RESUMEN
Non-coding genetic variation is a major driver of phenotypic diversity and allows the investigation of mechanisms that control gene expression. Here, we systematically investigated the effects of >50 million variations from five strains of mice on mRNA, nascent transcription, transcription start sites, and transcription factor binding in resting and activated macrophages. We observed substantial differences associated with distinct molecular pathways. Evaluating genetic variation provided evidence for roles of â¼100 TFs in shaping lineage-determining factor binding. Unexpectedly, a substantial fraction of strain-specific factor binding could not be explained by local mutations. Integration of genomic features with chromatin interaction data provided evidence for hundreds of connected cis-regulatory domains associated with differences in transcription factor binding and gene expression. This system and the >250 datasets establish a substantial new resource for investigation of how genetic variation affects cellular phenotypes.
Asunto(s)
Variación Genética , Macrófagos/metabolismo , Factores de Transcripción/metabolismo , Animales , Sitios de Unión , Células de la Médula Ósea/citología , Proteína beta Potenciadora de Unión a CCAAT/genética , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Análisis por Conglomerados , Elementos de Facilitación Genéticos/genética , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Lipopolisacáridos/farmacología , Macrófagos/citología , Macrófagos/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Regiones Promotoras Genéticas , Unión Proteica , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/genéticaRESUMEN
The lung is inhabited by resident alveolar and interstitial macrophages as well as monocytic cells that survey lung tissues. Each cell type plays distinct functional roles under homeostatic and inflammatory conditions, but mechanisms establishing their molecular identities and functional potential remain poorly understood. In the present study, systematic evaluation of transcriptomes and open chromatin of alveolar macrophages (AMs), interstitial macrophages (IMs) and lung monocytes from two mouse strains enabled inference of common and cell-specific transcriptional regulators. We provide evidence that these factors drive selection of regulatory landscapes that specify distinct phenotypes of AMs and IMs and entrain qualitatively different responses to toll-like receptor 4 signaling in vivo. These studies reveal a striking divergence in a fundamental innate immune response pathway in AMs and establish a framework for further understanding macrophage diversity in the lung.
Asunto(s)
Inmunidad Innata/inmunología , Pulmón/inmunología , Macrófagos/inmunología , Monocitos/inmunología , Animales , Epigénesis Genética/inmunología , Macrófagos/citología , Ratones , Monocitos/citología , Transcriptoma/inmunologíaRESUMEN
Macrophages reside in essentially all tissues of the body and play key roles in innate and adaptive immune responses. Distinct populations of tissue macrophages also acquire context-specific functions that are important for normal tissue homeostasis. To investigate mechanisms responsible for tissue-specific functions, we analyzed the transcriptomes and enhancer landscapes of brain microglia and resident macrophages of the peritoneal cavity. In addition, we exploited natural genetic variation as a genome-wide "mutagenesis" strategy to identify DNA recognition motifs for transcription factors that promote common or subset-specific binding of the macrophage lineage-determining factor PU.1. We find that distinct tissue environments drive divergent programs of gene expression by differentially activating a common enhancer repertoire and by inducing the expression of divergent secondary transcription factors that collaborate with PU.1 to establish tissue-specific enhancers. These findings provide insights into molecular mechanisms by which tissue environment influences macrophage phenotypes that are likely to be broadly applicable to other cell types.
Asunto(s)
Elementos de Facilitación Genéticos , Macrófagos/metabolismo , Animales , Variación Genética , Código de Histonas , Macrófagos/citología , Macrófagos/inmunología , Masculino , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones Endogámicos , Factores de Transcripción/metabolismoRESUMEN
Δ9 -Tetrahydrocannabinol (Δ9 -THC) and cannabidiol (CBD) are cannabinoids found in Cannabis sativa. While research supports cannabinoids reduce inflammation, the consensus surrounding receptor(s)-mediated effects has yet to be established. Here, we investigated the receptor-mediated properties of Δ9 -THC and CBD on alveolar macrophages, an important pulmonary immune cell in direct contact with cannabinoids inhaled by cannabis smokers. MH-S cells, a mouse alveolar macrophage cell line, were exposed to Δ9 -THC and CBD, with and without lipopolysaccharide (LPS). Outcomes included RNA-sequencing and cytokine analysis. Δ9 -THC and CBD alone did not affect the basal transcriptional response of MH-S cells. In response to LPS, Δ9 -THC and CBD significantly reduced the expression of numerous proinflammatory cytokines including tumor necrosis factor-alpha, interleukin (IL)-1ß and IL-6, an effect that was dependent on CB2 . The anti-inflammatory effects of CBD but not Δ9 -THC were mediated through a reduction in signaling through nuclear factor-kappa B and extracellular signal-regulated protein kinase 1/2. These results suggest that CBD and Δ9 -THC have potent immunomodulatory properties in alveolar macrophages, a cell type important in immune homeostasis in the lungs. Further investigation into the effects of cannabinoids on lung immune cells could lead to the identification of therapies that may ameliorate conditions characterized by inflammation.
Asunto(s)
Cannabidiol , Cannabinoides , Cannabis , Ratones , Animales , Cannabidiol/farmacología , Dronabinol/farmacología , Macrófagos Alveolares/metabolismo , Lipopolisacáridos/farmacología , Cannabis/metabolismo , Citocinas/metabolismo , Inflamación/metabolismoRESUMEN
The COVID-19 pandemic is associated with severe pneumonia and acute respiratory distress syndrome leading to death in susceptible individuals. For those who recover, post-COVID-19 complications may include development of pulmonary fibrosis. Factors contributing to disease severity or development of complications are not known. Using computational analysis with experimental data, we report that idiopathic pulmonary fibrosis (IPF)- and chronic obstructive pulmonary disease (COPD)-derived lung fibroblasts express higher levels of angiotensin-converting enzyme 2 (ACE2), the receptor for SARS-CoV-2 entry and part of the renin-angiotensin system that is antifibrotic and anti-inflammatory. In preclinical models, we found that chronic exposure to cigarette smoke, a risk factor for both COPD and IPF and potentially for SARS-CoV-2 infection, significantly increased pulmonary ACE2 protein expression. Further studies are needed to understand the functional implications of ACE2 on lung fibroblasts, a cell type that thus far has received relatively little attention in the context of COVID-19.
Asunto(s)
Enzima Convertidora de Angiotensina 2/biosíntesis , COVID-19/patología , Fibroblastos/metabolismo , Fibrosis Pulmonar Idiopática/patología , Enfermedad Pulmonar Obstructiva Crónica/patología , Adulto , Animales , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Receptores Virales/biosíntesis , Síndrome de Dificultad Respiratoria/patología , Síndrome de Dificultad Respiratoria/virología , SARS-CoV-2/metabolismo , Humo/efectos adversosRESUMEN
The oncoproteins of the small DNA tumor viruses interact with a plethora of cellular regulators to commandeer control of the infected cell. During infection, adenovirus E1A deregulates cAMP signalling and repurposes it for activation of viral gene expression. We show that E1A structurally and functionally mimics a cellular A-kinase anchoring protein (AKAP). E1A interacts with and relocalizes protein kinase A (PKA) to the nucleus, likely to virus replication centres, via an interaction with the regulatory subunits of PKA. Binding to PKA requires the N-terminus of E1A, which bears striking similarity to the amphipathic α-helical domain present in cellular AKAPs. E1A also targets the same docking-dimerization domain of PKA normally bound by cellular AKAPs. In addition, the AKAP like motif within E1A could restore PKA interaction to a cellular AKAP in which its normal interaction motif was deleted. During infection, E1A successfully competes with endogenous cellular AKAPs for PKA interaction. E1A's role as a viral AKAP contributes to viral transcription, protein expression and progeny production. These data establish HAdV E1A as the first known viral AKAP. This represents a unique example of viral subversion of a crucial cellular regulatory pathway via structural mimicry of the PKA interaction domain of cellular AKAPs.
Asunto(s)
Proteínas de Anclaje a la Quinasa A/metabolismo , Proteínas E1A de Adenovirus/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Imitación Molecular , Proteínas de Anclaje a la Quinasa A/química , Adenoviridae/química , Adenoviridae/metabolismo , Proteínas E1A de Adenovirus/química , Secuencia de Aminoácidos , Línea Celular , Inmunoprecipitación de Cromatina , Proteínas Quinasas Dependientes de AMP Cíclico/química , Técnica del Anticuerpo Fluorescente , Técnicas de Silenciamiento del Gen , Humanos , Procesamiento de Imagen Asistido por Computador , Inmunoprecipitación , Simulación del Acoplamiento Molecular , Unión Proteica , Estructura Secundaria de Proteína , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
UNLABELLED: During infection by human adenovirus (HAdV), the proteins encoded by the early region 1A (E1A) gene bind and appropriate components of the cellular transcriptional machinery to activate the transcription of viral early genes. Previously, we identified roles for the human Bre1 (hBre1) and hPaf1 complexes in E1A-mediated transcriptional activation of HAdV early genes. Here we show that E1A binds hBre1 directly and that this complex targets the hPaf1 complex via the Rtf1 subunit. Depletion of hPaf1 reduces E1A-dependent activation of transcription from the E2e, E3, and E4 viral transcription units, and this does not result from a reduced ability of RNA polymerase II to be recruited to the promoter-proximal regions of these genes. In contrast, depletion of hPaf1 reduces the occupancy of RNA polymerase II across these transcription units. This is accompanied by reductions in the level of H3K36 trimethylation, a posttranslational histone modification associated with efficient transcriptional elongation, and the number of full-length transcripts from these genes. Together, these results indicate that E1A uses hBre1 to recruit the hPaf1 complex in order to optimally activate viral early transcription by enhancing transcriptional elongation. IMPORTANCE: This work provides the mechanism by which the hPaf1 complex contributes to E1A-dependent activation of early gene transcription. The work also demonstrates that E1A induces gene expression by stimulating transcriptional elongation, in addition to its better-characterized effects on transcriptional initiation.
Asunto(s)
Proteínas E1A de Adenovirus/metabolismo , Adenovirus Humanos/fisiología , Interacciones Huésped-Patógeno , Proteínas Nucleares/metabolismo , Transcripción Genética , Adenovirus Humanos/genética , Línea Celular , Células Epiteliales/virología , Regulación Viral de la Expresión Génica , Humanos , Unión Proteica , Factores de Transcripción/metabolismo , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Upon infection, human adenovirus (HAdV) must activate the expression of its early genes to reprogram the cellular environment to support virus replication. This activation is orchestrated in large part by the first HAdV gene expressed during infection, early region 1A (E1A). E1A binds and appropriates components of the cellular transcriptional machinery to modulate cellular gene transcription and activate viral early genes transcription. Previously, we identified hBre1/RNF20 as a target for E1A. The interaction between E1A and hBre1 antagonizes the innate antiviral response by blocking H2B monoubiquitination, a chromatin modification necessary for the interferon (IFN) response. Here, we describe a second distinct role for the interaction of E1A with hBre1 in transcriptional activation of HAdV early genes. Furthermore, we show that E1A changes the function of hBre1 from a ubiquitin ligase involved in substrate selection to a scaffold which recruits hPaf1 as a means to stimulate transcription and transcription-coupled histone modifications. By using hBre1 to recruit hPaf1, E1A is able to optimally activate viral early transcription and begin the cycle of viral replication. The ability of E1A to target hBre1 to simultaneously repress cellular IFN dependent transcription while activating viral transcription, represents an elegant example of the incredible economy of action accomplished by a viral regulatory protein through a single protein interaction.
Asunto(s)
Infecciones por Adenoviridae/metabolismo , Adenoviridae/fisiología , Proteínas E1A de Adenovirus/metabolismo , Inmunidad Innata , Proteínas Nucleares/metabolismo , Activación Transcripcional , Ubiquitina-Proteína Ligasas/metabolismo , Replicación Viral/fisiología , Infecciones por Adenoviridae/genética , Infecciones por Adenoviridae/inmunología , Proteínas E1A de Adenovirus/genética , Proteínas E1A de Adenovirus/inmunología , Línea Celular Tumoral , Histonas/genética , Histonas/inmunología , Histonas/metabolismo , Humanos , Interferones/genética , Interferones/inmunología , Interferones/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/inmunología , Procesamiento Proteico-Postraduccional/genética , Procesamiento Proteico-Postraduccional/inmunología , Factores de Transcripción , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/inmunologíaRESUMEN
Single-cell sequencing is a crucial tool for dissecting the cellular intricacies of complex diseases. Its prohibitive cost, however, hampers its application in expansive biomedical studies. Traditional cellular deconvolution approaches can infer cell type proportions from more affordable bulk sequencing data, yet they fall short in providing the detailed resolution required for single-cell-level analyses. To overcome this challenge, we introduce "scSemiProfiler", an innovative computational framework that marries deep generative models with active learning strategies. This method adeptly infers single-cell profiles across large cohorts by fusing bulk sequencing data with targeted single-cell sequencing from a few rigorously chosen representatives. Extensive validation across heterogeneous datasets verifies the precision of our semi-profiling approach, aligning closely with true single-cell profiling data and empowering refined cellular analyses. Originally developed for extensive disease cohorts, "scSemiProfiler" is adaptable for broad applications. It provides a scalable, cost-effective solution for single-cell profiling, facilitating in-depth cellular investigation in various biological domains.
Asunto(s)
Análisis de la Célula Individual , Análisis de la Célula Individual/métodos , Humanos , Aprendizaje Profundo , Biología Computacional/métodos , Perfilación de la Expresión Génica/métodos , Aprendizaje Automático SupervisadoRESUMEN
In vitro evolution and whole genome analysis has proven to be a powerful method for studying the mechanism of action of small molecules in many haploid microbes but has generally not been applied to human cell lines in part because their diploid state complicates the identification of variants that confer drug resistance. To determine if haploid human cells could be used in MOA studies, we evolved resistance to five different anticancer drugs (doxorubicin, gemcitabine, etoposide, topotecan, and paclitaxel) using a near-haploid cell line (HAP1) and then analyzed the genomes of the drug resistant clones, developing a bioinformatic pipeline that involved filtering for high frequency alleles predicted to change protein sequence, or alleles which appeared in the same gene for multiple independent selections with the same compound. Applying the filter to sequences from 28 drug resistant clones identified a set of 21 genes which was strongly enriched for known resistance genes or known drug targets (TOP1, TOP2A, DCK, WDR33, SLCO3A1). In addition, some lines carried structural variants that encompassed additional known resistance genes (ABCB1, WWOX and RRM1). Gene expression knockdown and knockout experiments of 10 validation targets showed a high degree of specificity and accuracy in our calls and demonstrates that the same drug resistance mechanisms found in diverse clinical samples can be evolved, discovered and studied in an isogenic background.
Asunto(s)
Antineoplásicos , Resistencia a Antineoplásicos , Haploidia , Humanos , Resistencia a Antineoplásicos/genética , Antineoplásicos/farmacología , Genoma Humano , Secuenciación Completa del Genoma/métodos , Línea CelularRESUMEN
The largest isoform of adenovirus early region 1A (E1A) contains a unique region termed conserved region 3 (CR3). This region activates viral gene expression by recruiting cellular transcription machinery to the early viral promoters. Recent studies have suggested that there is an optimal level of E1A-dependent transactivation required by human adenovirus (hAd) during infection and that this may be achieved via functional cross talk between the N termini of E1A and CR3. The N terminus of E1A binds GCN5, a cellular lysine acetyltransferase (KAT). We have identified a second independent interaction of E1A with GCN5 that is mediated by CR3, which requires residues 178 to 188 in hAd5 E1A. GCN5 was recruited to the viral genome during infection in an E1A-dependent manner, and this required both GCN5 interaction sites on E1A. Ectopic expression of GCN5 repressed transactivation by both E1A CR3 and full-length E1A. In contrast, RNA interference (RNAi) depletion of GCN5 or treatment with the KAT inhibitor cyclopentylidene-[4-(4'-chlorophenyl)thiazol-2-yl]hydrazone (CPTH2) resulted in increased E1A CR3 transactivation. Moreover, activation of the adenovirus E4 promoter by E1A was increased during infection of homozygous GCN5 KAT-defective (hat/hat) mouse embryonic fibroblasts (MEFs) compared to wild-type control MEFs. Enhanced histone H3 K9/K14 acetylation at the viral E4 promoter required the newly identified binding site for GCN5 within CR3 and correlated with repression and reduced occupancy by phosphorylated RNA polymerase II. Treatment with CPTH2 during infection also reduced virus yield. These data identify GCN5 as a new negative regulator of transactivation by E1A and suggest that its KAT activity is required for optimal virus replication.
Asunto(s)
Proteínas E1A de Adenovirus/metabolismo , Infecciones por Adenovirus Humanos/metabolismo , Adenovirus Humanos/fisiología , Regulación Viral de la Expresión Génica/fisiología , Regiones Promotoras Genéticas , Activación Transcripcional/fisiología , Replicación Viral/fisiología , Factores de Transcripción p300-CBP/metabolismo , Acetilación/efectos de los fármacos , Proteínas E1A de Adenovirus/genética , Infecciones por Adenovirus Humanos/genética , Animales , Embrión de Mamíferos/metabolismo , Embrión de Mamíferos/patología , Embrión de Mamíferos/virología , Inhibidores Enzimáticos/farmacología , Fibroblastos/metabolismo , Fibroblastos/patología , Fibroblastos/virología , Regulación Viral de la Expresión Génica/efectos de los fármacos , Células HeLa , Histonas/genética , Histonas/metabolismo , Humanos , Ratones , Ratones Mutantes , Fosforilación/efectos de los fármacos , Fosforilación/genética , Estructura Terciaria de Proteína , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Activación Transcripcional/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Factores de Transcripción p300-CBP/genéticaRESUMEN
Early region 1A (E1A) of human adenovirus (HAdV) has been the focus of over 30 years of investigation and is required for the oncogenic capacity of HAdV in rodents. Alternative splicing of the E1A transcript generates mRNAs encoding multiple E1A proteins. The 55-residue (55R) E1A protein, which is encoded by the 9S mRNA, is particularly interesting due to the unique properties it displays relative to all other E1A isoforms. 55R E1A does not contain any of the conserved regions (CRs) present in the other E1A isoforms. The C-terminal region of the 55R E1A protein contains a unique sequence compared to all other E1A isoforms, which results from a frameshift generated by alternative splicing. The 55R E1A protein is thought to be produced preferentially at the late stages of infection. Here we report the first study to directly investigate the function of the species C HAdV 55R E1A protein during infection. Polyclonal rabbit antibodies (Abs) have been generated that are capable of immunoprecipitating HAdV-2 55R E1A. These Abs can also detect HAdV-2 55R E1A by immunoblotting and indirect immunofluorescence assay. These studies indicate that 55R E1A is expressed late and is localized to the cytoplasm and to the nucleus. 55R E1A was able to activate the expression of viral genes during infection and could also promote productive replication of species C HAdV. 55R E1A was also found to interact with the S8 component of the proteasome, and knockdown of S8 was detrimental to viral replication dependent on 55R E1A.
Asunto(s)
Proteínas E1A de Adenovirus/genética , Proteínas E1A de Adenovirus/metabolismo , Adenovirus Humanos/genética , ARN Mensajero/química , ARN Viral/química , Adenosina Trifosfatasas/metabolismo , Proteínas E1A de Adenovirus/inmunología , Adenovirus Humanos/inmunología , Secuencia de Aminoácidos , Anticuerpos Antivirales/inmunología , Línea Celular , Núcleo Celular/metabolismo , Inhibición de Contacto , Citoplasma/metabolismo , Regulación Viral de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Unión Proteica , Transporte de Proteínas , Transcripción Genética , Replicación Viral/genéticaRESUMEN
Epstein-Barr virus (EBV) is a gamma-herpesvirus associated with nearly 10% of gastric cancers (GCs). These EBV-associated GCs (EBVaGCs) are molecularly, histopathologically, and clinically distinct from EBV-negative GCs (EBVnGCs). While viral genes in EBVaGCs contribute to the carcinogenesis process, viral proteins also represent foreign antigens that could trigger enhanced immune responses compared to EBVnGCs. Despite prior investigations of the EBVaGC tumor microenvironment (TME), the cellular composition has not been thoroughly explored. In this study, cellular subpopulations overrepresented in EBVaGCs were identified and molecularly characterized. Genes consistently expressed across both bulk tumor and single-cell RNA sequencing data were highlighted, with the expression across the identified cellular subpopulations analyzed. As expected, based on existing histopathological analysis, EBVaGC is characterized by abundant lymphocytic infiltration of the stroma. Our molecular analysis identified three unique immune cell subpopulations in EBVaGC: T and B cells expressing high levels of proliferation markers and B cells expressing T cell features. The proliferating T cell cluster also expressed markers of follicular T helper cells. Overall, EBVaGC also exhibited unique features indicative of a higher inflammatory response. These substantial differences within the TME suggest that further detailed exploration of the cellular composition of EBVaGCs is needed, which may identify cellular subpopulations and phenotypes associated with patient outcomes.
RESUMEN
The adenovirus E1A proteins function via protein-protein interactions. By making many connections with the cellular protein network, individual modules of this virally encoded hub reprogram numerous aspects of cell function and behavior. Although many of these interactions have been thoroughly studied, those mediated by the C-terminal region of E1A are less well understood. This review focuses on how this region of E1A affects cell cycle progression, apoptosis, senescence, transformation, and conversion of cells to an epithelial state through interactions with CTBP1/2, DYRK1A/B, FOXK1/2, and importin-α. Furthermore, novel potential pathways that the C-terminus of E1A influences through these connections with the cellular interaction network are discussed.
Asunto(s)
Proteínas E1A de Adenovirus/metabolismo , Mapeo de Interacción de Proteínas , Adenoviridae/fisiología , Proteínas E1A de Adenovirus/química , Proteínas E1B de Adenovirus/metabolismo , Animales , Fenómenos Fisiológicos Celulares , Transformación Celular Neoplásica , Interacciones Huésped-Patógeno , Humanos , Unión Proteica , Dominios y Motivos de Interacción de ProteínasRESUMEN
Dysregulation of the balance between pro-inflammatory and anti-inflammatory macrophages has a key function in the pathogenesis of Duchenne muscular dystrophy (DMD), a fatal genetic disease. We postulate that an evolutionarily ancient protective mechanism against infection, known as trained immunity, drives pathological inflammation in DMD. Here we show that bone marrow-derived macrophages from a murine model of DMD (mdx) exhibit cardinal features of trained immunity, consisting of transcriptional hyperresponsiveness associated with metabolic and epigenetic remodeling. The hyperresponsive phenotype is transmissible by bone marrow transplantation to previously healthy mice and persists for up to 11 weeks post-transplant. Mechanistically, training is induced by muscle extract in vitro. The functional and epigenetic changes in bone marrow-derived macrophages from dystrophic mice are TLR4-dependent. Adoptive transfer experiments further support the TLR4-dependence of trained macrophages homing to damaged muscles from the bone marrow. Collectively, this suggests that a TLR4-regulated, memory-like capacity of innate immunity induced at the level of the bone marrow promotes dysregulated inflammation in DMD.
Asunto(s)
Trasplante de Médula Ósea , Inmunidad Innata/inmunología , Macrófagos/inmunología , Músculo Esquelético/patología , Distrofia Muscular de Duchenne/patología , Receptor Toll-Like 4/inmunología , Animales , Células de la Médula Ósea/inmunología , Línea Celular , Modelos Animales de Enfermedad , Inflamación/inmunología , Células L , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos mdx , Ratones Noqueados , Músculo Esquelético/inmunología , Distrofia Muscular de Duchenne/inmunología , Extractos de Tejidos/farmacología , Transcripción Genética/genéticaRESUMEN
Regulation of gene expression requires the combinatorial binding of sequence-specific transcription factors (TFs) at promoters and enhancers. Prior studies showed that alterations in the spacing between TF binding sites can influence promoter and enhancer activity. However, the relative importance of TF spacing alterations resulting from naturally occurring insertions and deletions (InDels) has not been systematically analyzed. To address this question, we first characterized the genome-wide spacing relationships of 73 TFs in human K562 cells as determined by ChIP-seq (chromatin immunoprecipitation sequencing). We found a dominant pattern of a relaxed range of spacing between collaborative factors, including 45 TFs exclusively exhibiting relaxed spacing with their binding partners. Next, we exploited millions of InDels provided by genetically diverse mouse strains and human individuals to investigate the effects of altered spacing on TF binding and local histone acetylation. These analyses suggested that spacing alterations resulting from naturally occurring InDels are generally tolerated in comparison to genetic variants directly affecting TF binding sites. To experimentally validate this prediction, we introduced synthetic spacing alterations between PU.1 and C/EBPß binding sites at six endogenous genomic loci in a macrophage cell line. Remarkably, collaborative binding of PU.1 and C/EBPß at these locations tolerated changes in spacing ranging from 5 bp increase to >30 bp decrease. Collectively, these findings have implications for understanding mechanisms underlying enhancer selection and for the interpretation of non-coding genetic variation.
Asunto(s)
Regulación de la Expresión Génica , Genómica/métodos , Factores de Transcripción/genética , Animales , Proteína beta Potenciadora de Unión a CCAAT/genética , Inmunoprecipitación de Cromatina , Elementos de Facilitación Genéticos , Humanos , Células K562 , Masculino , Ratones , Unión Proteica , Proteínas Proto-Oncogénicas/genética , Transactivadores/genéticaRESUMEN
Enteric helminths form intimate physical connections with the intestinal epithelium, yet their ability to directly alter epithelial stem cell fate has not been resolved. Here we demonstrate that infection of mice with the parasite Heligmosomoides polygyrus bakeri (Hpb) reprograms the intestinal epithelium into a fetal-like state marked by the emergence of Clusterin-expressing revival stem cells (revSCs). Organoid-based studies using parasite-derived excretory-secretory products reveal that Hpb-mediated revSC generation occurs independently of host-derived immune signals and inhibits type 2 cytokine-driven differentiation of secretory epithelial lineages that promote their expulsion. Reciprocally, type 2 cytokine signals limit revSC differentiation and, consequently, Hpb fitness, indicating that helminths compete with their host for control of the intestinal stem cell compartment to promote continuation of their life cycle.
Asunto(s)
Nematospiroides dubius , Infecciones por Strongylida , Animales , Citocinas , Mucosa Intestinal , Intestinos , Ratones , Células MadreRESUMEN
ß-Amyloid (Aß) plaques can trigger chronic inflammation in the cellular environment that recruits infiltrating macrophages during the course of Alzheimer disease (AD). Activated macrophages release pro-inflammatory cytokines that increase neurotoxicity associated with AD. A major impediment to investigating neuroinflammation involving macrophage activity is the inability to discriminate resident microglial macrophages (mMÏ) from hematogenous macrophages (hMÏ), as they are morphologically and phenotypically similar when activated. To distinguish between mMÏ and hMÏ and to determine their respective roles in chronic inflammation associated with the progression of amyloidosis, we used lys-EGFP-ki transgenic mice that express enhanced green fluorescent protein in hMÏ, but not in mMÏ. These mice were crossed with 5XFAD mice. The offspring demonstrated robust AD pathology and enabled visual discrimination of mMÏ from hMÏ. Mutant mice demonstrated robust increases in Aß1-42, area of Aß plaques, gliosis and deficits in spatial learning by age 5 months. The time-course of Aß accumulation, paralleled by the accumulation of hMÏ around Aß plaques, was more robust in female compared with male mice and preceded behavioral changes. Thus, the accumulation of infiltrating hMÏ around Aß plaques was age- and sex-dependent and preceded cognitive impairment.
Asunto(s)
Enfermedad de Alzheimer/patología , Encéfalo/patología , Macrófagos/patología , Placa Amiloide/patología , Enfermedad de Alzheimer/inmunología , Péptidos beta-Amiloides/inmunología , Péptidos beta-Amiloides/metabolismo , Animales , Encéfalo/inmunología , Modelos Animales de Enfermedad , Femenino , Inflamación/inmunología , Inflamación/patología , Masculino , Ratones , Ratones Transgénicos , Placa Amiloide/inmunologíaRESUMEN
Viruses alter a multitude of host-cell processes to create a more optimal environment for viral replication. This includes altering metabolism to provide adequate substrates and energy required for replication. Typically, viral infections induce a metabolic phenotype resembling the Warburg effect, with an upregulation of glycolysis and a concurrent decrease in cellular respiration. Human adenovirus (HAdV) has been observed to induce the Warburg effect, which can be partially attributed to the adenovirus protein early region 4, open reading frame 1 (E4orf1). E4orf1 regulates a multitude of host-cell processes to benefit viral replication and can influence cellular metabolism through the transcription factor avian myelocytomatosis viral oncogene homolog (MYC). However, E4orf1 does not explain the full extent of Warburg-like HAdV metabolic reprogramming, especially the accompanying decrease in cellular respiration. The HAdV protein early region 1A (E1A) also modulates the function of the infected cell to promote viral replication. E1A can interact with a wide variety of host-cell proteins, some of which have been shown to interact with metabolic enzymes independently of an interaction with E1A. To determine if the HAdV E1A proteins are responsible for reprogramming cell metabolism, we measured the extracellular acidification rate and oxygen consumption rate of A549 human lung epithelial cells with constitutive endogenous expression of either of the two major E1A isoforms. This was followed by the characterization of transcript levels for genes involved in glycolysis and cellular respiration, and related metabolic pathways. Cells expressing the 13S encoded E1A isoform had drastically increased baseline glycolysis and lower maximal cellular respiration than cells expressing the 12S encoded E1A isoform. Cells expressing the 13S encoded E1A isoform exhibited upregulated expression of glycolysis genes and downregulated expression of cellular respiration genes. However, tricarboxylic acid cycle genes were upregulated, resembling anaplerotic metabolism employed by certain cancers. Upregulation of glycolysis and tricarboxylic acid cycle genes was also apparent in IMR-90 human primary lung fibroblast cells infected with a HAdV-5 mutant virus that expressed the 13S, but not the 12S encoded E1A isoform. In conclusion, it appears that the two major isoforms of E1A differentially influence cellular glycolysis and oxidative phosphorylation and this is at least partially due to the altered regulation of mRNA expression for the genes in these pathways.
Asunto(s)
Proteínas E1A de Adenovirus/metabolismo , Infecciones por Adenovirus Humanos/metabolismo , Adenovirus Humanos/metabolismo , Células Epiteliales/virología , Pulmón/virología , Células A549 , Proteínas E1A de Adenovirus/genética , Infecciones por Adenovirus Humanos/genética , Infecciones por Adenovirus Humanos/virología , Adenovirus Humanos/genética , Células Epiteliales/metabolismo , Glucólisis , Humanos , Pulmón/metabolismo , Fosforilación Oxidativa , Oxígeno/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMEN
C-terminal binding protein (CtBP) binds to adenovirus early region 1A (AdE1A) through a highly conserved PXDLS motif close to the C terminus. We now have demonstrated that CtBP1 also interacts directly with the transcriptional activation domain (conserved region 3 [CR3]) of adenovirus type 5 E1A (Ad5E1A) and requires the integrity of the entire CR3 region for optimal binding. The interaction appears to be at least partially mediated through a sequence ((161)RRNTGDP(167)) very similar to a recently characterized novel CtBP binding motif in ZNF217 as well as other regions of CR3. Using reporter assays, we further demonstrated that CtBP1 represses Ad5E1A CR3-dependent transcriptional activation. Ad5E1A also appears to be recruited to the E-cadherin promoter through its interaction with CtBP. Significantly, Ad5E1A, CtBP1, and ZNF217 form a stable complex which requires CR3 and the PLDLS motif. It has been shown that Ad513SE1A, containing the CR3 region, is able to overcome the transcriptional repressor activity of a ZNF217 polypeptide fragment in a GAL4 reporter assay through recruitment of CtBP1. These results suggest a hitherto-unsuspected complexity in the association of Ad5E1A with CtBP, with the interaction resulting in transcriptional activation by recruitment of CR3-bound factors to CtBP1-containing complexes.