RESUMEN

OTX2 is a transcription factor and known driver in medulloblastoma (MB), where it is amplified in a subset of tumours and overexpressed in most cases of group 3 and group 4 MB. Here we demonstrate a noncanonical role for OTX2 in group 3 MB alternative splicing. OTX2 associates with the large assembly of splicing regulators complex through protein-protein interactions and regulates a stem cell splicing program. OTX2 can directly or indirectly bind RNA and this may be partially independent of its DNA regulatory functions. OTX2 controls a pro-tumorigenic splicing program that is mirrored in human cerebellar rhombic lip origins. Among the OTX2-regulated differentially spliced genes, PPHLN1 is expressed in the most primitive rhombic lip stem cells, and targeting PPHLN1 splicing reduces tumour growth and enhances survival in vivo. These findings identify OTX2-mediated alternative splicing as a major determinant of cell fate decisions that drive group 3 MB progression.

Asunto(s)

Empalme Alternativo , Neoplasias Cerebelosas , Meduloblastoma , Células Madre Neoplásicas , Factores de Transcripción Otx , Factores de Transcripción Otx/metabolismo , Factores de Transcripción Otx/genética , Meduloblastoma/genética , Meduloblastoma/patología , Meduloblastoma/metabolismo , Empalme Alternativo/genética , Humanos , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Neoplasias Cerebelosas/genética , Neoplasias Cerebelosas/patología , Neoplasias Cerebelosas/metabolismo , Animales , Regulación Neoplásica de la Expresión Génica , Línea Celular Tumoral , Ratones , Proliferación Celular

RESUMEN

Background: Next-generation sequencing (NGS) of tumor genomes has changed and improved cancer treatment over the past few decades. It can inform clinicians on the optimal therapeutic approach in many of the solid and hematologic cancers, including non-small lung cancer (NSCLC). Our study aimed to determine the costs of NGS assays for NSCLC diagnostics. Methods: We performed a micro-costing study of four NGS assays (Trusight Tumor 170 Kit (Illumina), Oncomine Focus (Thermo Fisher), QIAseq Targeted DNA Custom Panel and QIASeq Targeted RNAscan Custom Panel (Qiagen), and KAPA HyperPlus/SeqCap EZ (Roche)) at the StemCore Laboratories, the Ottawa Hospital, Canada. We used a time-and-motion approach to measure personnel time and a pre-defined questionnaire to collect resource utilization. The unit costs were based on market prices. The cost data were reported in 2019 Canadian dollars. Results: Based on a case throughput of 500 cases per year, the per-sample cost for TruSight Tumor 170 Kit, QIASeq Targeted DNA Custom Panel and QIASeq Targeted RNAscan Custom Panel, Oncomine Focus, and HyperPlus/SeqCap EZ were CAD 1778, CAD 599, CAD 1100 and CAD 1270, respectively. The key cost drivers were library preparation (34-60%) and sequencing (31-51%), followed by data analysis (6-13%) and administrative support (2-7%). Conclusions: Trusight Tumor 170 Kit was the most expensive NGS assay for NSCLC diagnostics; however, an economic evaluation is required to identify the most cost-effective NGS assay. Our study results could help inform decisions to select a robust platform for NSCLC diagnostics from fine needle aspirates, and future economic evaluations of the NGS platforms to guide treatment selections for NSCLC patients.

Asunto(s)

Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Canadá , Carcinoma de Pulmón de Células no Pequeñas/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Neoplasias Pulmonares/genética

RESUMEN

It is challenging to identify the causes and consequences of retrotransposon expression in human disease due to the hundreds of active genomic copies and their poor conservation across species. We profiled genomic insertions of retrotransposons in ovarian cancer. In addition, in ovarian and breast cancer we analyzed RNAs exhibiting Bayesian correlation with retrotransposon RNA to identify causes and consequences of retrotransposon expression. This strategy finds divergent inflammatory responses associated with retrotransposon expression in ovarian and breast cancer and identifies new factors inducing expression of endogenous retrotransposons including anti-viral responses and the common tumor suppressor BRCA1. In cell lines, mouse ovarian epithelial cells and patient-derived tumor spheroids, BRCA1 promotes accumulation of retrotransposon RNA. BRCA1 promotes transcription of active families of retrotransposons and their insertion into the genome. Intriguingly, elevated retrotransposon expression predicts survival in ovarian cancer patients. Retrotransposons are part of a complex regulatory network in ovarian cancer including BRCA1 that contributes to patient survival. The described strategy can be used to identify the regulators and impacts of retrotransposons in various contexts of biology and disease in humans.

RESUMEN

BACKGROUND: Treating cancer depends in part on identifying the mutations driving each patient's disease. Many clinical laboratories are adopting high-throughput sequencing for assaying patients' tumours, applying targeted panels to formalin-fixed paraffin-embedded tumour tissues to detect clinically-relevant mutations. While there have been some benchmarking and best practices studies of this scenario, much variant calling work focuses on whole-genome or whole-exome studies, with fresh or fresh-frozen tissue. Thus, definitive guidance on best choices for sequencing platforms, sequencing strategies, and variant calling for clinical variant detection is still being developed. METHODS: Because ground truth for clinical specimens is rarely known, we used the well-characterized Coriell cell lines GM12878 and GM12877 to generate data. We prepared samples to mimic as closely as possible clinical biopsies, including formalin fixation and paraffin embedding. We evaluated two well-known targeted sequencing panels, Illumina's TruSight 170 hybrid-capture panel and the amplification-based Oncomine Focus panel. Sequencing was performed on an Illumina NextSeq500 and an Ion Torrent PGM respectively. We performed multiple replicates of each assay, to test reproducibility. Finally, we applied four different freely-available somatic single-nucleotide variant (SNV) callers to the data, along with the vendor-recommended callers for each sequencing platform. RESULTS: We did not observe major differences in variant calling success within the regions that each panel covers, but there were substantial differences between callers. All had high sensitivity for true SNVs, but numerous and non-overlapping false positives. Overriding certain default parameters to make them consistent between callers substantially reduced discrepancies, but still resulted in high false positive rates. Intersecting results from multiple replicates or from different variant callers eliminated most false positives, while maintaining sensitivity. CONCLUSIONS: Reproducibility and accuracy of targeted clinical sequencing results depend less on sequencing platform and panel than on variability between replicates and downstream bioinformatics. Differences in variant callers' default parameters are a greater influence on algorithm disagreement than other differences between the algorithms. Contrary to typical clinical practice, we recommend employing multiple variant calling pipelines and/or analyzing replicate samples, as this greatly decreases false positive calls.

Asunto(s)

Algoritmos , Biomarcadores de Tumor/genética , Análisis Mutacional de ADN/métodos , Mutación , Neoplasias/genética , Neoplasias/patología , Polimorfismo de Nucleótido Simple , Biología Computacional , Formaldehído , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Humanos , Adhesión en Parafina , Reproducibilidad de los Resultados , Células Tumorales Cultivadas

RESUMEN

OTX2 is a potent oncogene that promotes tumor growth in Group 3 medulloblastoma. However, the mechanisms by which OTX2 represses neural differentiation are not well characterized. Here, we perform extensive multiomic analyses to identify an OTX2 regulatory network that controls Group 3 medulloblastoma cell fate. OTX2 silencing modulates the repressive chromatin landscape, decreases levels of PRC2 complex genes and increases the expression of neurodevelopmental transcription factors including PAX3 and PAX6. Expression of PAX3 and PAX6 is significantly lower in Group 3 medulloblastoma patients and is correlated with reduced survival, yet only PAX3 inhibits self-renewal in vitro and increases survival in vivo. Single cell RNA sequencing of Group 3 medulloblastoma tumorspheres demonstrates expression of an undifferentiated progenitor program observed in primary tumors and characterized by translation/elongation factor genes. Identification of mTORC1 signaling as a downstream effector of OTX2-PAX3 reveals roles for protein synthesis pathways in regulating Group 3 medulloblastoma pathogenesis.

Asunto(s)

Carcinogénesis/genética , Neoplasias Cerebelosas , Meduloblastoma , Factores de Transcripción Otx/metabolismo , Factor de Transcripción PAX3/genética , Animales , Carcinogénesis/metabolismo , Diferenciación Celular/genética , Línea Celular Tumoral , Proliferación Celular/genética , Neoplasias Cerebelosas/genética , Neoplasias Cerebelosas/metabolismo , Regulación Neoplásica de la Expresión Génica , Humanos , Meduloblastoma/genética , Meduloblastoma/metabolismo , Células Madre Neoplásicas/metabolismo , Oncogenes , Factor de Transcripción PAX3/metabolismo , Factor de Transcripción PAX6/genética , Factor de Transcripción PAX6/metabolismo , Transducción de Señal/genética

RESUMEN

Muscle-specific transcription factor MyoD orchestrates the myogenic gene expression program by binding to short DNA motifs called E-boxes within myogenic cis-regulatory elements (CREs). Genome-wide analyses of MyoD cistrome by chromatin immnunoprecipitation sequencing shows that MyoD-bound CREs contain multiple E-boxes of various sequences. However, how E-box numbers, sequences and their spatial arrangement within CREs collectively regulate the binding affinity and transcriptional activity of MyoD remain largely unknown. Here, by an integrative analysis of MyoD cistrome combined with genome-wide analysis of key regulatory histones and gene expression data we show that the affinity landscape of MyoD is driven by multiple E-boxes, and that the overall binding affinity-and associated nucleosome positioning and epigenetic features of the CREs-crucially depend on the variant sequences and positioning of the E-boxes within the CREs. By comparative genomic analysis of single nucleotide polymorphism (SNPs) across publicly available data from 17 strains of laboratory mice, we show that variant sequences within the MyoD-bound motifs, but not their genome-wide counterparts, are under selection. At last, we show that the quantitative regulatory effect of MyoD binding on the nearby genes can, in part, be predicted by the motif composition of the CREs to which it binds. Taken together, our data suggest that motif numbers, sequences and their spatial arrangement within the myogenic CREs are important determinants of the cis-regulatory code of myogenic CREs.

Asunto(s)

Elementos E-Box/genética , Desarrollo de Músculos/genética , Proteína MioD/genética , Proteína MioD/metabolismo , Transcripción Genética/genética , Activación Transcripcional/genética , Animales , Secuencia de Bases/genética , Inmunoprecipitación de Cromatina , Proteínas de Unión al ADN/genética , Expresión Génica/genética , Regulación de la Expresión Génica , Estudio de Asociación del Genoma Completo , Ratones , Desarrollo de Músculos/fisiología , Motivos de Nucleótidos/genética , Polimorfismo de Nucleótido Simple/genética , Regiones Promotoras Genéticas/genética

RESUMEN

Mutations in proteins like FUS which cause Amyotrophic Lateral Sclerosis (ALS) result in the aberrant formation of stress granules while ALS-linked mutations in other proteins impede elimination of stress granules. Repeat expansions in C9ORF72, the major cause of ALS, reduce C9ORF72 levels but how this impacts stress granules is uncertain. Here, we demonstrate that C9ORF72 associates with the autophagy receptor p62 and controls elimination of stress granules by autophagy. This requires p62 to associate via the Tudor protein SMN with proteins, including FUS, that are symmetrically methylated on arginines. Mice lacking p62 accumulate arginine-methylated proteins and alterations in FUS-dependent splicing. Patients with C9ORF72 repeat expansions accumulate symmetric arginine dimethylated proteins which co-localize with p62. This suggests that C9ORF72 initiates a cascade of ALS-linked proteins (C9ORF72, p62, SMN, FUS) to recognize stress granules for degradation by autophagy and hallmarks of a defect in this process are observable in ALS patients.

Asunto(s)

Esclerosis Amiotrófica Lateral/genética , Autofagia/genética , Proteína C9orf72/genética , Proteína FUS de Unión a ARN/genética , Proteína Sequestosoma-1/genética , Proteína 1 para la Supervivencia de la Neurona Motora/genética , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Animales , Arginina/metabolismo , Proteína C9orf72/metabolismo , Línea Celular Tumoral , Gránulos Citoplasmáticos/metabolismo , Gránulos Citoplasmáticos/patología , Embrión de Mamíferos , Células HeLa , Humanos , Metilación , Ratones , Ratones Noqueados , Neuronas Motoras/citología , Neuronas Motoras/metabolismo , Cultivo Primario de Células , Proteína-Arginina N-Metiltransferasas/genética , Proteína-Arginina N-Metiltransferasas/metabolismo , Proteína FUS de Unión a ARN/metabolismo , Proteína Sequestosoma-1/metabolismo , Estrés Fisiológico , Proteína 1 para la Supervivencia de la Neurona Motora/metabolismo

RESUMEN

Polycomb repressive complex 2 (PRC2) accessory proteins play substoichiometric, tissue-specific roles to recruit PRC2 to specific genomic loci or increase enzymatic activity, while PRC2 core proteins are required for complex stability and global levels of trimethylation of histone 3 at lysine 27 (H3K27me3). Here, we demonstrate a role for the classical PRC2 accessory protein Mtf2/Pcl2 in the hematopoietic system that is more akin to that of a core PRC2 protein. Mtf2-/- erythroid progenitors demonstrate markedly decreased core PRC2 protein levels and a global loss of H3K27me3 at promoter-proximal regions. The resulting de-repression of transcriptional and signaling networks blocks definitive erythroid development, culminating in Mtf2-/- embryos dying by e15.5 due to severe anemia. Gene regulatory network (GRN) analysis demonstrated Mtf2 directly regulates Wnt signaling in erythroblasts, leading to activated canonical Wnt signaling in Mtf2-deficient erythroblasts, while chemical inhibition of canonical Wnt signaling rescued Mtf2-deficient erythroblast differentiation in vitro. Using a combination of in vitro, in vivo and systems analyses, we demonstrate that Mtf2 is a critical epigenetic regulator of Wnt signaling during erythropoiesis and recast the role of polycomb accessory proteins in a tissue-specific context.

RESUMEN

Medulloblastoma (MB) is the most common malignant primary pediatric brain cancer. Among the most aggressive subtypes, Group 3 and Group 4 originate from stem/progenitor cells, frequently metastasize, and often display the worst prognosis, yet we know the least about the molecular mechanisms driving their progression. Here, we show that the transcription factor orthodenticle homeobox 2 (OTX2) promotes self-renewal while inhibiting differentiation in vitro and increases tumor initiation from MB stem/progenitor cells in vivo. To determine how OTX2 contributes to these processes, we employed complementary bioinformatic approaches to characterize the OTX2 regulatory network and identified novel relationships between OTX2 and genes associated with neuronal differentiation and axon guidance signaling in Group 3 and Group 4 MB stem/progenitor cells. In particular, OTX2 levels were negatively correlated with semaphorin (SEMA) signaling, as expression of 9 SEMA pathway genes is upregulated following OTX2 knockdown with some being potential direct OTX2 targets. Importantly, this negative correlation was also observed in patient samples, with lower expression of SEMA4D associated with poor outcome specifically in Group 4 tumors. Functional proof-of-principle studies demonstrated that increased levels of select SEMA pathway genes are associated with decreased self-renewal and growth in vitro and in vivo and that RHO signaling, known to mediate the effects of SEMA genes, is contributing to the OTX2 KD phenotype. Our study provides mechanistic insight into the networks controlled by OTX2 in MB stem/progenitor cells and reveals novel roles for axon guidance genes and their downstream effectors as putative tumor suppressors in MB.

Asunto(s)

Neoplasias Encefálicas/metabolismo , Regulación Neoplásica de la Expresión Génica , Meduloblastoma/metabolismo , Proteínas de Neoplasias/metabolismo , Células Madre Neoplásicas/metabolismo , Factores de Transcripción Otx/metabolismo , Transducción de Señal , Animales , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Humanos , Meduloblastoma/genética , Meduloblastoma/patología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteínas de Neoplasias/genética , Células Madre Neoplásicas/patología , Factores de Transcripción Otx/genética

RESUMEN

Ideally, disease modeling using patient-derived induced pluripotent stem cells (iPSCs) enables analysis of disease initiation and progression. This requires any pathological features of the patient cells used for reprogramming to be eliminated during iPSC generation. Hutchinson-Gilford progeria syndrome (HGPS) is a segmental premature aging disorder caused by the accumulation of the truncated form of Lamin A known as Progerin within the nuclear lamina. Cellular hallmarks of HGPS include nuclear blebbing, loss of peripheral heterochromatin, defective epigenetic inheritance, altered gene expression, and senescence. To model HGPS using iPSCs, detailed genome-wide and structural analysis of the epigenetic landscape is required to assess the initiation and progression of the disease. We generated a library of iPSC lines from fibroblasts of patients with HGPS and controls, including one family trio. HGPS patient-derived iPSCs are nearly indistinguishable from controls in terms of pluripotency, nuclear membrane integrity, as well as transcriptional and epigenetic profiles, and can differentiate into affected cell lineages recapitulating disease progression, despite the nuclear aberrations, altered gene expression, and epigenetic landscape inherent to the donor fibroblasts. These analyses demonstrate the power of iPSC reprogramming to reset the epigenetic landscape to a revitalized pluripotent state in the face of widespread epigenetic defects, validating their use to model the initiation and progression of disease in affected cell lineages.

Asunto(s)

Reprogramación Celular , Epigénesis Genética , Fibroblastos/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Lamina Tipo A/genética , Progeria/genética , Secuencia de Bases , Estudios de Casos y Controles , Diferenciación Celular , Senescencia Celular , Fibroblastos/patología , Perfilación de la Expresión Génica , Heterocromatina/metabolismo , Heterocromatina/ultraestructura , Histonas/genética , Histonas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/patología , Cariotipo , Lamina Tipo A/metabolismo , Miocitos del Músculo Liso/metabolismo , Miocitos del Músculo Liso/patología , Cultivo Primario de Células , Progeria/metabolismo , Progeria/patología

RESUMEN

BACKGROUND: Unraveling transcriptional regulatory networks is a central problem in molecular biology and, in this quest, chromatin immunoprecipitation and sequencing (ChIP-seq) technology has given us the unprecedented ability to identify sites of protein-DNA binding and histone modification genome wide. However, multiple systemic and procedural biases hinder harnessing the full potential of this technology. Previous studies have addressed this problem, but a thorough characterization of different, interacting biases on ChIP-seq signals is still lacking. RESULTS: Here, we present a novel framework where the genome-wide ChIP-seq signal is viewed as being quantifiably influenced by different, measurable sources of bias, which can then be computationally subtracted away. We use a compendium of 123 human ENCODE ChIP-seq datasets to build regression models that tell us how much of a ChIP-seq signal can be attributed to mappability, GC-content, chromatin accessibility, and factors represented in input DNA and IgG controls. When we use the model to separate out these non-binding influences from the ChIP-seq signal, we obtain a purified signal that associates better to TF-DNA-binding motifs than do other measures of peak significance. We also carry out a multiscale analysis that reveals how ChIP-seq signal biases differ across different scales. Finally, we investigate previously reported associations between gene expression and ChIP-seq signals at transcription start sites. We show that our model can be used to discriminate ChIP-seq signals that are truly related to gene expression from those that are merely correlated by virtue of bias-in particular, chromatin accessibility bias, which shows up in ChIP-seq signals and also relates to gene expression. CONCLUSIONS: Our study provides new insights into the behavior of ChIP-seq signal biases and proposes a novel mitigation framework that improves results compared to existing techniques. With ChIP-seq now being the central technology for studying transcriptional regulation, it is most crucial to accurately characterize, quantify, and adjust for the genome-wide effects of biases affecting ChIP-seq. Our study also emphasizes that properly accounting for confounders in ChIP-seq data is of paramount importance for obtaining biologically accurate insights into the workings of the complex regulatory mechanisms in living organisms. R and MATLAB packages implementing the framework can be obtained from http://www.perkinslab.ca/Software.html.

RESUMEN

Alpha-solenoids are flexible protein structural domains formed by ensembles of alpha-helical repeats (Armadillo and HEAT repeats among others). While homology can be used to detect many of these repeats, some alpha-solenoids have very little sequence homology to proteins of known structure and we expect that many remain undetected. We previously developed a method for detection of alpha-helical repeats based on a neural network trained on a dataset of protein structures. Here we improved the detection algorithm and updated the training dataset using recently solved structures of alpha-solenoids. Unexpectedly, we identified occurrences of alpha-solenoids in solved protein structures that escaped attention, for example within the core of the catalytic subunit of PI3KC. Our results expand the current set of known alpha-solenoids. Application of our tool to the protein universe allowed us to detect their significant enrichment in proteins interacting with many proteins, confirming that alpha-solenoids are generally involved in protein-protein interactions. We then studied the taxonomic distribution of alpha-solenoids to discuss an evolutionary scenario for the emergence of this type of domain, speculating that alpha-solenoids have emerged in multiple taxa in independent events by convergent evolution. We observe a higher rate of alpha-solenoids in eukaryotic genomes and in some prokaryotic families, such as Cyanobacteria and Planctomycetes, which could be associated to increased cellular complexity. The method is available at http://cbdm.mdc-berlin.de/~ard2/.

Asunto(s)

Genómica , Proteínas/fisiología , Conformación Proteica , Proteínas/química , Proteínas/genética

RESUMEN

Chromatin immunoprecipitation coupled with ultra-high-throughput sequencing (ChIP-seq) is a widely used method for mapping the interactions of proteins with DNA. However, the requirements for ChIP-grade antibodies impede wider application of this method, and variations in results can be high owing to differences in affinity and cross-reactivity of antibodies. Therefore, we developed chromatin tandem affinity purification (ChTAP) as an effective alternative to ChIP. Through the use of affinity tags and reagents that are identical for all proteins investigated, ChTAP enables one to directly compare the binding between different transcription factors and to directly assess the background in control experiments. Thus, ChTAP-seq can be used to rapidly map the genome-wide binding of multiple DNA-binding proteins in a wide range of cell types. ChTAP can be completed in 3-4 d, starting from cross-linking of chromatin to purification of ChIP DNA.

Asunto(s)

Cromatina/metabolismo , Cromatografía de Afinidad/métodos , Proteínas de Unión al ADN/metabolismo , Sitios de Unión , Inmunoprecipitación de Cromatina , Células HEK293 , Humanos , Sonicación

RESUMEN

MOTIVATION: Reliable estimation of the mean fragment length for next-generation short-read sequencing data is an important step in next-generation sequencing analysis pipelines, most notably because of its impact on the accuracy of the enriched regions identified by peak-calling algorithms. Although many peak-calling algorithms include a fragment-length estimation subroutine, the problem has not been adequately solved, as demonstrated by the variability of the estimates returned by different algorithms. RESULTS: In this article, we investigate the use of strand cross-correlation to estimate mean fragment length of single-end data and show that traditional estimation approaches have mixed reliability. We observe that the mappability of different parts of the genome can introduce an artificial bias into cross-correlation computations, resulting in incorrect fragment-length estimates. We propose a new approach, called mappability-sensitive cross-correlation (MaSC), which removes this bias and allows for accurate and reliable fragment-length estimation. We analyze the computational complexity of this approach, and evaluate its performance on a test suite of NGS datasets, demonstrating its superiority to traditional cross-correlation analysis. AVAILABILITY: An open-source Perl implementation of our approach is available at http://www.perkinslab.ca/Software.html.

Asunto(s)

Algoritmos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Mapeo Cromosómico , Interpretación Estadística de Datos , Genómica , Humanos , Reproducibilidad de los Resultados

RESUMEN

BACKGROUND: Reviewer and editor selection for peer review is getting harder for authors and publishers due to the specialization onto narrower areas of research carried by the progressive growth of the body of knowledge. Examination of the literature facilitates finding appropriate reviewers but is time consuming and complicated by author name ambiguities. RESULTS: We have developed a method called peer2ref to support authors and editors in selecting suitable reviewers for scientific manuscripts. Peer2ref works from a text input, usually the abstract of the manuscript, from which important concepts are extracted as keywords using a fuzzy binary relations approach. The keywords are searched on indexed profiles of words constructed from the bibliography attributed to authors in MEDLINE. The names of these scientists have been previously disambiguated by coauthors identified across the whole MEDLINE. The methods have been implemented in a web server that automatically suggests experts for peer-review among scientists that have authored manuscripts published during the last decade in more than 3,800 journals indexed in MEDLINE. CONCLUSION: peer2ref web server is publicly available at http://www.ogic.ca/projects/peer2ref/.

RESUMEN

Pax3 and Pax7 regulate stem cell function in skeletal myogenesis. However, molecular insight into their distinct roles has remained elusive. Using gene expression data combined with genome-wide binding-site analysis, we show that both Pax3 and Pax7 bind identical DNA motifs and jointly activate a large panel of genes involved in muscle stem cell function. Surprisingly, in adult myoblasts Pax3 binds a subset (6.4%) of Pax7 targets. Despite a significant overlap in their transcriptional network, Pax7 regulates distinct panels of genes involved in the promotion of proliferation and inhibition of myogenic differentiation. We show that Pax7 has a higher binding affinity to the homeodomain-binding motif relative to Pax3, suggesting that intrinsic differences in DNA binding contribute to the observed functional difference between Pax3 and Pax7 binding in myogenesis. Together, our data demonstrate distinct attributes of Pax7 function and provide mechanistic insight into the nonredundancy of Pax3 and Pax7 in muscle development.

Asunto(s)

Secuencias de Aminoácidos/fisiología , Proteínas de Homeodominio/metabolismo , Desarrollo de Músculos/fisiología , Músculo Esquelético/metabolismo , Factor de Transcripción PAX7/metabolismo , Transcripción Genética , Animales , Diferenciación Celular , Proliferación Celular , Perfilación de la Expresión Génica , Ratones , Factor de Transcripción PAX3 , Factores de Transcripción Paired Box/metabolismo

RESUMEN

BACKGROUND: In spite of extensive research on the effect of mutation and selection on codon usage, a general model of codon usage bias due to mutational bias has been lacking. Because most amino acids allow synonymous GC content changing substitutions in the third codon position, the overall GC bias of a genome or genomic region is highly correlated with GC3, a measure of third position GC content. For individual amino acids as well, G/C ending codons usage generally increases with increasing GC bias and decreases with increasing AT bias. Arginine and leucine, amino acids that allow GC-changing synonymous substitutions in the first and third codon positions, have codons which may be expected to show different usage patterns. PRINCIPAL FINDINGS: In analyzing codon usage bias in hundreds of prokaryotic and plant genomes and in human genes, we find that two G-ending codons, AGG (arginine) and TTG (leucine), unlike all other G/C-ending codons, show overall usage that decreases with increasing GC bias, contrary to the usual expectation that G/C-ending codon usage should increase with increasing genomic GC bias. Moreover, the usage of some codons appears nonlinear, even nonmonotone, as a function of GC bias. To explain these observations, we propose a continuous-time Markov chain model of GC-biased synonymous substitution. This model correctly predicts the qualitative usage patterns of all codons, including nonlinear codon usage in isoleucine, arginine and leucine. The model accounts for 72%, 64% and 52% of the observed variability of codon usage in prokaryotes, plants and human respectively. When codons are grouped based on common GC content, 87%, 80% and 68% of the variation in usage is explained for prokaryotes, plants and human respectively. CONCLUSIONS: The model clarifies the sometimes-counterintuitive effects that GC mutational bias can have on codon usage, quantifies the influence of GC mutational bias and provides a natural null model relative to which other influences on codon bias may be measured.

Asunto(s)

Codón , Modelos Teóricos , Mutación , Composición de Base , Citosina/química , Guanina/química , Cadenas de Markov

RESUMEN

The MEDLINE database of medical literature is routinely used by researchers and doctors to find articles pertaining to their area of interest. Insight into historical changes in research areas may be gained by chronological analysis of the 18 million records currently in the database, however such analysis is generally complex and time consuming. The authors' MLTrends web application graphs term usage in MEDLINE over time, allowing the determination of emergence dates for biomedical terms and historical variations in term usage intensity. MLTrends may be used at: http://www.ogic.ca/mltrends.

RESUMEN

Skeletal muscle ageing is characterized by faulty degenerative/regenerative processes that promote the decline of its mass, strength, and endurance. In this study, we used a transcriptional profiling method to better understand the molecular pathways and factors that contribute to these processes. To more appropriately contrast the differences in regenerative capacity of old muscle, we compared it with young muscle, where robust growth and efficient myogenic differentiation is ongoing. Notably, in old mice, we found a severe deficit in satellite cells activation. We performed expression analyses on RNA from the gastrocnemius muscle of young (3-week-old) and old (24-month-old) mice. The differential expression highlighted genes that are involved in the efficient functioning of satellite cells. Indeed, the greatest number of up-regulated genes in young mice encoded components of the extracellular matrix required for the maintenance of the satellite cell niche. Moreover, other genes included Wnt inhibitors (Wif1 and Sfrp2) and Notch activator (Dner), which are putatively involved in the interconnected signalling networks that control satellite cell function. The widespread expression differences for inhibitors of TGFbeta signalling further emphasize the shortcomings in satellite cell performance. Therefore, we draw attention to the breakdown of features required to maintain satellite cell integrity during the ageing process.

Asunto(s)

Envejecimiento/genética , Senescencia Celular/genética , Perfilación de la Expresión Génica/métodos , Desarrollo de Músculos/genética , Músculo Esquelético/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Células Satélite del Músculo Esquelético/metabolismo , Factores de Edad , Animales , Células Cultivadas , Regulación de la Expresión Génica , Masculino , Ratones , Ratones Endogámicos C57BL , Músculo Esquelético/crecimiento & desarrollo , Reacción en Cadena de la Polimerasa , Reproducibilidad de los Resultados , Transducción de Señal/genética

RESUMEN

BACKGROUND: Currently one of the largest online repositories for human and mouse stem cell gene expression data, StemBase was first designed as a simple web-interface to DNA microarray data generated by the Canadian Stem Cell Network to facilitate the discovery of gene functions relevant to stem cell control and differentiation. FINDINGS: Since its creation, StemBase has grown in both size and scope into a system with analysis tools that examine either the whole database at once, or slices of data, based on tissue type, cell type or gene of interest. As of September 1, 2008, StemBase contains gene expression data (microarray and Serial Analysis of Gene Expression) from 210 stem cell samples in 60 different experiments. CONCLUSION: StemBase can be used to study gene expression in human and murine stem cells and is available at http://www.stembase.ca.