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Adult autologous human epidermal stem cells can be extensively expanded ex vivo for cell and gene therapy. Identifying the mechanisms involved in stem cell maintenance and defining culture conditions to maintain stemness is critical, because an inadequate environment can result in the rapid conversion of stem cells into progenitors/transient amplifying cells (clonal conversion), with deleterious consequences on the quality of the transplants and their ability to engraft. Here, we demonstrate that cultured human epidermal stem cells respond to a small drop in temperature through thermoTRP channels via mTOR signaling. Exposure of cells to rapamycin or a small drop in temperature induces the nuclear translocation of mTOR with an impact on gene expression. We also demonstrate by single-cell analysis that long-term inhibition of mTORC1 reduces clonal conversion and favors the maintenance of stemness. Taken together, our results demonstrate that human keratinocyte stem cells can adapt to environmental changes (e.g., small variations in temperature) through mTOR signaling and constant inhibition of mTORC1 favors stem cell maintenance, a finding of high importance for regenerative medicine applications.
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Queratinocitos , Serina-Treonina Quinasas TOR , Adulto , Humanos , Temperatura , Queratinocitos/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Células Madre/metabolismo , Diana Mecanicista del Complejo 1 de la RapamicinaRESUMEN
Upon replication stress, budding yeast checkpoint kinase Mec1ATR triggers the downregulation of transcription, thereby reducing the level of RNA polymerase (RNAP) on chromatin to facilitate replication fork progression. Here, we identify a hydroxyurea-induced phosphorylation site on Mec1, Mec1-S1991, that contributes to the eviction of RNAPII and RNAPIII during replication stress. The expression of the non-phosphorylatable mec1-S1991A mutant reduces replication fork progression genome-wide and compromises survival on hydroxyurea. This defect can be suppressed by destabilizing chromatin-bound RNAPII through a TAP fusion to its Rpb3 subunit, suggesting that lethality in mec1-S1991A mutants arises from replication-transcription conflicts. Coincident with a failure to repress gene expression on hydroxyurea in mec1-S1991A cells, highly transcribed genes such as GAL1 remain bound at nuclear pores. Consistently, we find that nuclear pore proteins and factors controlling RNAPII and RNAPIII are phosphorylated in a Mec1-dependent manner on hydroxyurea. Moreover, we show that Mec1 kinase also contributes to reduced RNAPII occupancy on chromatin during an unperturbed S phase by promoting degradation of the Rpb1 subunit.
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Replicación del ADN , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Polimerasa III/genética , ARN Polimerasa II/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Cromatina/química , Cromatina/efectos de los fármacos , Cromatina/metabolismo , Galactoquinasa/genética , Galactoquinasa/metabolismo , Regulación Fúngica de la Expresión Génica , Hidroxiurea/farmacología , Péptidos y Proteínas de Señalización Intracelular/genética , Fosfoproteínas , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , ARN Polimerasa II/metabolismo , ARN Polimerasa III/metabolismo , Fase S/efectos de los fármacos , Fase S/genética , Saccharomyces cerevisiae/genética , Estrés Fisiológico/efectos de los fármacos , Estrés Fisiológico/genética , Transcripción GenéticaRESUMEN
Each day, approximately 27,000 people become ill with tuberculosis (TB), and 4,000 die from this disease. Pulmonary TB is the main clinical form of TB, and affects the lungs with a considerably heterogeneous manifestation among patients. Immunomodulation by an interplay of host-, environment-, and pathogen-associated factors partially explains such heterogeneity. Microbial communities residing in the host's airways have immunomodulatory effects, but it is unclear if the inter-individual variability of these microbial communities is associated with the heterogeneity of pulmonary TB. Here, we investigated this possibility by characterizing the microbial composition in the sputum of 334 TB patients from Tanzania, and by assessing its association with three aspects of disease manifestations: sputum mycobacterial load, severe clinical findings, and chest x-ray (CXR) findings. Compositional data analysis of taxonomic profiles based on 16S-rRNA gene amplicon sequencing and on whole metagenome shotgun sequencing, and graph-based inference of microbial associations revealed that the airway microbiome of TB patients was shaped by inverse relationships between Streptococcus and two anaerobes: Selenomonas and Fusobacterium. Specifically, the strength of these microbial associations was negatively correlated with Faith's phylogenetic diversity (PD) and with the accumulation of transient genera. Furthermore, low body mass index (BMI) determined the association between abnormal CXRs and community diversity and composition. These associations were mediated by increased abundance of Selenomonas and Fusobacterium, relative to the abundance of Streptococcus, in underweight patients with lung parenchymal infiltrates and in comparison to those with normal chest x-rays. And last, the detection of herpesviruses and anelloviruses in sputum microbial assemblage was linked to co-infection with HIV. Given the anaerobic metabolism of Selenomonas and Fusobacterium, and the hypoxic environment of lung infiltrates, our results suggest that in underweight TB patients, lung tissue remodeling toward anaerobic conditions favors the growth of Selenomonas and Fusobacterium at the expense of Streptococcus. These new insights into the interplay among particular members of the airway microbiome, BMI, and lung parenchymal lesions in TB patients, add a new dimension to the long-known association between low BMI and pulmonary TB. Our results also drive attention to the airways virome in the context of HIV-TB coinfection.
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Eukaryotic cells package their genomes around histone octamers. In response to DNA damage, checkpoint activation in yeast induces core histone degradation resulting in 20%-40% reduction in nucleosome occupancy. To gain insight into this process, we developed a new approach to analyze the chromatin-associated proteome comprehensively before and after damage. This revealed extensive changes in protein composition after Zeocin-induced damage. First, core histones and the H1 homolog Hho1 were partially lost from chromatin along with replication, transcription, and chromatin remodeling machineries, while ubiquitin ligases and the proteasome were recruited. We found that the checkpoint- and INO80C-dependent recruitment of five ubiquitin-conjugating factors (Rad6, Bre1, Pep5, Ufd4, and Rsp5) contributes to core and linker histone depletion, reducing chromatin compaction and enhancing DNA locus mobility. Importantly, loss of Rad6/Bre1, Ufd4/TRIP12, and Pep5/VPS11 compromise DNA strand invasion kinetics during homology-driven repair. Thus we provide a comprehensive overview of a functionally relevant genome-wide chromatin response to DNA damage.
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Ensamble y Desensamble de Cromatina , Reparación del ADN , ADN de Hongos/metabolismo , Histonas/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , ADN de Hongos/genética , Histonas/genética , Complejo de la Endopetidasa Proteasomal/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
In fission yeast and plants, RNA processing and degradation contribute to heterochromatin silencing, alongside conserved pathways of transcriptional repression. It has not been known whether similar pathways exist in metazoans. Here, we describe a pathway of silencing in Caenorhabditis elegans somatic cells, in which the highly conserved RNA-binding complex LSM2-8 contributes selectively to the repression of heterochromatic reporters and endogenous genes bearing the Polycomb mark, histone H3K27me3. This acts by degrading selected transcripts through the XRN-2 exoribonuclease. Disruption of the LSM2-8 pathway leads to mRNA stabilization. Unlike previously described pathways of heterochromatic RNA degradation, LSM2-8-mediated RNA degradation does not target nor require H3K9 methylation. Intriguingly, loss of this pathway coincides with a localized reduction in H3K27me3 at lsm-8-sensitive loci. We have thus uncovered a mechanism of RNA degradation that selectively contributes to the silencing of a subset of H3K27me3-marked genes, revealing a previously unrecognized layer of post-transcriptional control in metazoan heterochromatin.
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Proteínas de Caenorhabditis elegans/genética , Exorribonucleasas/genética , Histonas/genética , Estabilidad del ARN/genética , Ribonucleoproteínas Nucleares Pequeñas/genética , Animales , Caenorhabditis elegans/genética , Silenciador del Gen/fisiología , Heterocromatina/genética , Metilación , Proteínas del Grupo Polycomb/genética , Interferencia de ARN/fisiología , ARN Mensajero/genética , ARN Interferente Pequeño/genéticaRESUMEN
Mutations in the nuclear structural protein lamin A produce rare, tissue-specific diseases called laminopathies. The introduction of a human Emery-Dreifuss muscular dystrophy (EDMD)-inducing mutation into the C. elegans lamin (LMN-Y59C), recapitulates many muscular dystrophy phenotypes, and correlates with hyper-sequestration of a heterochromatic array at the nuclear periphery in muscle cells. Using muscle-specific emerin Dam-ID in worms, we monitored the effects of the mutation on endogenous chromatin. An increased contact with the nuclear periphery along chromosome arms, and an enhanced release of chromosomal centers, coincided with the disease phenotypes of reduced locomotion and compromised sarcomere integrity. The coupling of the LMN-Y59C mutation with the ablation of CEC-4, a chromodomain protein that anchors H3K9-methylated chromatin at the nuclear envelope (NE), suppressed the muscle-associated disease phenotypes. Deletion of cec-4 also rescued LMN-Y59C-linked alterations in chromatin organization and some changes in transcription. Sequences that changed position in the LMN-Y59C mutant, are enriched for E2F (EFL-2)-binding sites, consistent with previous studies suggesting that altered Rb-E2F interaction with lamin A may contribute to muscle dysfunction. In summary, we were able to counteract the dominant muscle-specific defects provoked by LMNA mutation by the ablation of a lamin-associated H3K9me anchor, suggesting a novel therapeutic pathway for EDMD.
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Proteínas de Caenorhabditis elegans/genética , Núcleo Celular/genética , Proteínas Cromosómicas no Histona/genética , Eliminación de Gen , Distrofia Muscular de Emery-Dreifuss/genética , Animales , Sitios de Unión/genética , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Núcleo Celular/patología , Cromatina/genética , Modelos Animales de Enfermedad , Genoma de los Helmintos/genética , Laminina/genética , Laminina/metabolismo , Músculos/fisiopatología , Distrofia Muscular de Emery-Dreifuss/fisiopatología , Mutación , Estructura Terciaria de Proteína/genética , Sarcómeros/química , Sarcómeros/genética , Transcripción Genética/genéticaRESUMEN
Regulation of mRNA stability by RNA-protein interactions contributes significantly to quantitative aspects of gene expression. We have identified potential mRNA targets of the AU-rich element binding protein AUF1. Myc-tagged AUF1 p42 was induced in mouse NIH/3T3 cells and RNA-protein complexes isolated using anti-myc tag antibody beads. Bound mRNAs were analyzed with Affymetrix microarrays. We have identified 508 potential target mRNAs that were at least 3-fold enriched compared to control cells without myc-AUF1. 22.3% of the enriched mRNAs had an AU-rich cluster in the ARED Organism database, against 16.3% of non-enriched control mRNAs. The enrichment towards AU-rich elements was also visible by AREScore with an average value of 5.2 in the enriched mRNAs versus 4.2 in the control group. Yet, numerous mRNAs were enriched without a high ARE score. The enrichment of tetrameric and pentameric sequences suggests a broad AUF1 p42-binding spectrum at short U-rich sequences flanked by A or G. Still, some enriched mRNAs were highly unstable, as those of TNFSF11 (known as RANKL), KLF10, HES1, CCNT2, SMAD6, and BCL6. We have mapped some of the instability determinants. HES1 mRNA appeared to have a coding region determinant. Detailed analysis of the RANKL and BCL6 3'UTR revealed for both that full instability required two elements, which are conserved in evolution. In RANKL mRNA both elements are AU-rich and separated by 30 bases, while in BCL6 mRNA one is AU-rich and 60 bases from a non AU-rich element that potentially forms a stem-loop structure.
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Ribonucleoproteína Heterogénea-Nuclear Grupo D/metabolismo , Proteínas Proto-Oncogénicas c-bcl-6/genética , Ligando RANK/genética , Estabilidad del ARN/genética , Regiones no Traducidas 3'/genética , Elementos Ricos en Adenilato y Uridilato/genética , Animales , Sitios de Unión/genética , Células HEK293 , Ribonucleoproteína Nuclear Heterogénea D0 , Ribonucleoproteína Heterogénea-Nuclear Grupo D/genética , Humanos , Ratones , Células 3T3 NIH , Análisis de Secuencia por Matrices de Oligonucleótidos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Proto-Oncogénicas c-bcl-6/metabolismo , Ligando RANK/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
Combinatorial chemotherapy is necessary for the treatment of malaria. However, finding a suitable partner drug for a new candidate is challenging. Here we develop an algorithm that identifies all of the gene pairs of Plasmodium falciparum that possess orthologues in yeast that have a synthetic lethal interaction but are absent in humans. This suggests new options for drug combinations, particularly for inhibitors of targets such as P. falciparum calcineurin, cation ATPase 4, or phosphatidylinositol 4-kinase.
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Antimaláricos/uso terapéutico , Malaria Falciparum/tratamiento farmacológico , 1-Fosfatidilinositol 4-Quinasa/metabolismo , Adenosina Trifosfatasas/metabolismo , Algoritmos , Calcineurina/metabolismo , Combinación de Medicamentos , Humanos , Malaria Falciparum/prevención & control , Plasmodium falciparum/efectos de los fármacosRESUMEN
Extremely-low-frequency magnetic fields (ELF-MF) have been classified as "possibly carcinogenic" to humans on the grounds of an epidemiological association of ELF-MF exposure with an increased risk of childhood leukaemia. Yet, underlying mechanisms have remained obscure. Genome instability seems an unlikely reason as the energy transmitted by ELF-MF is too low to damage DNA and induce cancer-promoting mutations. ELF-MF, however, may perturb the epigenetic code of genomes, which is well-known to be sensitive to environmental conditions and generally deranged in cancers, including leukaemia. We examined the potential of ELF-MF to influence key epigenetic modifications in leukaemic Jurkat cells and in human CD34+ haematopoietic stem cells undergoing in vitro differentiation into the neutrophilic lineage. During granulopoiesis, sensitive genome-wide profiling of multiple replicate experiments did not reveal any statistically significant, ELF-MF-dependent alterations in the patterns of active (H3K4me2) and repressive (H3K27me3) histone marks nor in DNA methylation. However, ELF-MF exposure showed consistent effects on the reproducibility of these histone and DNA modification profiles (replicate variability), which appear to be of a stochastic nature but show preferences for the genomic context. The data indicate that ELF-MF exposure stabilizes active chromatin, particularly during the transition from a repressive to an active state during cell differentiation.
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Epigénesis Genética/efectos de la radiación , Células Madre Hematopoyéticas/efectos de la radiación , Células Jurkat/efectos de la radiación , Campos Magnéticos , Diferenciación Celular/efectos de la radiación , ADN/metabolismo , Histonas/metabolismo , Humanos , MetilaciónRESUMEN
Exposure to extremely low-frequency magnetic fields (ELF-MF) was evaluated in an International Agency for Research on Cancer (IARC) Monographs as "possibly carcinogenic to humans" in 2001, based on increased childhood leukemia risk observed in epidemiological studies. We conducted a hazard assessment using available scientific evidence published before March 2015, with inclusion of new research findings from the Advanced Research on Interaction Mechanisms of electroMagnetic exposures with Organisms for Risk Assessment (ARIMMORA) project. The IARC Monograph evaluation scheme was applied to hazard identification. In ARIMMORA for the first time, a transgenic mouse model was used to mimic the most common childhood leukemia: new pathogenic mechanisms were indicated, but more data are needed to draw definitive conclusions. Although experiments in different animal strains showed exposure-related decreases of CD8+ T-cells, a role in carcinogenesis must be further established. No direct damage of DNA by exposure was observed. Overall in the literature, there is limited evidence of carcinogenicity in humans and inadequate evidence of carcinogenicity in experimental animals, with only weak supporting evidence from mechanistic studies. New exposure data from ARIMMORA confirmed that if the association is nevertheless causal, up to 2% of childhood leukemias in Europe, as previously estimated, may be attributable to ELF-MF. In summary, ARIMMORA concludes that the relationship between ELF-MF and childhood leukemia remains consistent with possible carcinogenicity in humans. While this scientific uncertainty is dissatisfactory for science and public health, new mechanistic insight from ARIMMORA experiments points to future research that could provide a step-change in future assessments. Bioelectromagnetics. 37:183-189, 2016. © 2016 Wiley Periodicals, Inc.
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The Gram-negative bacterium Neisseria meningitidis features extensive genetic variability. To present, proposed virulence genotypes are also detected in isolates from asymptomatic carriers, indicating more complex mechanisms underlying variable colonization modes of N. meningitidis. We applied the Single Molecule, Real-Time (SMRT) sequencing method from Pacific Biosciences to assess the genome-wide DNA modification profiles of two genetically related N. meningitidis strains, both of serogroup A. The resulting DNA methylomes revealed clear divergences, represented by the detection of shared and of strain-specific DNA methylation target motifs. The positional distribution of these methylated target sites within the genomic sequences displayed clear biases, which suggest a functional role of DNA methylation related to the regulation of genes. DNA methylation in N. meningitidis has a likely underestimated potential for variability, as evidenced by a careful analysis of the ORF status of a panel of confirmed and predicted DNA methyltransferase genes in an extended collection of N. meningitidis strains of serogroup A. Based on high coverage short sequence reads, we find phase variability as a major contributor to the variability in DNA methylation. Taking into account the phase variable loci, the inferred functional status of DNA methyltransferase genes matched the observed methylation profiles. Towards an elucidation of presently incompletely characterized functional consequences of DNA methylation in N. meningitidis, we reveal a prominent colocalization of methylated bases with Single Nucleotide Polymorphisms (SNPs) detected within our genomic sequence collection. As a novel observation we report increased mutability also at 6mA methylated nucleotides, complementing mutational hotspots previously described at 5mC methylated nucleotides. These findings suggest a more diverse role of DNA methylation and Restriction-Modification (RM) systems in the evolution of prokaryotic genomes.
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ADN Bacteriano/metabolismo , Epigénesis Genética , Genoma Bacteriano , Mutación , Neisseria meningitidis/genética , Neisseria meningitidis/patogenicidad , Adenina/metabolismo , Citosina/metabolismo , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN , ADN Bacteriano/genética , Expresión Génica , Infecciones Meningocócicas/microbiología , Infecciones Meningocócicas/patología , Datos de Secuencia Molecular , Neisseria meningitidis/metabolismo , Motivos de Nucleótidos , Polimorfismo de Nucleótido Simple , Alineación de Secuencia , Análisis de Secuencia de ADN/métodos , Serogrupo , VirulenciaRESUMEN
Sensorineural hearing loss is a common and currently irreversible disorder, because mammalian hair cells (HCs) do not regenerate and current stem cell and gene delivery protocols result only in immature HC-like cells. Importantly, although the transcriptional regulators of embryonic HC development have been described, little is known about the postnatal regulators of maturating HCs. Here we apply a cell type-specific functional genomic analysis to the transcriptomes of auditory and vestibular sensory epithelia from early postnatal mice. We identify RFX transcription factors as essential and evolutionarily conserved regulators of the HC-specific transcriptomes, and detect Rfx1,2,3,5 and 7 in the developing HCs. To understand the role of RFX in hearing, we generate Rfx1/3 conditional knockout mice. We show that these mice are deaf secondary to rapid loss of initially well-formed outer HCs. These data identify an essential role for RFX in hearing and survival of the terminally differentiating outer HCs.
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Proteínas de Unión al ADN/metabolismo , Células Ciliadas Auditivas/metabolismo , Audición/fisiología , Factores de Transcripción/metabolismo , Animales , Animales Recién Nacidos , Evolución Biológica , Inmunoprecipitación de Cromatina , Femenino , Regulación de la Expresión Génica , Células Ciliadas Auditivas/ultraestructura , Masculino , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Noqueados , Familia de Multigenes , Factores de Transcripción del Factor Regulador X , Factor Regulador X1 , Análisis de Secuencia de ADN , Transcriptoma , Pez CebraRESUMEN
The activation, or maturation, of dendritic cells (DCs) is crucial for the initiation of adaptive T-cell mediated immune responses. Research on the molecular mechanisms implicated in DC maturation has focused primarily on inducible gene-expression events promoting the acquisition of new functions, such as cytokine production and enhanced T-cell-stimulatory capacity. In contrast, mechanisms that modulate DC function by inducing widespread gene-silencing remain poorly understood. Yet the termination of key functions is known to be critical for the function of activated DCs. Genome-wide analysis of activation-induced histone deacetylation, combined with genome-wide quantification of activation-induced silencing of nascent transcription, led us to identify a novel inducible transcriptional-repression pathway that makes major contributions to the DC-maturation process. This silencing response is a rapid primary event distinct from repression mechanisms known to operate at later stages of DC maturation. The repressed genes function in pivotal processes--including antigen-presentation, extracellular signal detection, intracellular signal transduction and lipid-mediator biosynthesis--underscoring the central contribution of the silencing mechanism to rapid reshaping of DC function. Interestingly, promoters of the repressed genes exhibit a surprisingly high frequency of PU.1-occupied sites, suggesting a novel role for this lineage-specific transcription factor in marking genes poised for inducible repression.
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Células Dendríticas/metabolismo , Silenciador del Gen , Proteínas Nucleares/genética , Transactivadores/genética , Transcripción Genética , Animales , Humanos , Ratones , Proteínas Proto-Oncogénicas/metabolismo , Transactivadores/metabolismoRESUMEN
STAT1 is an indispensable component of a heterotrimer (ISGF3) and a STAT1 homodimer (GAF) that function as transcription regulators in type 1 and type 2 interferon signaling, respectively. To investigate the importance of STAT1-cooperative DNA binding, we generated gene-targeted mice expressing cooperativity-deficient STAT1 with alanine substituted for Phe77. Neither ISGF3 nor GAF bound DNA cooperatively in the STAT1F77A mouse strain, but type 1 and type 2 interferon responses were affected differently. Type 2 interferon-mediated transcription and antibacterial immunity essentially disappeared owing to defective promoter recruitment of GAF. In contrast, STAT1 recruitment to ISGF3 binding sites and type 1 interferon-dependent responses, including antiviral protection, remained intact. We conclude that STAT1 cooperativity is essential for its biological activity and underlies the cellular responses to type 2, but not type 1 interferon.
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Interferón Tipo I/metabolismo , Interferón gamma/metabolismo , Proteínas Mutantes/metabolismo , Factor de Transcripción STAT1/metabolismo , Animales , Células Cultivadas , ADN/metabolismo , Factor 3 de Genes Estimulados por el Interferón/metabolismo , Listeriosis/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Mutantes/genética , Unión Proteica/genética , Ingeniería de Proteínas , Factor de Transcripción STAT1/genética , Transducción de Señal/genética , Transgenes/genética , Virus de la Estomatitis Vesicular IndianaRESUMEN
BACKGROUND: The Nuclear Factor I (NFI) family of DNA binding proteins (also called CCAAT box transcription factors or CTF) is involved in both DNA replication and gene expression regulation. Using chromatin immuno-precipitation and high throughput sequencing (ChIP-Seq), we performed a genome-wide mapping of NFI DNA binding sites in primary mouse embryonic fibroblasts. RESULTS: We found that in vivo and in vitro NFI DNA binding specificities are indistinguishable, as in vivo ChIP-Seq NFI binding sites matched predictions based on previously established position weight matrix models of its in vitro binding specificity. Combining ChIP-Seq with mRNA profiling data, we found that NFI preferentially associates with highly expressed genes that it up-regulates, while binding sites were under-represented at expressed but unregulated genes. Genomic binding also correlated with markers of transcribed genes such as histone modifications H3K4me3 and H3K36me3, even outside of annotated transcribed loci, implying NFI in the control of the deposition of these modifications. Positional correlation between + and - strand ChIP-Seq tags revealed that, in contrast to other transcription factors, NFI associates with a nucleosomal length of cleavage-resistant DNA, suggesting an interaction with positioned nucleosomes. In addition, NFI binding prominently occurred at boundaries displaying discontinuities in histone modifications specific of expressed and silent chromatin, such as loci submitted to parental allele-specific imprinted expression. CONCLUSIONS: Our data thus suggest that NFI nucleosomal interaction may contribute to the partitioning of distinct chromatin domains and to epigenetic gene expression regulation.NFI ChIP-Seq and input control DNA data were deposited at Gene Expression Omnibus (GEO) repository under accession number GSE15844. Gene expression microarray data for mouse embryonic fibroblasts are on GEO accession number GSE15871.
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Epigénesis Genética , Factores de Transcripción NFI/genética , Nucleosomas/genética , Regiones Promotoras Genéticas , Animales , Sitios de Unión , Cromatina/genética , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina , Mapeo Cromosómico , Metilación de ADN , Replicación del ADN/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Regulación de la Expresión Génica , Genoma , Ratones , Factores de Transcripción NFI/metabolismo , Nucleosomas/metabolismo , Activación Transcripcional/genéticaRESUMEN
Plasmodium falciparum is responsible for the most severe form of malaria in humans. Antigenic variation of P. falciparum erythrocyte membrane protein 1 leads to immune evasion and occurs through switches in mutually exclusive var gene transcription. The recent progress in Plasmodium epigenetics notwithstanding, the mechanisms by which singularity of var activation is achieved are unknown. Here, we employed a functional approach to dissect the role of var gene upstream regions in mutually exclusive activation. Besides identifying sequence elements involved in activation and initiation of transcription, we mapped a region downstream of the transcriptional start site that is required to maintain singular var gene choice. Activation of promoters lacking this sequence occurs no longer in competition with endogenous var genes. Within this region we pinpointed a sequence-specific DNA-protein interaction involving a cis-acting sequence motif that is conserved in the majority of var loci. These results suggest an important role for this interaction in mutually exclusive locus recognition. Our findings are furthermore consistent with a novel mechanism for the control of singular gene choice in eukaryotes. In addition to their importance in P. falciparum antigenic variation, our results may also help to explain similar processes in other systems.
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Variación Antigénica , ADN Protozoario/metabolismo , Regulación de la Expresión Génica , Plasmodium falciparum/genética , Plasmodium falciparum/fisiología , Proteínas Protozoarias/biosíntesis , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Protozoarias/genética , Transcripción GenéticaRESUMEN
The heartworm Dirofilaria immitis is an important parasite of dogs. Transmitted by mosquitoes in warmer climatic zones, it is spreading across southern Europe and the Americas at an alarming pace. There is no vaccine, and chemotherapy is prone to complications. To learn more about this parasite, we have sequenced the genomes of D. immitis and its endosymbiont Wolbachia. We predict 10,179 protein coding genes in the 84.2 Mb of the nuclear genome, and 823 genes in the 0.9-Mb Wolbachia genome. The D. immitis genome harbors neither DNA transposons nor active retrotransposons, and there is very little genetic variation between two sequenced isolates from Europe and the United States. The differential presence of anabolic pathways such as heme and nucleotide biosynthesis hints at the intricate metabolic interrelationship between the heartworm and Wolbachia. Comparing the proteome of D. immitis with other nematodes and with mammalian hosts, we identify families of potential drug targets, immune modulators, and vaccine candidates. This genome sequence will support the development of new tools against dirofilariasis and aid efforts to combat related human pathogens, the causative agents of lymphatic filariasis and river blindness.
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Antihelmínticos/farmacología , Dirofilaria immitis/genética , Dirofilariasis/parasitología , Enfermedades de los Perros/parasitología , Genoma de los Helmintos , Vacunas/inmunología , Animales , Antihelmínticos/uso terapéutico , Dirofilaria immitis/efectos de los fármacos , Dirofilaria immitis/inmunología , Dirofilaria immitis/microbiología , Dirofilariasis/tratamiento farmacológico , Dirofilariasis/prevención & control , Enfermedades de los Perros/tratamiento farmacológico , Enfermedades de los Perros/prevención & control , Perros , Femenino , Variación Genética , Genoma Bacteriano , Masculino , Filogenia , Proteoma , ARN de Helminto/química , Simbiosis , Transcriptoma/genética , Wolbachia/genética , Wolbachia/fisiologíaRESUMEN
The Plasmodium falciparum genome is equipped with several subtelomeric gene families that are implicated in parasite virulence and immune evasion. Members of these families are uniformly positioned within heterochromatic domains and are thus subject to variegated expression. The best-studied example is that of the var family encoding the major parasite virulence factor P. falciparum erythrocyte membrane protein 1 (PfEMP1). PfEMP1 undergoes antigenic variation through switches in mutually exclusive var gene transcription. var promoters function as crucial regulatory elements in the underlying epigenetic control strategy. Here, we analysed promoters of upsA, upsB and upsC var, rifA1-type rif, stevor, phist and pfmc-2tm genes and investigated their role in endogenous gene transcription by comparative genome-wide expression profiling of transgenic parasite lines. We find that the three major var promoter types are functionally equal and play an essential role in singular gene choice. Unlike var promoters, promoters of non-var families are not silenced by default, and transcription of non-var families is not subject to the same mode of mutually exclusive transcription as has been observed for var genes. Our findings identified a differential logic in the regulation of var and other subtelomeric virulence gene families, which will have important implications for our understanding and future analyses of phenotypic variation in malaria parasites.
Asunto(s)
Regulación de la Expresión Génica , Genes Protozoarios , Plasmodium falciparum/genética , Plasmodium falciparum/patogenicidad , Factores de Virulencia/biosíntesis , Factores de Virulencia/genética , Perfilación de la Expresión Génica , Regiones Promotoras Genéticas , Transcripción GenéticaRESUMEN
One of the major virulence factors of the malaria causing parasite is the Plasmodium falciparum encoded erythrocyte membrane protein 1 (PfEMP1). It is translocated to It the membrane of infected erythrocytes and expressed from approximately 60 var genes in a mutually exclusive manner. Switching of var genes allows the parasite to alter functional and antigenic properties of infected erythrocytes, to escape the immune defense and to establish chronic infections. We have developed an efficient method for isolating VAR genes from telomeric and other genome locations by adapting transformation-associated recombination (TAR) cloning, which can then be analyzed and sequenced. For this purpose, three plasmids each containing a homologous sequence representing the upstream regions of the group A, B, and C var genes and a sequence homologous to the conserved acidic terminal segment (ATS) of var genes were generated. Co-transfection with P. falciparum strain ITG2F6 genomic DNA in yeast cells yielded 200 TAR clones. The relative frequencies of clones from each group were not biased. Clones were screened by PCR, as well as Southern blotting, which revealed clones missed by PCR due to sequence mismatches with the primers. Selected clones were transformed into E. coli and further analyzed by RFLP and end sequencing. Physical analysis of 36 clones revealed 27 distinct types potentially representing 50% of the var gene repertoire. Three clones were selected for sequencing and assembled into single var gene containing contigs. This study demonstrates that it is possible to rapidly obtain the repertoire of var genes from P. falciparum within a single set of cloning experiments. This technique can be applied to individual isolates which will provide a detailed picture of the diversity of var genes in the field. This is a powerful tool to overcome the obstacles with cloning and assembly of multi-gene families by simultaneously cloning each member.
Asunto(s)
Genes Protozoarios/genética , Plasmodium falciparum/genética , Proteínas Protozoarias/genética , Recombinación Genética/genética , Transformación Genética , Secuencia de Bases , Southern Blotting , Clonación Molecular , Biblioteca de Genes , Vectores Genéticos/genética , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción/genética , Análisis de Secuencia de ADNRESUMEN
The current understanding of the regulation of transcription does not keep the pace with the spectacular advances in the determination of genomic sequences. Chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-seq) promises to give better insight into transcription regulation by locating sites of protein-DNA interactions. Such loci of putative interactions can be inferred from the genome-wide distributions of ChIP-seq data by peak-calling software. The analysis of ChIP-seq data critically depends on this step and a multitude of these peak-callers have been deployed in the recent years. A recent study reported severe variation among peak-calling results. Yet, peak-calling still lacks systematic quantitative benchmarking. Here, we summarize benchmarking efforts and explain potential drawbacks of each benchmarking method.