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Microscopy-based spatially resolved omic methods are transforming the life sciences. However, these methods rely on high numerical aperture objectives and cannot resolve crowded molecular targets, limiting the amount of extractable biological information. To overcome these limitations, here we develop Deconwolf, an open-source, user-friendly software for high-performance deconvolution of widefield fluorescence microscopy images, which efficiently runs on laptop computers. Deconwolf enables accurate quantification of crowded diffraction limited fluorescence dots in DNA and RNA fluorescence in situ hybridization images and allows robust detection of individual transcripts in tissue sections imaged with ×20 air objectives. Deconvolution of in situ spatial transcriptomics images with Deconwolf increased the number of transcripts identified more than threefold, while the application of Deconwolf to images obtained by fluorescence in situ sequencing of barcoded Oligopaint probes drastically improved chromosome tracing. Deconwolf greatly facilitates the use of deconvolution in many bioimaging applications.
Assuntos
Processamento de Imagem Assistida por Computador , Hibridização in Situ Fluorescente , Microscopia de Fluorescência , Software , Microscopia de Fluorescência/métodos , Hibridização in Situ Fluorescente/métodos , Processamento de Imagem Assistida por Computador/métodos , Animais , Camundongos , HumanosRESUMO
Single-cell RNA sequencing studies on gene co-expression patterns could yield important regulatory and functional insights, but have so far been limited by the confounding effects of differentiation and cell cycle. We apply a tailored experimental design that eliminates these confounders, and report thousands of intrinsically covarying gene pairs in mouse embryonic stem cells. These covariations form a network with biological properties, outlining known and novel gene interactions. We provide the first evidence that miRNAs naturally induce transcriptome-wide covariations and compare the relative importance of nuclear organization, transcriptional and post-transcriptional regulation in defining covariations. We find that nuclear organization has the greatest impact, and that genes encoding for physically interacting proteins specifically tend to covary, suggesting importance for protein complex formation. Our results lend support to the concept of post-transcriptional RNA operons, but we further present evidence that nuclear proximity of genes may provide substantial functional regulation in mammalian single cells.
Assuntos
Núcleo Celular/genética , Redes Reguladoras de Genes , Mapas de Interação de Proteínas , Animais , Linhagem Celular , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Variação Genética , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , RNA-Seq , Ribonuclease III/deficiência , Ribonuclease III/genética , Ribonuclease III/metabolismo , Análise de Célula Única , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , TranscriptomaRESUMO
With the exception of lamina-associated domains, the radial organization of chromatin in mammalian cells remains largely unexplored. Here we describe genomic loci positioning by sequencing (GPSeq), a genome-wide method for inferring distances to the nuclear lamina all along the nuclear radius. GPSeq relies on gradual restriction digestion of chromatin from the nuclear lamina toward the nucleus center, followed by sequencing of the generated cut sites. Using GPSeq, we mapped the radial organization of the human genome at 100-kb resolution, which revealed radial patterns of genomic and epigenomic features and gene expression, as well as A and B subcompartments. By combining radial information with chromosome contact frequencies measured by Hi-C, we substantially improved the accuracy of whole-genome structure modeling. Finally, we charted the radial topography of DNA double-strand breaks, germline variants and cancer mutations and found that they have distinctive radial arrangements in A and B subcompartments. We conclude that GPSeq can reveal fundamental aspects of genome architecture.
Assuntos
Núcleo Celular/genética , Cromatina/genética , Epigenômica , Genoma Humano/genética , Regulação da Expressão Gênica/genética , Sequenciamento de Nucleotídeos em Larga Escala , HumanosRESUMO
DNA fluorescence in situ hybridization (DNA FISH) is a powerful method to study chromosomal organization in single cells. At present, there is a lack of free resources of DNA FISH probes and probe design tools which can be readily applied. Here, we describe iFISH, an open-source repository currently comprising 380 DNA FISH probes targeting multiple loci on the human autosomes and chromosome X, as well as a genome-wide database of optimally designed oligonucleotides and a freely accessible web interface ( http://ifish4u.org ) that can be used to design DNA FISH probes. We individually validate 153 probes and take advantage of our probe repository to quantify the extent of intermingling between multiple heterologous chromosome pairs, showing a much higher extent of intermingling in human embryonic stem cells compared to fibroblasts. In conclusion, iFISH is a versatile and expandable resource, which can greatly facilitate the use of DNA FISH in research and diagnostics.
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Sondas de DNA/genética , Bases de Dados de Ácidos Nucleicos , Genoma Humano/genética , Hibridização in Situ Fluorescente/métodos , Células A549 , Mapeamento Cromossômico/métodos , Cromossomos Humanos/genética , Fibroblastos , Células-Tronco Embrionárias Humanas , Humanos , Oligonucleotídeos/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Projetos de PesquisaRESUMO
DNA fluorescence in situ hybridization (DNA FISH) has emerged as a powerful microscopy technique that allows a unique view into the composition and arrangement of the genetic material in its natural context-be it the cell nucleus in interphase, or chromosomes in metaphase spreads. The core principle of DNA FISH is the ability of fluorescently labeled DNA probes (either double- or single-stranded DNA fragments) to bind to their complementary sequences in situ in cells or tissues, revealing the location of their target as fluorescence signals detectable with a fluorescence microscope. Numerous variants and improvements of the original DNA FISH method as well as a vast repertoire of applications have been described since its inception more than 4 decades ago. In recent years, the development of many new fluorescent dyes together with drastic advancements in methods for probe generation (Boyle et al., Chromosome Res 19:901-909, 2011; Beliveau et al., Proc Natl Acad Sci U S A 109:21301-21306, 2012; Bienko et al., Nat Methods 10:122-124, 2012), as well as improvements in the resolution of microscopy technologies, have boosted the number of DNA FISH applications, particularly in the field of genome architecture (Markaki et al., Bioessays 34:412-426, 2012; Beliveau et al., Nat Commun 6:7147, 2015). However, despite these remarkable steps forward, choosing which type of DNA FISH sample preparation protocol, probe design, hybridization procedure, and detection method is best suited for a given application remains still challenging for many research labs, preventing a more widespread use of this powerful technology. Here, we present a comprehensive platform to help researchers choose which DNA FISH protocol is most suitable for their particular application. In addition, we describe computational pipelines that can be implemented for efficient DNA FISH probe design and for signal quantification. Our goal is to make DNA FISH a versatile and streamlined technique that can be easily implemented by both research and diagnostic labs.
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Hibridização in Situ Fluorescente , Animais , Sequência de Bases , Núcleo Celular/genética , DNA/química , DNA/metabolismo , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Genoma/genética , Humanos , Sensibilidade e Especificidade , Análise de Célula Única , Software , Design de SoftwareRESUMO
The original version of the chapter was inadvertently published with some errors and this has been corrected now.
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Emerging data demonstrate homologous recombination (HR) defects in castration-resistant prostate cancers, rendering these tumours sensitive to PARP inhibition. Here we demonstrate a direct requirement for the androgen receptor (AR) to maintain HR gene expression and HR activity in prostate cancer. We show that PARP-mediated repair pathways are upregulated in prostate cancer following androgen-deprivation therapy (ADT). Furthermore, upregulation of PARP activity is essential for the survival of prostate cancer cells and we demonstrate a synthetic lethality between ADT and PARP inhibition in vivo. Our data suggest that ADT can functionally impair HR prior to the development of castration resistance and that, this potentially could be exploited therapeutically using PARP inhibitors in combination with androgen-deprivation therapy upfront in advanced or high-risk prostate cancer.Tumours with homologous recombination (HR) defects become sensitive to PARPi. Here, the authors show that androgen receptor (AR) regulates HR and AR inhibition activates the PARP pathway in vivo, thus inhibition of both AR and PARP is required for effective treatment of high risk prostate cancer.
Assuntos
Colágeno Tipo XI/metabolismo , Neoplasias de Próstata Resistentes à Castração/genética , Receptores Androgênicos/metabolismo , Mutações Sintéticas Letais , Colágeno Tipo XI/genética , Recombinação Homóloga , Humanos , Masculino , Neoplasias de Próstata Resistentes à Castração/enzimologia , Neoplasias de Próstata Resistentes à Castração/metabolismo , Receptores Androgênicos/genética , Transdução de SinaisRESUMO
Tyrosine kinase inhibitors targeting the BCR-ABL oncoprotein in chronic myeloid leukemia (CML) are remarkably effective inducing deep molecular remission in most patients. However, they are less effective to eradicate the leukemic stem cells (LSC), resulting in disease persistence. Therefore, there is great need to develop novel therapeutic strategies to specifically target the LSC. In an experimental mouse CML model system, the leukotriene pathway, and specifically, the expression ALOX5, encoding 5-lipoxygenase (5-LO), has been reported as a critical regulator of the LSC. Based on these results, the 5-LO inhibitor zileuton has been introduced in clinical trials as a therapeutic option to target the LSC although its effect on primary human CML LSC has not been studied. We have here by using multiplex single cell PCR analyzed the expression of the mediators of the leukotriene pathway in bone marrow (BM) BCR-ABL+CD34+CD38- cells at diagnosis, and found low or undetectable expression of ALOX5. In line with this, zileuton did not exert significant overall growth inhibition in the long-term culture-initiating cell (LTC-IC) and colony (CFU-C) assays of BM CD34+CD38- cells from 7 CML patients. The majority of the single leukemic BCR-ABL+CD34+CD38- cells expressed cysteinyl leukotriene receptors CYSLT1 and CYSLT2. However, montelukast, an inhibitor of CYSLT1, also failed to significantly suppress CFU-C and LTC-IC growth. These findings indicate that targeting ALOX5 or CYSLT1 signaling with leukotriene antagonists, introduced into the clinical practice primarily as prophylaxis and treatment for asthma, may not be a promising pharmacological strategy to eradicate persisting LSC in CML patients.