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1.
bioRxiv ; 2024 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-38915658

RESUMO

Studying protein isoforms is an essential step in biomedical research; at present, the main approach for analyzing proteins is via bottom-up mass spectrometry proteomics, which return peptide identifications, that are indirectly used to infer the presence of protein isoforms. However, the detection and quantification processes are noisy; in particular, peptides may be erroneously detected, and most peptides, known as shared peptides, are associated to multiple protein isoforms. As a consequence, studying individual protein isoforms is challenging, and inferred protein results are often abstracted to the gene-level or to groups of protein isoforms. Here, we introduce IsoBayes, a novel statistical method to perform inference at the isoform level. Our method enhances the information available, by integrating mass spectrometry proteomics and transcriptomics data in a Bayesian probabilistic framework. To account for the uncertainty in the measurement process, we propose a two-layer latent variable approach: first, we sample if a peptide has been correctly detected (or, alternatively filter peptides); second, we allocate the abundance of such selected peptides across the protein(s) they are compatible with. This enables us, starting from peptide-level data, to recover protein-level data; in particular, we: i) infer the presence/absence of each protein isoform (via a posterior probability), ii) estimate its abundance (and credible interval), and iii) target isoforms where transcript and protein relative abundances significantly differ. We benchmarked our approach in simulations, and in two multi-protease real datasets: our method displays good sensitivity and specificity when detecting protein isoforms, its estimated abundances highly correlate with the ground truth, and can detect changes between protein and transcript relative abundances. IsoBayes is freely distributed as a Bioconductor R package, and is accompanied by an example usage vignette.

2.
iScience ; 27(4): 109532, 2024 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-38577110

RESUMO

Wound healing is impaired by infection; however, how microbe-induced inflammation modulates tissue repair remains unclear. We took advantage of the optical transparency of zebrafish and a genetically tractable microbe, Listeria monocytogenes, to probe the role of infection and inflammation in wound healing. Infection with bacteria engineered to activate the inflammasome, Lm-Pyro, induced persistent inflammation and impaired healing despite low bacterial burden. Inflammatory infections induced il1b expression and blocking IL-1R signaling partially rescued wound healing in the presence of persistent infection. We found a critical window of microbial clearance necessary to limit persistent inflammation and enable efficient wound repair. Taken together, our findings suggest that the dynamics of microbe-induced tissue inflammation impacts repair in complex tissue damage independent of bacterial load, with a critical early window for efficient tissue repair.

3.
Blood Adv ; 7(4): 586-601, 2023 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-36161469

RESUMO

The RNA-regulatory exosome complex (EC) posttranscriptionally and cotranscriptionally processes and degrades RNAs in a context-dependent manner. Although the EC functions in diverse cell types, its contributions to stem and progenitor cell development are not well understood. Previously, we demonstrated that the transcriptional regulator of erythrocyte development, GATA1, represses EC subunit genes, and the EC maintains erythroid progenitors in vitro. To determine if this mechanism operates in vivo, we used the hematopoietic-specific Vav1-Cre and "conditional by inversion" mouse system to ablate Exosc3, encoding an EC structural subunit. Although Exosc3C/C Cre+ embryos developed normally until embryonic day 14.5, Exosc3 ablation was embryonic lethal and severely reduced erythromyeloid progenitor activity. RNA sequencing analysis of Exosc3-ablated burst-forming unit-erythroid revealed elevated transcripts encoding multiple proapoptotic factors, and the mutant erythroid progenitors exhibited increased apoptosis. We propose that the EC controls an ensemble of apoptosis-regulatory RNAs, thereby promoting erythroid progenitor survival and developmental erythropoiesis in vivo.


Assuntos
Células Precursoras Eritroides , Exossomos , Camundongos , Animais , Complexo Multienzimático de Ribonucleases do Exossomo , Apoptose , RNA
4.
Comput Biol Chem ; 102: 107795, 2023 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-36436489

RESUMO

RNA sequencing (RNA-seq) has been a widely used high-throughput method to characterize transcriptomic dynamics spatiotemporally. However, RNA-seq data analysis pipelines typically depend on either a sequenced genome and/or corresponding reference transcripts. This limitation is a challenge for species lacking sequenced genomes and corresponding reference transcripts. The Nile rat (Arvicanthis niloticus) has two key features - it is daytime active, and it is prone to diet-induced diabetes, which makes it more similar to humans than regular laboratory rodents. However, at the time of this study, neither a Nile rat genome nor a reference transcript set were available, making it technically challenging to perform large-scale RNA-seq based transcriptomic studies. This genome-independent work progressed concurrently with our generation of a Nile rat genome. A well-annotated genome requires several iterations of manually reviewing curated transcripts and takes years to achieve. Here, we developed a Comparative RNA-Seq Pipeline (CRSP), integrating a comparative species strategy independent of a specific sequenced genome or species-matched reference transcripts. We performed benchmarking to validate that our CRSP tool can accurately quantify gene expression levels. In this study, we generated the first ultra-deep (2.3 billion × 2 paired-end) Nile rat RNA-seq data from 59 biopsy samples representing 22 major organs, providing a unique resource and spatial gene expression reference for Nile rat researchers. Importantly, CRSP is not limited to the Nile rat species and can be applied to any species without prior genomic knowledge. To facilitate a general use of CRSP, we also characterized the number of RNA-seq reads required for accurate estimation via simulation studies. CRSP and documents are available at: https://github.com/pjiang1105/CRSP.


Assuntos
Murinae , Transcriptoma , Humanos , Animais , Transcriptoma/genética , RNA-Seq , Perfilação da Expressão Gênica , Genoma , Análise de Sequência de RNA/métodos , Sequenciamento de Nucleotídeos em Larga Escala
5.
iScience ; 25(9): 104934, 2022 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-36060075

RESUMO

Memory T cells underpin vaccine-induced immunity but are not yet fully understood. To distinguish features of memory cells that confer protective immunity, we used single cell transcriptome analysis to compare antigen-specific CD4+T cells recalled to lungs of mice that received a protective or nonprotective subunit vaccine followed by challenge with a fungal pathogen. We unexpectedly found populations specific to protection that expressed a strong type I interferon response signature, whose distinctive transcriptional signature appeared unconventionally dependent on IFN-γ receptor. We also detected a unique population enriched in protection that highly expressed the gene for the natural killer cell marker NKG7. Lastly, we detected differences in TCR gene use and in Th1- and Th17-skewed responses after protective and nonprotective vaccine, respectively, reflecting heterogeneous Ifng- and Il17a-expressing populations. Our findings highlight key features of transcriptionally diverse and distinctive antigen-specific T cells associated with protective vaccine-induced immunity.

6.
Front Immunol ; 13: 818893, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35250998

RESUMO

Neutrophils in the tumor microenvironment exhibit altered functions. However, the changes in neutrophil behavior during tumor initiation remain unclear. Here we used Translating Ribosomal Affinity Purification (TRAP) and RNA sequencing to identify neutrophil, macrophage and transformed epithelial cell transcriptional changes induced by oncogenic RasG12V in larval zebrafish. We found that transformed epithelial cells and neutrophils, but not macrophages, had significant changes in gene expression in larval zebrafish. Interestingly, neutrophils had more significantly down-regulated genes, whereas gene expression was primarily upregulated in transformed epithelial cells. The antioxidant, thioredoxin (txn), a small thiol that regulates reduction-oxidation (redox) balance, was upregulated in transformed keratinocytes and neutrophils in response to oncogenic Ras. To determine the role of thioredoxin during tumor initiation, we generated a zebrafish thioredoxin mutant. We observed an increase in wound-induced reactive oxygen species signaling and neutrophil recruitment in thioredoxin-deficient zebrafish. Transformed keratinocytes also showed increased proliferation and reduced apoptosis in thioredoxin-deficient larvae. Using live imaging, we visualized neutrophil behavior near transformed cells and found increased neutrophil recruitment and altered motility dynamics. Finally, in the absence of neutrophils, transformed keratinocytes no longer exhibited increased proliferation in thioredoxin mutants. Taken together, our findings demonstrate that tumor initiation induces changes in neutrophil gene expression and behavior that can impact proliferation of transformed cells in the early tumor microenvironment.


Assuntos
Proteínas de Peixe-Zebra , Peixe-Zebra , Animais , Transformação Celular Neoplásica , Perfilação da Expressão Gênica , Larva/genética , Larva/metabolismo , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Microambiente Tumoral/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
7.
STAR Protoc ; 2(3): 100705, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34458864

RESUMO

Cell type annotation is important in the analysis of single-cell RNA-seq data. CellO is a machine-learning-based tool for annotating cells using the Cell Ontology, a rich hierarchy of known cell types. We provide a protocol for using the CellO Python package to annotate human cells. We demonstrate how to use CellO in conjunction with Scanpy, a Python library for performing single-cell analysis, annotate a lung tissue data set, interpret its hierarchically structured cell type annotations, and create publication-ready figures. For complete details on the use and execution of this protocol, please refer to Bernstein et al. (2021).


Assuntos
Curadoria de Dados/métodos , RNA-Seq/métodos , Análise de Sequência de RNA/métodos , Ontologias Biológicas , Biologia Computacional/métodos , Humanos , Aprendizado de Máquina , Análise de Célula Única/métodos , Software , Transcriptoma/genética , Sequenciamento do Exoma/métodos
8.
Nucleic Acids Res ; 49(16): 9007-9025, 2021 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-34059908

RESUMO

Cellular differentiation requires vast remodeling of transcriptomes, and therefore machinery mediating remodeling controls differentiation. Relative to transcriptional mechanisms governing differentiation, post-transcriptional processes are less well understood. As an important post-transcriptional determinant of transcriptomes, the RNA exosome complex (EC) mediates processing and/or degradation of select RNAs. During erythropoiesis, the erythroid transcription factor GATA1 represses EC subunit genes. Depleting EC structural subunits prior to GATA1-mediated repression is deleterious to erythroid progenitor cells. To assess the importance of the EC catalytic subunits Dis3 and Exosc10 in this dynamic process, we asked if these subunits function non-redundantly to control erythropoiesis. Dis3 or Exosc10 depletion in primary murine hematopoietic progenitor cells reduced erythroid progenitors and their progeny, while sparing myeloid cells. Dis3 loss severely compromised erythroid progenitor and erythroblast survival, rendered erythroblasts hypersensitive to apoptosis-inducing stimuli and induced γ-H2AX, indicative of DNA double-stranded breaks. Dis3 loss-of-function phenotypes were more severe than those caused by Exosc10 depletion. We innovated a genetic rescue system to compare human Dis3 with multiple myeloma-associated Dis3 mutants S447R and R750K, and only wild type Dis3 was competent to rescue progenitors. Thus, Dis3 establishes a disease mutation-sensitive, cell type-specific survival mechanism to enable a differentiation program.


Assuntos
Eritropoese , Exorribonucleases/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Exossomos/metabolismo , Processamento Pós-Transcricional do RNA , Animais , Apoptose , Células Cultivadas , Quebras de DNA de Cadeia Dupla , Eritroblastos/citologia , Eritroblastos/metabolismo , Exorribonucleases/genética , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Exossomos/genética , Fator de Transcrição GATA1/metabolismo , Humanos , Mutação com Perda de Função , Camundongos , Camundongos Endogâmicos C57BL , Transcriptoma
9.
iScience ; 24(1): 101913, 2021 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-33364592

RESUMO

Cell type annotation is a fundamental task in the analysis of single-cell RNA-sequencing data. In this work, we present CellO, a machine learning-based tool for annotating human RNA-seq data with the Cell Ontology. CellO enables accurate and standardized cell type classification of cell clusters by considering the rich hierarchical structure of known cell types. Furthermore, CellO comes pre-trained on a comprehensive data set of human, healthy, untreated primary samples in the Sequence Read Archive. CellO's comprehensive training set enables it to run out of the box on diverse cell types and achieves competitive or even superior performance when compared to existing state-of-the-art methods. Lastly, CellO's linear models are easily interpreted, thereby enabling exploration of cell-type-specific expression signatures across the ontology. To this end, we also present the CellO Viewer: a web application for exploring CellO's models across the ontology.

10.
Mol Cancer Ther ; 20(1): 161-172, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33177155

RESUMO

Melanoma is one of the most serious forms of skin cancer, and its increasing incidence coupled with nonlasting therapeutic options for metastatic disease highlights the need for additional novel approaches for its management. In this study, we determined the potential interactions between polo-like kinase 1 (PLK1, a serine/threonine kinase involved in mitotic regulation) and NOTCH1 (a type I transmembrane protein deciding cell fate during development) in melanoma. Employing an in-house human melanoma tissue microarray (TMA) containing multiple cases of melanomas and benign nevi, coupled with high-throughput, multispectral quantitative fluorescence imaging analysis, we found a positive correlation between PLK1 and NOTCH1 in melanoma. Furthermore, The Cancer Genome Atlas database analysis of patients with melanoma showed an association of higher mRNA levels of PLK1 and NOTCH1 with poor overall, as well as disease-free, survival. Next, utilizing small-molecule inhibitors of PLK1 and NOTCH (BI 6727 and MK-0752, respectively), we found a synergistic antiproliferative response of combined treatment in multiple human melanoma cells. To determine the molecular targets of the overall and synergistic responses of combined PLK1 and NOTCH inhibition, we conducted RNA-sequencing analysis employing a unique regression model with interaction terms. We identified the modulations of several key genes relevant to melanoma progression/metastasis, including MAPK, PI3K, and RAS, as well as some new genes such as Apobec3G, BTK, and FCER1G, which have not been well studied in melanoma. In conclusion, our study demonstrated a synergistic antiproliferative response of concomitant targeting of PLK1 and NOTCH in melanoma, unraveling a potential novel therapeutic approach for detailed preclinical/clinical evaluation.


Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/metabolismo , Melanoma/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Receptores Notch/antagonistas & inibidores , Receptores Notch/metabolismo , Transdução de Sinais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Ontologia Genética , Pleiotropia Genética , Humanos , Melanoma/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Bibliotecas de Moléculas Pequenas/farmacologia , Análise de Sobrevida , Quinase 1 Polo-Like
11.
Sci Rep ; 10(1): 15716, 2020 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-32973200

RESUMO

Tissue damage induces rapid recruitment of leukocytes and changes in the transcriptional landscape that influence wound healing. However, the cell-type specific transcriptional changes that influence leukocyte function and tissue repair have not been well characterized. Here, we employed translating ribosome affinity purification (TRAP) and RNA sequencing, TRAP-seq, in larval zebrafish to identify genes differentially expressed in neutrophils, macrophages, and epithelial cells in response to wounding. We identified the complement pathway and c3a.1, homologous to the C3 component of human complement, as significantly increased in neutrophils in response to wounds. c3a.1-/- zebrafish larvae have impaired neutrophil directed migration to tail wounds with an initial lag in recruitment early after wounding. Moreover, c3a.1-/- zebrafish larvae have impaired recruitment to localized bacterial infections and reduced survival that is, at least in part, neutrophil mediated. Together, our findings support the power of TRAP-seq to identify cell type specific changes in gene expression that influence neutrophil behavior in response to tissue damage.


Assuntos
Complemento C3/genética , Neutrófilos/metabolismo , Cicatrização/genética , Proteínas de Peixe-Zebra/genética , Animais , Complemento C3/metabolismo , Perfilação da Expressão Gênica , Larva/metabolismo , Análise de Sequência de RNA , Transdução de Sinais/genética , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismo
12.
Genome Res ; 30(11): 1655-1666, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32958497

RESUMO

Publicly available RNA-seq data is routinely used for retrospective analysis to elucidate new biology. Novel transcript discovery enabled by joint analysis of large collections of RNA-seq data sets has emerged as one such analysis. Current methods for transcript discovery rely on a '2-Step' approach where the first step encompasses building transcripts from individual data sets, followed by the second step that merges predicted transcripts across data sets. To increase the power of transcript discovery from large collections of RNA-seq data sets, we developed a novel '1-Step' approach named Pooling RNA-seq and Assembling Models (PRAM) that builds transcript models from pooled RNA-seq data sets. We demonstrate in a computational benchmark that 1-Step outperforms 2-Step approaches in predicting overall transcript structures and individual splice junctions, while performing competitively in detecting exonic nucleotides. Applying PRAM to 30 human ENCODE RNA-seq data sets identified unannotated transcripts with epigenetic and RAMPAGE signatures similar to those of recently annotated transcripts. In a case study, we discovered and experimentally validated new transcripts through the application of PRAM to mouse hematopoietic RNA-seq data sets. We uncovered new transcripts that share a differential expression pattern with a neighboring gene Pik3cg implicated in human hematopoietic phenotypes, and we provided evidence for the conservation of this relationship in human. PRAM is implemented as an R/Bioconductor package.


Assuntos
RNA-Seq/métodos , Animais , Classe Ib de Fosfatidilinositol 3-Quinase/genética , DNA Intergênico , Genômica , Células-Tronco Hematopoéticas/metabolismo , Humanos , Camundongos , RNA/metabolismo , Software
13.
Genome Biol Evol ; 12(8): 1277-1301, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32531054

RESUMO

Island populations repeatedly evolve extreme body sizes, but the genomic basis of this pattern remains largely unknown. To understand how organisms on islands evolve gigantism, we compared genome-wide patterns of gene expression in Gough Island mice, the largest wild house mice in the world, and mainland mice from the WSB/EiJ wild-derived inbred strain. We used RNA-seq to quantify differential gene expression in three key metabolic organs: gonadal adipose depot, hypothalamus, and liver. Between 4,000 and 8,800 genes were significantly differentially expressed across the evaluated organs, representing between 20% and 50% of detected transcripts, with 20% or more of differentially expressed transcripts in each organ exhibiting expression fold changes of at least 2×. A minimum of 73 candidate genes for extreme size evolution, including Irs1 and Lrp1, were identified by considering differential expression jointly with other data sets: 1) genomic positions of published quantitative trait loci for body weight and growth rate, 2) whole-genome sequencing of 16 wild-caught Gough Island mice that revealed fixed single-nucleotide differences between the strains, and 3) publicly available tissue-specific regulatory elements. Additionally, patterns of differential expression across three time points in the liver revealed that Arid5b potentially regulates hundreds of genes. Functional enrichment analyses pointed to cell cycling, mitochondrial function, signaling pathways, inflammatory response, and nutrient metabolism as potential causes of weight accumulation in Gough Island mice. Collectively, our results indicate that extensive gene regulatory evolution in metabolic organs accompanied the rapid evolution of gigantism during the short time house mice have inhabited Gough Island.


Assuntos
Evolução Biológica , Tamanho Corporal/genética , Expressão Gênica , Camundongos/genética , Camundongos/metabolismo , Animais , Feminino , Hipotálamo/metabolismo , Fígado/crescimento & desenvolvimento , Fígado/metabolismo , Masculino , Camundongos/crescimento & desenvolvimento , Locos de Características Quantitativas
14.
Methods Mol Biol ; 1910: 121-147, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31278663

RESUMO

Whole-genome alignment (WGA) is the prediction of evolutionary relationships at the nucleotide level between two or more genomes. It combines aspects of both colinear sequence alignment and gene orthology prediction and is typically more challenging to address than either of these tasks due to the size and complexity of whole genomes. Despite the difficulty of this problem, numerous methods have been developed for its solution because WGAs are valuable for genome-wide analyses such as phylogenetic inference, genome annotation, and function prediction. In this chapter, we discuss the meaning and significance of WGA and present an overview of the methods that address it. We also examine the problem of evaluating whole-genome aligners and offer a set of methodological challenges that need to be tackled in order to make most effective use of our rapidly growing databases of whole genomes.


Assuntos
Biologia Computacional , Genoma , Genômica , Alinhamento de Sequência , Algoritmos , Biologia Computacional/métodos , Bases de Dados Genéticas , Evolução Molecular , Estudo de Associação Genômica Ampla , Genômica/métodos , Alinhamento de Sequência/métodos
15.
Proc Natl Acad Sci U S A ; 116(17): 8310-8319, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30971496

RESUMO

The second messenger nucleotide ppGpp dramatically alters gene expression in bacteria to adjust cellular metabolism to nutrient availability. ppGpp binds to two sites on RNA polymerase (RNAP) in Escherichia coli, but it has also been reported to bind to many other proteins. To determine the role of the RNAP binding sites in the genome-wide effects of ppGpp on transcription, we used RNA-seq to analyze transcripts produced in response to elevated ppGpp levels in strains with/without the ppGpp binding sites on RNAP. We examined RNAs rapidly after ppGpp production without an accompanying nutrient starvation. This procedure enriched for direct effects of ppGpp on RNAP rather than for indirect effects on transcription resulting from starvation-induced changes in metabolism or on secondary events from the initial effects on RNAP. The transcriptional responses of all 757 genes identified after 5 minutes of ppGpp induction depended on ppGpp binding to RNAP. Most (>75%) were not reported in earlier studies. The regulated transcripts encode products involved not only in translation but also in many other cellular processes. In vitro transcription analysis of more than 100 promoters from the in vivo dataset identified a large collection of directly regulated promoters, unambiguously demonstrated that most effects of ppGpp on transcription in vivo were direct, and allowed comparison of DNA sequences from inhibited, activated, and unaffected promoter classes. Our analysis greatly expands our understanding of the breadth of the stringent response and suggests promoter sequence features that contribute to the specific effects of ppGpp.


Assuntos
Sítios de Ligação/genética , RNA Polimerases Dirigidas por DNA , Escherichia coli/genética , Guanosina Tetrafosfato , Transcrição Gênica/genética , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , Genoma Bacteriano/genética , Guanosina Tetrafosfato/química , Guanosina Tetrafosfato/genética , Guanosina Tetrafosfato/metabolismo , Regiões Promotoras Genéticas/genética , Transcriptoma
16.
Dev Cell ; 42(3): 213-225.e4, 2017 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-28787589

RESUMO

An enhancer with amalgamated E-box and GATA motifs (+9.5) controls expression of the regulator of hematopoiesis GATA-2. While similar GATA-2-occupied elements are common in the genome, occupancy does not predict function, and GATA-2-dependent genetic networks are incompletely defined. A "+9.5-like" element resides in an intron of Samd14 (Samd14-Enh) encoding a sterile alpha motif (SAM) domain protein. Deletion of Samd14-Enh in mice strongly decreased Samd14 expression in bone marrow and spleen. Although steady-state hematopoiesis was normal, Samd14-Enh-/- mice died in response to severe anemia. Samd14-Enh stimulated stem cell factor/c-Kit signaling, which promotes erythrocyte regeneration. Anemia activated Samd14-Enh by inducing enhancer components and enhancer chromatin accessibility. Thus, a GATA-2/anemia-regulated enhancer controls expression of an SAM domain protein that confers survival in anemia. We propose that Samd14-Enh and an ensemble of anemia-responsive enhancers are essential for erythrocyte regeneration in stress erythropoiesis, a vital process in pathologies, including ß-thalassemia, myelodysplastic syndrome, and viral infection.


Assuntos
Anemia/metabolismo , Elementos Facilitadores Genéticos , Eritrócitos/metabolismo , Eritropoese , Fatores de Transcrição GATA/metabolismo , Proteínas/genética , Motivos de Aminoácidos , Animais , Proliferação de Células , Sobrevivência Celular , Eritrócitos/citologia , Fatores de Transcrição GATA/genética , Camundongos , Proteínas/metabolismo , Ativação Transcricional
17.
Dev Biol ; 429(1): 92-104, 2017 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-28689736

RESUMO

The vertebrate retina develops in close proximity to the forebrain and neural crest-derived cartilages of the face and jaw. Coloboma, a congenital eye malformation, is associated with aberrant forebrain development (holoprosencephaly) and with craniofacial defects (frontonasal dysplasia) in humans, suggesting a critical role for cross-lineage interactions during retinal morphogenesis. ZIC2, a zinc-finger transcription factor, is linked to human holoprosencephaly. We have previously used morpholino assays to show zebrafish zic2 functions in the developing forebrain, retina and craniofacial cartilage. We now report that zebrafish with genetic lesions in zebrafish zic2 orthologs, zic2a and zic2b, develop with retinal coloboma and craniofacial anomalies. We demonstrate a requirement for zic2 in restricting pax2a expression and show evidence that zic2 function limits Hh signaling. RNA-seq transcriptome analysis identified an early requirement for zic2 in periocular neural crest as an activator of alx1, a transcription factor with essential roles in craniofacial and ocular morphogenesis in human and zebrafish. Collectively, these data establish zic2 mutant zebrafish as a powerful new genetic model for in-depth dissection of cell interactions and genetic controls during craniofacial complex development.


Assuntos
Corioide/embriologia , Corioide/metabolismo , Morfogênese , Crista Neural/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Cartilagem/efeitos dos fármacos , Cartilagem/metabolismo , Linhagem da Célula/efeitos dos fármacos , Linhagem da Célula/genética , Coloboma/patologia , Face/embriologia , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Morfogênese/efeitos dos fármacos , Morfogênese/genética , Mutação/genética , Crista Neural/citologia , Crista Neural/efeitos dos fármacos , Fator de Transcrição PAX2/genética , Fator de Transcrição PAX2/metabolismo , Retina/efeitos dos fármacos , Retina/embriologia , Análise de Sequência de RNA , Homologia de Sequência de Aminoácidos , Crânio/embriologia , Fatores de Transcrição/genética , Alcaloides de Veratrum/farmacologia , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética
18.
Bioinformatics ; 33(18): 2914-2923, 2017 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-28535296

RESUMO

MOTIVATION: The NCBI's Sequence Read Archive (SRA) promises great biological insight if one could analyze the data in the aggregate; however, the data remain largely underutilized, in part, due to the poor structure of the metadata associated with each sample. The rules governing submissions to the SRA do not dictate a standardized set of terms that should be used to describe the biological samples from which the sequencing data are derived. As a result, the metadata include many synonyms, spelling variants and references to outside sources of information. Furthermore, manual annotation of the data remains intractable due to the large number of samples in the archive. For these reasons, it has been difficult to perform large-scale analyses that study the relationships between biomolecular processes and phenotype across diverse diseases, tissues and cell types present in the SRA. RESULTS: We present MetaSRA, a database of normalized SRA human sample-specific metadata following a schema inspired by the metadata organization of the ENCODE project. This schema involves mapping samples to terms in biomedical ontologies, labeling each sample with a sample-type category, and extracting real-valued properties. We automated these tasks via a novel computational pipeline. AVAILABILITY AND IMPLEMENTATION: The MetaSRA is available at metasra.biostat.wisc.edu via both a searchable web interface and bulk downloads. Software implementing our computational pipeline is available at http://github.com/deweylab/metasra-pipeline. CONTACT: cdewey@biostat.wisc.edu. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Assuntos
Ontologias Biológicas , Bases de Dados Genéticas , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Metadados , Software , Humanos , Análise de Sequência de DNA/métodos , Análise de Sequência de RNA/métodos , Vocabulário Controlado
19.
Dev Biol ; 426(2): 143-154, 2017 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-27475628

RESUMO

The axolotl (Ambystoma mexicanum) has long been the subject of biological research, primarily owing to its outstanding regenerative capabilities. However, the gene expression programs governing its embryonic development are particularly underexplored, especially when compared to other amphibian model species. Therefore, we performed whole transcriptome polyA+ RNA sequencing experiments on 17 stages of embryonic development. As the axolotl genome is unsequenced and its gene annotation is incomplete, we built de novo transcriptome assemblies for each stage and garnered functional annotation by comparing expressed contigs with known genes in other organisms. In evaluating the number of differentially expressed genes over time, we identify three waves of substantial transcriptome upheaval each followed by a period of relative transcriptome stability. The first wave of upheaval is between the one and two cell stage. We show that the number of differentially expressed genes per unit time is higher between the one and two cell stage than it is across the mid-blastula transition (MBT), the period of zygotic genome activation. We use total RNA sequencing to demonstrate that the vast majority of genes with increasing polyA+ signal between the one and two cell stage result from polyadenylation rather than de novo transcription. The first stable phase begins after the two cell stage and continues until the mid-blastula transition, corresponding with the pre-MBT phase of transcriptional quiescence in amphibian development. Following this is a peak of differential gene expression corresponding with the activation of the zygotic genome and a phase of transcriptomic stability from stages 9-11. We observe a third wave of transcriptomic change between stages 11 and 14, followed by a final stable period. The last two stable phases have not been documented in amphibians previously and correspond to times of major morphogenic change in the axolotl embryo: gastrulation and neurulation. These results yield new insights into global gene expression during early stages of amphibian embryogenesis and will help to further develop the axolotl as a model species for developmental and regenerative biology.


Assuntos
Ambystoma mexicanum/embriologia , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Transcriptoma , Animais , Mapeamento de Sequências Contíguas , Ontologia Genética , Morfogênese/genética , Família Multigênica , RNA Mensageiro/genética , Análise de Sequência de RNA , Transcrição Gênica
20.
Genome Res ; 26(8): 1124-33, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27405803

RESUMO

RNA-seq is currently the technology of choice for global measurement of transcript abundances in cells. Despite its successes, isoform-level quantification remains difficult because short RNA-seq reads are often compatible with multiple alternatively spliced isoforms. Existing methods rely heavily on uniquely mapping reads, which are not available for numerous isoforms that lack regions of unique sequence. To improve quantification accuracy in such difficult cases, we developed a novel computational method, prior-enhanced RSEM (pRSEM), which uses a complementary data type in addition to RNA-seq data. We found that ChIP-seq data of RNA polymerase II and histone modifications were particularly informative in this approach. In qRT-PCR validations, pRSEM was shown to be superior than competing methods in estimating relative isoform abundances within or across conditions. Data-driven simulations suggested that pRSEM has a greatly decreased false-positive rate at the expense of a small increase in false-negative rate. In aggregate, our study demonstrates that pRSEM transforms existing capacity to precisely estimate transcript abundances, especially at the isoform level.


Assuntos
Processamento Alternativo/genética , RNA/genética , Análise de Sequência de RNA/métodos , Algoritmos , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , RNA Polimerase II/genética , Software
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