RESUMEN
Somatic stem cells contribute to normal tissue homeostasis, and their epigenomic features play an important role in regulating tissue identities or developing disease states. Enhancers are one of the key players controlling chromatin context-specific gene expression in a spatial and temporal manner while maintaining tissue homeostasis, and their dysregulation leads to tumorigenesis. Here, epigenomic and transcriptomic analyses reveal that forkhead box protein D2 (FOXD2) is a hub for the gene regulatory network exclusive to large intestinal stem cells, and its overexpression plays a significant role in colon cancer regression. FOXD2 is positioned at the closed chromatin and facilitates mixed-lineage leukemia protein-4 (MLL4/KMT2D) binding to deposit H3K4 monomethylation. De novo FOXD2-mediated chromatin interactions rewire the regulation of p53-responsive genes and induction of apoptosis. Taken together, our findings illustrate the novel mechanistic details of FOXD2 in suppressing colorectal cancer growth and suggest its function as a chromatin-tuning factor and a potential therapeutic target for colorectal cancer.
Asunto(s)
Neoplasias Colorrectales , Histonas , Humanos , Cromatina/genética , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Elementos de Facilitación Genéticos , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Histonas/genética , Histonas/metabolismoRESUMEN
BACKGROUND: Plants memorize previous pathogen attacks and are "primed" to produce a faster and stronger defense response, which is critical for defense against pathogens. In plants, cytosines in transposons and gene bodies are reported to be frequently methylated. Demethylation of transposons can affect disease resistance by regulating the transcription of nearby genes during defense response, but the role of gene body methylation (GBM) in defense responses remains unclear. RESULTS: Here, we find that loss of the chromatin remodeler decrease in DNA methylation 1 (ddm1) synergistically enhances resistance to a biotrophic pathogen under mild chemical priming. DDM1 mediates gene body methylation at a subset of stress-responsive genes with distinct chromatin properties from conventional gene body methylated genes. Decreased gene body methylation in loss of ddm1 mutant is associated with hyperactivation of these gene body methylated genes. Knockout of glyoxysomal protein kinase 1 (gpk1), a hypomethylated gene in ddm1 loss-of-function mutant, impairs priming of defense response to pathogen infection in Arabidopsis. We also find that DDM1-mediated gene body methylation is prone to epigenetic variation among natural Arabidopsis populations, and GPK1 expression is hyperactivated in natural variants with demethylated GPK1. CONCLUSIONS: Based on our collective results, we propose that DDM1-mediated GBM provides a possible regulatory axis for plants to modulate the inducibility of the immune response.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Metilación de ADN , Factores de Transcripción/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cromatina/metabolismo , Regulación de la Expresión Génica de las PlantasRESUMEN
Different from the canonical right-handed B-DNA, a left-handed Z-DNA forms an alternating syn- and anti-base conformations along the double-stranded helix under physiological conditions. Z-DNA structure plays a role in transcriptional regulation, chromatin remodeling, and genome stability. To understand the biological function of Z-DNA and map the genome-wide Z-DNA-forming sites (ZFSs), a ChIP-Seq strategy is applied, which is a combination of chromatin immunoprecipitation (ChIP) and high-throughput DNA sequencing analysis. Cross-linked chromatin is sheared and its fragments associated with Z-DNA-binding proteins are mapped onto the reference genome sequence. The global information of ZFSs positioning can provide a useful resource for better understanding of DNA structure-dependent biological mechanism.
Asunto(s)
ADN de Forma Z , Humanos , Genoma Humano , Secuenciación de Inmunoprecipitación de Cromatina , Proteínas de Unión al ADN/metabolismo , ADN/genética , Análisis de Secuencia de ADN , Secuenciación de Nucleótidos de Alto Rendimiento , Sitios de UniónRESUMEN
DNA methylation is a crucial epigenetic modification in the establishment of cell-type-specific characteristics. However, how DNA methylation is selectively reprogrammed at adipocyte-specific loci during adipogenesis remains unclear. Here, we show that the transcription factor, C/EBPδ, and the DNA methylation eraser, TET3, cooperatively control adipocyte differentiation. We perform whole-genome bisulfite sequencing to explore the dynamics and regulatory mechanisms of DNA methylation in adipocyte differentiation. During adipogenesis, DNA methylation selectively decreases at adipocyte-specific loci carrying the C/EBP binding motif, which correlates with the activity of adipogenic promoters and enhancers. Mechanistically, we find that C/EBPδ recruits a DNA methylation eraser, TET3, to catalyse DNA demethylation at the C/EBP binding motif and stimulate the expression of key adipogenic genes. Ectopic expression of TET3 potentiates in vitro and in vivo adipocyte differentiation and recovers downregulated adipogenic potential, which is observed in aged mice and humans. Taken together, our study highlights how targeted reprogramming of DNA methylation through cooperative action of the transcription factor C/EBPδ, and the DNA methylation eraser TET3, controls adipocyte differentiation.
Asunto(s)
Adipogénesis , Dioxigenasas , Adipogénesis/genética , Animales , Proteínas Potenciadoras de Unión a CCAAT , Diferenciación Celular/genética , Metilación de ADN , Dioxigenasas/genética , Epigénesis Genética , Humanos , Ratones , Factores de Transcripción/genéticaRESUMEN
Dietary antigens affect the adaptive immunity of the host by inducing regulatory T cells and IgE-producing B cells. However, their roles in innate immune compartments such as innate lymphoid cells (ILCs) and intestinal epithelial cells (IECs) are unclear. Here, using antigen-free (AF) mice, which are germ-free (GF) mice fed with amino-acid-based diet, we found dietary proteins suppress the development of GATA-3-expressing ILC2s independent of the adaptive immune cells. These cells produce more type 2 cytokines and upregulated proliferation and activation markers such as Ki-67, CD69, and CD25. With this, AF mice had increased expressions of tuft cell-specific transcripts such as Il25, Il33, Dclk1, Trpm5, and Pou2f3 in IECs. Accordingly, expanded ILC2s upregulated IL-17RB, a receptor of IL-25, and their proliferation was blocked by IL-25 neutralizing or IL-17RB blocking antibodies. These results suggest a new dialogue between dietary antigens, IECs, and ILCs in which dietary antigens suppress ILC2 activation and proliferation by restraining homeostatic IL-25 production, potentially limiting type 2 immunity by food antigens.
Asunto(s)
Inmunidad Innata , Linfocitos , Animales , Proliferación Celular , Citocinas , Dieta , RatonesRESUMEN
The advent of the assay for transposase-accessible chromatin using sequencing (ATAC-seq) has shown great potential as a leading method for analyzing the genome-wide profiling of chromatin accessibility. A comprehensive reference to the ATAC-seq dataset for disease progression is important for understanding the regulatory specificity caused by genetic or epigenetic changes. In this study, we present a genome-wide chromatin accessibility profile of 44 liver samples spanning the full histological spectrum of nonalcoholic fatty liver disease (NAFLD). We analyzed the ATAC-seq signal enrichment, fragment size distribution, and correlation coefficients according to the histological severity of NAFLD (healthy control vs steatosis vs fibrotic nonalcoholic steatohepatitis), demonstrating the high quality of the dataset. Consequently, 112,303 merged regions (genomic regions containing one or multiple overlapping peak regions) were identified. Additionally, we found differentially accessible regions (DARs) and performed transcription factor binding motif enrichment analysis and de novo motif analysis to determine new biomarker candidates. These data revealed the generegulatory interactions and noncoding factors that can affect NAFLD progression. In summary, our study provides a valuable resource for the human epigenome by applying an advanced approach to facilitate diagnosis and treatment by understanding the non-coding genome of NAFLD.
Asunto(s)
Cromatina , Enfermedad del Hígado Graso no Alcohólico , Cromatina/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Enfermedad del Hígado Graso no Alcohólico/genética , Análisis de Secuencia de ADN/métodos , Transposasas/genética , Transposasas/metabolismoRESUMEN
B-1 cell development mainly occurs via fetal and neonatal hematopoiesis and is suppressed in adult bone marrow hematopoiesis. However, little is known about the factors inhibiting B-1 cell development at the adult stage. We report that capicua (CIC) suppresses postnatal B-1a cell development and survival. CIC levels are high in B-1a cells and gradually increase in transitional B-1a (TrB-1a) cells with age. B-cell-specific Cic-null mice exhibit expansion of the B-1a cell population and a gradual increase in TrB-1a cell frequency with age but attenuated B-2 cell development. CIC deficiency enhances B cell receptor (BCR) signaling in transitional B cells and B-1a cell viability. Mechanistically, CIC-deficiency-mediated Per2 derepression upregulates Bhlhe41 levels by inhibiting CRY-mediated transcriptional repression for Bhlhe41, consequently promoting B-1a cell formation in Cic-null mice. Taken together, CIC is a key transcription factor that limits the B-1a cell population at the adult stage and balances B-1 versus B-2 cell formation.
Asunto(s)
Subgrupos de Linfocitos B/citología , Subgrupos de Linfocitos B/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Proteínas Circadianas Period/metabolismo , Proteínas Represoras/metabolismo , Transducción de Señal , Animales , Animales Recién Nacidos , Apoptosis , Secuencia de Bases , Médula Ósea/embriología , Diferenciación Celular , Supervivencia Celular , Niño , Preescolar , Feto/embriología , Células HEK293 , Humanos , Hígado/embriología , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Células 3T3 NIH , Receptores de Antígenos de Linfocitos B/metabolismoRESUMEN
BACKGROUND: Diesel exhaust particles (DEPs) are the main component of traffic-related air pollution and have been implicated in the pathogenesis and exacerbation of asthma. However, the mechanism by which DEP exposure aggravates asthma symptoms remains unclear. OBJECTIVE: This study aimed to identify a key cellular player of air pollutant-induced asthma exacerbation and development. METHODS: We examined the distribution of innate immune cells in the murine models of asthma induced by house dust mite and DEP. Changes in immune cell profiles caused by DEP exposure were confirmed by flow cytometry and RNA-Seq analysis. The roles of sialic acid-binding, Ig-like lectin F (SiglecF)-positive neutrophils were further evaluated by adoptive transfer experiment and in vitro functional studies. RESULTS: DEP exposure induced a unique population of lung granulocytes that coexpressed Ly6G and SiglecF. These cells differed phenotypically, morphologically, functionally, and transcriptionally from other SiglecF-expressing cells in the lungs. Our findings with murine models suggest that intratracheal challenge with DEPs induces the local release of adenosine triphosphate, which is a damage-associated molecular pattern signal. Adenosine triphosphate promotes the expression of SiglecF on neutrophils, and these SiglecF+ neutrophils worsen type 2 and 3 airway inflammation by producing high levels of cysteinyl leukotrienes and neutrophil extracellular traps. We also found Siglec8- (which corresponds to murine SiglecF) expressing neutrophils, and we found it in patients with asthma-chronic obstructive pulmonary disease overlap. CONCLUSION: The SiglecF+ neutrophil is a novel and critical player in airway inflammation and targeting this population could reverse or ameliorate asthma.
Asunto(s)
Contaminantes Atmosféricos , Asma , Adenosina Trifosfato/metabolismo , Contaminantes Atmosféricos/toxicidad , Animales , Humanos , Inflamación/metabolismo , Pulmón , Ratones , Neutrófilos/patología , Emisiones de Vehículos/toxicidadRESUMEN
Amino-terminal acetylation is catalyzed by a set of N-terminal acetyltransferases (NATs). The NatA complex (including X-linked Naa10 and Naa15) is the major acetyltransferase, with 40-50% of all mammalian proteins being potential substrates. However, the overall role of amino-terminal acetylation on a whole-organism level is poorly understood, particularly in mammals. Male mice lacking Naa10 show no globally apparent in vivo amino-terminal acetylation impairment and do not exhibit complete embryonic lethality. Rather Naa10 nulls display increased neonatal lethality, and the majority of surviving undersized mutants exhibit a combination of hydrocephaly, cardiac defects, homeotic anterior transformation, piebaldism, and urogenital anomalies. Naa12 is a previously unannotated Naa10-like paralog with NAT activity that genetically compensates for Naa10. Mice deficient for Naa12 have no apparent phenotype, whereas mice deficient for Naa10 and Naa12 display embryonic lethality. The discovery of Naa12 adds to the currently known machinery involved in amino-terminal acetylation in mice.
Asunto(s)
Acetiltransferasa A N-Terminal/genética , Acetiltransferasa E N-Terminal/genética , Acetilación , Animales , Femenino , Masculino , Ratones , Ratones Noqueados , Acetiltransferasa A N-Terminal/deficiencia , Acetiltransferasa A N-Terminal/metabolismo , Acetiltransferasa E N-Terminal/deficiencia , Acetiltransferasa E N-Terminal/metabolismoRESUMEN
Retinoic acid-related orphan receptor γ (RORγ) maintains the circadian rhythms of its downstream genes. However, the mechanism behind the transcriptional activation of RORγ itself remains unclear. Here, we demonstrate that transcription of RORγ is activated by heterogeneous nuclear ribonucleoprotein K (hnRNP K) via the poly(C) motif within its proximal promoter. Interestingly, we confirmed the binding of endogenous hnRNP K within RORγ1 and RORγ2 promoter along with the recruitment of RNA polymerase 2 through chromatin immunoprecipitation (ChIP). Furthermore, an assay for transposase accessible chromatin (ATAC)-qPCR showed that hnRNP K induced higher chromatin accessibility within the RORγ1 and RORγ2 promoter. Then we found that the knockdown of hnRNP K lowers RORγ mRNA oscillation amplitude in both RORγ and RORγ-dependent metabolic genes. Moreover, we demonstrated that time-dependent extracellular signal-regulated kinase (ERK) activation controls mRNA oscillation of RORγ and RORγ-dependent metabolic genes through hnRNP K. Taken together, our results provide new insight into the regulation of RORγ by hnRNP K as a transcriptional activator, along with its physiological significance in metabolism.
Asunto(s)
Cromatina/metabolismo , Ritmo Circadiano/fisiología , Ribonucleoproteína Heterogénea-Nuclear Grupo K/metabolismo , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Animales , Inmunoprecipitación de Cromatina/métodos , Ritmo Circadiano/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo K/genética , Ratones , Factores de Transcripción/metabolismo , Activación Transcripcional/fisiologíaRESUMEN
Current organoid models are limited by their inability to mimic mature organ architecture and associated tissue microenvironments1,2. Here we create multilayer bladder 'assembloids' by reconstituting tissue stem cells with stromal components to represent an organized architecture with an epithelium surrounding stroma and an outer muscle layer. These assembloids exhibit characteristics of mature adult bladders in cell composition and gene expression at the single-cell transcriptome level, and recapitulate in vivo tissue dynamics of regenerative responses to injury. We also develop malignant counterpart tumour assembloids to recapitulate the in vivo pathophysiological features of urothelial carcinoma. Using the genetically manipulated tumour-assembloid platform, we identify tumoural FOXA1, induced by stromal bone morphogenetic protein (BMP), as a master pioneer factor that drives enhancer reprogramming for the determination of tumour phenotype, suggesting the importance of the FOXA1-BMP-hedgehog signalling feedback axis between tumour and stroma in the control of tumour plasticity.
Asunto(s)
Organoides/patología , Organoides/fisiología , Regeneración , Neoplasias de la Vejiga Urinaria/patología , Neoplasias de la Vejiga Urinaria/fisiopatología , Vejiga Urinaria/patología , Vejiga Urinaria/fisiología , Adulto , Animales , Proteínas Morfogenéticas Óseas/metabolismo , Femenino , Erizos/metabolismo , Factor Nuclear 3-alfa del Hepatocito/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Organoides/fisiopatología , Análisis de la Célula Individual , Células Madre/citología , Células Madre/patología , Células Madre/fisiología , Transcriptoma , Vejiga Urinaria/citología , Infecciones Urinarias/metabolismo , Infecciones Urinarias/patologíaRESUMEN
BACKGROUND: Macrophages produce many inflammation-associated molecules, released by matrix metalloproteinases, such as adhesion molecules, and cytokines, as well, which play a crucial role in atherosclerosis. In this context, we investigated the relationship between Ninjurin-1 (Ninj1 [nerve injury-induced protein]), a novel matrix metalloproteinase 9 substrate, expression, and atherosclerosis progression. METHODS: Ninj1 expression and atherosclerosis progression were assessed in atherosclerotic aortic tissue and serum samples from patients with coronary artery disease and healthy controls, and atheroprone apolipoprotein e-deficient (Apoe-/-) and wild-type mice, as well. Apoe-/- mice lacking systemic Ninj1 expression (Ninj1-/-Apoe-/-) were generated to assess the functional effects of Ninj1. Bone marrow transplantation was also used to generate low-density lipoprotein receptor-deficient (Ldlr-/-) mice that lack Ninj1 specifically in bone marrow-derived cells. Mice were fed a Western diet for 5 to 23 weeks, and atherosclerotic lesions were investigated. The anti-inflammatory role of Ninj1 was verified by treating macrophages and mice with the peptides Ninj11-56 (ML56) and Ninj126-37 (PN12), which mimic the soluble form of Ninj1 (sNinj1). RESULTS: Our in vivo results conclusively showed a correlation between Ninj1 expression in aortic macrophages and the extent of human and mouse atherosclerotic lesions. Ninj1-deficient macrophages promoted proinflammatory gene expression by activating mitogen-activated protein kinase and inhibiting the phosphoinositide 3-kinase/Akt signaling pathway. Whole-body and bone marrow-specific Ninj1 deficiencies significantly increased monocyte recruitment and macrophage accumulation in atherosclerotic lesions through elevated macrophage-mediated inflammation. Macrophage Ninj1 was directly cleaved by matrix metalloproteinase 9 to generate a soluble form that exhibited antiatherosclerotic effects, as assessed in vitro and in vivo. Treatment with the sNinj1-mimetic peptides, ML56 and PN12, reduced proinflammatory gene expression in human and mouse classically activated macrophages, thereby attenuating monocyte transendothelial migration. Moreover, continuous administration of mPN12 alleviated atherosclerosis by inhibiting the enhanced monocyte recruitment and inflammation characteristics of this disorder in mice, regardless of the presence of Ninj1. CONCLUSIONS: Ninj1 is a novel matrix metalloproteinase 9 substrate in macrophages, and sNinj1 is a secreted atheroprotective protein that regulates macrophage inflammation and monocyte recruitment in atherosclerosis. Moreover, sNinj1-mediated anti-inflammatory effects are conserved in human macrophages and likely contribute to human atherosclerosis.
Asunto(s)
Antiinflamatorios/farmacología , Aterosclerosis , Moléculas de Adhesión Celular Neuronal , Macrófagos/metabolismo , Factores de Crecimiento Nervioso , Peptidomiméticos/farmacología , Transducción de Señal/efectos de los fármacos , Animales , Aterosclerosis/tratamiento farmacológico , Aterosclerosis/genética , Aterosclerosis/metabolismo , Moléculas de Adhesión Celular Neuronal/genética , Moléculas de Adhesión Celular Neuronal/metabolismo , Moléculas de Adhesión Celular Neuronal/farmacología , Femenino , Masculino , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Noqueados para ApoE , Factores de Crecimiento Nervioso/genética , Factores de Crecimiento Nervioso/metabolismo , Factores de Crecimiento Nervioso/farmacología , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/genéticaRESUMEN
BACKGROUND: The essential roles of gut microbiome have been emphasized in modulating human health and disease. Fusobacterium nucleatum (F. nucleatum), an obligate Gram-negative microorganism residing in oral cavity, gastrointestinal tract and elsewhere, has been recently considered as a potential oncobacterium associated with human cancers. However, the consequence of its enrichment was not extensively explored in terms of microbial homeostasis and stability at the early stage of disease development. RESULT: Our analysis on longitudinal metagenomic data generated by the Integrative Human Microbiome Project (iHMP) showed that F. nucleatum was frequently found in inflammatory bowel diseases (IBD) subjects with reduced microbial diversity. Using non-parametric logarithmic linear discriminant analysis (LDA) effect size (LEfSe) algorithm, 12 IBD- and 14 non-IBD-specific bacterial species were identified in the fecal metagenome and the IBD-specific ones were over-represented in the F. nucleatum-experienced subjects during long-term surveillance. In addition, F. nucleatum experience severely abrogated intra-personal stability of microbiome in IBD patients and induced highly variable gut microbiome between subjects. From the longitudinal comparison between microbial distributions prior and posterior to F. nucleatum detection, 41 species could be proposed as indicative "classifiers" for dysbiotic gut state. By multiple logistic regression models established on these classifiers, the high probability of experiencing F. nucleatum was significantly correlated with decreased alpha-diversity and increased number of biomarker species for IBD and colorectal cancer (CRC). Finally, microbial clustering confirmed that biomarker species for IBD and non-IBD conditions as well as CRC signature markers were well distinguishable and could be utilized for explaining gut symbiosis and dysbiosis. CONCLUSION: F. nucleatum opportunistically appeared under early dysbiotic condition in gut, and discriminative classifier species associated with F. nucleatum were successfully applied to predict microbial alterations in both IBD and non-IBD conditions. Our prediction model and microbial classifier biomarkers for estimating gut dysbiosis should provide a novel aspect of microbial homeostasis/dynamics and useful information on non-invasive biomarker screening.
Asunto(s)
Bacterias/clasificación , Disbiosis/diagnóstico , Fusobacterium nucleatum/aislamiento & purificación , Enfermedades Inflamatorias del Intestino/microbiología , Bacterias/aislamiento & purificación , Análisis Discriminante , Heces/microbiología , Femenino , Microbioma Gastrointestinal , Humanos , Modelos Logísticos , Estudios Longitudinales , Masculino , FilogeniaRESUMEN
Indoor pollutants are important problems to public health. Among indoor pollutants, indoor dust contains extracellular vesicles (EVs), which are associated with pulmonary inflammation. However, it has not been reported whether indoor dust EVs affect the cancer lung metastasis. In this study, we isolated indoor dust EVs and investigated their roles in cancer lung metastasis. Upon intranasal administration, indoor dust EVs enhanced mouse melanoma lung metastasis in a dose-dependent manner in mice. Pre-treatment or co-treatment of indoor dust EVs significantly promoted melanoma lung metastasis, whereas post-treatment of the EVs did not. In addition, the lung lysates from indoor dust EV-treated mice significantly increased tumour cell migration in vitro. We observed that tumour necrosis factor-α played important roles in indoor dust EV-mediated promotion of tumour cell migration in vitro and cancer lung metastasis in vivo. Furthermore, Pseudomonas EVs, the main components of indoor dust EVs, and indoor dust EVs showed comparable effects in promoting tumour cell migration in vitro and cancer lung metastasis in vivo. Taken together, our results suggest that indoor dust EVs, at least partly contributed by Pseudomonas EVs, are potential promoting agents of cancer lung metastasis.
RESUMEN
The explosive growth of next-generation sequencing data has resulted in ultra-large-scale datasets and ensuing computational problems. In Korea, the amount of genomic data has been increasing rapidly in the recent years. Leveraging these big data requires researchers to use large-scale computational resources and analysis pipelines. A promising solution for addressing this computational challenge is cloud computing, where CPUs, memory, storage, and programs are accessible in the form of virtual machines. Here, we present a cloud computing-based system, Bio-Express, that provides user-friendly, cost-effective analysis of massive genomic datasets. Bio-Express is loaded with predefined multi-omics data analysis pipelines, which are divided into genome, transcriptome, epigenome, and metagenome pipelines. Users can employ predefined pipelines or create a new pipeline for analyzing their own omics data. We also developed several web-based services for facilitating downstream analysis of genome data. Bio-Express web service is freely available at https://www.bioexpress.re.kr/.
RESUMEN
The oligosaccharyl transferase (OST) protein complex mediates the N-linked glycosylation of substrate proteins in the endoplasmic reticulum (ER), which regulates stability, activity, and localization of its substrates. Although many OST substrate proteins have been identified, the physiological role of the OST complex remains incompletely understood. Here we show that the OST complex in C. elegans is crucial for ER protein homeostasis and defense against infection with pathogenic bacteria Pseudomonas aeruginosa (PA14), via immune-regulatory PMK-1/p38 MAP kinase. We found that genetic inhibition of the OST complex impaired protein processing in the ER, which in turn up-regulated ER unfolded protein response (UPRER). We identified vitellogenin VIT-6 as an OST-dependent glycosylated protein, critical for maintaining survival on PA14. We also showed that the OST complex was required for up-regulation of PMK-1 signaling upon infection with PA14. Our study demonstrates that an evolutionarily conserved OST complex, crucial for ER homeostasis, regulates host defense mechanisms against pathogenic bacteria.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Retículo Endoplásmico/metabolismo , Proteostasis/fisiología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Hexosiltransferasas/metabolismo , Inmunidad Innata/fisiología , Sistema de Señalización de MAP Quinasas/fisiología , Proteínas de la Membrana/metabolismo , Pseudomonas aeruginosa/metabolismo , Transducción de Señal/fisiología , Factores de Transcripción/metabolismo , Respuesta de Proteína Desplegada/fisiología , Regulación hacia Arriba/fisiología , Vitelogeninas/metabolismoRESUMEN
Circadian gene expression is defined by the gene-specific phase and amplitude of daily oscillations in mRNA and protein levels. D site-binding protein mRNA (Dbp mRNA) shows high-amplitude oscillation; however, the underlying mechanism remains elusive. Here, we demonstrate that heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a key regulator that activates Dbp transcription via the poly(C) motif within its proximal promoter. Biochemical analyses identified hnRNP K as a specific protein that directly associates with the poly(C) motif in vitro Interestingly, we further confirmed the rhythmic binding of endogenous hnRNP K within the Dbp promoter through chromatin immunoprecipitation as well as the cycling expression of hnRNP K. Finally, knockdown of hnRNP K decreased mRNA oscillation in both Dbp and Dbp-dependent clock genes. Taken together, our results show rhythmic protein expression of hnRNP K and provide new insights into its function as a transcriptional amplifier of Dbp.
Asunto(s)
Ritmo Circadiano/genética , Proteínas de Unión al ADN/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo K/genética , Regiones Promotoras Genéticas/genética , Factores de Transcripción/genética , Activación Transcripcional/genética , Células 3T3 , Animales , Línea Celular , Células HEK293 , Humanos , Ratones , Poli C/genética , Interferencia de ARN , ARN Mensajero/genética , ARN Interferente Pequeño/genéticaRESUMEN
Chromatin immunoprecipitation coupled with high-throughput DNA sequencing (ChIP-Seq) is a powerful technology to profile the location of proteins of interest on a whole-genome scale. To identify the enrichment location of proteins, many programs and algorithms have been proposed. However, none of the commonly used peak calling programs could accurately explain the binding features of target proteins detected by ChIP-Seq. Here, publicly available data on 12 histone modifications, including H3K4ac/me1/me2/me3, H3K9ac/me3, H3K27ac/me3, H3K36me3, H3K56ac, and H3K79me1/me2, generated from a human embryonic stem cell line (H1), were profiled with five peak callers (CisGenome, MACS1, MACS2, PeakSeq, and SISSRs). The performance of the peak calling programs was compared in terms of reproducibility between replicates, examination of enriched regions to variable sequencing depths, the specificity-to-noise signal, and sensitivity of peak prediction. There were no major differences among peak callers when analyzing point source histone modifications. The peak calling results from histone modifications with low fidelity, such as H3K4ac, H3K56ac, and H3K79me1/me2, showed low performance in all parameters, which indicates that their peak positions might not be located accurately. Our comparative results could provide a helpful guide to choose a suitable peak calling program for specific histone modifications.
RESUMEN
In yeast, Hda1 histone deacetylase complex (Hda1C) preferentially deacetylates histones H3 and H2B, and functionally interacts with Tup1 to repress transcription. However, previous studies identified global increases in histone H4 acetylation in cells lacking Hda1, a component of Hda1C. Here, we find that Hda1C binds to hyperactive genes, likely via the interaction between the Arb2 domain of Hda1 and RNA polymerase II. Additionally, we report that Hda1C specifically deacetylates H4, but not H3, at hyperactive genes to partially inhibit elongation. This role is contrast to that of the Set2-Rpd3S pathway deacetylating histones at infrequently transcribed genes. We also find that Hda1C deacetylates H3 at inactive genes to delay the kinetics of gene induction. Therefore, in addition to fine-tuning of transcriptional response via H3-specific deacetylation, Hda1C may modulate elongation by specifically deacetylating H4 at highly transcribed regions.
Asunto(s)
Regulación Fúngica de la Expresión Génica/genética , Histona Desacetilasas/metabolismo , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Transcripción Genética/genética , Proteínas de Unión al ADN/metabolismo , Histona Desacetilasas/genética , Proteínas Nucleares/metabolismo , Dominios Proteicos/genética , Proteínas Represoras/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The D site-binding protein (Dbp) supports the rhythmic transcription of downstream genes, in part by displaying high-amplitude cycling of its own transcripts compared to other circadian-clock genes. However, the underlying mechanism remains elusive. Here, we demonstrated that the poly(C) motif within the Dbp proximal promoter, in addition to an E-box element, provoked transcriptional activation. Furthermore, we generated a cell line with poly(C) deleted to demonstrate the endogenous effect of the poly(C) motif within the Dbp promoter. We investigated whether RNA polymerase 2 (Pol2) recruitment on the Dbp promoter was decreased in the cell line with poly(C) deleted. Next, assay for transposase-accessible chromatin (ATAC)-quantitative PCR (qPCR) showed that the poly(C) motif induced greater chromatin accessibility within the region of the Dbp promoter. Finally, we determined that the oscillation amplitude of endogenous Dbp mRNA of the cell line with poly(C) deleted was decreased, which affected the oscillation of other clock genes that are controlled by Dbp Taken together, our results provide new insights into the function of the poly(C) motif as a novel cis-acting element of Dbp, along with its significance in the regulation of circadian rhythms.