), whereas patients with active TB do not. OBJECTIVES: We investigated the mechanism of this difference and evaluated the possible involvement of regulatory T (Treg) cells and/or cytokines in the low HBHA T-cell responses of patients with active TB. METHODS: The impact of anti-transforming growth factor (TGF)-beta and anti-IL-10 antibodies and of Treg cell depletion on the HBHA-induced IFN-gamma secretion was analyzed, and the Treg cell phenotype was characterized by flow cytometry. MEASUREMENTS AND MAIN RESULTS: Although the addition of anti-TGF-beta or anti-IL-10 antibodies had no effect on the HBHA-induced IFN-gamma secretion in patients with active TB, depletion of CD4(+)CD25(high)FOXP3(+) T lymphocytes resulted in the induction by HBHA of IFN-gamma concentrations that reached levels similar to those obtained for latent TB infection. No effect was noted on the early-secreted antigen target-6 or candidin T-cell responses. CONCLUSIONS: Specific CD4(+)CD25(high)FOXP3(+) T cells depress the T-cell-mediated immune responses to the protective mycobacterial antigen HBHA during active TB in humans.

Bordetella pertussis from functional genomics to intranasal vaccination.

Whooping cough still represents a major health problem, despite the use of effective vaccines for several decades. Being classically a typical childhood disease, whooping cough in young adults is now more common than it used to be, suggesting that protection after vaccination wanes during adolescence. As an alternative to the current vaccines, we wish to develop live attenuated vaccines to be delivered by the nasal route, such as to mimic the natural route of infection and to induce long lasting immunity. Bordetella pertussis, the etiological agent of whooping cough, produces a number of virulence factors, including toxins. Its recently determined genome sequence makes it now possible to apply functional genomics, such as transcriptomics and systematic knock-out mutagenesis. The expression of most known B. pertussis virulence genes is controlled by the two-component system BvgA/S. DNA microarray analyses have led to the identification of novel genes in the BvgA/S regulon, some of which are activated by BvgA/S and others are repressed by BvgA/S. In addition, some genes appear to be differentially modulated by nicotinic acid and MgSO4, both known to modulate the expression of BvgA/S-regulated genes. Among others, the functional genomics approach has uncovered two strongly BvgA/S-activated genes, named hotA and hotB (for 'homolog of toxin'), the products of which show high sequence similarities to pertussis toxin subunits. The identification of the full array of virulence factors, as well as an integrated understanding of the bacterial physiology should allow us to design attenuated B. pertussis strains useful for intranasal vaccination. A first generation of attenuated strains has already shown full protection in mice after a single intranasal administration. Such strains may also serve as vaccine carriers for heterologous antigens, in order to vaccinate against several different pathogens simultaneously.

SLAT regulates Th1 and Th2 inflammatory responses by controlling Ca2+/NFAT signaling

SWAP-70-like adapter of T cells (SLAT) is a novel guanine nucleotide exchange factor for Rho GTPases that is upregulated in Th2 cells, but whose physiological function is unclear. We show that SLAT-/- mice displayed a developmental defect at one of the earliest stages of thymocyte differentiation, the double-negative 1 (DN1) stage, leading to decreased peripheral T cell numbers. SLAT-/- peripheral CD4+ T cells demonstrated impaired TCR/CD28-induced proliferation and IL-2 production, which was rescued by the addition of exogenous IL-2. Importantly, SLAT-/- mice were grossly impaired in their ability to mount not only Th2, but also Th1-mediated lung inflammatory responses, as evidenced by reduced airway neutrophilia and eosinophilia, respectively. Levels of Th1 and Th2 cytokine in the lungs were also markedly reduced, paralleling the reduction in pulmonary inflammation. This defect in mounting Th1/Th2 responses, which was also evident in vitro, was traced to a severe reduction in Ca2+ mobilization from ER stores, which consequently led to defective TCR/CD28-induced translocation of nuclear factor of activated T cells 1/2 (NFATc1/2). Thus, SLAT is required for thymic DN1 cell expansion, T cell activation, and Th1 and Th2 inflammatory responses.