RESUMEN
BACKGROUND: Human T cell lymphotropic virus type 1 (HTLV-1) infection remains a largely neglected public health problem, particularly in resource-poor areas with high burden of communicable and non-communicable diseases, such as some remote populations in Central Australia where an estimated 37% of adults are infected with HTLV-1. Most of our understanding of HTLV-1 infection comes from studies of the globally spread subtype-A (HTLV-1a), with few molecular studies reported with the Austral-Melanesian subtype-C (HTLV-1c) predominant in the Indo-Pacific and Oceania regions. RESULTS: Using a primer walking strategy and direct sequencing, we constructed HTLV-1c genomic consensus sequences from 22 First Nations participants living with HTLV-1c in Central Australia. Phylogenetic and pairwise analysis of this subtype-C proviral gDNA showed higher levels of genomic divergence in comparison to previously published HTLV-1a genomes. While the overall genomic homology between subtypes was 92.5%, the lowest nucleotide and amino acid sequence identity occurred near the 3' end of the proviral genome coding regulatory genes, especially overlapping hbz (85.37%, 77.46%, respectively) and orf-I product p12 (82.00%, 70.30%, respectively). Strikingly, the HTLV-1c genomic consensus sequences uniformly showed a defective translation start codon for the immune regulatory proteins p12/p8 encoded by the HTLV-1A orf-I. Deletions in the proviral genome were detected in many subjects, particularly in the structural gag, pol and env genes. Similarly, using a droplet digital PCR assay measuring the copies of gag and tax per reference host genome, we quantitatively confirmed that provirus retains the tax gene region at higher levels than gag. CONCLUSIONS: Our genomic analysis of HTLV-1c in Central Australia in conjunction with earlier Melanesian HTLV-1c sequences, elucidate substantial differences with respect to the globally spread HTLV-1a. Future studies should address the impact these genomic differences have on infection and the regionally distinctive frequency of associated pulmonary disease. Understanding the host and virus subtype factors which contribute to the differential morbidity observed, is crucial for the development of much needed therapeutics and vaccine strategies against this highly endemic infection in remote First Nations communities in Central Australia.
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Infecciones por HTLV-I , Virus Linfotrópico T Tipo 1 Humano , Filogenia , Proteínas de los Retroviridae , Virus Linfotrópico T Tipo 1 Humano/genética , Virus Linfotrópico T Tipo 1 Humano/clasificación , Humanos , Infecciones por HTLV-I/virología , Australia , Proteínas de los Retroviridae/genética , Variación Genética , Adulto , Genoma Viral , Proteínas Reguladoras y Accesorias Virales/genética , Análisis de Secuencia de ADN , Masculino , Femenino , Persona de Mediana Edad , ADN Viral/genética , Proteínas Virales/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter BásicoRESUMEN
Retrotransposons are mobile DNA sequences duplicated via transcription and reverse transcription of an RNA intermediate. Cis-regulatory elements encoded by retrotransposons can also promote the transcription of adjacent genes. Somatic LINE-1 (L1) retrotransposon insertions have been detected in mammalian neurons. It is, however, unclear whether L1 sequences are mobile in only some neuronal lineages or therein promote neurodevelopmental gene expression. Here we report programmed L1 activation by SOX6, a transcription factor critical for parvalbumin (PV) interneuron development. Mouse PV interneurons permit L1 mobilization in vitro and in vivo, harbor unmethylated L1 promoters and express full-length L1 mRNAs and proteins. Using nanopore long-read sequencing, we identify unmethylated L1s proximal to PV interneuron genes, including a novel L1 promoter-driven Caps2 transcript isoform that enhances neuron morphological complexity in vitro. These data highlight the contribution made by L1 cis-regulatory elements to PV interneuron development and transcriptome diversity, uncovered due to L1 mobility in this milieu.
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Interneuronas , Elementos de Nucleótido Esparcido Largo , Parvalbúminas , Animales , Interneuronas/metabolismo , Interneuronas/fisiología , Ratones , Elementos de Nucleótido Esparcido Largo/genética , Parvalbúminas/metabolismo , Retroelementos/genética , Masculino , Neurogénesis/fisiología , Neurogénesis/genética , Ratones Endogámicos C57BL , Regulación del Desarrollo de la Expresión Génica/genéticaRESUMEN
Whole genome sequencing of viruses provides high-resolution molecular insights, enhancing our understanding of viral genome function and phylogeny. Beyond fundamental research, viral sequencing is increasingly vital for pathogen surveillance, epidemiology, and clinical applications. As sequencing methods rapidly evolve, the diversity of viral genomics applications and catalogued genomes continues to expand. Advances in long-read, single molecule, real-time sequencing methodologies present opportunities to sequence contiguous, haplotype resolved viral genomes in a range of research and applied settings. Here we present an overview of nucleic acid sequencing methods and their applications in studying viral genomes. We emphasise the advantages of different viral sequencing approaches, with a particular focus on the benefits of third-generation sequencing technologies in elucidating viral evolution, transmission networks, and pathogenesis.
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Genoma Viral , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Virus/genética , Genómica/métodos , Filogenia , Secuenciación Completa del Genoma/métodos , Análisis de Secuencia de ADN/métodosRESUMEN
The O- GlcNAc transferase OGT interacts robustly with all three mammalian TET methylcytosine dioxygenases. We show here that deletion of the Ogt gene in mouse embryonic stem cells (mESC) results in a widespread increase in the TET product 5-hydroxymethylcytosine (5hmC) in both euchromatic and heterochromatic compartments, with concomitant reduction of the TET substrate 5-methylcytosine (5mC) at the same genomic regions. mESC engineered to abolish the TET1-OGT interaction likewise displayed a genome-wide decrease of 5mC. DNA hypomethylation in OGT-deficient cells was accompanied by de-repression of transposable elements (TEs) predominantly located in heterochromatin, and this increase in TE expression was sometimes accompanied by increased cis -expression of genes and exons located 3' of the expressed TE. Thus, the TET-OGT interaction prevents DNA demethylation and TE expression in heterochromatin by restraining TET activity genome-wide. We suggest that OGT protects the genome against DNA hypomethylation and impaired heterochromatin integrity, preventing the aberrant increase in TE expression observed in cancer, autoimmune-inflammatory diseases, cellular senescence and ageing.
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The interplay between 3D chromatin architecture and gene silencing is incompletely understood. Here, we report a novel point mutation in the non-canonical SMC protein SMCHD1 that enhances its silencing capacity at endogenous developmental targets. Moreover, it also results in enhanced silencing at the facioscapulohumeral muscular dystrophy associated macrosatellite-array, D4Z4, resulting in enhanced repression of DUX4 encoded by this repeat. Heightened SMCHD1 silencing perturbs developmental Hox gene activation, causing a homeotic transformation in mice. Paradoxically, the mutant SMCHD1 appears to enhance insulation against other epigenetic regulators, including PRC2 and CTCF, while depleting long range chromatin interactions akin to what is observed in the absence of SMCHD1. These data suggest that SMCHD1's role in long range chromatin interactions is not directly linked to gene silencing or insulating the chromatin, refining the model for how the different levels of SMCHD1-mediated chromatin regulation interact to bring about gene silencing in normal development and disease.
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Cromatina , Proteínas Cromosómicas no Histona , Distrofia Muscular Facioescapulohumeral , Animales , Ratones , Cromatina/genética , Epigenómica , Silenciador del Gen , Genes Homeobox , Distrofia Muscular Facioescapulohumeral/genética , Proteínas Cromosómicas no Histona/genéticaRESUMEN
Female mouse embryonic stem cells (mESCs) present differently from male mESCs in several fundamental ways; however, complications with their in vitro culture have resulted in an under-representation of female mESCs in the literature. Recent studies show that the second X chromosome in female, and more specifically the transcriptional activity from both of these chromosomes due to absent X chromosome inactivation, sets female and male mESCs apart. To avoid this undesirable state, female mESCs in culture preferentially adopt an XO karyotype, with this adaption leading to loss of their unique properties in favour of a state that is near indistinguishable from male mESCs. If female pluripotency is to be studied effectively in this system, it is crucial that high-quality cultures of XX mESCs are available. Here, we report a method for better maintaining XX female mESCs in culture that also stabilises the male karyotype and makes study of female-specific pluripotency more feasible.
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Células Madre Embrionarias de Ratones , Inactivación del Cromosoma X , Masculino , Animales , Femenino , Ratones , Diferenciación Celular/fisiología , Inactivación del Cromosoma X/genética , CariotipoRESUMEN
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.
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Encéfalo , Elementos de Nucleótido Esparcido Largo , Retroelementos , Animales , Proteínas de Unión al ADN/genética , Macaca mulatta/genética , Neuronas , Retroelementos/genética , Factores de Transcripción/genéticaRESUMEN
The process of epigenetic silencing, while fundamentally important, is not yet completely understood. Here we report a replenishable female mouse embryonic stem cell (mESC) system, Xmas, that allows rapid assessment of X chromosome inactivation (XCI), the epigenetic silencing mechanism of one of the two X chromosomes that enables dosage compensation in female mammals. Through a targeted genetic screen in differentiating Xmas mESCs, we reveal that the BAF complex is required to create nucleosome-depleted regions at promoters on the inactive X chromosome during the earliest stages of establishment of XCI. Without this action gene silencing fails. Xmas mESCs provide a tractable model for screen-based approaches that enable the discovery of unknown facets of the female-specific process of XCI and epigenetic silencing more broadly.
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ARN Largo no Codificante , Inactivación del Cromosoma X , Animales , Cromatina/genética , Compensación de Dosificación (Genética) , Epigénesis Genética , Femenino , Ratones , ARN Largo no Codificante/genética , Cromosoma X/genética , Inactivación del Cromosoma X/genéticaRESUMEN
Endogenous retroviruses (ERVs) are emerging as promising therapeutic targets in cancer. As remnants of ancient retroviral infections, ERV-derived regulatory elements coordinate expression from gene networks, including those underpinning embryogenesis and immune cell function. ERV activation can promote an interferon response, a phenomenon termed viral mimicry. Although ERV expression is associated with cancer, and provisionally with autoimmune and neurodegenerative diseases, ERV-mediated inflammation is being explored as a way to sensitize tumors to immunotherapy. Here we review ERV co-option in development and innate immunity, the aberrant contribution of ERVs to tumorigenesis, and the wider biomedical potential of therapies directed at ERVs.
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Retrovirus Endógenos/fisiología , Neoplasias/terapia , Neoplasias/virología , Animales , Carcinogénesis/genética , Carcinogénesis/patología , Elementos Transponibles de ADN/genética , Redes Reguladoras de Genes , Humanos , Inmunidad Innata , Neoplasias/inmunologíaRESUMEN
Transposable elements dominate the mammalian genome, but their contribution to genetic and epigenetic regulation has been largely overlooked. This was in part due to technical limitations, which made the study of repetitive sequences at single copy resolution difficult. The advancement of next-generation sequencing assays in the last decade has greatly enhanced our understanding of transposable element function. In some instances, specific transposable elements are thought to have been co-opted into regulatory roles during both mouse and human development, while in disease such regulatory potential can contribute to malignancy. DNA methylation is arguably the best characterised regulator of transposable element activity. DNA methylation is associated with transposable element repression, and acts to limit their genotoxic potential. In specific developmental contexts, erasure of DNA methylation is associated with a burst of transposable element expression. Developmental regulation of DNA methylation enables transposon activation, ensuring their survival and propagation throughout the host genome, and also allows the host access to regulatory sequences encoded within the elements. Here I discuss DNA methylation at transposable elements, describing its function and dynamic regulation throughout murine and human development.
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Metilación de ADN/fisiología , Elementos Transponibles de ADN/fisiología , Animales , Represión Epigenética/fisiología , HumanosRESUMEN
Over the last decade, our understanding of how the genome is packaged in three dimension within the nucleus has grown considerably. This is largely due to advances in high-throughput genomics assays to study higher order chromatin organization. Our knowledge of the structures adopted by the chromatin has far preceded our understanding of function and mechanism. An outstanding question has been how are such structures established. Recently, a suite of genomics assays has been adapted for low-input material, making it possible to apply them to the pre-implantation mammalian embryo. For the first time, chromatin topology and associations with the nuclear lamina have been described in the earliest stages of murine development. These studies have revealed the dynamics with which higher-order chromatin architecture is established in vivo. Additionally, they have yielded some intriguing findings that will pave the way for futures studies into the mechanisms underlying the establishment of three dimensional genome organization. Here, we discuss findings on how embryonic chromatin is dynamically organized within the nucleus throughout preimplantation development, and the outline a number of outstanding questions that will be exciting to address in the future.
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Núcleo Celular/metabolismo , Cromatina/metabolismo , Embrión de Mamíferos/fisiología , Desarrollo Embrionario , Regulación del Desarrollo de la Expresión Génica , Genoma , Animales , Núcleo Celular/genética , Cromatina/genética , Embrión de Mamíferos/citología , Epigénesis Genética , HumanosRESUMEN
Neural tube defects (NTDs) are common birth defects in humans and show an unexplained female bias. Female mice lacking the tumor suppressor p53 display NTDs with incomplete penetrance. We found that the combined loss of pro-apoptotic BIM and p53 caused 100% penetrant, female-exclusive NTDs, which allowed us to investigate the female-specific functions of p53. We report that female p53-/- embryonic neural tube samples show fewer cells with inactive X chromosome markers Xist and H3K27me3 and a concomitant increase in biallelic expression of the X-linked genes, Huwe1 and Usp9x. Decreased Xist and increased X-linked gene expression was confirmed by RNA sequencing. Moreover, we found that p53 directly bound response elements in the X chromosome inactivation center (XIC). Together, these findings suggest p53 directly activates XIC genes, without which there is stochastic failure in X chromosome inactivation, and that X chromosome inactivation failure may underlie the female bias in neural tube closure defects.
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Defectos del Tubo Neural/genética , Proteína p53 Supresora de Tumor/deficiencia , Animales , Células Madre Embrionarias/patología , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Defectos del Tubo Neural/patología , Embarazo , Procesos Estocásticos , Proteína p53 Supresora de Tumor/genética , Inactivación del Cromosoma XRESUMEN
We and others have recently reported that the SMC protein Smchd1 is a regulator of chromosome conformation. Smchd1 is critical for the structure of the inactive X chromosome and at autosomal targets such as the Hox genes. However, it is unknown how Smchd1 is recruited to these sites. Here, we report that Smchd1 localizes to the inactive X via the Xist-HnrnpK-PRC1 (polycomb repressive complex 1) pathway. Contrary to previous reports, Smchd1 does not bind Xist or other RNA molecules with any specificity. Rather, the localization of Smchd1 to the inactive X is H2AK119ub dependent. Following perturbation of this interaction, Smchd1 is destabilized, which has consequences for gene silencing genome-wide. Our work adds Smchd1 to the PRC1 silencing pathway for X chromosome inactivation.
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Proteínas Cromosómicas no Histona/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo K/metabolismo , Complejo Represivo Polycomb 1/metabolismo , ARN Largo no Codificante/metabolismo , Inactivación del Cromosoma X/genética , Animales , Secuencia de Bases , Diferenciación Celular , Femenino , Genoma , Histonas/metabolismo , Lisina/metabolismo , Ratones , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , Oligonucleótidos/metabolismo , Transporte de ProteínasRESUMEN
The regulation of higher-order chromatin structure is complex and dynamic, and a full understanding of the suite of mechanisms governing this architecture is lacking. Here, we reveal the noncanonical SMC protein Smchd1 to be a novel regulator of long-range chromatin interactions in mice, and we add Smchd1 to the canon of epigenetic proteins required for Hox-gene regulation. The effect of losing Smchd1-dependent chromatin interactions has varying outcomes that depend on chromatin context. At autosomal targets transcriptionally sensitive to Smchd1 deletion, we found increased short-range interactions and ectopic enhancer activation. In contrast, the inactive X chromosome was transcriptionally refractive to Smchd1 ablation, despite chromosome-wide increases in short-range interactions. In the inactive X, we observed spreading of trimethylated histone H3 K27 (H3K27me3) domains into regions not normally decorated by this mark. Together, these data suggest that Smchd1 is able to insulate chromatin, thereby limiting access to other chromatin-modifying proteins.
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Cromatina/metabolismo , Proteínas Cromosómicas no Histona/fisiología , Genes Homeobox , Familia de Multigenes , Cromosoma X , Animales , Proteínas Cromosómicas no Histona/genética , Elementos de Facilitación Genéticos , Eliminación de Gen , Silenciador del Gen , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
Recent reports on the characteristics of naive human pluripotent stem cells (hPSCs) obtained using independent methods differ. Naive hPSCs have been mainly derived by conversion from primed hPSCs or by direct derivation from human embryos rather than by somatic cell reprogramming. To provide an unbiased molecular and functional reference, we derived genetically matched naive hPSCs by direct reprogramming of fibroblasts and by primed-to-naive conversion using different naive conditions (NHSM, RSeT, 5iLAF and t2iLGöY). Our results show that hPSCs obtained in these different conditions display a spectrum of naive characteristics. Furthermore, our characterization identifies KLF4 as sufficient for conversion of primed hPSCs into naive t2iLGöY hPSCs, underscoring the role that reprogramming factors can play for the derivation of bona fide naive hPSCs.
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Reprogramación Celular , Células Madre Pluripotentes/citología , Diferenciación Celular , Fibroblastos/citología , Inestabilidad Genómica , Humanos , Factor 4 Similar a KruppelRESUMEN
It has very recently become clear that the epigenetic modifier SMCHD1 has a role in two distinct disorders: facioscapulohumoral muscular dystrophy (FSHD) and Bosma arhinia and micropthalmia (BAMS). In the former there are heterozygous loss-of-function mutations, while both gain- and loss-of-function mutations have been proposed to underlie the latter. These findings have led to much interest in SMCHD1 and how it works at the molecular level. We summarise here current understanding of the mechanism of action of SMCHD1, its role in these diseases, and what has been learnt from study of mouse models null for Smchd1 in the decade since the discovery of SMCHD1.
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Atresia de las Coanas/genética , Proteínas Cromosómicas no Histona/genética , Epigénesis Genética , Microftalmía/genética , Distrofia Muscular Facioescapulohumeral/genética , Nariz/anomalías , Animales , Metilación de ADN/genética , Heterocigoto , Humanos , Ratones , MutaciónRESUMEN
BACKGROUND: The presence of histone 3 lysine 9 (H3K9) methylation on the mouse inactive X chromosome has been controversial over the last 15 years, and the functional role of H3K9 methylation in X chromosome inactivation in any species has remained largely unexplored. RESULTS: Here we report the first genomic analysis of H3K9 di- and tri-methylation on the inactive X: we find they are enriched at the intergenic, gene poor regions of the inactive X, interspersed between H3K27 tri-methylation domains found in the gene dense regions. Although H3K9 methylation is predominantly non-genic, we find that depletion of H3K9 methylation via depletion of H3K9 methyltransferase Set domain bifurcated 1 (Setdb1) during the establishment of X inactivation, results in failure of silencing for around 150 genes on the inactive X. By contrast, we find a very minor role for Setdb1-mediated H3K9 methylation once X inactivation is fully established. In addition to failed gene silencing, we observed a specific failure to silence X-linked long-terminal repeat class repetitive elements. CONCLUSIONS: Here we have shown that H3K9 methylation clearly marks the murine inactive X chromosome. The role of this mark is most apparent during the establishment phase of gene silencing, with a more muted effect on maintenance of the silent state. Based on our data, we hypothesise that Setdb1-mediated H3K9 methylation plays a role in epigenetic silencing of the inactive X via silencing of the repeats, which itself facilitates gene silencing through alterations to the conformation of the whole inactive X chromosome.
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Smchd1 is an epigenetic repressor with important functions in healthy cellular processes and disease. To elucidate its role in transcriptional regulation, we performed two independent genome-wide RNA-sequencing studies comparing wild-type and Smchd1 null samples in neural stem cells and lymphoma cell lines. Using an R-based analysis pipeline that accommodates observational and sample-specific weights in the linear modeling, we identify key genes dysregulated by Smchd1 deletion such as clustered protocadherins in the neural stem cells and imprinted genes in both experiments. Here we provide a detailed description of this analysis, from quality control to read mapping and differential expression analysis. These data sets are publicly available from the Gene Expression Omnibus database (accession numbers GSE64099 and GSE65747).
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Variations in sample quality are frequently encountered in small RNA-sequencing experiments, and pose a major challenge in a differential expression analysis. Removal of high variation samples reduces noise, but at a cost of reducing power, thus limiting our ability to detect biologically meaningful changes. Similarly, retaining these samples in the analysis may not reveal any statistically significant changes due to the higher noise level. A compromise is to use all available data, but to down-weight the observations from more variable samples. We describe a statistical approach that facilitates this by modelling heterogeneity at both the sample and observational levels as part of the differential expression analysis. At the sample level this is achieved by fitting a log-linear variance model that includes common sample-specific or group-specific parameters that are shared between genes. The estimated sample variance factors are then converted to weights and combined with observational level weights obtained from the mean-variance relationship of the log-counts-per-million using 'voom'. A comprehensive analysis involving both simulations and experimental RNA-sequencing data demonstrates that this strategy leads to a universally more powerful analysis and fewer false discoveries when compared to conventional approaches. This methodology has wide application and is implemented in the open-source 'limma' package.