ABSTRACT
Antibody-secreting plasma cells (PCs) are generated in secondary lymphoid organs but are reported to reside in an emerging range of anatomical sites. Analysis of the transcriptome of different tissue-resident (Tr)PC populations revealed that they each have their own transcriptional signature indicative of functional adaptation to the host tissue environment. In contrast to expectation, all TrPCs were extremely long-lived, regardless of their organ of residence, with longevity influenced by intrinsic factors like the immunoglobulin isotype. Analysis at single-cell resolution revealed that the bone marrow is unique in housing a compendium of PCs generated all over the body that retain aspects of the transcriptional program indicative of their tissue of origin. This study reveals that extreme longevity is an intrinsic property of TrPCs whose transcriptome is imprinted by signals received both at the site of induction and within the tissue of residence.
Subject(s)
Bone Marrow , Plasma Cells , Bone Marrow CellsABSTRACT
Group 3 innate lymphoid cell (ILC3)-mediated production of the cytokine interleukin-22 (IL-22) is critical for the maintenance of immune homeostasis in the gastrointestinal tract. Here, we find that the function of ILC3s is not constant across the day, but instead oscillates between active phases and resting phases. Coordinate responsiveness of ILC3s in the intestine depended on the food-induced expression of the neuropeptide vasoactive intestinal peptide (VIP). Intestinal ILC3s had high expression of the G protein-coupled receptor vasoactive intestinal peptide receptor 2 (VIPR2), and activation by VIP markedly enhanced the production of IL-22 and the barrier function of the epithelium. Conversely, deficiency in signaling through VIPR2 led to impaired production of IL-22 by ILC3s and increased susceptibility to inflammation-induced gut injury. Thus, intrinsic cellular rhythms acted in synergy with the cyclic patterns of food intake to drive the production of IL-22 and synchronize protection of the intestinal epithelium through a VIP-VIPR2 pathway in ILC3s.
Subject(s)
Immunity, Mucosal/immunology , Lymphocyte Subsets/immunology , Lymphocytes/immunology , Periodicity , Vasoactive Intestinal Peptide/immunology , Animals , Eating/immunology , Immunity, Innate/immunology , Lymphocyte Subsets/metabolism , Lymphocytes/metabolism , Mice , Mice, Inbred C57BL , Vasoactive Intestinal Peptide/metabolismABSTRACT
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
ABSTRACT
Recent studies have elucidated cell-lineage-specific three-dimensional genome organization; however, how such specific architecture is established or maintained is unclear. We hypothesized that lineage-defining transcription factors maintain cell identity via global control of genome organization. These factors bind many genomic sites outside of the genes that they directly regulate and thus are potentially implicated in three-dimensional genome organization. Using chromosome-conformation-capture techniques, we show that the transcription factor Paired box 5 (Pax5) is critical for the establishment and maintenance of the global lineage-specific architecture of B cells. Pax5 was found to supervise genome architecture throughout B cell differentiation, until the plasmablast stage, in which Pax5 is naturally silenced and B cell-specific genome structure is lost. Crucially, Pax5 did not rely on ongoing transcription to organize the genome. These results implicate sequence-specific DNA-binding proteins in global genome organization to establish and maintain lineage fidelity.
Subject(s)
B-Lymphocytes/cytology , Cell Differentiation/genetics , Cell Lineage/genetics , PAX5 Transcription Factor/genetics , Animals , B-Lymphocytes/metabolism , Male , Mice , Mice, Inbred C57BL , PAX5 Transcription Factor/metabolismABSTRACT
To separate causal effects of histone acetylation on chromatin accessibility and transcriptional output, we used integrated epigenomic and transcriptomic analyses following acute inhibition of major cellular lysine acetyltransferases P300 and CBP in hematological malignancies. We found that catalytic P300/CBP inhibition dynamically perturbs steady-state acetylation kinetics and suppresses oncogenic transcriptional networks in the absence of changes to chromatin accessibility. CRISPR-Cas9 screening identified NCOR1 and HDAC3 transcriptional co-repressors as the principal antagonists of P300/CBP by counteracting acetylation turnover kinetics. Finally, deacetylation of H3K27 provides nucleation sites for reciprocal methylation switching, a feature that can be exploited therapeutically by concomitant KDM6A and P300/CBP inhibition. Overall, this study indicates that the steady-state histone acetylation-methylation equilibrium functions as a molecular rheostat governing cellular transcription that is amenable to therapeutic exploitation as an anti-cancer regimen.
Subject(s)
Biocatalysis , Histones/metabolism , Oncogenes , Transcription, Genetic , p300-CBP Transcription Factors/metabolism , Acetylation , Cell Line , Chromatin/metabolism , Co-Repressor Proteins/metabolism , Conserved Sequence , Evolution, Molecular , Gene Regulatory Networks , Genome , Histone Deacetylases/metabolism , Humans , Kinetics , Methylation , Models, Biological , RNA Polymerase II/metabolismABSTRACT
Investigating how transcription factors control complex cellular processes requires tools that enable responses to be visualised at the single-cell level and their cell fate to be followed over time. For example, the tumour suppressor p53 (also called TP53 in humans and TRP53 in mice) can initiate diverse cellular responses by transcriptional activation of its target genes: Puma to induce apoptotic cell death and p21 to induce cell cycle arrest/cell senescence. However, it is not known how these processes are regulated and initiated in different cell types. Also, the context-dependent interaction partners and binding loci of p53 remain largely elusive. To be able to examine these questions, we here developed knock-in mice expressing triple-FLAG-tagged p53 to facilitate p53 pull-down and two p53 response reporter mice, knocking tdTomato and GFP into the Puma/Bbc3 and p21 gene loci, respectively. By crossing these reporter mice into a p53-deficient background, we show that the new reporters reliably inform on p53-dependent and p53-independent initiation of both apoptotic or cell cycle arrest/senescence programs, respectively, in vitro and in vivo.
ABSTRACT
Plasma cell differentiation requires silencing of B cell transcription, while it establishes antibody-secretory function and long-term survival. The transcription factors Blimp-1 and IRF4 are essential for the generation of plasma cells; however, their function in mature plasma cells has remained elusive. We found that while IRF4 was essential for the survival of plasma cells, Blimp-1 was dispensable for this. Blimp-1-deficient plasma cells retained their transcriptional identity but lost the ability to secrete antibody. Blimp-1 regulated many components of the unfolded protein response (UPR), including XBP-1 and ATF6. The overlap in the functions of Blimp-1 and XBP-1 was restricted to that response, with Blimp-1 uniquely regulating activity of the kinase mTOR and the size of plasma cells. Thus, Blimp-1 was required for the unique physiological ability of plasma cells that enables the secretion of protective antibody.
Subject(s)
Cell Differentiation/immunology , Immunoglobulins/immunology , Interferon Regulatory Factors/immunology , Plasma Cells/immunology , Transcription Factors/immunology , Unfolded Protein Response/immunology , Activating Transcription Factor 6/genetics , Activating Transcription Factor 6/immunology , Animals , Cell Size , Chromatin Immunoprecipitation , DNA-Binding Proteins/genetics , DNA-Binding Proteins/immunology , Enzyme-Linked Immunosorbent Assay , Flow Cytometry , Gene Expression Profiling , Gene Expression Regulation , High-Throughput Nucleotide Sequencing , Immunoglobulins/metabolism , Interferon Regulatory Factors/genetics , Mice , Mice, Knockout , Microscopy, Electron, Transmission , Plasma Cells/metabolism , Positive Regulatory Domain I-Binding Factor 1 , Regulatory Factor X Transcription Factors , Sequence Analysis, DNA , TOR Serine-Threonine Kinases/genetics , TOR Serine-Threonine Kinases/immunology , Transcription Factors/genetics , Unfolded Protein Response/genetics , X-Box Binding Protein 1ABSTRACT
T cell responses are guided by cytokines that induce transcriptional regulators, which ultimately control differentiation of effector and memory T cells. However, it is unknown how the activities of these molecular regulators are coordinated and integrated during the differentiation process. Using genetic approaches and transcriptional profiling of antigen-specific CD8(+) T cells, we reveal a common program of effector differentiation that is regulated by IL-2 and IL-12 signaling and the combined activities of the transcriptional regulators Blimp-1 and T-bet. The loss of both T-bet and Blimp-1 leads to abrogated cytotoxic function and ectopic IL-17 production in CD8(+) T cells. Overall, our data reveal two major overlapping pathways of effector differentiation governed by the availability of Blimp-1 and T-bet and suggest a model for cytokine-induced transcriptional changes that combine, quantitatively and qualitatively, to promote robust effector CD8(+) T cell differentiation.
Subject(s)
CD8-Positive T-Lymphocytes/immunology , Cell Differentiation/immunology , Interleukin-12/immunology , Interleukin-2/immunology , T-Box Domain Proteins/immunology , Transcription Factors/immunology , Animals , Arenaviridae Infections/immunology , Chromatin Immunoprecipitation , Cytokines/immunology , Flow Cytometry , Gene Expression Profiling , Influenza A Virus, H1N1 Subtype , Interleukin-17/immunology , Lymphocytic choriomeningitis virus , Mice , Orthomyxoviridae Infections/immunology , Positive Regulatory Domain I-Binding Factor 1 , Real-Time Polymerase Chain Reaction , STAT4 Transcription Factor/immunology , STAT5 Transcription Factor/immunology , Sequence Analysis, RNA , Signal TransductionABSTRACT
Intestinal T cells and group 3 innate lymphoid cells (ILC3 cells) control the composition of the microbiota and gut immune responses. Within the gut, ILC3 subsets coexist that either express or lack the natural cytoxicity receptor (NCR) NKp46. We identified here the transcriptional signature associated with the transcription factor T-bet-dependent differentiation of NCR(-) ILC3 cells into NCR(+) ILC3 cells. Contrary to the prevailing view, we found by conditional deletion of the key ILC3 genes Stat3, Il22, Tbx21 and Mcl1 that NCR(+) ILC3 cells were redundant for the control of mouse colonic infection with Citrobacter rodentium in the presence of T cells. However, NCR(+) ILC3 cells were essential for cecal homeostasis. Our data show that interplay between intestinal ILC3 cells and adaptive lymphocytes results in robust complementary failsafe mechanisms that ensure gut homeostasis.
Subject(s)
Immunity, Innate , Interleukins/biosynthesis , Lymphocytes/immunology , Lymphocytes/metabolism , Animals , Citrobacter rodentium/immunology , Cluster Analysis , Disease Models, Animal , Enterobacteriaceae Infections/genetics , Enterobacteriaceae Infections/immunology , Enterobacteriaceae Infections/metabolism , Enterobacteriaceae Infections/mortality , Enterobacteriaceae Infections/pathology , Female , Gene Expression Profiling , Gene Expression Regulation , Homeostasis , Lymphocyte Subsets/immunology , Lymphocyte Subsets/metabolism , Male , Mice , Mice, Knockout , Mice, Transgenic , Myeloid Cell Leukemia Sequence 1 Protein/deficiency , Myeloid Cell Leukemia Sequence 1 Protein/genetics , Myeloid Cell Leukemia Sequence 1 Protein/metabolism , Natural Cytotoxicity Triggering Receptor 1/metabolism , Signal Transduction , T-Box Domain Proteins/deficiency , T-Box Domain Proteins/genetics , T-Box Domain Proteins/metabolism , Transcriptome , Interleukin-22ABSTRACT
Dendritic cells (DCs) are can be broadly divided into conventional (cDC) and plasmacytoid (pDC) subsets. Despite the importance of this lineage diversity, its genetic basis is not fully understood. We found that conditional ablation of the Ets-family transcription factor PU.1 in DC-restricted progenitors led to increased pDC production at the expense of cDCs. PU.1 controlled many of the cardinal functions of DCs, such as antigen presentation by cDCs and type I interferon production by pDCs. Conditional ablation of PU.1 de-repressed the pDC transcriptional signature in cDCs. The combination of genome-wide mapping of PU.1 binding and gene expression analysis revealed a key role for PU.1 in maintaining cDC identity through the induction of the transcriptional regulator DC-SCRIPT. PU.1 activated DC-SCRIPT expression, which in turn promoted cDC formation, particularly of cDC1s, and repressed pDC development. Thus, cDC identity is regulated by a transcriptional node requiring PU.1 and DC-SCRIPT.
Subject(s)
DNA-Binding Proteins/metabolism , Dendritic Cells/physiology , Nuclear Proteins/metabolism , Proto-Oncogene Proteins/metabolism , Trans-Activators/metabolism , Transcription Factors/metabolism , Animals , Antigen Presentation , Cell Differentiation , Cell Lineage , DNA-Binding Proteins/genetics , Gene Expression Regulation , HEK293 Cells , Humans , Interferon Type I/metabolism , Mice , Mice, Transgenic , Nuclear Proteins/genetics , Proto-Oncogene Proteins/genetics , Signal Transduction , Trans-Activators/genetics , Transcription Factors/genetics , TranscriptomeABSTRACT
H3K9me3-dependent heterochromatin is critical for the silencing of repeat-rich pericentromeric regions and also has key roles in repressing lineage-inappropriate protein-coding genes in differentiation and development. Here, we investigate the molecular consequences of heterochromatin loss in cells deficient in both SUV39H1 and SUV39H2 (Suv39DKO), the major mammalian histone methyltransferase enzymes that catalyze heterochromatic H3K9me3 deposition. We reveal a paradoxical repression of protein-coding genes in Suv39DKO cells, with these differentially expressed genes principally in euchromatic (Tn5-accessible, H3K4me3- and H3K27ac-marked) rather than heterochromatic (H3K9me3-marked) or polycomb (H3K27me3-marked) regions. Examination of the three-dimensional (3D) nucleome reveals that transcriptomic dysregulation occurs in euchromatic regions close to the nuclear periphery in 3D space. Moreover, this transcriptomic dysregulation is highly correlated with altered 3D genome organization in Suv39DKO cells. Together, our results suggest that the nuclear lamina-tethering of Suv39-dependent H3K9me3 domains provides an essential scaffold to support euchromatic genome organization and the maintenance of gene transcription for healthy cellular function.
Subject(s)
Euchromatin , Heterochromatin , Histone-Lysine N-Methyltransferase , Histones , Methyltransferases , Transcription, Genetic , Animals , Mice , Cell Line , Euchromatin/metabolism , Euchromatin/genetics , Gene Expression Regulation , Heterochromatin/metabolism , Heterochromatin/genetics , Histone-Lysine N-Methyltransferase/metabolism , Histone-Lysine N-Methyltransferase/genetics , Histones/metabolism , Histones/genetics , Methyltransferases/metabolism , Methyltransferases/genetics , Repressor Proteins/metabolism , Repressor Proteins/geneticsABSTRACT
Inhibitor of growth 4 and 5 (ING4, ING5) are structurally similar chromatin-binding proteins in the KAT6A, KAT6B and KAT7 histone acetyltransferase protein complexes. Heterozygous mutations in the KAT6A or KAT6B gene cause human disorders with cardiac defects, but the contribution of their chromatin-adaptor proteins to development is unknown. We found that Ing5-/- mice had isolated cardiac ventricular septal defects. Ing4-/-Ing5-/- embryos failed to undergo chorioallantoic fusion and arrested in development at embryonic day 8.5, displaying loss of histone H3 lysine 14 acetylation, reduction in H3 lysine 23 acetylation levels and reduced developmental gene expression. Embryonic day 12.5 Ing4+/-Ing5-/- hearts showed a paucity of epicardial cells and epicardium-derived cells, failure of myocardium compaction, and coronary vasculature defects, accompanied by reduced expression of epicardium genes. Cell adhesion gene expression and proepicardium outgrowth were defective in the ING4- and ING5-deficient state. Our findings suggest that ING4 and ING5 are essential for heart development and promote epicardium and epicardium-derived cell fates and imply mutation of the human ING5 gene as a possible cause of isolated ventricular septal defects.
Subject(s)
Carrier Proteins , Heart Septal Defects, Ventricular , Lysine , Humans , Animals , Mice , Cell Lineage , Histones , Acetylation , Chromatin , Transcription Factors , Tumor Suppressor Proteins , Homeodomain Proteins/genetics , Cell Cycle Proteins , Histone AcetyltransferasesABSTRACT
When B cells encounter an antigen, they alter their physiological state and anatomical localization and initiate a differentiation process that ultimately produces antibody-secreting cells (ASCs). We have defined the transcriptomes of many mature B cell populations and stages of plasma cell differentiation in mice. We provide a molecular signature of ASCs that highlights the stark transcriptional divide between B cells and plasma cells and enables the demarcation of ASCs on the basis of location and maturity. Changes in gene expression correlated with cell-division history and the acquisition of permissive histone modifications, and they included many regulators that had not been previously implicated in B cell differentiation. These findings both highlight and expand the core program that guides B cell terminal differentiation and the production of antibodies.
Subject(s)
Cell Differentiation/genetics , Plasma Cells/cytology , Plasma Cells/immunology , Transcriptome , Animals , B-Cell Maturation Antigen/genetics , Cell Division/genetics , Cell Movement/genetics , Cells, Cultured , Gene Expression Profiling , Histone Code/genetics , Lymphocyte Activation/genetics , Mice , Mice, Inbred C57BL , Mice, Knockout , Positive Regulatory Domain I-Binding Factor 1 , RNA/analysis , Suppressor of Cytokine Signaling Proteins/genetics , Transcription Factors/geneticsABSTRACT
Foxp3(+) regulatory T (Treg) cells in visceral adipose tissue (VAT-Treg cells) are functionally specialized tissue-resident cells that prevent obesity-associated inflammation and preserve insulin sensitivity and glucose tolerance. Their development depends on the transcription factor PPAR-γ; however, the environmental cues required for their differentiation are unknown. Here we show that interleukin 33 (IL-33) signaling through the IL-33 receptor ST2 and myeloid differentiation factor MyD88 is essential for development and maintenance of VAT-Treg cells and sustains their transcriptional signature. Furthermore, the transcriptional regulators BATF and IRF4 were necessary for VAT-Treg differentiation through direct regulation of ST2 and PPAR-γ expression. IL-33 administration induced vigorous population expansion of VAT-Treg cells, which tightly correlated with improvements in metabolic parameters in obese mice. Human omental adipose tissue Treg cells also showed high ST2 expression, suggesting an evolutionarily conserved requirement for IL-33 in VAT-Treg cell homeostasis.
Subject(s)
Adipose Tissue/cytology , Basic-Leucine Zipper Transcription Factors/metabolism , Interferon Regulatory Factors/metabolism , Interleukins/metabolism , T-Lymphocytes, Regulatory/cytology , Adipose Tissue/metabolism , Animals , Cell Differentiation/physiology , Humans , Interleukin-33 , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Myeloid Differentiation Factor 88/metabolism , Obesity/metabolism , PPAR gamma/metabolism , Receptors, Cell Surface/metabolism , T-Lymphocytes, Regulatory/metabolismABSTRACT
Polymorphisms in NFKB1 that diminish its expression have been linked to human inflammatory diseases and increased risk for epithelial cancers. The underlying mechanisms are unknown, and the link is perplexing given that NF-κB signaling reportedly typically exerts pro-tumorigenic activity. Here we have shown that NF-κB1 deficiency, even loss of a single allele, resulted in spontaneous invasive gastric cancer (GC) in mice that mirrored the histopathological progression of human intestinal-type gastric adenocarcinoma. Bone marrow chimeras revealed that NF-κB1 exerted tumor suppressive functions in both epithelial and hematopoietic cells. RNA-seq analysis showed that NF-κB1 deficiency resulted in aberrant JAK-STAT signaling, which dysregulated expression of effectors of inflammation, antigen presentation, and immune checkpoints. Concomitant loss of STAT1 prevented these immune abnormalities and GC development. These findings provide mechanistic insight into how polymorphisms that attenuate NFKB1 expression predispose humans to epithelial cancers, highlighting the pro-tumorigenic activity of STAT1 and identifying targetable vulnerabilities in GC.
Subject(s)
Gene Expression Regulation, Neoplastic , Inflammation/genetics , Inflammation/metabolism , NF-kappa B/deficiency , STAT1 Transcription Factor/metabolism , Stomach Neoplasms/genetics , Stomach Neoplasms/metabolism , Animals , Antigen Presentation/immunology , Bone Marrow Cells/metabolism , Bone Marrow Cells/pathology , Cell Transformation, Neoplastic/genetics , Cell Transformation, Neoplastic/metabolism , Disease Models, Animal , Epithelial Cells/metabolism , Epithelial Cells/pathology , Gene Regulatory Networks , Humans , Inflammation/pathology , Mice , Mice, Knockout , STAT1 Transcription Factor/deficiency , Stomach Neoplasms/immunology , Stomach Neoplasms/pathologyABSTRACT
The lack of benchmark data sets with inbuilt ground-truth makes it challenging to compare the performance of existing long-read isoform detection and differential expression analysis workflows. Here, we present a benchmark experiment using two human lung adenocarcinoma cell lines that were each profiled in triplicate together with synthetic, spliced, spike-in RNAs (sequins). Samples were deeply sequenced on both Illumina short-read and Oxford Nanopore Technologies long-read platforms. Alongside the ground-truth available via the sequins, we created in silico mixture samples to allow performance assessment in the absence of true positives or true negatives. Our results show that StringTie2 and bambu outperformed other tools from the six isoform detection tools tested, DESeq2, edgeR and limma-voom were best among the five differential transcript expression tools tested and there was no clear front-runner for performing differential transcript usage analysis between the five tools compared, which suggests further methods development is needed for this application.
Subject(s)
Gene Expression Profiling , High-Throughput Nucleotide Sequencing , Humans , Gene Expression Profiling/methods , High-Throughput Nucleotide Sequencing/methods , Benchmarking/methods , RNA , Protein IsoformsABSTRACT
FoxP3-expressing regulatory T (Treg) cells are essential for maintaining immune homeostasis. Activated Treg cells undergo further differentiation into an effector state that highly expresses genes critical for Treg cell function, although how this process is coordinated on a transcriptional level is poorly understood. Here, we demonstrate that mice lacking the transcription factor Myb in Treg cells succumbed to a multi-organ inflammatory disease. Myb was specifically expressed in, and required for the differentiation of, thymus-derived effector Treg cells. The combination of transcriptome and genomic footprint analyses revealed that Myb directly regulated a large proportion of the gene expression specific to effector Treg cells, identifying Myb as a critical component of the gene regulatory network controlling effector Treg cell differentiation and function.
Subject(s)
Gene Regulatory Networks/immunology , Homeostasis/immunology , Lymphocyte Activation/immunology , Proto-Oncogene Proteins c-myb/immunology , T-Lymphocytes, Regulatory/immunology , Animals , Cell Differentiation/immunology , Disease Models, Animal , Flow Cytometry , Gene Expression Profiling , Mice , Mice, Inbred C57BL , Mice, Knockout , TranscriptomeABSTRACT
Double-stranded RNA (dsRNA) is a common by-product of viral infections and acts as a potent trigger of antiviral immunity. In the nematode C. elegans, sid-1 encodes a dsRNA transporter that is highly conserved throughout animal evolution, but the physiological role of SID-1 and its orthologs remains unclear. Here, we show that the mammalian SID-1 ortholog, SIDT2, is required to transport internalized extracellular dsRNA from endocytic compartments into the cytoplasm for immune activation. Sidt2-deficient mice exposed to extracellular dsRNA, encephalomyocarditis virus (EMCV), and herpes simplex virus 1 (HSV-1) show impaired production of antiviral cytokines and-in the case of EMCV and HSV-1-reduced survival. Thus, SIDT2 has retained the dsRNA transport activity of its C. elegans ortholog, and this transport is important for antiviral immunity.
Subject(s)
Immunity, Innate , Membrane Proteins/metabolism , RNA Transport , RNA, Double-Stranded/immunology , RNA, Double-Stranded/metabolism , Animals , Cardiovirus Infections/genetics , Cardiovirus Infections/immunology , Cell Line , Cytoplasm , DEAD Box Protein 58/metabolism , Disease Models, Animal , Encephalomyocarditis virus/genetics , Encephalomyocarditis virus/immunology , Endosomes/metabolism , Female , Gene Expression , Gene Knockout Techniques , Herpes Simplex/genetics , Herpes Simplex/immunology , Herpesvirus 1, Human/genetics , Herpesvirus 1, Human/immunology , Host-Pathogen Interactions/genetics , Host-Pathogen Interactions/immunology , Lysosomes/metabolism , Membrane Proteins/genetics , Mice , Mice, Knockout , Nucleotide Transport Proteins , Protein Binding , Protein Transport , RNA, Viral/genetics , RNA, Viral/metabolism , Signal Transduction , Toll-Like Receptor 3/metabolismABSTRACT
Differential expression analysis of RNA-seq is one of the most commonly performed bioinformatics analyses. Transcript-level quantifications are inherently more uncertain than gene-level read counts because of ambiguous assignment of sequence reads to transcripts. While sequence reads can usually be assigned unambiguously to a gene, reads are very often compatible with multiple transcripts for that gene, particularly for genes with many isoforms. Software tools designed for gene-level differential expression do not perform optimally on transcript counts because the read-to-transcript ambiguity (RTA) disrupts the mean-variance relationship normally observed for gene level RNA-seq data and interferes with the efficiency of the empirical Bayes dispersion estimation procedures. The pseudoaligners kallisto and Salmon provide bootstrap samples from which quantification uncertainty can be assessed. We show that the overdispersion arising from RTA can be elegantly estimated by fitting a quasi-Poisson model to the bootstrap counts for each transcript. The technical overdispersion arising from RTA can then be divided out of the transcript counts, leading to scaled counts that can be input for analysis by established gene-level software tools with full statistical efficiency. Comprehensive simulations and test data show that an edgeR analysis of the scaled counts is more powerful and efficient than previous differential transcript expression pipelines while providing correct control of the false discovery rate. Simulations explore a wide range of scenarios including the effects of paired vs single-end reads, different read lengths and different numbers of replicates.
Subject(s)
Gene Expression Profiling , Software , Gene Expression Profiling/methods , Bayes Theorem , Uncertainty , Sequence Analysis, RNA/methodsABSTRACT
Mutations in Trp53, prevalent in human cancer, are reported to drive tumorigenesis through dominant-negative effects (DNEs) over wild-type TRP53 function as well as neomorphic gain-of-function (GOF) activity. We show that five TRP53 mutants do not accelerate lymphomagenesis on a TRP53-deficient background but strongly synergize with c-MYC overexpression in a manner that distinguishes the hot spot Trp53 mutations. RNA sequencing revealed that the mutant TRP53 DNE does not globally repress wild-type TRP53 function but disproportionately impacts a subset of wild-type TRP53 target genes. Accordingly, TRP53 mutant proteins impair pathways for DNA repair, proliferation, and metabolism in premalignant cells. This reveals that, in our studies of lymphomagenesis, mutant TRP53 drives tumorigenesis primarily through the DNE, which modulates wild-type TRP53 function in a manner advantageous for neoplastic transformation.