RESUMEN
Cellular dynamics, the transition of a cell from one state to another, is central to understanding developmental processes and disease progression. Single-cell transcriptomics has been pushing the frontiers of cellular dynamics studies into a genome-wide and single-cell level. While most single-cell RNA sequencing approaches are disruptive and only provide a snapshot of cell states, the dynamics of a cell could be reconstructed by either exploiting temporal information hiding in the transcriptomics data or integrating additional information. In this review, we describe these approaches, highlighting their underlying principles, key assumptions, and the rationality to interpret the results as models. We also discuss the recently emerging nondisruptive live-cell transcriptomics methods, which are highly complementary to the computational models for their assumption-free nature.
Asunto(s)
Análisis de la Célula Individual , Transcriptoma , Transcriptoma/genética , Análisis de la Célula Individual/métodos , Perfilación de la Expresión GénicaRESUMEN
Gut-draining mesenteric lymph nodes (LN) provide the framework to shape intestinal adaptive immune responses. Based on the transcriptional signatures established by our previous work, the composition and immunomodulatory function of LN stromal cells (SC) vary according to location. Here, we describe the single-cell composition and development of the SC compartment within mesenteric LNs derived from postnatal to aged mice. We identify CD34+ SC and fibroblastic reticular stromal cell (FRC) progenitors as putative progenitors, both supplying the typical rapid postnatal mesenteric LN expansion. We further establish the location-specific chromatin accessibility and DNA methylation landscape of non-endothelial SCs and identify a microbiota-independent core epigenomic signature, showing characteristic differences between SCs from mesenteric and skin-draining peripheral LNs. The epigenomic landscape of SCs points to dynamic expression of Irf3 along the differentiation trajectories of FRCs. Accordingly, a mesenchymal stem cell line acquires a Cxcl9+ FRC molecular phenotype upon lentiviral overexpression of Irf3, and the relevance of Irf3 for SC biology is further underscored by the diminished proportion of Ccl19+ and Cxcl9+ FRCs in LNs of Irf3-/- mice. Together, our data constitute a comprehensive transcriptional and epigenomic map of mesenteric LNSC development in early life and dissect location-specific, microbiota-independent properties of non-endothelial SCs.
Asunto(s)
Ganglios Linfáticos , Células del Estroma , Ratones , Animales , Ratones Endogámicos C57BL , Células del Estroma/metabolismo , Ganglios Linfáticos/patología , Moléculas de Adhesión Celular/metabolismo , Antígenos CD34/metabolismoRESUMEN
Single-cell transcriptomics (scRNA-seq) has greatly advanced our ability to characterize cellular heterogeneity1. However, scRNA-seq requires lysing cells, which impedes further molecular or functional analyses on the same cells. Here, we established Live-seq, a single-cell transcriptome profiling approach that preserves cell viability during RNA extraction using fluidic force microscopy2,3, thus allowing to couple a cell's ground-state transcriptome to its downstream molecular or phenotypic behaviour. To benchmark Live-seq, we used cell growth, functional responses and whole-cell transcriptome read-outs to demonstrate that Live-seq can accurately stratify diverse cell types and states without inducing major cellular perturbations. As a proof of concept, we show that Live-seq can be used to directly map a cell's trajectory by sequentially profiling the transcriptomes of individual macrophages before and after lipopolysaccharide (LPS) stimulation, and of adipose stromal cells pre- and post-differentiation. In addition, we demonstrate that Live-seq can function as a transcriptomic recorder by preregistering the transcriptomes of individual macrophages that were subsequently monitored by time-lapse imaging after LPS exposure. This enabled the unsupervised, genome-wide ranking of genes on the basis of their ability to affect macrophage LPS response heterogeneity, revealing basal Nfkbia expression level and cell cycle state as important phenotypic determinants, which we experimentally validated. Thus, Live-seq can address a broad range of biological questions by transforming scRNA-seq from an end-point to a temporal analysis approach.
Asunto(s)
Supervivencia Celular , Perfilación de la Expresión Génica , Macrófagos , RNA-Seq , Análisis de la Célula Individual , Transcriptoma , Tejido Adiposo/citología , Ciclo Celular/efectos de los fármacos , Ciclo Celular/genética , Diferenciación Celular , Perfilación de la Expresión Génica/métodos , Perfilación de la Expresión Génica/normas , Genoma/efectos de los fármacos , Genoma/genética , Lipopolisacáridos/inmunología , Lipopolisacáridos/farmacología , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Inhibidor NF-kappaB alfa/genética , Especificidad de Órganos , Fenotipo , ARN/genética , ARN/aislamiento & purificación , RNA-Seq/métodos , RNA-Seq/normas , Reproducibilidad de los Resultados , Análisis de Secuencia de ARN/métodos , Análisis de Secuencia de ARN/normas , Análisis de la Célula Individual/métodos , Células del Estroma/citología , Células del Estroma/metabolismo , Factores de Tiempo , Transcriptoma/genéticaRESUMEN
Krüppel-associated box zinc finger proteins (KZFPs) constitute the largest family of mammalian transcription factors, but most remain completely uncharacterized. While initially proposed to primarily repress transposable elements, recent reports have revealed that KFZPs contribute to a wide variety of other biological processes. Using murine and human in vitro and in vivo models, we demonstrate here that one poorly studied KZFP, ZFP30, promotes adipogenesis by directly targeting and activating a retrotransposon-derived Pparg2 enhancer. Through mechanistic studies, we further show that ZFP30 recruits the co-regulator KRAB-associated protein 1 (KAP1), which, surprisingly, acts as a ZFP30 co-activator in this adipogenic context. Our findings provide an understanding of both adipogenic and KZFP-KAP1 complex-mediated gene regulation, showing that the KZFP-KAP1 axis can also function in a non-repressive manner.
Asunto(s)
Adipogénesis/genética , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción/metabolismo , Proteína 28 que Contiene Motivos Tripartito/metabolismo , Dedos de Zinc/fisiología , Células 3T3 , Adipocitos/fisiología , Animales , Biología Computacional , Proteínas de Unión al ADN/genética , Elementos de Facilitación Genéticos , Femenino , Regulación de la Expresión Génica/fisiología , Técnicas de Inactivación de Genes , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , PPAR gamma/genética , Regiones Promotoras Genéticas/genética , Retroelementos/genética , Factores de Transcripción/genéticaRESUMEN
Adipose tissue in the mammary gland undergoes dramatic remodeling during reproduction. Adipocytes are replaced by mammary alveolar structures during pregnancy and lactation, then reappear upon weaning. The fate of the original adipocytes during lactation and the developmental origin of the re-appearing adipocyte post involution are unclear. Here, we reveal that adipocytes in the mammary gland de-differentiate into Pdgfrα+ preadipocyte- and fibroblast-like cells during pregnancy and remain de-differentiated during lactation. Upon weaning, de-differentiated fibroblasts proliferate and re-differentiate into adipocytes. This cycle occurs over multiple pregnancies. These observations reveal the potential of terminally differentiated adipocytes to undergo repeated cycles of de-differentiation and re-differentiation in a physiological setting.
Asunto(s)
Adipocitos Blancos/metabolismo , Adipogénesis , Tejido Adiposo , Lactancia/metabolismo , Glándulas Mamarias Animales , Adipocitos Blancos/citología , Tejido Adiposo/citología , Tejido Adiposo/metabolismo , Animales , Femenino , Glándulas Mamarias Animales/citología , Glándulas Mamarias Animales/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Embarazo , DesteteRESUMEN
The c-Jun gene encodes a transcription factor that has been implicated in many physiological and pathological processes. c-Jun is a highly unstable protein that is degraded through a ubiquitination/proteasome-dependent mechanism. However, the deubiquitinating enzyme (DUB) that regulates the stability of the c-Jun protein requires further investigation. Here, by screening a DUB expression library, we identified ubiquitin-specific protease 6 (USP6) and showed that it regulates the stability of the c-Jun protein in a manner depending on its enzyme activity. USP6 interacts with c-Jun and antagonizes its ubiquitination. USP6 overexpression upregulates the activity of the downstream signaling pathway mediated by c-Jun/AP-1 and promotes cell invasion. Moreover, many aberrant genes that are upregulated in USP6 translocated nodular fasciitis are great potential targets regulated by c-Jun. Based on our data, USP6 is an enzyme that deubiquitinates c-Jun and regulates its downstream cellular functions.
Asunto(s)
Fascitis/genética , Proteínas Proto-Oncogénicas c-jun/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Movimiento Celular , Femenino , Regulación de la Expresión Génica , Células HeLa , Humanos , Células MCF-7 , Estabilidad Proteica , Transporte de Proteínas , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-jun/genética , Transducción de Señal , Factor de Transcripción AP-1/metabolismo , Ubiquitina/metabolismo , Ubiquitina Tiolesterasa/genética , UbiquitinaciónRESUMEN
Complex biological systems are composed of multiple cell types whose transcriptional activity can vary due to differences in cell state, environmental stimulation, or intrinsic programs. Conventional bulk analysis methods capture the average transcriptional programs of the cell population, thus missing the unique cellular signature of each single cell. In recent years, the development of single-cell RNA-sequencing (scRNA-seq) technologies has provided a powerful approach to dissect the cellular heterogeneity of complex biological systems. However, such approaches require specialized equipment or are costly. In this article, we describe an improved Smart-seq2-based method to profile the transcriptome of hundreds of single cells simultaneously, without utilizing commercial kits or requiring any specialized single-cell capture/library preparation tools. Moreover, we introduce the Automated Single-cell Analysis Pipeline (ASAP), which allows researchers without strong computational expertise to explore scRNA-seq data using a wide range of commonly used algorithms and sophisticated visualization tools. © 2017 by John Wiley & Sons, Inc.
Asunto(s)
Análisis de la Célula Individual , Transcriptoma , Animales , Secuencia de Bases , Biblioteca de Genes , Humanos , Ratones , Análisis de Secuencia de ARNRESUMEN
Brown adipocytes regulate energy expenditure via mitochondrial uncoupling, which makes them attractive therapeutic targets to tackle obesity. However, the regulatory mechanisms underlying brown adipogenesis are still poorly understood. To address this, we profiled the transcriptome and chromatin state during mouse brown fat cell differentiation, revealing extensive gene expression changes and chromatin remodeling, especially during the first day post-differentiation. To identify putatively causal regulators, we performed transcription factor binding site overrepresentation analyses in active chromatin regions and prioritized factors based on their expression correlation with the bona-fide brown adipogenic marker Ucp1 across multiple mouse and human datasets. Using loss-of-function assays, we evaluated both the phenotypic effect as well as the transcriptomic impact of several putative regulators on the differentiation process, uncovering ZFP467, HOXA4 and Nuclear Factor I A (NFIA) as novel transcriptional regulators. Of these, NFIA emerged as the regulator yielding the strongest molecular and cellular phenotypes. To examine its regulatory function, we profiled the genomic localization of NFIA, identifying it as a key early regulator of terminal brown fat cell differentiation.
Asunto(s)
Adipocitos Marrones/metabolismo , Metabolismo Energético/genética , Factores de Transcripción NFI/genética , Proteína Desacopladora 1/genética , Adipogénesis/genética , Animales , Diferenciación Celular/genética , Ensamble y Desensamble de Cromatina/genética , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica , Genómica , Proteínas de Homeodominio , Humanos , Ratones , Factores de Transcripción , Transcriptoma/genéticaRESUMEN
Clnk, as a third member of the Blnk/SLP-76 adapter family, is involved in the positive regulation of immunoreceptor signaling. Here we provide findings that Clnk may be is required for TNF induced cell death, it functions downstream of RIP3 and promotes TNF- induced ROS generation and MLKL tetramer formation and subsequent necrosis of L929 cells. Therefore, Clnk, as an adaptor protein, may take part in the other cellular processes.
Asunto(s)
Proteínas Portadoras/inmunología , Fibrosarcoma/inmunología , Especies Reactivas de Oxígeno/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Proteínas Adaptadoras Transductoras de Señales , Animales , Muerte Celular , Línea Celular Tumoral , Fibrosarcoma/patología , Células HeLa , Humanos , Ratones , Necrosis/inmunología , Proteínas Quinasas/química , Proteínas Quinasas/inmunología , Multimerización de Proteína , Proteína Serina-Treonina Quinasas de Interacción con Receptores/inmunología , Transducción de SeñalRESUMEN
The auto-phosphorylation of murine receptor-interacting protein 3 (Rip3) on Thr 231 and Ser 232 in the necrosome is required to trigger necroptosis. However, how Rip3 phosphorylation is regulated is still largely unknown. Here we identified protein phosphatase 1B (Ppm1b) as a Rip3 phosphatase and found that Ppm1b restricts necroptosis in two settings: spontaneous necroptosis caused by Rip3 auto-phosphorylation in resting cells, and tumour necrosis factor-α (TNF)-induced necroptosis in cultured cells. We revealed that Ppm1b selectively suppresses necroptosis through the dephosphorylation of Rip3, which then prevents the recruitment of mixed lineage kinase domain-like protein (Mlkl) to the necrosome. We further showed that Ppm1b deficiency (Ppm1b(d/d)) in mice enhanced TNF-induced death in a Rip3-dependent manner, and the role of Ppm1b in inhibiting necroptosis was evidenced by elevated Rip3 phosphorylation and tissue damage in the caecum of TNF-treated Ppm1b(d/d) mice. These data indicate that Ppm1b negatively regulates necroptosis through dephosphorylating Rip3 in vitro and in vivo.
Asunto(s)
Apoptosis/genética , Fosfoproteínas Fosfatasas/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Células 3T3 , Animales , Ciego/citología , Línea Celular , Técnicas de Inactivación de Genes , Células HeLa , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosforilación , Proteínas Quinasas/metabolismo , Proteína Fosfatasa 2C , Interferencia de ARN , ARN Interferente Pequeño , Transducción de Señal , Factor de Necrosis Tumoral alfaRESUMEN
Necroptosis is a form of programmed necrosis that is mediated by signaling complexes containing the receptor-interacting protein 3 (RIP3) and RIP1 kinases. We show that RIP3 and its interaction with the herpes simplex virus type 1 (HSV-1) protein ICP6 triggers necroptosis in infected mouse cells and limits viral propagation in mice. ICP6 interacts with RIP1/RIP3 through its RHIM domain and forms dimers/oliogmers by its C-terminal R1 domain. These binding events result in RIP1-RIP3 hetero- and RIP3-RIP3 homo-interactions and subsequent necroptosis of HSV-1-infected mouse cells. However, ICP6 RHIM cannot trigger necroptosis and even inhibits TNF-induced necroptosis in human cells. As the RHIM domain in murine cytomegalovirus protein vIRA can inhibit necroptosis in both human and mouse cells, these data suggest that both viral and host RHIM sequences determine whether the virus-host RHIM interaction is pro- or anti-necroptotic and that some viruses may evolve to escape this restriction.
Asunto(s)
Muerte Celular , Proteínas Activadoras de GTPasa/metabolismo , Herpesvirus Humano 1/inmunología , Interacciones Huésped-Patógeno , Mapas de Interacción de Proteínas , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Proteínas Virales/metabolismo , Animales , Línea Celular , Herpesvirus Humano 1/fisiología , Humanos , Evasión Inmune , Ratones , Unión Proteica , Replicación ViralRESUMEN
Viruses hijack host factors for their high speed protein synthesis, but information about these factors is largely unknown. In searching for genes that are involved in viral replication, we carried out a forward genetic screen for Drosophila mutants that are more resistant or sensitive to Drosophila C virus (DCV) infection-caused death, and found a virus-resistant line in which the expression of pelo gene was deficient. Our mechanistic studies excluded the viral resistance of pelo deficient flies resulting from the known Drosophila anti-viral pathways, and revealed that pelo deficiency limits the high level synthesis of the DCV capsid proteins but has no or very little effect on the expression of some other viral proteins, bulk cellular proteins, and transfected exogenous genes. The restriction of replication of other types of viruses in pelo deficient flies was also observed, suggesting pelo is required for high level production of capsids of all kinds of viruses. We show that both pelo deficiency and high level DCV protein synthesis increase aberrant 80S ribosomes, and propose that the preferential requirement of pelo for high level synthesis of viral capsids is at least partly due to the role of pelo in dissociation of stalled 80S ribosomes and clearance of aberrant viral RNA and proteins. Our data demonstrated that pelo is a host factor that is required for high efficiency translation of viral capsids and targeting pelo could be a strategy for general inhibition of viral infection.
Asunto(s)
Dicistroviridae/fisiología , Proteínas de Drosophila/metabolismo , Regulación Viral de la Expresión Génica/fisiología , Proteínas Nucleares/metabolismo , Biosíntesis de Proteínas/fisiología , Proteínas Virales/biosíntesis , Replicación Viral/fisiología , Animales , Cápside/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster , Mutación , Proteínas Nucleares/genética , Proteínas Virales/genéticaRESUMEN
Formation of multi-component signaling complex necrosomes is essential for tumor necrosis factor α (TNF)-induced programmed necrosis (also called necroptosis). However, the mechanisms of necroptosis are still largely unknown. We isolated a TNF-resistant L929 mutant cell line generated by retrovirus insertion and identified that disruption of the guanine nucleotide-binding protein γ 10 (Gγ10) gene is responsible for this phenotype. We further show that Gγ10 is involved in TNF-induced necroptosis and Gß2 is the partner of Gγ10. Src is the downstream effector of Gß2γ10 in TNF-induced necroptosis because TNF-induced Src activation was impaired upon Gγ10 knockdown. Gγ10 does not affect TNF-induced activation of NF-κB and MAPKs and the formation of necrosomes, but is required for trafficking of necrosomes to their potential functioning site, an unidentified subcellular organelle that can be fractionated into heterotypic membrane fractions. The TNF-induced Gßγ-Src signaling pathway is independent of RIP1/RIP3 kinase activity and necrosome formation, but is required for the necrosome to function.
Asunto(s)
Apoptosis/efectos de los fármacos , Apoptosis/genética , Vesículas Citoplasmáticas/efectos de los fármacos , Subunidades beta de la Proteína de Unión al GTP/fisiología , Subunidades gamma de la Proteína de Unión al GTP/fisiología , Factor de Necrosis Tumoral alfa/farmacología , Familia-src Quinasas/fisiología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Transporte Biológico/efectos de los fármacos , Transporte Biológico/genética , Vesículas Citoplasmáticas/metabolismo , Células HEK293 , Humanos , Ratones , Datos de Secuencia Molecular , Necrosis/inducido químicamente , Necrosis/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Células Tumorales CultivadasRESUMEN
Mixed lineage kinase domain-like protein (Mlkl) was recently found to interact with receptor interacting protein 3 (Rip3) and to be essential for tumor necrosis factor (TNF)-induced programmed necrosis (necroptosis) in cultured cell lines. We have generated Mlkl-deficient mice by transcription activator-like effector nucleases (TALENs)-mediated gene disruption and found Mlkl to be dispensable for normal mouse development as well as immune cell development. Mlkl-deficient mouse embryonic fibroblasts (MEFs) and macrophages both showed resistance to necrotic but not apoptotic stimuli. Mlkl-deficient MEFs and macrophages were indistinguishable from wild-type cells in their ability to activate NF-κB, ERK, JNK, and p38 in response to TNF and lipopolysaccharides (LPS), respectively. Consistently, Mlkl-deficient macrophages and mice exhibited normal interleukin-1ß (IL-1ß), IL-6, and TNF production after LPS treatment. Mlkl deficiency protects mice from cerulean-induced acute pancreatitis, a necrosis-related disease, but has no effect on polymicrobial septic shock-induced animal death. Our results provide genetic evidence for the role of Mlkl in necroptosis.
Asunto(s)
Apoptosis , Proteínas Quinasas/metabolismo , Clorometilcetonas de Aminoácidos/farmacología , Animales , Secuencia de Bases , Línea Celular , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Lipopolisacáridos/toxicidad , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , FN-kappa B/metabolismo , Necrosis , Proteínas Quinasas/deficiencia , Proteínas Quinasas/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/deficiencia , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/farmacología , Factores de Necrosis Tumoral/metabolismoRESUMEN
Receptor interacting protein 3 (RIP3) is a protein kinase essential for TNF-induced necroptosis. Phosphorylation on Ser-227 in human RIP3 (hRIP3) is required for its interaction with human mixed lineage kinase domain-like (MLKL) in the necrosome, a signaling complex induced by TNF stimulation. RIP1 and RIP3 mediate necrosome aggregation leading to the formation of amyloid-like signaling complexes. We found that TNF induces Thr-231 and Ser-232 phosphorylation in mouse RIP3 (mRIP3) and this phosphorylation is required for mRIP3 to interact with mMLKL. Ser-232 in mRIP3 corresponds to Ser-227 in hRIP3, whereas Thr-231 is not conserved in hRIP3. Although the RIP3-MLKL interaction is required for necroptosis in both human and mouse cells, hRIP3 does not interact with mMLKL and mRIP3 cannot bind to hMLKL. The species specificity of the RIP3-MLKL interaction is primarily determined by the sequence differences in the phosphorylation sites and the flanking sequence around the phosphorylation sites in hRIP3 and mRIP3. It appears that the RIP3-MLKL interaction has been selected as an evolutionarily conserved mechanism in mediating necroptosis signaling despite that differing structural and mechanistic bases for this interaction emerged simultaneously in different organisms. In addition, we further revealed that the interaction of RIP3 with MLKL prevented massive abnormal RIP3 aggregation, and therefore should be crucial for formation of the amyloid signaling complex of necrosomes. We also found that the interaction between RIP3 and MLKL is required for the translocation of necrosomes to mitochondria-associated membranes. Our data demonstrate the importance of the RIP3-MLKL interaction in the formation of functional necrosomes and suggest that translocation of necrosomes to mitochondria-associated membranes is essential for necroptosis signaling.
Asunto(s)
Células Musculares/enzimología , Proteínas Musculares/metabolismo , Proteínas Quinasas/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Transducción de Señal , Amiloide/genética , Amiloide/metabolismo , Animales , Línea Celular , Humanos , Ratones , Mitocondrias Musculares/metabolismo , Mitocondrias Musculares/patología , Células Musculares/patología , Proteínas Musculares/genética , Necrosis/enzimología , Necrosis/genética , Necrosis/patología , Fosforilación/genética , Proteínas Quinasas/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genéticaRESUMEN
Mitochondrial adenine nucleotide translocase (ANT) is believed to be a component or a regulatory component of the mitochondrial permeability transition pore (mtPTP), which controls mitochondrial permeability transition during apoptosis. However, the role of ANT in apoptosis is still uncertain, because hepatocytes isolated from ANT knockout and wild-type mice are equally sensitive to TNF- and Fas-induced apoptosis. In a screen for genes required for tumor necrosis factor alpha (TNF-alpha)-induced apoptosis in MCF-7 human breast cancer cells using retrovirus insertion-mediated random mutagenesis, we discovered that the ANT3 gene is involved in TNF-alpha-induced cell death in MCF-7 cells. We further found that ANT3 is selectively required for TNF- and oxidative stress-induced cell death in MCF-7 cells, but it is dispensable for cell death induced by several other inducers. This data supplements previous data obtained from ANT knockout studies, indicating that ANT is involved in some apoptotic processes. We found that the resistance to TNF-alpha-induced apoptosis observed in ANT3 mutant (ANT3(mut)) cells is associated with a deficiency in the regulation of the mitochondrial membrane potential and cytochrome c release. It is not related to intracellular ATP levels or survival pathways, supporting a previous model in which ANT regulates mtPTP. Our study provides genetic evidence supporting a role of ANT in apoptosis and suggests that the involvement of ANT in cell death is cell type- and stimulus-dependent.