RESUMO
Transposable elements (TEs) drive genome evolution and are a notable source of pathogenesis, including cancer. While CpG methylation regulates TE activity, the locus-specific methylation landscape of mobile human TEs has to date proven largely inaccessible. Here, we apply new computational tools and long-read nanopore sequencing to directly infer CpG methylation of novel and extant TE insertions in hippocampus, heart, and liver, as well as paired tumor and non-tumor liver. As opposed to an indiscriminate stochastic process, we find pronounced demethylation of young long interspersed element 1 (LINE-1) retrotransposons in cancer, often distinct to the adjacent genome and other TEs. SINE-VNTR-Alu (SVA) retrotransposons, including their internal tandem repeat-associated CpG island, are near-universally methylated. We encounter allele-specific TE methylation and demethylation of aberrantly expressed young LINE-1s in normal tissues. Finally, we recover the complete sequences of tumor-specific LINE-1 insertions and their retrotransposition hallmarks, demonstrating how long-read sequencing can simultaneously survey the epigenome and detect somatic TE mobilization.
Assuntos
Metilação de DNA , Elementos de DNA Transponíveis , DNA de Neoplasias , Epigênese Genética , Epigenoma , Regulação Neoplásica da Expressão Gênica , Elementos Nucleotídeos Longos e Dispersos , Sequenciamento por Nanoporos , Neoplasias , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Pessoa de Meia-Idade , Neoplasias/genética , Neoplasias/metabolismo , Especificidade de ÓrgãosRESUMO
Epigenetic silencing defends against LINE-1 (L1) retrotransposition in mammalian cells. However, the mechanisms that repress young L1 families and how L1 escapes to cause somatic genome mosaicism in the brain remain unclear. Here we report that a conserved Yin Yang 1 (YY1) transcription factor binding site mediates L1 promoter DNA methylation in pluripotent and differentiated cells. By analyzing 24 hippocampal neurons with three distinct single-cell genomic approaches, we characterized and validated a somatic L1 insertion bearing a 3' transduction. The source (donor) L1 for this insertion was slightly 5' truncated, lacked the YY1 binding site, and was highly mobile when tested in vitro. Locus-specific bisulfite sequencing revealed that the donor L1 and other young L1s with mutated YY1 binding sites were hypomethylated in embryonic stem cells, during neurodifferentiation, and in liver and brain tissue. These results explain how L1 can evade repression and retrotranspose in the human body.
Assuntos
Repressão Epigenética/genética , Elementos Nucleotídeos Longos e Dispersos/genética , Retroelementos/genética , Fator de Transcrição YY1/genética , Sítios de Ligação/genética , Metilação de DNA/genética , Proteínas de Ligação a DNA/genética , Genoma Humano/genética , Hipocampo/metabolismo , Humanos , Fígado/metabolismo , Neurônios/metabolismo , Análise de Célula ÚnicaRESUMO
Pseudogenes are defined as regions of the genome that contain defective copies of genes. They exist across almost all forms of life, and in mammalian genomes are annotated in similar numbers to recognized protein-coding genes. Although often presumed to lack function, growing numbers of pseudogenes are being found to play important biological roles. In consideration of their evolutionary origins and inherent limitations in genome annotation practices, we posit that pseudogenes have been classified on a scientifically unsubstantiated basis. We reflect that a broad misunderstanding of pseudogenes, perpetuated in part by the pejorative inference of the 'pseudogene' label, has led to their frequent dismissal from functional assessment and exclusion from genomic analyses. With the advent of technologies that simplify the study of pseudogenes, we propose that an objective reassessment of these genomic elements will reveal valuable insights into genome function and evolution.
Assuntos
Pseudogenes , Animais , Evolução Molecular , Genômica , HumanosRESUMO
The retrotransposon LINE-1 (L1) is central to the recent evolutionary history of the human genome and continues to drive genetic diversity and germline pathogenesis. However, the spatiotemporal extent and biological significance of somatic L1 activity are poorly defined and are virtually unexplored in other primates. From a single L1 lineage active at the divergence of apes and Old World monkeys, successive L1 subfamilies have emerged in each descendant primate germline. As revealed by case studies, the presently active human L1 subfamily can also mobilize during embryonic and brain development in vivo. It is unknown whether nonhuman primate L1s can similarly generate somatic insertions in the brain. Here we applied approximately 40× single-cell whole-genome sequencing (scWGS), as well as retrotransposon capture sequencing (RC-seq), to 20 hippocampal neurons from two rhesus macaques (Macaca mulatta). In one animal, we detected and PCR-validated a somatic L1 insertion that generated target site duplications, carried a short 5' transduction, and was present in â¼7% of hippocampal neurons but absent from cerebellum and nonbrain tissues. The corresponding donor L1 allele was exceptionally mobile in vitro and was embedded in PRDM4, a gene expressed throughout development and in neural stem cells. Nanopore long-read methylome and RNA-seq transcriptome analyses indicated young retrotransposon subfamily activation in the early embryo, followed by repression in adult tissues. These data highlight endogenous macaque L1 retrotransposition potential, provide prototypical evidence of L1-mediated somatic mosaicism in a nonhuman primate, and allude to L1 mobility in the brain over the past 30 million years of human evolution.
Assuntos
Encéfalo , Elementos Nucleotídeos Longos e Dispersos , Retroelementos , Animais , Proteínas de Ligação a DNA/genética , Macaca mulatta/genética , Neurônios , Retroelementos/genética , Fatores de Transcrição/genéticaRESUMO
SUMMARY: Methylartist is a consolidated suite of tools for processing, visualizing and analysing nanopore-derived modified base calls. All detectable methylation types (e.g. 5mCpG, 5hmC, 6mA) are supported, enabling integrated study of base pairs when modified naturally or as part of an experimental protocol. AVAILABILITY AND IMPLEMENTATION: Methylartist is implemented in Python and is installable via PyPI and bioconda. Source code and test data are available at https://github.com/adamewing/methylartist. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Nanoporos , SoftwareRESUMO
Processed pseudogenes may serve as a genetic reservoir for evolutionary innovation. Here, we argue that through the activity of long interspersed element-1 retrotransposons, processed pseudogenes disperse coding and noncoding sequences rich with regulatory potential throughout the human genome. While these sequences may appear to be non-functional, a lack of contemporary function does not prohibit future development of biological activity. Here, we discuss the dynamic evolution of certain processed pseudogenes into coding and noncoding genes and regulatory elements, and their implication in wide-ranging biological and pathological processes. Also see the video abstract here: https://youtu.be/iUY_mteVoPI.
Assuntos
Pseudogenes , Retroelementos , Evolução Molecular , Genoma Humano/genética , Humanos , Elementos Nucleotídeos Longos e Dispersos , Pseudogenes/genética , Retroelementos/genéticaRESUMO
The interaction of proteins and RNA with chromatin underlies the regulation of gene expression. The ability to profile easily these interactions is fundamental for understanding chromatin biology in vivo DNA adenine methyltransferase identification (DamID) profiles genome-wide protein-DNA interactions without antibodies, fixation or protein pull-downs. Recently, DamID has been adapted for applications beyond simple assaying of protein-DNA interactions, such as for studying RNA-chromatin interactions, chromatin accessibility and long-range chromosome interactions. Here, we provide an overview of DamID and introduce improvements to the technology, discuss their applications and compare alternative methodologies.
Assuntos
Cromatina/metabolismo , Metilação de DNA , Técnicas Genéticas , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , Animais , Sítios de Ligação/genética , Drosophila melanogaster/metabolismo , Regulação da Expressão Gênica , Genômica , Humanos , Camundongos , Ligação Proteica , Ribossomos/metabolismo , Análise de Sequência de DNARESUMO
The precise control of gene expression by transcription factor networks is crucial to organismal development. The predominant approach for mapping transcription factor-chromatin interactions has been chromatin immunoprecipitation (ChIP). However, ChIP requires a large number of homogeneous cells and antisera with high specificity. A second approach, DamID, has the drawback that high levels of Dam methylase are toxic. Here, we modify our targeted DamID approach (TaDa) to enable cell type-specific expression in mammalian systems, generating an inducible system (mammalian TaDa or MaTaDa) to identify genome-wide protein/DNA interactions in 100 to 1000 times fewer cells than ChIP-based approaches. We mapped the binding sites of two key pluripotency factors, OCT4 and PRDM14, in mouse embryonic stem cells, epiblast-like cells and primordial germ cell-like cells (PGCLCs). PGCLCs are an important system for elucidating primordial germ cell development in mice. We monitored PRDM14 binding during the specification of PGCLCs, identifying direct targets of PRDM14 that are key to understanding its crucial role in PGCLC development. We show that MaTaDa is a sensitive and accurate method for assessing cell type-specific transcription factor binding in limited numbers of cells.
Assuntos
Metilação de DNA/genética , Células-Tronco Pluripotentes/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sítios de Ligação , Cromatina/metabolismo , Proteínas de Ligação a DNA , Genoma , Células Germinativas/metabolismo , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Ligação Proteica , Proteínas de Ligação a RNARESUMO
Basal cell nevus syndrome (also known as Gorlin Syndrome; MIM109400) is an autosomal dominant disorder characterized by recurrent pathological features such as basal cell carcinomas and odontogenic keratocysts as well as skeletal abnormalities. Most affected individuals have point mutations or small insertions or deletions within the PTCH1 gene on human chromosome 9, but there are some cases with more extensive deletion of the region, usually including the neighboring FANCC and/or ERCC6L2 genes. We report a 16-year-old patient with a deletion of approximately 400,000 bases which removes only PTCH1 and some non-coding RNA genes but leaves FANCC and ERCC6L2 intact. In spite of the small amount of DNA for which he is haploid, his phenotype is more extreme than many individuals with longer deletions in the region. This includes early presentation with a large number of basal cell nevi and other skin lesions, multiple jaw keratocysts, and macrosomia. We found that the deletion was in the paternal chromosome, in common with other macrosomia cases. Using public databases, we have examined possible interactions between sequences within and outside the deletion and speculate that a regulatory relationship exists with flanking genes, which is unbalanced by the deletion, resulting in abnormal activation or repression of the target genes and hence the severity of the phenotype.
Assuntos
Síndrome do Nevo Basocelular/genética , DNA Helicases/genética , Proteína do Grupo de Complementação C da Anemia de Fanconi/genética , Receptor Patched-1/genética , Adolescente , Síndrome do Nevo Basocelular/epidemiologia , Síndrome do Nevo Basocelular/patologia , Criança , Pré-Escolar , Transtornos Cromossômicos/genética , Transtornos Cromossômicos/patologia , Cromossomos Humanos Par 9/genética , Predisposição Genética para Doença , Humanos , Lactente , Recém-Nascido , Masculino , Recidiva Local de Neoplasia/epidemiologia , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/patologia , Cistos Odontogênicos/genética , Cistos Odontogênicos/patologia , Fenótipo , Índice de Gravidade de DoençaRESUMO
Genomic regulation of cardiomyocyte differentiation is central to heart development and function. This study uses genetic loss-of-function human-induced pluripotent stem cell-derived cardiomyocytes to evaluate the genomic regulatory basis of the non-DNA-binding homeodomain protein HOPX. We show that HOPX interacts with and controls cardiac genes and enhancer networks associated with diverse aspects of heart development. Using perturbation studies in vitro, we define how upstream cell growth and proliferation control HOPX transcription to regulate cardiac gene programs. We then use cell, organoid, and zebrafish regeneration models to demonstrate that HOPX-regulated gene programs control cardiomyocyte function in development and disease. Collectively, this study mechanistically links cell signaling pathways as upstream regulators of HOPX transcription to control gene programs underpinning cardiomyocyte identity and function.
Assuntos
Células-Tronco Pluripotentes Induzidas , Miócitos Cardíacos , Animais , Humanos , Miócitos Cardíacos/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Peixe-Zebra/metabolismo , Diferenciação Celular/genética , Proliferação de CélulasRESUMO
A mechanistic connection between aging and development is largely unexplored. Through profiling age-related chromatin and transcriptional changes across 22 murine cell types, analyzed alongside previous mouse and human organismal maturation datasets, we uncovered a transcription factor binding site (TFBS) signature common to both processes. Early-life candidate cis-regulatory elements (cCREs), progressively losing accessibility during maturation and aging, are enriched for cell-type identity TFBSs. Conversely, cCREs gaining accessibility throughout life have a lower abundance of cell identity TFBSs but elevated activator protein 1 (AP-1) levels. We implicate TF redistribution toward these AP-1 TFBS-rich cCREs, in synergy with mild downregulation of cell identity TFs, as driving early-life cCRE accessibility loss and altering developmental and metabolic gene expression. Such remodeling can be triggered by elevating AP-1 or depleting repressive H3K27me3. We propose that AP-1-linked chromatin opening drives organismal maturation by disrupting cell identity TFBS-rich cCREs, thereby reprogramming transcriptome and cell function, a mechanism hijacked in aging through ongoing chromatin opening.
Assuntos
Envelhecimento , Cromatina , Fator de Transcrição AP-1 , Animais , Envelhecimento/genética , Envelhecimento/metabolismo , Fator de Transcrição AP-1/metabolismo , Cromatina/metabolismo , Camundongos , Humanos , Camundongos Endogâmicos C57BL , Sítios de LigaçãoRESUMO
MOTIVATION: Comparing transcriptomic data with proteomic data to identify protein-coding sequences is a long-standing challenge in molecular biology, one that is exacerbated by the increasing size of high-throughput datasets. To address this challenge, and thereby to improve the quality of genome annotation and understanding of genome biology, we have developed an integrated suite of programs, called Pinstripe. We demonstrate its application, utility and discovery power using transcriptomic and proteomic data from publicly available datasets. RESULTS: To demonstrate the efficacy of Pinstripe for large-scale analysis, we applied Pinstripe's reverse peptide mapping pipeline to a transcript library including de novo assembled transcriptomes from the human Illumina Body Atlas (IBA2) and GENCODE v10 gene annotations, and the EBI Proteomics Identifications Database (PRIDE) peptide database. This analysis identified 736 canonical open reading frames (ORFs) supported by three or more PRIDE peptide fragments that are positioned outside any known coding DNA sequence (CDS). Because of the unfiltered nature of the PRIDE database and high probability of false discovery, we further refined this list using independent evidence for translation, including the presence of a Kozak sequence or functional domains, synonymous/non-synonymous substitution ratios and ORF length. Using this integrative approach, we observed evidence of translation from a previously unknown let7e primary transcript, the archetypical lncRNA H19, and a homolog of RD3. Reciprocally, by exclusion of transcripts with mapped peptides or significant ORFs (>80 codon), we identify 32 187 loci with RNAs longer than 2000 nt that are unlikely to encode proteins. AVAILABILITY AND IMPLEMENTATION: Pinstripe (pinstripe.matticklab.com) is freely available as source code or a Mono binary. Pinstripe is written in C# and runs under the Mono framework on Linux or Mac OS X, and both under Mono and .Net under Windows. CONTACT: m.dinger@garvan.org.au or j.mattick@garvan.org.au SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Perfilação da Expressão Gênica/métodos , Genômica/métodos , Proteômica/métodos , Software , Biologia Computacional/métodos , Bases de Dados de Proteínas , Éxons , Biblioteca Gênica , Genoma , Humanos , Anotação de Sequência Molecular , Fases de Leitura Aberta , Proteínas/genética , RNA Longo não Codificante/genética , RNA Mensageiro/genética , Análise de Sequência de RNARESUMO
Over the past decades, there has been an exponential increase in the development of preclinical and clinical nanodelivery systems, and recently, an accelerating demand to deliver RNA and protein-based therapeutics. Organ-specific vasculature provides a promising intermediary for site-specific delivery of nanoparticles and extracellular vesicles to interstitial cells. Endothelial cells express organ-specific surface marker repertoires that can be used for targeted delivery. This article highlights organ-specific vasculature properties, nanodelivery strategies that exploit vasculature organotropism, and overlooked challenges and opportunities in targeting and simultaneously overcoming the endothelial barrier. Impediments in the clinical translation of vasculature organotropism in drug delivery are also discussed.
Assuntos
Portadores de Fármacos , Nanopartículas , Humanos , Células Endoteliais , Sistemas de Liberação de Medicamentos , Sistemas de Liberação de Fármacos por NanopartículasRESUMO
The success of mRNA vaccines has been realised, in part, by advances in manufacturing that enabled billions of doses to be produced at sufficient quality and safety. However, mRNA vaccines must be rigorously analysed to measure their integrity and detect contaminants that reduce their effectiveness and induce side-effects. Currently, mRNA vaccines and therapies are analysed using a range of time-consuming and costly methods. Here we describe a streamlined method to analyse mRNA vaccines and therapies using long-read nanopore sequencing. Compared to other industry-standard techniques, VAX-seq can comprehensively measure key mRNA vaccine quality attributes, including sequence, length, integrity, and purity. We also show how direct RNA sequencing can analyse mRNA chemistry, including the detection of nucleoside modifications. To support this approach, we provide supporting software to automatically report on mRNA and plasmid template quality and integrity. Given these advantages, we anticipate that RNA sequencing methods, such as VAX-seq, will become central to the development and manufacture of mRNA drugs.
Assuntos
Comércio , Vacinas de mRNA , RNA Mensageiro/genética , Análise de Sequência de RNARESUMO
The Notch signalling pathway is a master regulator of cell fate transitions in development and disease. In the brain, Notch promotes neural stem cell (NSC) proliferation, regulates neuronal migration and maturation and can act as an oncogene or tumour suppressor. How NOTCH and its transcription factor RBPJ activate distinct gene regulatory networks in closely related cell types in vivo remains to be determined. Here we use Targeted DamID (TaDa), requiring only thousands of cells, to identify NOTCH and RBPJ binding in NSCs and their progeny in the mouse embryonic cerebral cortex in vivo. We find that NOTCH and RBPJ associate with a broad network of NSC genes. Repression of NSC-specific Notch target genes in intermediate progenitors and neurons correlates with decreased chromatin accessibility, suggesting that chromatin compaction may contribute to restricting NOTCH-mediated transactivation.
Assuntos
Cromatina , Células-Tronco Neurais , Animais , Diferenciação Celular/fisiologia , Camundongos , Células-Tronco Neurais/metabolismo , Receptores Notch/metabolismo , Transdução de SinaisRESUMO
Pseudogenes are gene copies presumed to mainly be functionless relics of evolution due to acquired deleterious mutations or transcriptional silencing. Using deep full-length PacBio cDNA sequencing of normal human tissues and cancer cell lines, we identify here hundreds of novel transcribed pseudogenes expressed in tissue-specific patterns. Some pseudogene transcripts have intact open reading frames and are translated in cultured cells, representing unannotated protein-coding genes. To assess the biological impact of noncoding pseudogenes, we CRISPR-Cas9 delete the nucleus-enriched pseudogene PDCL3P4 and observe hundreds of perturbed genes. This study highlights pseudogenes as a complex and dynamic component of the human transcriptional landscape.
Assuntos
DNA Complementar/genética , Pseudogenes , Análise de Sequência de DNA , Transcriptoma/genética , Linhagem Celular , Deleção de Genes , Haploidia , Humanos , Regiões Promotoras Genéticas/genéticaRESUMO
Genetic studies of autism have revealed causal roles for chromatin remodeling gene mutations. Chromodomain helicase DNA binding protein 8 (CHD8) encodes a chromatin remodeler with significant de novo mutation rates in sporadic autism. However, relationships between CHD8 genomic function and autism-relevant biology remain poorly elucidated. Published studies utilizing ChIP-seq to map CHD8 protein-DNA interactions have high variability, consistent with technical challenges and limitations associated with this method. Thus, complementary approaches are needed to establish CHD8 genomic targets and regulatory functions in developing brain. We used in utero CHD8 Targeted DamID followed by sequencing (TaDa-seq) to characterize CHD8 binding in embryonic mouse cortex. CHD8 TaDa-seq reproduced interaction patterns observed from ChIP-seq and further highlighted CHD8 distal interactions associated with neuronal loci. This study establishes TaDa-seq as a useful alternative for mapping protein-DNA interactions in vivo and provides insights into the regulatory targets of CHD8 and autism-relevant pathophysiology associated with CHD8 mutations.
RESUMO
Long-noncoding RNAs (lncRNAs) are emerging as regulators of development and disease. lncRNAs are expressed in exquisitely precise expression patterns in vivo and many interact with chromatin to regulate gene expression. However, the limited sensitivity of RNA-purification techniques has precluded the identification of genomic targets of cell-type specific lncRNAs. RNA-DamID is a powerful new approach to understand the mechanisms by which lncRNAs act in vivo. RNA-DamID is highly sensitive and accurate, and can resolve cell-type-specific chromatin binding patterns without cell isolation. The determinants of RNA-chromatin interactions can be identified with RNA-DamID by analyzing RNA and protein cofactor mutants. Here we describe how to implement RNA-DamID and the design considerations to take into account to accurately identify lncRNA-chromatin interactions in vivo.
Assuntos
Cromatina/metabolismo , RNA Longo não Codificante/química , Análise de Sequência/métodos , Animais , Linhagem Celular , Células Cultivadas , Cromatina/química , Metilação de DNA , Humanos , Ligação Proteica , Mapeamento de Interação de Proteínas , RNA Longo não Codificante/metabolismoRESUMO
Thousands of long noncoding RNAs (lncRNAs) have been identified in eukaryotic genomes, many of which are expressed in spatially and temporally restricted patterns. Nonetheless, the roles of the majority of these transcripts are still unknown. One of the mechanisms by which lncRNAs function is through the modulation of chromatin states. To assess the functions of lncRNAs, we developed RNA-DamID, a novel approach that detects lncRNA-genome interactions in a cell-type-specific manner in vivo with high sensitivity and accuracy. Identifying the cell-type-specific genome occupancy of lncRNAs is vital to understanding their mechanisms of action in development and disease. We used RNA-DamID to investigate targeting of the lncRNAs in the Drosophila dosage-compensation complex (DCC) and show that initial targeting is cell-type specific.
Assuntos
Cromatina/química , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Proteínas Nucleares/genética , RNA Longo não Codificante/química , Proteínas de Ligação a RNA/genética , Fatores de Transcrição/genética , Aminoidrolases/metabolismo , Animais , Proteínas de Ligação a DNA/química , Mecanismo Genético de Compensação de Dose , Proteínas de Drosophila/química , Drosophila melanogaster/genética , Feminino , Genótipo , Masculino , Células-Tronco Neurais/metabolismo , Proteínas Nucleares/química , Glândulas Salivares/metabolismo , Fatores de Transcrição/químicaRESUMO
Cellular responses to stimuli are rapid and continuous and yet the vast majority of investigations of transcriptional responses during developmental transitions typically use long interval time courses; limiting the available interpretive power. Moreover, such experiments typically focus on protein-coding transcripts, ignoring the important impact of long noncoding RNAs. We therefore evaluated coding and noncoding expression dynamics at unprecedented temporal resolution (6-hourly) in differentiating mouse embryonic stem cells and report new insight into molecular processes and genome organization. We present a highly resolved differentiation cascade that exhibits coding and noncoding transcriptional alterations, transcription factor network interactions and alternative splicing events, little of which can be resolved by long-interval developmental time-courses. We describe novel short lived and cycling patterns of gene expression and dissect temporally ordered gene expression changes in response to transcription factors. We elucidate patterns in gene co-expression across the genome, describe asynchronous transcription at bidirectional promoters and functionally annotate known and novel regulatory lncRNAs. These findings highlight the complex and dynamic molecular events underlying mammalian differentiation that can only be observed though a temporally resolved time course.