ABSTRACT
The past decade has seen an explosion in the use of DNA-based microarrays. These techniques permit assessment of RNA abundance on a genome-wide scale. Medical applications emerged in the field of cancer, with studies of both solid tumors and hematological malignancies leading to the development of tests that are now used to personalize therapeutic options. Microarrays have also been used to analyze the blood transcriptome in a wide range of diseases. In human autoimmune diseases, these studies are showing potential for identifying therapeutic targets as well as biomarkers for diagnosis, assessment of disease activity, and response to treatment. More quantitative and sensitive high-throughput RNA profiling methods are starting to be available and will be necessary for transcriptome analyses to become routine tests in the clinical setting. We expect this to crystallize within the coming decade, as these methods become part of the personalized medicine armamentarium.
Subject(s)
Autoimmune Diseases/genetics , Autoimmune Diseases/immunology , Animals , Autoimmune Diseases/blood , Autoimmune Diseases/drug therapy , Biomarkers/blood , Genomics , High-Throughput Screening Assays , Humans , Oligonucleotide Array Sequence AnalysisABSTRACT
Invasive lobular carcinoma (ILC) is the second most prevalent histologic subtype of invasive breast cancer. Here, we comprehensively profiled 817 breast tumors, including 127 ILC, 490 ductal (IDC), and 88 mixed IDC/ILC. Besides E-cadherin loss, the best known ILC genetic hallmark, we identified mutations targeting PTEN, TBX3, and FOXA1 as ILC enriched features. PTEN loss associated with increased AKT phosphorylation, which was highest in ILC among all breast cancer subtypes. Spatially clustered FOXA1 mutations correlated with increased FOXA1 expression and activity. Conversely, GATA3 mutations and high expression characterized luminal A IDC, suggesting differential modulation of ER activity in ILC and IDC. Proliferation and immune-related signatures determined three ILC transcriptional subtypes associated with survival differences. Mixed IDC/ILC cases were molecularly classified as ILC-like and IDC-like revealing no true hybrid features. This multidimensional molecular atlas sheds new light on the genetic bases of ILC and provides potential clinical options.
Subject(s)
Breast Neoplasms/genetics , Breast Neoplasms/pathology , Carcinoma, Lobular/genetics , Carcinoma, Lobular/pathology , Antigens, CD , Breast Neoplasms/metabolism , Cadherins/chemistry , Cadherins/genetics , Cadherins/metabolism , Carcinoma, Ductal, Breast/genetics , Carcinoma, Ductal, Breast/pathology , Carcinoma, Lobular/metabolism , Female , Hepatocyte Nuclear Factor 3-alpha/chemistry , Hepatocyte Nuclear Factor 3-alpha/genetics , Hepatocyte Nuclear Factor 3-alpha/metabolism , Humans , Models, Molecular , Mutation , Oligonucleotide Array Sequence Analysis , Oncogene Protein v-akt/metabolism , TranscriptomeABSTRACT
Rapid activation of memory CD4(+) T helper 2 (TH2) cells during allergic inflammation requires their recruitment into the affected tissue. Here we demonstrate that group 2 innate lymphoid (ILC2) cells have a crucial role in memory TH2 cell responses, with targeted depletion of ILC2 cells profoundly impairing TH2 cell localization to the lungs and skin of sensitized mice after allergen re-challenge. ILC2-derived interleukin 13 (IL-13) is critical for eliciting production of the TH2 cell-attracting chemokine CCL17 by IRF4(+)CD11b(+)CD103(-) dendritic cells (DCs). Consequently, the sentinel function of DCs is contingent on ILC2 cells for the generation of an efficient memory TH2 cell response. These results elucidate a key innate mechanism in the regulation of the immune memory response to allergens.
Subject(s)
Dendritic Cells/immunology , Hypersensitivity/immunology , Immunologic Memory/immunology , Lymphocyte Activation/immunology , Lymphocytes/immunology , Th2 Cells/immunology , Animals , Disease Models, Animal , Flow Cytometry , Immunity, Innate/immunology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Oligonucleotide Array Sequence AnalysisABSTRACT
We used high-resolution mass spectrometry to map the cytotoxic T lymphocyte (CTL) proteome and the effect of the metabolic checkpoint kinase mTORC1 on CTLs. The CTL proteome was dominated by metabolic regulators and granzymes, and mTORC1 selectively repressed and promoted expression of a subset of CTL proteins (~10%). These included key CTL effector molecules, signaling proteins and a subset of metabolic enzymes. Proteomic data highlighted the potential for negative control of the production of phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) by mTORC1 in CTLs. mTORC1 repressed PtdIns(3,4,5)P3 production and determined the requirement for mTORC2 in activation of the kinase Akt. Our unbiased proteomic analysis thus provides comprehensive understanding of CTL identity and the control of CTL function by mTORC1.
Subject(s)
Multiprotein Complexes/metabolism , Proteome/immunology , T-Lymphocytes, Cytotoxic/metabolism , TOR Serine-Threonine Kinases/metabolism , Animals , Cells, Cultured , Chromatography , Enzyme-Linked Immunosorbent Assay , Female , Immunoblotting , Male , Mass Spectrometry , Mechanistic Target of Rapamycin Complex 1 , Mice , Multiprotein Complexes/immunology , Oligonucleotide Array Sequence Analysis , T-Lymphocytes, Cytotoxic/immunology , TOR Serine-Threonine Kinases/immunologyABSTRACT
Aberrant nucleic acids generated during viral replication are the main trigger for antiviral immunity, and mutations that disrupt nucleic acid metabolism can lead to autoinflammatory disorders. Here we investigated the etiology of X-linked reticulate pigmentary disorder (XLPDR), a primary immunodeficiency with autoinflammatory features. We discovered that XLPDR is caused by an intronic mutation that disrupts the expression of POLA1, which encodes the catalytic subunit of DNA polymerase-α. Unexpectedly, POLA1 deficiency resulted in increased production of type I interferons. This enzyme is necessary for the synthesis of RNA:DNA primers during DNA replication and, strikingly, we found that POLA1 is also required for the synthesis of cytosolic RNA:DNA, which directly modulates interferon activation. Together this work identifies POLA1 as a critical regulator of the type I interferon response.
Subject(s)
DNA Polymerase I/metabolism , DNA/biosynthesis , Interferon Type I/metabolism , RNA/biosynthesis , Base Sequence , Cells, Cultured , Cytosol/metabolism , DNA/genetics , DNA Polymerase I/genetics , Family Health , Female , Fibroblasts/cytology , Fibroblasts/metabolism , Gene Expression Profiling , Genetic Diseases, X-Linked/genetics , Genetic Diseases, X-Linked/metabolism , HEK293 Cells , HeLa Cells , Humans , Immunoblotting , Male , Microscopy, Confocal , Mutation , Oligonucleotide Array Sequence Analysis , Pedigree , Pigmentation Disorders/genetics , Pigmentation Disorders/metabolism , RNA/genetics , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Persistent viral infections are characterized by the simultaneous presence of chronic inflammation and T cell dysfunction. In prototypic models of chronicity--infection with human immunodeficiency virus (HIV) or lymphocytic choriomeningitis virus (LCMV)--we used transcriptome-based modeling to reveal that CD4(+) T cells were co-exposed not only to multiple inhibitory signals but also to tumor-necrosis factor (TNF). Blockade of TNF during chronic infection with LCMV abrogated the inhibitory gene-expression signature in CD4(+) T cells, including reduced expression of the inhibitory receptor PD-1, and reconstituted virus-specific immunity, which led to control of infection. Preventing signaling via the TNF receptor selectively in T cells sufficed to induce these effects. Targeted immunological interventions to disrupt the TNF-mediated link between chronic inflammation and T cell dysfunction might therefore lead to therapies to overcome persistent viral infection.
Subject(s)
CD4-Positive T-Lymphocytes/immunology , HIV Infections/immunology , HIV/immunology , Lymphocytic Choriomeningitis/immunology , Lymphocytic choriomeningitis virus/immunology , Tumor Necrosis Factor-alpha/immunology , Adolescent , Adult , Aged , Animals , CD4-Positive T-Lymphocytes/metabolism , CD4-Positive T-Lymphocytes/virology , Flow Cytometry , HEK293 Cells , HIV/physiology , HIV Infections/genetics , HIV Infections/virology , Host-Pathogen Interactions/immunology , Humans , Immunoblotting , Lymphocytic Choriomeningitis/genetics , Lymphocytic Choriomeningitis/virology , Lymphocytic choriomeningitis virus/physiology , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Middle Aged , Oligonucleotide Array Sequence Analysis , Programmed Cell Death 1 Receptor/genetics , Programmed Cell Death 1 Receptor/immunology , Programmed Cell Death 1 Receptor/metabolism , Receptors, Tumor Necrosis Factor/genetics , Receptors, Tumor Necrosis Factor/immunology , Receptors, Tumor Necrosis Factor/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Transcriptome/drug effects , Transcriptome/genetics , Transcriptome/immunology , Tumor Necrosis Factor-alpha/metabolism , Tumor Necrosis Factor-alpha/pharmacology , Young AdultABSTRACT
Positive selection occurs in the thymic cortex, but critical maturation events occur later in the medulla. Here we defined the precise stage at which T cells acquired competence to proliferate and emigrate. Transcriptome analysis of late gene changes suggested roles for the transcription factor NF-κB and interferon signaling. Mice lacking the inhibitor of NF-κB (IκB) kinase (IKK) kinase TAK1 underwent normal positive selection but exhibited a specific block in functional maturation. NF-κB signaling provided protection from death mediated by the cytokine TNF and was required for proliferation and emigration. The interferon signature was independent of NF-κB; however, thymocytes deficient in the interferon-α (IFN-α) receptor IFN-αR showed reduced expression of the transcription factor STAT1 and phenotypic abnormality but were able to proliferate. Thus, both NF-κB and tonic interferon signals are involved in the final maturation of thymocytes into naive T cells.
Subject(s)
Cell Differentiation/immunology , NF-kappa B/immunology , Receptor, Interferon alpha-beta/immunology , T-Lymphocytes/immunology , Thymus Gland/immunology , Animals , Cell Differentiation/genetics , Cell Movement/genetics , Cell Movement/immunology , Cell Proliferation/genetics , Flow Cytometry , MAP Kinase Kinase Kinases/genetics , MAP Kinase Kinase Kinases/immunology , MAP Kinase Kinase Kinases/metabolism , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , NF-kappa B/genetics , NF-kappa B/metabolism , Oligonucleotide Array Sequence Analysis , Receptor, Interferon alpha-beta/metabolism , Reverse Transcriptase Polymerase Chain Reaction , STAT1 Transcription Factor/genetics , STAT1 Transcription Factor/immunology , STAT1 Transcription Factor/metabolism , T-Lymphocytes/metabolism , Thymocytes/immunology , Thymocytes/metabolism , Thymus Gland/cytology , Thymus Gland/metabolism , Transcriptome/genetics , Transcriptome/immunology , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/immunology , Tumor Necrosis Factor-alpha/metabolismABSTRACT
To elucidate the transcriptional 'landscape' that regulates human lymphoid commitment during postnatal life, we used RNA sequencing to assemble the long non-coding transcriptome across human bone marrow and thymic progenitor cells spanning the earliest stages of B lymphoid and T lymphoid specification. Over 3,000 genes encoding previously unknown long non-coding RNAs (lncRNAs) were revealed through the analysis of these rare populations. Lymphoid commitment was characterized by lncRNA expression patterns that were highly stage specific and were more lineage specific than those of protein-coding genes. Protein-coding genes co-expressed with neighboring lncRNA genes showed enrichment for ontologies related to lymphoid differentiation. The exquisite cell-type specificity of global lncRNA expression patterns independently revealed new developmental relationships among the earliest progenitor cells in the human bone marrow and thymus.
Subject(s)
B-Lymphocytes/metabolism , Cell Lineage/genetics , Lymphoid Progenitor Cells/metabolism , RNA, Long Noncoding/genetics , T-Lymphocytes/metabolism , Transcriptome , Bayes Theorem , Bone Marrow Cells/metabolism , Cluster Analysis , Gene Expression Profiling/methods , Gene Ontology , Humans , Oligonucleotide Array Sequence Analysis , Reverse Transcriptase Polymerase Chain Reaction , Sequence Analysis, RNA/methods , Thymus Gland/cytology , Thymus Gland/metabolismABSTRACT
Foxm1 is known as a typical proliferation-associated transcription factor. Here we found that Foxm1 was essential for maintenance of the quiescence and self-renewal capacity of hematopoietic stem cells (HSCs) in vivo in mice. Reducing expression of FOXM1 also decreased the quiescence of human CD34(+) HSCs and progenitor cells, and its downregulation was associated with a subset of myelodysplastic syndrome (MDS). Mechanistically, Foxm1 directly bound to the promoter region of the gene encoding the receptor Nurr1 (Nr4a2; called 'Nurr1' here), inducing transcription, while forced expression of Nurr1 reversed the loss of quiescence observed in Foxm1-deficient cells in vivo. Thus, our studies reveal a previously unrecognized role for Foxm1 as a critical regulator of the quiescence and self-renewal of HSCs mediated at least in part by control of Nurr1 expression.
Subject(s)
Cell Proliferation/genetics , Forkhead Transcription Factors/genetics , Gene Expression Regulation , Hematopoietic Stem Cells/metabolism , Animals , Cells, Cultured , Flow Cytometry , Forkhead Box Protein M1 , Forkhead Transcription Factors/metabolism , Gene Expression Profiling , HEK293 Cells , Humans , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Myelodysplastic Syndromes/genetics , Myelodysplastic Syndromes/metabolism , Nuclear Receptor Subfamily 4, Group A, Member 2/genetics , Nuclear Receptor Subfamily 4, Group A, Member 2/metabolism , Oligonucleotide Array Sequence Analysis , Promoter Regions, Genetic/genetics , Protein Binding , RNA Interference , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Immune responses are tightly regulated to ensure efficient pathogen clearance while avoiding tissue damage. Here we report that Setdb2 was the only protein lysine methyltransferase induced during infection with influenza virus. Setdb2 expression depended on signaling via type I interferons, and Setdb2 repressed expression of the gene encoding the neutrophil attractant CXCL1 and other genes that are targets of the transcription factor NF-κB. This coincided with occupancy by Setdb2 at the Cxcl1 promoter, which in the absence of Setdb2 displayed diminished trimethylation of histone H3 Lys9 (H3K9me3). Mice with a hypomorphic gene-trap construct of Setdb2 exhibited increased infiltration of neutrophils during sterile lung inflammation and were less sensitive to bacterial superinfection after infection with influenza virus. This suggested that a Setdb2-mediated regulatory crosstalk between the type I interferons and NF-κB pathways represents an important mechanism for virus-induced susceptibility to bacterial superinfection.
Subject(s)
Histone-Lysine N-Methyltransferase/immunology , NF-kappa B/immunology , Orthomyxoviridae Infections/immunology , Orthomyxoviridae/immunology , Pneumonia/immunology , Superinfection/immunology , Animals , Chemokine CXCL1/immunology , Disease Susceptibility , Female , Interferon Type I/immunology , Male , Mice, Inbred C57BL , Mice, Knockout , Oligonucleotide Array Sequence Analysis , Orthomyxoviridae Infections/enzymology , Orthomyxoviridae Infections/virology , Pneumonia/enzymology , Pneumonia/virology , RNA/chemistry , RNA/genetics , Real-Time Polymerase Chain Reaction , Specific Pathogen-Free Organisms , Superinfection/enzymology , Superinfection/microbiologyABSTRACT
Aire is a transcriptional regulator that induces the promiscuous expression of thousands of tissue-restricted antigens (TRAs) in medullary thymic epithelial cells (mTECs), a step critical for the induction of immunological self-tolerance. Studies have offered molecular insights into how Aire operates, but more comprehensive understanding of this process still remains elusive. Here we found abundant expression of the protein deacetylase Sirtuin-1 (Sirt1) in mature Aire(+) mTECs, wherein it was required for the expression of Aire-dependent TRA-encoding genes and the subsequent induction of immunological self-tolerance. Our study elucidates a previously unknown molecular mechanism for Aire-mediated transcriptional regulation and identifies a unique function for Sirt1 in preventing organ-specific autoimmunity.
Subject(s)
Central Tolerance/immunology , Sirtuin 1/immunology , Transcription Factors/immunology , Transcriptional Activation/immunology , Acetylation , Animals , Antigens/immunology , Central Tolerance/genetics , Epithelial Cells/immunology , Epithelial Cells/metabolism , Flow Cytometry , HEK293 Cells , Humans , Immunoblotting , Interleukin Receptor Common gamma Subunit/deficiency , Interleukin Receptor Common gamma Subunit/genetics , Mice, Inbred C57BL , Mice, Inbred NOD , Mice, Knockout , Mice, SCID , Mice, Transgenic , Oligonucleotide Array Sequence Analysis , Organ Specificity/immunology , Protein Binding/immunology , Reverse Transcriptase Polymerase Chain Reaction , Sirtuin 1/genetics , Sirtuin 1/metabolism , Thymus Gland/cytology , Thymus Gland/immunology , Thymus Gland/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Transcriptome/immunology , AIRE ProteinABSTRACT
The transcription factors Batf3 and IRF8 are required for the development of CD8α(+) conventional dendritic cells (cDCs), but the basis for their actions has remained unclear. Here we identified two progenitor cells positive for the transcription factor Zbtb46 that separately generated CD8α(+) cDCs and CD4(+) cDCs and arose directly from the common DC progenitor (CDP). Irf8 expression in CDPs required prior autoactivation of Irf8 that was dependent on the transcription factor PU.1. Specification of the clonogenic progenitor of CD8α(+) cDCs (the pre-CD8 DC) required IRF8 but not Batf3. However, after specification of pre-CD8 DCs, autoactivation of Irf8 became Batf3 dependent at a CD8α(+) cDC-specific enhancer with multiple transcription factor AP1-IRF composite elements (AICEs) within the Irf8 superenhancer. CDPs from Batf3(-/-) mice that were specified toward development into pre-CD8 DCs failed to complete their development into CD8α(+) cDCs due to decay of Irf8 autoactivation and diverted to the CD4(+) cDC lineage.
Subject(s)
Basic-Leucine Zipper Transcription Factors/immunology , Dendritic Cells/immunology , Interferon Regulatory Factors/immunology , Repressor Proteins/immunology , Stem Cells/immunology , Animals , Base Sequence , Basic-Leucine Zipper Transcription Factors/genetics , Basic-Leucine Zipper Transcription Factors/metabolism , Bone Marrow Cells/immunology , Bone Marrow Cells/metabolism , CD24 Antigen/immunology , CD24 Antigen/metabolism , CD8 Antigens/immunology , CD8 Antigens/metabolism , Cells, Cultured , Clone Cells/immunology , Clone Cells/metabolism , Dendritic Cells/metabolism , Flow Cytometry , Interferon Regulatory Factors/genetics , Interferon Regulatory Factors/metabolism , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Molecular Sequence Data , Oligonucleotide Array Sequence Analysis , Protein Binding , Receptors, Immunologic/immunology , Receptors, Immunologic/metabolism , Repressor Proteins/genetics , Repressor Proteins/metabolism , Sequence Homology, Nucleic Acid , Stem Cells/metabolism , Transcriptome/genetics , Transcriptome/immunologyABSTRACT
Mouse conventional dendritic cells (cDCs) can be classified into two functionally distinct lineages: the CD8α(+) (CD103(+)) cDC1 lineage, and the CD11b(+) cDC2 lineage. cDCs arise from a cascade of bone marrow (BM) DC-committed progenitor cells that include the common DC progenitors (CDPs) and pre-DCs, which exit the BM and seed peripheral tissues before differentiating locally into mature cDCs. Where and when commitment to the cDC1 or cDC2 lineage occurs remains poorly understood. Here we found that transcriptional signatures of the cDC1 and cDC2 lineages became evident at the single-cell level from the CDP stage. We also identified Siglec-H and Ly6C as lineage markers that distinguished pre-DC subpopulations committed to the cDC1 lineage (Siglec-H(-)Ly6C(-) pre-DCs) or cDC2 lineage (Siglec-H(-)Ly6C(+) pre-DCs). Our results indicate that commitment to the cDC1 or cDC2 lineage occurs in the BM and not in the periphery.
Subject(s)
Bone Marrow Cells/immunology , Cell Lineage/immunology , Dendritic Cells/immunology , Stem Cells/immunology , Animals , Antigens, CD/immunology , Antigens, CD/metabolism , Antigens, Ly/genetics , Antigens, Ly/immunology , Antigens, Ly/metabolism , Bone Marrow Cells/metabolism , CD11b Antigen/immunology , CD11b Antigen/metabolism , CD8 Antigens/immunology , CD8 Antigens/metabolism , Cell Lineage/genetics , Cells, Cultured , Cluster Analysis , Dendritic Cells/metabolism , Dendritic Cells/ultrastructure , Flow Cytometry , Integrin alpha Chains/immunology , Integrin alpha Chains/metabolism , Lectins/genetics , Lectins/immunology , Lectins/metabolism , Mice, Inbred C57BL , Mice, Transgenic , Microscopy, Electron, Scanning , Oligonucleotide Array Sequence Analysis , Receptors, Cell Surface/genetics , Receptors, Cell Surface/immunology , Receptors, Cell Surface/metabolism , Single-Cell Analysis/methods , Stem Cells/metabolism , Transcriptome/genetics , Transcriptome/immunologyABSTRACT
The receptor NLRP3 is involved in the formation of the NLRP3 inflammasome that activates caspase-1 and mediates the release of interleukin 1ß (IL-1ß) and IL-18. Whether NLRP3 can shape immunological function independently of inflammasomes is unclear. We found that NLRP3 expression in CD4(+) T cells specifically supported a T helper type 2 (TH2) transcriptional program in a cell-intrinsic manner. NLRP3, but not the inflammasome adaptor ASC or caspase-1, positively regulated a TH2 program. In TH2 cells, NLRP3 bound the Il4 promoter and transactivated it in conjunction with the transcription factor IRF4. Nlrp3-deficient TH2 cells supported melanoma tumor growth in an IL-4-dependent manner and also promoted asthma-like symptoms. Our results demonstrate the ability of NLRP3 to act as a key transcription factor in TH2 differentiation.
Subject(s)
Carrier Proteins/immunology , Cell Differentiation/immunology , Th2 Cells/immunology , Trans-Activators/immunology , Animals , Asthma/genetics , Asthma/immunology , Asthma/metabolism , Blotting, Western , CD4-Positive T-Lymphocytes/immunology , CD4-Positive T-Lymphocytes/metabolism , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Differentiation/genetics , Cell Line, Tumor , Female , Gene Expression Profiling , Gene Expression Regulation, Neoplastic/immunology , Inflammasomes/genetics , Inflammasomes/immunology , Inflammasomes/metabolism , Interferon Regulatory Factors/genetics , Interferon Regulatory Factors/immunology , Interferon Regulatory Factors/metabolism , Interleukin-4/genetics , Interleukin-4/immunology , Interleukin-4/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , NIH 3T3 Cells , NLR Family, Pyrin Domain-Containing 3 Protein , Neoplasms, Experimental/genetics , Neoplasms, Experimental/immunology , Neoplasms, Experimental/metabolism , Oligonucleotide Array Sequence Analysis , Promoter Regions, Genetic/genetics , Promoter Regions, Genetic/immunology , Protein Binding/immunology , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/genetics , Signal Transduction/immunology , Th2 Cells/metabolism , Trans-Activators/genetics , Trans-Activators/metabolismABSTRACT
Gene expression analysis is a widely used and powerful method for investigating the transcriptional behavior of biological systems, for classifying cell states in disease, and for many other purposes. Recent studies indicate that common assumptions currently embedded in experimental and analytical practices can lead to misinterpretation of global gene expression data. We discuss these assumptions and describe solutions that should minimize erroneous interpretation of gene expression data from multiple analysis platforms.
Subject(s)
Gene Expression Profiling/methods , Genome-Wide Association Study , Humans , Oligonucleotide Array Sequence Analysis , Proto-Oncogene Proteins c-myc/genetics , Sequence Analysis, RNA , Transcription, GeneticABSTRACT
The mechanistic underpinnings of metastatic dormancy and reactivation are poorly understood. A gain-of-function cDNA screen reveals that Coco, a secreted antagonist of TGF-ß ligands, induces dormant breast cancer cells to undergo reactivation in the lung. Mechanistic studies indicate that Coco exerts this effect by blocking lung-derived BMP ligands. Whereas Coco enhances the manifestation of traits associated with cancer stem cells, BMP signaling suppresses it. Coco induces a discrete gene expression signature, which is strongly associated with metastatic relapse to the lung, but not to the bone or brain in patients. Experiments in mouse models suggest that these latter organs contain niches devoid of bioactive BMP. These findings reveal that metastasis-initiating cells need to overcome organ-specific antimetastatic signals in order to undergo reactivation.
Subject(s)
Breast Neoplasms/pathology , Intercellular Signaling Peptides and Proteins/metabolism , Lung Neoplasms/secondary , Animals , Bone Morphogenetic Proteins/metabolism , Cell Line, Tumor , Humans , Lung Neoplasms/metabolism , Mice , Mice, Inbred BALB C , Neoplasm Metastasis , Oligonucleotide Array Sequence AnalysisABSTRACT
The Drosophila auditory organ shares equivalent transduction mechanisms with vertebrate hair cells, and both are specified by atonal family genes. Using a whole-organ knockout strategy based on atonal, we have identified 274 Drosophila auditory organ genes. Only four of these genes had previously been associated with fly hearing, yet one in five of the genes that we identified has a human cognate that is implicated in hearing disorders. Mutant analysis of 42 genes shows that more than half of them contribute to auditory organ function, with phenotypes including hearing loss, auditory hypersusceptibility, and ringing ears. We not only discover ion channels and motors important for hearing, but also show that auditory stimulus processing involves chemoreceptor proteins as well as phototransducer components. Our findings demonstrate mechanosensory roles for ionotropic receptors and visual rhodopsins and indicate that different sensory modalities utilize common signaling cascades.
Subject(s)
Drosophila/physiology , Signal Transduction , Animals , Axonemal Dyneins/metabolism , Basic Helix-Loop-Helix Transcription Factors/metabolism , Drosophila/anatomy & histology , Drosophila/genetics , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Hair Cells, Auditory/metabolism , Hearing/physiology , Ion Channels/genetics , Ion Channels/metabolism , Nerve Tissue Proteins/metabolism , Oligonucleotide Array Sequence Analysis , Rhodopsin/genetics , Rhodopsin/metabolism , Transient Receptor Potential Channels/genetics , Transient Receptor Potential Channels/metabolismABSTRACT
In this issue of Molecular Cell, Layton et al. (2019) repurpose a common next-generation DNA sequencer to enable high-throughput protein biochemical studies, identifying improved sequence variants for stronger protein-protein interactions and dissecting the contributions of specific amino acids to enzymatic activity.
Subject(s)
DNA , High-Throughput Nucleotide Sequencing , Oligonucleotide Array Sequence Analysis , Sequence Analysis, DNAABSTRACT
High-throughput DNA sequencing techniques have enabled diverse approaches for linking DNA sequence to biochemical function. In contrast, assays of protein function have substantial limitations in terms of throughput, automation, and widespread availability. We have adapted an Illumina high-throughput sequencing chip to display an immense diversity of ribosomally translated proteins and peptides and then carried out fluorescence-based functional assays directly on this flow cell, demonstrating that a single, widely available high-throughput platform can perform both sequencing-by-synthesis and protein assays. We quantified the binding of the M2 anti-FLAG antibody to a library of 1.3 × 104 variant FLAG peptides, exploring non-additive effects of combinations of mutations and discovering a "superFLAG" epitope variant. We also measured the enzymatic activity of 1.56 × 105 molecular variants of full-length human O6-alkylguanine-DNA alkyltransferase (SNAP-tag). This comprehensive corpus of catalytic rates revealed amino acid interaction networks and cooperativity, linked positive cooperativity to structural proximity, and revealed ubiquitous positively cooperative interactions with histidine residues.
Subject(s)
Antibodies/metabolism , DNA Mutational Analysis/methods , High-Throughput Nucleotide Sequencing/methods , O(6)-Methylguanine-DNA Methyltransferase/metabolism , Oligonucleotide Array Sequence Analysis/methods , Oligopeptides/metabolism , Protein Array Analysis/methods , Antibody Affinity , Antibody Specificity , Automation, Laboratory , Binding Sites, Antibody , Catalysis , DNA Mutational Analysis/instrumentation , High-Throughput Nucleotide Sequencing/instrumentation , Kinetics , Mutation , O(6)-Methylguanine-DNA Methyltransferase/genetics , Oligonucleotide Array Sequence Analysis/instrumentation , Oligopeptides/genetics , Protein Array Analysis/instrumentation , Protein Binding , Protein Engineering , WorkflowABSTRACT
Mutations that lead to splicing defects can have severe consequences on gene function and cause disease. Here, we explore how human genetic variation affects exon recognition by developing a multiplexed functional assay of splicing using Sort-seq (MFASS). We assayed 27,733 variants in the Exome Aggregation Consortium (ExAC) within or adjacent to 2,198 human exons in the MFASS minigene reporter and found that 3.8% (1,050) of variants, most of which are extremely rare, led to large-effect splice-disrupting variants (SDVs). Importantly, we find that 83% of SDVs are located outside of canonical splice sites, are distributed evenly across distinct exonic and intronic regions, and are difficult to predict a priori. Our results indicate extant, rare genetic variants can have large functional effects on splicing at appreciable rates, even outside the context of disease, and MFASS enables their empirical assessment at scale.