RESUMEN
The size of the human head is highly heritable, but genetic drivers of its variation within the general population remain unmapped. We perform a genome-wide association study on head size (N = 80,890) and identify 67 genetic loci, of which 50 are novel. Neuroimaging studies show that 17 variants affect specific brain areas, but most have widespread effects. Gene set enrichment is observed for various cancers and the p53, Wnt, and ErbB signaling pathways. Genes harboring lead variants are enriched for macrocephaly syndrome genes (37-fold) and high-fidelity cancer genes (9-fold), which is not seen for human height variants. Head size variants are also near genes preferentially expressed in intermediate progenitor cells, neural cells linked to evolutionary brain expansion. Our results indicate that genes regulating early brain and cranial growth incline to neoplasia later in life, irrespective of height. This warrants investigation of clinical implications of the link between head size and cancer.
Asunto(s)
Estudio de Asociación del Genoma Completo , Cabeza , Neoplasias , Humanos , Cabeza/anatomía & histología , Neoplasias/genética , Neoplasias/patología , Femenino , Masculino , Polimorfismo de Nucleótido Simple/genética , Variación Genética , Tamaño de los Órganos/genética , Transducción de Señal/genética , Adulto , Predisposición Genética a la EnfermedadRESUMEN
A wide range of nuclear proteins are involved in the spatio-temporal organization of the genome through diverse biological processes such as gene transcription and DNA replication. Upon stimulation by testosterone and translocation to the nucleus, multiple androgen receptors (ARs) accumulate in microscopically discernable foci which are irregularly distributed in the nucleus. Here, we investigated the formation and physical nature of these foci, by combining novel fluorescent labeling techniques to visualize a defined chromatin locus of AR-regulated genes-PTPRN2 or BANP-simultaneously with either AR foci or individual AR molecules. Quantitative colocalization analysis showed evidence of AR foci formation induced by R1881 at both PTPRN2 and BANP loci. Furthermore, single-particle tracking (SPT) revealed three distinct subdiffusive fractional Brownian motion (fBm) states: immobilized ARs were observed near the labeled genes likely as a consequence of DNA-binding, while the intermediate confined state showed a similar spatial behavior but with larger displacements, suggesting compartmentalization by liquid-liquid phase separation (LLPS), while freely mobile ARs were diffusing in the nuclear environment. All together, we show for the first time in living cells the presence of AR-regulated genes in AR foci.
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Núcleo Celular , Receptores Androgénicos , Animales , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas Nucleares/metabolismo , Receptores Androgénicos/metabolismo , Humanos , Ratones , Línea Celular TumoralRESUMEN
Biallelic loss-of-function variants in SMPD4 cause a rare and severe neurodevelopmental disorder with progressive congenital microcephaly and early death. SMPD4 encodes a sphingomyelinase that hydrolyses sphingomyelin into ceramide at neutral pH and can thereby affect membrane lipid homeostasis. SMPD4 localizes to the membranes of the endoplasmic reticulum and nuclear envelope and interacts with nuclear pore complexes (NPC). We refine the clinical phenotype of loss-of-function SMPD4 variants by describing five individuals from three unrelated families with longitudinal data due to prolonged survival. All individuals surviving beyond infancy developed insulin-dependent diabetes, besides presenting with a severe neurodevelopmental disorder and microcephaly, making diabetes one of the most frequent age-dependent non-cerebral abnormalities. We studied the function of SMPD4 at the cellular and organ levels. Knock-down of SMPD4 in human neural stem cells causes reduced proliferation rates and prolonged mitosis. Moreover, SMPD4 depletion results in abnormal nuclear envelope breakdown and reassembly during mitosis and decreased post-mitotic NPC insertion. Fibroblasts from affected individuals show deficient SMPD4-specific neutral sphingomyelinase activity, without changing (sub)cellular lipidome fractions, which suggests a local function of SMPD4 on the nuclear envelope. In embryonic mouse brain, knockdown of Smpd4 impairs cortical progenitor proliferation and induces premature differentiation by altering the balance between neurogenic and proliferative progenitor cell divisions. We hypothesize that, in individuals with SMPD4-related disease, nuclear envelope bending, which is needed to insert NPCs in the nuclear envelope, is impaired in the absence of SMPD4 and interferes with cerebral corticogenesis and survival of pancreatic beta cells.
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Diabetes Mellitus , Microcefalia , Humanos , Animales , Ratones , Membrana Nuclear/química , Membrana Nuclear/metabolismo , Microcefalia/genética , Microcefalia/metabolismo , Esfingomielina Fosfodiesterasa/análisis , Esfingomielina Fosfodiesterasa/genética , Esfingomielina Fosfodiesterasa/metabolismo , Poro Nuclear/metabolismo , Mitosis , Diabetes Mellitus/metabolismoRESUMEN
TBR1 is a neuron-specific transcription factor involved in brain development and implicated in a neurodevelopmental disorder (NDD) combining features of autism spectrum disorder (ASD), intellectual disability (ID) and speech delay. TBR1 has been previously shown to interact with a small number of transcription factors and co-factors also involved in NDDs (including CASK, FOXP1/2/4 and BCL11A), suggesting that the wider TBR1 interactome may have a significant bearing on normal and abnormal brain development. Here, we have identified approximately 250 putative TBR1-interaction partners by affinity purification coupled to mass spectrometry. As well as known TBR1-interactors such as CASK, the identified partners include transcription factors and chromatin modifiers, along with ASD- and ID-related proteins. Five interaction candidates were independently validated using bioluminescence resonance energy transfer assays. We went on to test the interaction of these candidates with TBR1 protein variants implicated in cases of NDD. The assays uncovered disturbed interactions for NDD-associated variants and identified two distinct protein-binding domains of TBR1 that have essential roles in protein-protein interaction.
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Trastornos del Neurodesarrollo , Proteínas de Dominio T Box , Humanos , Trastorno del Espectro Autista/genética , Trastorno del Espectro Autista/metabolismo , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Discapacidad Intelectual/genética , Discapacidad Intelectual/metabolismo , Trastornos del Neurodesarrollo/genética , Trastornos del Neurodesarrollo/metabolismo , Unión Proteica/genética , Unión Proteica/fisiología , Proteínas/genética , Proteínas/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Proteínas de Dominio T Box/genética , Proteínas de Dominio T Box/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Congenital diaphragmatic hernia is a structural birth defect of the diaphragm, with lung hypoplasia and persistent pulmonary hypertension. Aside from vascular defects, the lungs show a disturbed balance of differentiated airway epithelial cells. The Sry related HMG box protein SOX2 is an important transcription factor for proper differentiation of the lung epithelium. The transcriptional activity of SOX2 depends on interaction with other proteins and the identification of SOX2-associating factors may reveal important complexes involved in the disturbed differentiation in CDH. To identify SOX2-associating proteins, we purified SOX2 complexes from embryonic mouse lungs at 18.5 days of gestation. Mass spectrometry analysis of SOX2-associated proteins identified several potential candidates, among which were the Chromodomain Helicase DNA binding protein 4 (CHD4), Cut-Like Homeobox1 (CUX1), and the Forkhead box proteins FOXP2 and FOXP4. We analyzed the expression patterns of FOXP2, FOXP4, CHD4, and CUX1 in lung during development and showed co-localization with SOX2. Co-immunoprecipitations validated the interactions of these four transcription factors with SOX2, and large-scale chromatin immunoprecipitation (ChIP) data indicated that SOX2 and CHD4 bound to unique sites in the genome, but also co-occupied identical regions, suggesting that these complexes could be involved in co-regulation of genes involved in the respiratory system.
RESUMEN
Marked by incomplete division of the embryonic forebrain, holoprosencephaly is one of the most common human developmental disorders. Despite decades of phenotype-driven research, 80-90% of aneuploidy-negative holoprosencephaly individuals with a probable genetic aetiology do not have a genetic diagnosis. Here we report holoprosencephaly associated with variants in the two X-linked cohesin complex genes, STAG2 and SMC1A, with loss-of-function variants in 10 individuals and a missense variant in one. Additionally, we report four individuals with variants in the cohesin complex genes that are not X-linked, SMC3 and RAD21. Using whole mount in situ hybridization, we show that STAG2 and SMC1A are expressed in the prosencephalic neural folds during primary neurulation in the mouse, consistent with forebrain morphogenesis and holoprosencephaly pathogenesis. Finally, we found that shRNA knockdown of STAG2 and SMC1A causes aberrant expression of HPE-associated genes ZIC2, GLI2, SMAD3 and FGFR1 in human neural stem cells. These findings show the cohesin complex as an important regulator of median forebrain development and X-linked inheritance patterns in holoprosencephaly.
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Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/genética , Holoprosencefalia/diagnóstico , Holoprosencefalia/genética , Adolescente , Animales , Niño , Preescolar , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Ratones , Ratones Endogámicos C57BL , CohesinasRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMEN
The Mediator complex regulates transcription by connecting enhancers to promoters. High Mediator binding density defines super enhancers, which regulate cell-identity genes and oncogenes. Protein interactions of Mediator may explain its role in these processes but have not been identified comprehensively. Here, we purify Mediator from neural stem cells (NSCs) and identify 75 protein-protein interaction partners. We identify super enhancers in NSCs and show that Mediator-interacting chromatin modifiers colocalize with Mediator at enhancers and super enhancers. Transcription factor families with high affinity for Mediator dominate enhancers and super enhancers and can explain genome-wide Mediator localization. We identify E-box transcription factor Tcf4 as a key regulator of NSCs. Tcf4 interacts with Mediator, colocalizes with Mediator at super enhancers and regulates neurogenic transcription factor genes with super enhancers and broad H3K4me3 domains. Our data suggest that high binding-affinity for Mediator is an important organizing feature in the transcriptional network that determines NSC identity.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/fisiología , Redes Reguladoras de Genes/fisiología , Complejo Mediador/metabolismo , Células-Madre Neurales/fisiología , Neurogénesis/genética , Factor de Transcripción 4/metabolismo , Línea Celular , Elementos de Facilitación Genéticos/genética , Histonas/metabolismo , Humanos , Histona Demetilasas con Dominio de Jumonji/metabolismo , Oxidorreductasas N-Desmetilantes/metabolismo , Regiones Promotoras Genéticas/genética , Mapeo de Interacción de Proteínas , Mapas de Interacción de Proteínas/genética , Proteína-Arginina N-Metiltransferasas/metabolismo , Transcripción Genética/fisiologíaRESUMEN
Recessive mutations in RTTN, encoding the protein rotatin, were originally identified as cause of polymicrogyria, a cortical malformation. With time, a wide variety of other brain malformations has been ascribed to RTTN mutations, including primary microcephaly. Rotatin is a centrosomal protein possibly involved in centriolar elongation and ciliogenesis. However, the function of rotatin in brain development is largely unknown and the molecular disease mechanism underlying cortical malformations has not yet been elucidated. We performed both clinical and cell biological studies, aimed at clarifying rotatin function and pathogenesis. Review of the 23 published and five unpublished clinical cases and genomic mutations, including the effect of novel deep intronic pathogenic mutations on RTTN transcripts, allowed us to extrapolate the core phenotype, consisting of intellectual disability, short stature, microcephaly, lissencephaly, periventricular heterotopia, polymicrogyria and other malformations. We show that the severity of the phenotype is related to residual function of the protein, not only the level of mRNA expression. Skin fibroblasts from eight affected individuals were studied by high resolution immunomicroscopy and flow cytometry, in parallel with in vitro expression of RTTN in HEK293T cells. We demonstrate that rotatin regulates different phases of the cell cycle and is mislocalized in affected individuals. Mutant cells showed consistent and severe mitotic failure with centrosome amplification and multipolar spindle formation, leading to aneuploidy and apoptosis, which could relate to depletion of neuronal progenitors often observed in microcephaly. We confirmed the role of rotatin in functional and structural maintenance of primary cilia and determined that the protein localized not only to the basal body, but also to the axoneme, proving the functional interconnectivity between ciliogenesis and cell cycle progression. Proteomics analysis of both native and exogenous rotatin uncovered that rotatin interacts with the neuronal (non-muscle) myosin heavy chain subunits, motors of nucleokinesis during neuronal migration, and in human induced pluripotent stem cell-derived bipolar mature neurons rotatin localizes at the centrosome in the leading edge. This illustrates the role of rotatin in neuronal migration. These different functions of rotatin explain why RTTN mutations can lead to heterogeneous cerebral malformations, both related to proliferation and migration defects.
Asunto(s)
Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/fisiología , Adulto , Encéfalo/patología , Proteínas Portadoras/genética , Ciclo Celular/fisiología , Cilios/metabolismo , Femenino , Estudios de Asociación Genética/métodos , Células HEK293 , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Lactante , Recién Nacido , Masculino , Malformaciones del Desarrollo Cortical/genética , Malformaciones del Desarrollo Cortical/metabolismo , Microcefalia/genética , Mutación , Malformaciones del Sistema Nervioso/genética , Polimicrogiria/etiología , Polimicrogiria/patologíaRESUMEN
In this study, we demonstrate that, among all five CBX Polycomb proteins, only CBX7 possesses the ability to control self-renewal of human hematopoietic stem and progenitor cells (HSPCs). Xenotransplantation of CBX7-overexpressing HSPCs resulted in increased multi-lineage long-term engraftment and myelopoiesis. Gene expression and chromatin analyses revealed perturbations in genes involved in differentiation, DNA and chromatin maintenance, and cell cycle control. CBX7 is upregulated in acute myeloid leukemia (AML), and its genetic or pharmacological repression in AML cells inhibited proliferation and induced differentiation. Mass spectrometry analysis revealed several non-histone protein interactions between CBX7 and the H3K9 methyltransferases SETDB1, EHMT1, and EHMT2. These CBX7-binding proteins possess a trimethylated lysine peptide motif highly similar to the canonical CBX7 target H3K27me3. Depletion of SETDB1 in AML cells phenocopied repression of CBX7. We identify CBX7 as an important regulator of self-renewal and uncover non-canonical crosstalk between distinct pathways, revealing therapeutic opportunities for leukemia.
Asunto(s)
Células Madre Hematopoyéticas/metabolismo , Complejo Represivo Polycomb 1/metabolismo , Células Madre/metabolismo , Animales , Femenino , Sangre Fetal/citología , Sangre Fetal/metabolismo , Células HEK293 , Células HL-60 , Células Madre Hematopoyéticas/citología , Xenoinjertos , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Células K562 , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Complejo Represivo Polycomb 1/biosíntesis , Complejo Represivo Polycomb 1/genética , Células Madre/citología , Transcripción GenéticaRESUMEN
Super-enhancers (SEs) are important for regulating cell identity genes and oncogenes, but correctly assigning target genes to SEs is difficult. Recently in Cell Reports, Lopes Novo et al. (2018) map interactions between SEs and promoters and observe a significant rewiring of complex SE-promoter networks between different pluripotent states.
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Células Madre Embrionarias de Ratones , Animales , Elementos de Facilitación Genéticos , RatonesRESUMEN
FOXP transcription factors play important roles in neurodevelopment, but little is known about how their transcriptional activity is regulated. FOXP proteins cooperatively regulate gene expression by forming homo- and hetero-dimers with each other. Physical associations with other transcription factors might also modulate the functions of FOXP proteins. However, few FOXP-interacting transcription factors have been identified so far. Therefore, we sought to discover additional transcription factors that interact with the brain-expressed FOXP proteins, FOXP1, FOXP2 and FOXP4, through affinity-purifications of protein complexes followed by mass spectrometry. We identified seven novel FOXP-interacting transcription factors (NR2F1, NR2F2, SATB1, SATB2, SOX5, YY1 and ZMYM2), five of which have well-estabslished roles in cortical development. Accordingly, we found that these transcription factors are co-expressed with FoxP2 in the deep layers of the cerebral cortex and also in the Purkinje cells of the cerebellum, suggesting that they may cooperate with the FoxPs to regulate neural gene expression in vivo. Moreover, we demonstrated that etiological mutations of FOXP1 and FOXP2, known to cause neurodevelopmental disorders, severely disrupted the interactions with FOXP-interacting transcription factors. Additionally, we pinpointed specific regions within FOXP2 sequence involved in mediating these interactions. Thus, by expanding the FOXP interactome we have uncovered part of a broader neural transcription factor network involved in cortical development, providing novel molecular insights into the transcriptional architecture underlying brain development and neurodevelopmental disorders.
Asunto(s)
Factores de Transcripción Forkhead , Regulación de la Expresión Génica , Trastornos del Neurodesarrollo , Células de Purkinje/metabolismo , Proteínas Represoras , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Células HEK293 , Humanos , Trastornos del Neurodesarrollo/genética , Trastornos del Neurodesarrollo/metabolismo , Trastornos del Neurodesarrollo/patología , Células de Purkinje/patología , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Chromatin in embryonic stem cells (ESCs) differs markedly from that in somatic cells, with ESCs exhibiting a more open chromatin configuration. Accordingly, ATP-dependent chromatin remodeling complexes are important regulators of ESC homeostasis. Depletion of the remodeler SMARCAD1, an ATPase of the SNF2 family, has been shown to affect stem cell state, but the mechanistic explanation for this effect is unknown. Here, we set out to gain further insights into the function of SMARCAD1 in mouse ESCs. We identified KRAB-associated protein 1 (KAP1) as the stoichiometric binding partner of SMARCAD1 in ESCs. We found that this interaction occurs on chromatin and that SMARCAD1 binds to different classes of KAP1 target genes, including zinc finger protein (ZFP) and imprinted genes. We also found that the RING B-box coiled-coil (RBCC) domain in KAP1 and the proximal coupling of ubiquitin conjugation to ER degradation (CUE) domain in SMARCAD1 mediate their direct interaction. Of note, retention of SMARCAD1 in the nucleus depended on KAP1 in both mouse ESCs and human somatic cells. Mutations in the CUE1 domain of SMARCAD1 perturbed the binding to KAP1 in vitro and in vivo Accordingly, an intact CUE1 domain was required for tethering this remodeler to the nucleus. Moreover, mutation of the CUE1 domain compromised SMARCAD1 binding to KAP1 target genes. Taken together, our results reveal a mechanism that localizes SMARCAD1 to genomic sites through the interaction of SMARCAD1's CUE1 motif with KAP1.
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Células Madre Adultas/metabolismo , Núcleo Celular/metabolismo , ADN Helicasas/metabolismo , Regulación de la Expresión Génica , Células Madre Embrionarias de Ratones/metabolismo , Proteína 28 que Contiene Motivos Tripartito/metabolismo , Células Madre Adultas/citología , Células Madre Adultas/enzimología , Sustitución de Aminoácidos , Animales , Línea Celular , Núcleo Celular/enzimología , Cromatina/química , Cromatina/enzimología , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina , ADN Helicasas/antagonistas & inhibidores , ADN Helicasas/química , ADN Helicasas/genética , Eliminación de Gen , Humanos , Cinética , Ratones , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/enzimología , Mutación , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Dominios y Motivos de Interacción de Proteínas , Interferencia de ARN , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Proteína 28 que Contiene Motivos Tripartito/antagonistas & inhibidores , Proteína 28 que Contiene Motivos Tripartito/química , Proteína 28 que Contiene Motivos Tripartito/genéticaRESUMEN
Xist is indispensable for X chromosome inactivation. However, how Xist RNA directs chromosome-wide silencing and why some regions are more efficiently silenced than others remains unknown. Here, we explore the function of Xist by inducing ectopic Xist expression from multiple different X-linked and autosomal loci in mouse aneuploid and female diploid embryonic stem cells in which Xist-mediated silencing does not lead to lethal functional monosomy. We show that ectopic Xist expression faithfully recapitulates endogenous X chromosome inactivation from any location on the X chromosome, whereas long-range silencing of autosomal genes is less efficient. Long interspersed elements facilitate inactivation of genes located far away from the Xist transcription locus, and genes escaping X chromosome inactivation show enrichment of CTCF on X chromosomal but not autosomal loci. Our findings highlight important genomic and epigenetic features acquired during sex chromosome evolution to facilitate an efficient X chromosome inactivation process.Xist RNA is required for X chromosome inactivation but it is not well understood how Xist silences some regions more efficiently than others. Here, the authors induce ectopic Xist expression from multiple different X-linked and autosomal loci in cells to explore Xist function.
Asunto(s)
Evolución Molecular , ARN Largo no Codificante/fisiología , Inactivación del Cromosoma X/genética , Animales , Factor de Unión a CCCTC/metabolismo , Diferenciación Celular/genética , Células Madre Embrionarias/metabolismo , Silenciador del Gen , Elementos de Nucleótido Esparcido Largo/fisiología , Ratones , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismoRESUMEN
Although hepatitis E has emerged as a global health issue, there is limited knowledge of its infection biology and no FDA-approved medication is available. Aiming to investigate the role of protein kinases in hepatitis E virus (HEV) infection and to identify potential antiviral targets, we screened a library of pharmacological kinase inhibitors in a cell culture model, a subgenomic HEV replicon containing luciferase reporter. We identified protein kinase C alpha (PKCα) as an essential cell host factor restricting HEV replication. Both specific inhibitor and shRNA-mediated knockdown of PKCα enhanced HEV replication. Conversely, over-expression of the activated form of PKCα or treatment with its pharmacological activator strongly inhibited HEV replication. Interestingly, upon the stimulation by its activator, PKCα efficiently activates its downstream Activator Protein 1 (AP-1) pathway, leading to the induction of antiviral interferon-stimulated genes (ISGs). This process is independent of the JAK-STAT machinery and interferon production. However, PKCα induced HEV inhibition appears independent of the AP1 cascade. The discovery that activated PKCα restricts HEV replication reveals new insight of HEV-host interactions and provides new target for antiviral drug development.
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Antivirales/farmacología , Virus de la Hepatitis E/fisiología , Proteína Quinasa C-alfa/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Replicación Viral/efectos de los fármacos , Antivirales/aislamiento & purificación , Técnicas de Cultivo de Célula , Replicación del ADN , Activación Enzimática , Hepatitis E/tratamiento farmacológico , Hepatocitos/virología , Interacciones Huésped-Patógeno , Humanos , Proteína Quinasa C-alfa/antagonistas & inhibidores , Proteína Quinasa C-alfa/deficiencia , Proteína Quinasa C-alfa/genética , Inhibidores de Proteínas Quinasas/aislamiento & purificación , Replicón , Transducción de Señal , Acetato de Tetradecanoilforbol/farmacología , Factor de Transcripción AP-1/genética , Factor de Transcripción AP-1/metabolismo , Activación TranscripcionalRESUMEN
IFN-α has been used for decades to treat chronic hepatitis B and C, and as an off-label treatment for some cases of hepatitis E virus (HEV) infection. TNF-α is another important cytokine involved in inflammatory disease, which can interact with interferon signaling. Because interferon-stimulated genes (ISGs) are the ultimate antiviral effectors of the interferon signaling, this study aimed to understand the regulation of ISG transcription and the antiviral activity by IFN-α and TNF-α. In this study, treatment of TNF-α inhibited replication of HCV by 71 ± 2.4% and HEV by 41 ± 4.9%. Interestingly, TNF-α induced the expression of a panel of antiviral ISGs (2-11 fold). Blocking the TNF-α signaling by Humira abrogated ISG induction and its antiviral activity. Chip-seq data analysis and mutagenesis assay further revealed that the NF-κB protein complex, a key downstream element of TNF-α signaling, directly binds to the ISRE motif in the ISG promoters and thereby drives their transcription. This process is independent of interferons and JAK-STAT cascade. Importantly, when combined with IFN-α, TNF-α works cooperatively on ISG induction, explaining their additive antiviral effects. Thus, our study reveals a novel mechanism of convergent transcription of ISGs by TNF-α and IFN-α, which augments their antiviral activity against HCV and HEV.
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Hepacivirus/inmunología , Virus de la Hepatitis E/inmunología , Hepatocitos/inmunología , Hepatocitos/virología , Interferón-alfa/metabolismo , Transcripción Genética , Factor de Necrosis Tumoral alfa/metabolismo , Antivirales/metabolismo , Línea Celular , Hepacivirus/fisiología , Virus de la Hepatitis E/fisiología , Humanos , Replicación ViralRESUMEN
The chromosomal protein SMCHD1 plays an important role in epigenetic silencing at diverse loci, including the inactive X chromosome, imprinted genes, and the facioscapulohumeral muscular dystrophy locus. Although homology with canonical SMC family proteins suggests a role in chromosome organization, the mechanisms underlying SMCHD1 function and target site selection remain poorly understood. Here we show that SMCHD1 forms an active GHKL-ATPase homodimer, contrasting with canonical SMC complexes, which exist as tripartite ring structures. Electron microscopy analysis demonstrates that SMCHD1 homodimers structurally resemble prokaryotic condensins. We further show that the principal mechanism for chromatin loading of SMCHD1 involves an LRIF1-mediated interaction with HP1γ at trimethylated histone H3 lysine 9 (H3K9me3)-modified chromatin sites on the chromosome arms. A parallel pathway accounts for chromatin loading at a minority of sites, notably the inactive X chromosome. Together, our results provide key insights into SMCHD1 function and target site selection.
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Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Histonas/metabolismo , Cromosoma X/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Cromatina/química , Homólogo de la Proteína Chromobox 5 , Proteínas Cromosómicas no Histona/química , Células HEK293 , Histonas/química , Humanos , Lisina/análisis , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Mapas de Interacción de Proteínas , Multimerización de Proteína , Estructura Terciaria de Proteína , Alineación de SecuenciaRESUMEN
The locations of transcriptional enhancers and promoters were recently mapped in many mammalian cell types. Proteins that bind those regulatory regions can determine cell identity but have not been systematically identified. Here we purify native enhancers, promoters or heterochromatin from embryonic stem cells by chromatin immunoprecipitations (ChIP) for characteristic histone modifications and identify associated proteins using mass spectrometry (MS). 239 factors are identified and predicted to bind enhancers or promoters with different levels of activity, or heterochromatin. Published genome-wide data indicate a high accuracy of location prediction by ChIP-MS. A quarter of the identified factors are important for pluripotency and includes Oct4, Esrrb, Klf5, Mycn and Dppa2, factors that drive reprogramming to pluripotent stem cells. We determined the genome-wide binding sites of Dppa2 and find that Dppa2 operates outside the classical pluripotency network. Our ChIP-MS method provides a detailed read-out of the transcriptional landscape representative of the investigated cell type.
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Inmunoprecipitación de Cromatina/métodos , Histonas/química , Animales , Sitios de Unión , Dominio Catalítico , Células Madre Embrionarias/citología , Elementos de Facilitación Genéticos , Genoma , Código de Histonas , Factores de Transcripción de Tipo Kruppel/química , Espectrometría de Masas/métodos , Ratones , Proteína Proto-Oncogénica N-Myc , Proteínas Nucleares/química , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Células Madre Pluripotentes/citología , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Proto-Oncogénicas/química , Receptores de Estrógenos/química , Secuencias Reguladoras de Ácidos Nucleicos , Reproducibilidad de los Resultados , Factores de TranscripciónRESUMEN
Chromatin compaction of deoxyribonucleic acid (DNA) presents a major challenge to the detection and removal of DNA damage. Helix-distorting DNA lesions that block transcription are specifically repaired by transcription-coupled nucleotide excision repair, which is initiated by binding of the CSB protein to lesion-stalled RNA polymerase II. Using live cell imaging, we identify a novel function for two distinct mammalian ISWI adenosine triphosphate (ATP)-dependent chromatin remodeling complexes in resolving lesion-stalled transcription. Human ISWI isoform SMARCA5/SNF2H and its binding partners ACF1 and WSTF are rapidly recruited to UV-C induced DNA damage to specifically facilitate CSB binding and to promote transcription recovery. SMARCA5 targeting to UV-C damage depends on transcription and histone modifications and requires functional SWI2/SNF2-ATPase and SLIDE domains. After initial recruitment to UV damage, SMARCA5 re-localizes away from the center of DNA damage, requiring its HAND domain. Our studies support a model in which SMARCA5 targeting to DNA damage-stalled transcription sites is controlled by an ATP-hydrolysis-dependent scanning and proofreading mechanism, highlighting how SWI2/SNF2 chromatin remodelers identify and bind nucleosomes containing damaged DNA.