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Enhancers, critical regulatory elements within the human genome, are often transcribed into enhancer RNAs. The dysregulation of enhancers leads to diseases collectively termed enhanceropathies. While it is known that enhancers play a role in diseases by regulating gene expression, the specific mechanisms by which individual enhancers cause diseases are not well understood. Studies of individual enhancers are needed to fill this gap. This study delves into the role of APOE-activating noncoding RNA, AANCR, in the central nervous system, elucidating its function as a genetic modifier in Alzheimer's Disease. We employed RNA interference, RNaseH-mediated degradation, and single-molecule RNA fluorescence in situ hybridization to demonstrate that mere transcription of AANCR is insufficient; rather, its transcripts are crucial for promoting APOE expression. Our findings revealed that AANCR is induced by ATM-mediated ERK phosphorylation and subsequent AP-1 transcription factor activation. Once activated, AANCR enhances APOE expression, which in turn imparts an inflammatory phenotype to astrocytes. These findings demonstrate that AANCR is a key enhancer RNA in some cell types within the nervous system, pivotal for regulating APOE expression and influencing inflammatory responses, underscoring its potential as a therapeutic target in neurodegenerative diseases.
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Background: m6A-related lncRNAs have demonstrated great potential tumor diagnostic and therapeutic targets. The goal of this work was to find m6A-regulated lncRNAs in osteosarcoma patients. Method: The Cancer Genome Atlas (TCGA) database was used to retrieve RNA sequencing and medical information from osteosarcoma sufferers. The Pearson's correlation test was used to identify the m6A-related lncRNAs. A risk model was built using univariate and multivariable Cox regression analysis. Kaplan-Meier survival analysis and receiver functional requirements were used to assess the risk model's performance (ROC). By using the CIBERSORT method, the associations between the relative risks and different immune cell infiltration were investigated. Lastly, the bioactivities of high-risk and low-risk subgroups were investigated using Gene Set Enrichment Analysis (GSEA). Result: A total of 531 m6A-related lncRNAs were obtained from TCGA. Seven lncRNAs have demonstrated prognostic values. A total of 88 OS patients were separated into cluster 1, cluster 2, and cluster 3. The overall survival rate of OS patients in cluster 3 was more favorable than that of those in cluster 1 and cluster 2. The average Stromal score was much higher in cluster 1 than in cluster 2 and cluster 3 (P < 0.05). The expression levels of lncRNAs used in the construction of the risk prediction model in the high-risk group were generally lower than those in the low-risk group. Analysis of patient survival indicated that the survival of the low-risk group was higher than that of the high-risk group (P < 0.0001) and the area under the curve (AUC) of the ROC curve was 0.719. Using the CIBERSORT algorithm, the results revealed that Macrophages M0, Macrophages M2, and T cells CD4 memory resting accounted for a large proportion of immune cell infiltration. By GSEA analysis, our results implied that the high-risk group was mainly involved in unfolded protein response, DNA repair signaling, and epithelial-mesenchymal transition signaling pathway and glycolysis pathway; meanwhile, the low-risk group was mainly involved in estrogen response early and KRAS signaling pathway. Conclusion: Our investigation showed that m6A-related lncRNAs remained tightly connected to the immunological microenvironment of osteosarcoma tumors, potentially influencing carcinogenesis and development. The immune microenvironment and immune-related biochemical pathways can be changed by regulating the transcription of M6A modulators or lncRNAs. In addition, we looked for risk-related signaling of m6A-related lncRNAs in osteosarcomas and built and validated the risk prediction system. The findings of our current analysis will facilitate the assessment of outcomes and the development of immunotherapies for sufferers of osteosarcomas.
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Osteosarcoma , ARN Largo no Codificante , Perfilación de la Expresión Génica/métodos , Humanos , Osteosarcoma/genética , ARN Largo no Codificante/análisis , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Microambiente Tumoral/genéticaRESUMEN
miRNAs are small noncoding RNAs that may contribute to common diseases through epigenetic regulation of gene expression. Little is known regarding the role of miRNAs in type 2 diabetes (T2D). We performed miRNA sequencing and transcriptomic profiling of peripheral monocytes from the longitudinal Multi-Ethnic Study of Atherosclerosis (MESA) (N = 1,154). We examined associations between miRNAs and prevalent impaired fasting glucose and T2D and evaluated the T2D-associated miRNA effect on incident T2D. Of 774 detected miRNAs, 6 (miR-22-3p, miR-33a-5p, miR-181c-5p, miR-92b-3p, miR-222-3p, and miR-944) were associated with prevalent T2D. For five of the six miRNAs (all but miR-222-3p), our findings suggest a dose-response relationship with impaired fasting glucose and T2D. Two of the six miRNAs were associated with incident T2D (miR-92b-3p: hazard ratio [HR] 1.64, P = 1.30E-03; miR-222-3p: HR 1.97, P = 9.10E-03) in the highest versus lowest tertile of expression. Most of the T2D-associated miRNAs were also associated with HDL cholesterol concentrations. The genes targeted by these miRNAs belong to key nodes of a cholesterol metabolism transcriptomic network. Higher levels of miRNA expression expected to increase intracellular cholesterol accumulation in monocytes are linked to an increase in T2D risk.
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Diabetes Mellitus Tipo 2 , MicroARNs , Diabetes Mellitus Tipo 2/genética , Epigénesis Genética , Perfilación de la Expresión Génica , Glucosa , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Monocitos/metabolismoRESUMEN
INTRODUCTION: Diabetes and bone health are closely related. We examined the incidence and risk factors of hip fractures in Chinese patients with type 2 diabetes (T2D). MATERIALS AND METHODS: In this prospective cohort, we consecutively enrolled 22,325 adults with T2D above the age of 40 years in the Hong Kong Diabetes Register between 1994 and 2015 with crude hip fracture incidence rate censored in 2017. RESULTS: At baseline, the mean age of this cohort was 60.9 ± 10.5 years (mean duration of diabetes 6 years, 52.4% male). During a mean ± standard deviation (SD) follow-up period of 8.7 ± 5.2 years with 193,553 person-years, 603 patients were hospitalized due to hip fractures with an incidence (95% confidence interval, CI) of 315.1 (290.4-341.3) per 100,000 person-years. On multivariable analysis with competing death risk adjusted, the independent hazard ratios (95% CI) for hip fractures in T2D were 2.01 (1.61-2.51) for female sex, 1.08 (1.07-1.09) for age, 0.93 (0.90-0.95) for body mass index, 1.52 (1.25-1.85) for albuminuria and 1.12 (1.02-1.23) for low density lipoprotein-cholesterol. In men, the 30-day, 1-year and 5-year post-hip fracture mortality rate (95% CI) were 5.8 (2.4-9.1) %, 29.2 (22.3-35.5) % and 65.9 (57.3-72.8) % respectively. The corresponding rates in women were 3.4 (1.6-5.1) %, 18.6 (14.7-22.4) %, and 46.8 (40.9-52.1) %. CONCLUSIONS: Southern Chinese patients with T2D have a high risk of hip fracture associated with suboptimal cardiometabolic-renal risk factors and a high post-fracture mortality rate. The effects of improving modifiable risk factors on bone health warrants further evaluation.
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Enfermedades Cardiovasculares/patología , Diabetes Mellitus Tipo 2/fisiopatología , Fracturas de Cadera/complicaciones , Enfermedades Renales/patología , Enfermedades Metabólicas/patología , Adulto , Anciano , Enfermedades Cardiovasculares/etiología , Femenino , Estudios de Seguimiento , Fracturas de Cadera/epidemiología , Hong Kong/epidemiología , Humanos , Enfermedades Renales/etiología , Masculino , Enfermedades Metabólicas/etiología , Persona de Mediana Edad , Pronóstico , Estudios Prospectivos , Factores de RiesgoRESUMEN
Tobacco smoke exposure contributes to the global burden of communicable and chronic diseases. To identify immune cells affected by smoking, we use single-cell RNA sequencing on peripheral blood from smokers and nonsmokers. Transcriptomes reveal a subpopulation of FCGR3A (CD16)-expressing Natural Killer (NK)-like CD8 T lymphocytes that increase in smokers. Mass cytometry confirms elevated CD16+ CD8 T cells in smokers. Inferred as highly differentiated by pseudotime analysis, NK-like CD8 T cells express markers characteristic of effector memory re-expressing CD45RA T (TEMRA) cells. Indicative of immune aging, smokers' CD8 T cells are biased toward differentiated cells and smokers have fewer naïve cells than nonsmokers. DNA methylation-based models show that smoking dose is associated with accelerated aging and decreased telomere length, a biomarker of T cell senescence. Immune aging accompanies T cell senescence, which can ultimately lead to impaired immune function. This suggests a role for smoking-induced, senescence-associated immune dysregulation in smoking-mediated pathologies.
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Linfocitos T CD8-positivos/efectos de los fármacos , Fumar Cigarrillos/efectos adversos , Receptores de IgG/metabolismo , Adulto , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Fumar Cigarrillos/inmunología , Femenino , Proteínas Ligadas a GPI/efectos de los fármacos , Proteínas Ligadas a GPI/inmunología , Proteínas Ligadas a GPI/metabolismo , Humanos , Enfermedades del Sistema Inmune/fisiopatología , Células Asesinas Naturales/inmunología , Antígenos Comunes de Leucocito , Masculino , Persona de Mediana Edad , Receptores de IgG/efectos de los fármacos , Receptores de IgG/inmunología , Análisis de la Célula Individual/métodos , Fumadores , Fumar/sangreRESUMEN
BACKGROUND: Cigarette smoke is a causal factor in cancers and cardiovascular disease. Smoking-associated differentially methylated regions (SM-DMRs) have been observed in disease studies, but the causal link between altered DNA methylation and transcriptional change is obscure. OBJECTIVE: Our objectives were to finely resolve SM-DMRs and to interrogate the mechanistic link between SM-DMRs and altered transcription of enhancer noncoding RNA (eRNA) and mRNA in human circulating monocytes. METHOD: We integrated SM-DMRs identified by reduced representation bisulfite sequencing (RRBS) of circulating CD14+ monocyte DNA collected from two independent human studies [n=38 from Clinical Research Unit (CRU) and n=55 from the Multi-Ethnic Study of Atherosclerosis (MESA), about half of whom were active smokers] with gene expression for protein-coding genes and noncoding RNAs measured by RT-PCR or RNA sequencing. Candidate SM-DMRs were compared with RRBS of purified CD4+ T cells, CD8+ T cells, CD15+ granulocytes, CD19+ B cells, and CD56+ NK cells (n=19 females, CRU). DMRs were validated using pyrosequencing or bisulfite amplicon sequencing in up to 85 CRU volunteers, who also provided saliva DNA. RESULTS: RRBS identified monocyte SM-DMRs frequently located in putative gene regulatory regions. The most significant monocyte DMR occurred at a poised enhancer in the aryl-hydrocarbon receptor repressor gene (AHRR) and it was also detected in both granulocytes and saliva DNA. To our knowledge, we identify for the first time that SM-DMRs in or near AHRR, C5orf55-EXOC-AS, and SASH1 were associated with increased noncoding eRNA as well as mRNA in monocytes. Functionally, the AHRR SM-DMR appeared to up-regulate AHRR mRNA through activating the AHRR enhancer, as suggested by increased eRNA in the monocytes, but not granulocytes, from smokers compared with nonsmokers. CONCLUSIONS: Our findings suggest that AHRR SM-DMR up-regulates AHRR mRNA in a monocyte-specific manner by activating the AHRR enhancer. Cell type-specific activation of enhancers at SM-DMRs may represent a mechanism driving smoking-related disease. https://doi.org/10.1289/EHP2395.
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Metilación de ADN/efectos de los fármacos , ARN Mensajero/efectos de los fármacos , ARN no Traducido/efectos de los fármacos , Fumar/efectos adversos , Sulfitos/efectos adversos , Transcripción Genética/efectos de los fármacos , Adulto , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , ARN Mensajero/metabolismo , ARN no Traducido/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos/efectos de los fármacos , Secuencias Reguladoras de Ácidos Nucleicos/genéticaRESUMEN
Alterations in DNA methylation and gene expression in blood leukocytes are potential biomarkers of harm and mediators of the deleterious effects of tobacco exposure. However, methodological issues, including the use of self-reported smoking status and mixed cell types have made previously identified alterations in DNA methylation and gene expression difficult to interpret. In this study, we examined associations of tobacco exposure with DNA methylation and gene expression, utilizing a biomarker of tobacco exposure (urine cotinine) and CD14+ purified monocyte samples from 934 participants of the community-based Multi-Ethnic Study of Atherosclerosis (MESA). Urine cotinine levels were measured using an immunoassay. DNA methylation and gene expression were measured with microarrays. Multivariate linear regression was used to test for associations adjusting for age, sex, race/ethnicity, education, and study site. Urine cotinine levels were associated with methylation of 176 CpGs [false discovery rate (FDR)<0.01]. Four CpGs not previously identified by studies of non-purified blood samples nominally replicated (P value<0.05) with plasma cotinine-associated methylation in 128 independent monocyte samples. Urine cotinine levels associated with expression of 12 genes (FDR<0.01), including increased expression of P2RY6 (Beta ± standard error = 0.078 ± 0.008, P = 1.99 × 10-22), a gene previously identified to be involved in the release of pro-inflammatory cytokines. No cotinine-associated (FDR<0.01) methylation profiles significantly (FDR<0.01) correlated with cotinine-associated (FDR<0.01) gene expression profiles. In conclusion, our findings i) identify potential monocyte-specific smoking-associated methylation patterns and ii) suggest that alterations in methylation may not be a main mechanism regulating gene expression in monocytes in response to cigarette smoking.
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Metilación de ADN , Fumar Tabaco/genética , Anciano , Anciano de 80 o más Años , Aterosclerosis/epidemiología , Cotinina/orina , Femenino , Humanos , Masculino , Persona de Mediana Edad , Monocitos/metabolismo , Fumar Tabaco/epidemiología , Fumar Tabaco/orinaRESUMEN
Little is known regarding the epigenetic basis of atherosclerosis. Here we present the CD14+ blood monocyte transcriptome and epigenome signatures associated with human atherosclerosis. The transcriptome signature includes transcription coactivator, ARID5B, which is known to form a chromatin derepressor complex with a histone H3K9Me2-specific demethylase and promote adipogenesis and smooth muscle development. ARID5B CpG (cg25953130) methylation is inversely associated with both ARID5B expression and atherosclerosis, consistent with this CpG residing in an ARID5B enhancer region, based on chromatin capture and histone marks data. Mediation analysis supports assumptions that ARID5B expression mediates effects of cg25953130 methylation and several cardiovascular disease risk factors on atherosclerotic burden. In lipopolysaccharide-stimulated human THP1 monocytes, ARID5B knockdown reduced expression of genes involved in atherosclerosis-related inflammatory and lipid metabolism pathways, and inhibited cell migration and phagocytosis. These data suggest that ARID5B expression, possibly regulated by an epigenetically controlled enhancer, promotes atherosclerosis by dysregulating immunometabolism towards a chronic inflammatory phenotype.The molecular mechanisms mediating the impact of environmental factors in atherosclerosis are unclear. Here, the authors examine CD14+ blood monocyte's transcriptome and epigenome signatures to find differential methylation and expression of ARID5B to be associated with human atherosclerosis.
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Aterosclerosis/genética , Proteínas de Unión al ADN/genética , Monocitos/metabolismo , Factores de Transcripción/genética , Transcriptoma , Anciano , Aterosclerosis/metabolismo , Metilación de ADN , Proteínas de Unión al ADN/metabolismo , Epigénesis Genética , Femenino , Histonas/genética , Histonas/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Monocitos/química , Factores de Transcripción/metabolismoRESUMEN
[This corrects the article DOI: 10.1371/journal.pone.0166486.].
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Tobacco smoke exposure dramatically alters DNA methylation in blood cells and may mediate smoking-associated complex diseases through effects on immune cell function. However, knowledge of smoking effects in specific leukocyte subtypes is limited. To better characterize smoking-associated methylation changes in whole blood and leukocyte subtypes, we used Illumina 450K arrays and Reduced Representation Bisulfite Sequencing (RRBS) to assess genome-wide DNA methylation. Differential methylation analysis in whole blood DNA from 172 smokers and 81 nonsmokers revealed 738 CpGs, including 616 previously unreported CpGs, genome-wide significantly associated with current smoking (p <1.2x10-7, Bonferroni correction). Several CpGs (MTSS1, NKX6-2, BTG2) were associated with smoking duration among heavy smokers (>22 cigarettes/day, n = 86) which might relate to long-term heavy-smoking pathology. In purified leukocyte subtypes from an independent group of 20 smokers and 14 nonsmokers we further examined methylation and gene expression for selected genes among CD14+ monocytes, CD15+ granulocytes, CD19+ B cells, and CD2+ T cells. In 10 smokers and 10 nonsmokers we used RRBS to fine map differential methylation in CD4+ T cells, CD8+ T cells, CD14+, CD15+, CD19+, and CD56+ natural killer cells. Distinct cell-type differences in smoking-associated methylation and gene expression were identified. AHRR (cg05575921), ALPPL2 (cg21566642), GFI1 (cg09935388), IER3 (cg06126421) and F2RL3 (cg03636183) showed a distinct pattern of significant smoking-associated methylation differences across cell types: granulocytes> monocytes>> B cells. In contrast GPR15 (cg19859270) was highly significant in T and B cells and ITGAL (cg09099830) significant only in T cells. Numerous other CpGs displayed distinctive cell-type responses to tobacco smoke exposure that were not apparent in whole blood DNA. Assessing the overlap between these CpG sites and differential methylated regions (DMRs) with RRBS in 6 cell types, we confirmed cell-type specificity in the context of DMRs. We identified new CpGs associated with current smoking, pack-years, duration, and revealed unique profiles of smoking-associated DNA methylation and gene expression among immune cell types, providing potential clues to hematopoietic lineage-specific effects in disease etiology.
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Metilación de ADN , Epigenómica/métodos , Leucocitos/metabolismo , Fumar , Adulto , Fosfatasa Alcalina/genética , Proteínas Reguladoras de la Apoptosis/genética , Linfocitos B/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Islas de CpG/genética , Proteínas de Unión al ADN/genética , Epigénesis Genética , Femenino , Proteínas Ligadas a GPI/genética , Expresión Génica , Estudio de Asociación del Genoma Completo/métodos , Granulocitos/metabolismo , Humanos , Leucocitos/clasificación , Masculino , Proteínas de la Membrana/genética , Persona de Mediana Edad , Monocitos/metabolismo , Receptores de Trombina/genética , Proteínas Represoras/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN/métodos , Linfocitos T/metabolismo , Factores de Transcripción/genética , Adulto JovenRESUMEN
The NRF2/sMAF protein complex regulates the oxidative stress response by occupying cis-acting enhancers containing an antioxidant response element (ARE). Integrating genome-wide maps of NRF2/sMAF occupancy with disease-susceptibility loci, we discovered eight polymorphic AREs linked to 14 highly ranked disease-risk SNPs in individuals of European ancestry. Among these SNPs was rs242561, located within a regulatory region of the MAPT gene (encoding microtubule-associated protein Tau). It was consistently occupied by NRF2/sMAF in multiple experiments and its strong-binding allele associated with higher mRNA levels in cell lines and human brain tissue. Induction of MAPT transcription by NRF2 was confirmed using a human neuroblastoma cell line and a Nrf2-deficient mouse model. Most importantly, rs242561 displayed complete linkage disequilibrium with a highly protective allele identified in multiple GWASs of progressive supranuclear palsy, Parkinson's disease, and corticobasal degeneration. These observations suggest a potential role for NRF2/sMAF in tauopathies and a possible role for NRF2 pathway activators in disease prevention.
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BACKGROUND: Tobacco smoke contains numerous agonists of the aryl hydrocarbon receptor (AhR) pathway, and activation of the AhR pathway was shown to promote atherosclerosis in mice. Intriguingly, cigarette smoking is most strongly and robustly associated with DNA modifications to an AhR pathway gene, the AhR repressor (AHRR). We hypothesized that altered AHRR methylation in monocytes, a cell type sensitive to cigarette smoking and involved in atherogenesis, may be a part of the biological link between cigarette smoking and atherosclerosis. METHODS AND RESULTS: DNA methylation profiles of AHRR in monocytes (542 CpG sites ± 150 kb of AHRR, using Illumina 450K array) were integrated with smoking habits and ultrasound-measured carotid plaque scores from 1256 participants of the Multi-Ethnic Study of Atherosclerosis (MESA). Methylation of cg05575921 significantly associated (P=6.1 × 10(-134)) with smoking status (current versus never). Novel associations between cg05575921 methylation and carotid plaque scores (P=3.1 × 10(-10)) were identified, which remained significant in current and former smokers even after adjusting for self-reported smoking habits, urinary cotinine, and well-known cardiovascular disease risk factors. This association replicated in an independent cohort using hepatic DNA (n=141). Functionally, cg05575921 was located in a predicted gene expression regulatory element (enhancer) and had methylation correlated with AHRR mRNA profiles (P=1.4 × 10(-17)) obtained from RNA sequencing conducted on a subset (n=373) of the samples. CONCLUSIONS: These findings suggest that AHRR methylation may be functionally related to AHRR expression in monocytes and represents a potential biomarker of subclinical atherosclerosis in smokers.
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Aterosclerosis/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Metilación de ADN , Receptores de Hidrocarburo de Aril/metabolismo , Proteínas Represoras/genética , Fumar , Anciano , Aterosclerosis/etnología , Aterosclerosis/genética , Población Negra/genética , Femenino , Estudios de Asociación Genética , Hispánicos o Latinos/genética , Humanos , Masculino , Monocitos/metabolismo , Fumar/etnología , Población Blanca/genéticaRESUMEN
Actl6a (actin-like protein 6A, also known as Baf53a or Arp4) is a subunit shared by multiple complexes including esBAF, INO80, and Tip60-p400, whose main components (Brg1, Ino80, and p400, respectively) are crucial for the maintenance of embryonic stem cells (ESCs). However, whether and how Actl6a functions in ESCs has not been investigated. ESCs originate from the epiblast (EPI) that is derived from the inner cell mass (ICM) in blastocysts, which also give rise to primitive endoderm (PrE). The molecular mechanisms for EPI/PrE specification remain unclear. In this study, we provide the first evidence that Actl6a can protect mouse ESCs (mESCs) from differentiating into PrE. While RNAi knockdown of Actl6a, which appeared highly expressed in mESCs and downregulated during differentiation, induced mESCs to differentiate towards the PrE lineage, ectopic expression of Actl6a was able to repress PrE differentiation. Our work also revealed that Actl6a could interact with Nanog and Sox2 and promote Nanog binding to pluripotency genes such as Oct4 and Sox2. Interestingly, cells depleted of p400, but not of Brg1 or Ino80, displayed similar PrE differentiation patterns. Mutant Actl6a with impaired ability to bind Tip60 and p400 failed to block PrE differentiation induced by Actl6a dysfunction. Finally, we showed that Actl6a could target to the promoters of key PrE regulators (e.g., Sall4 and Fgf4), repressing their expression and inhibiting PrE differentiation. Our findings uncover a novel function of Actl6a in mESCs, where it acts as a gatekeeper to prevent mESCs from entering into the PrE lineage through a Yin/Yang regulating pattern.
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Actinas/metabolismo , Blastocisto/citología , Diferenciación Celular/fisiología , Proteínas Cromosómicas no Histona/metabolismo , Proteínas de Unión al ADN/metabolismo , Endodermo/citología , Estratos Germinativos/citología , Células Madre Embrionarias de Ratones/citología , Animales , Linaje de la Célula/genética , Regulación del Desarrollo de la Expresión Génica/fisiología , Ratones , Factor 3 de Transcripción de Unión a Octámeros/metabolismoRESUMEN
As a member of the Tet (Ten-eleven translocation) family proteins that can convert 5-methylcytosine (5mC) to 5-hydroxylmethylcytosine (5hmC), Tet1 has been implicated in regulating global DNA demethylation and gene expression. Tet1 is highly expressed in embryonic stem (ES) cells and appears primarily to repress developmental genes for maintaining pluripotency. To understand how Tet1 may regulate gene expression, we conducted large scale immunoprecipitation followed by mass spectrometry of endogenous Tet1 in mouse ES cells. We found that Tet1 could interact with multiple chromatin regulators, including Sin3A and NuRD complexes. In addition, we showed that Tet1 could also interact with the O-GlcNAc transferase (Ogt) and be O-GlcNAcylated. Depletion of Ogt led to reduced Tet1 and 5hmC levels on Tet1-target genes, whereas ectopic expression of wild-type but not enzymatically inactive Ogt increased Tet1 levels. Mutation of the putative O-GlcNAcylation site on Tet1 led to decreased O-GlcNAcylation and level of the Tet1 protein. Our results suggest that O-GlcNAcylation can positively regulate Tet1 protein concentration and indicate that Tet1-mediated 5hmC modification and target repression is controlled by Ogt.
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Proteínas de Unión al ADN/metabolismo , Células Madre Embrionarias/enzimología , Regulación del Desarrollo de la Expresión Génica , N-Acetilglucosaminiltransferasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Represoras/metabolismo , Animales , Línea Celular , Cromatina/metabolismo , Proteínas de Unión al ADN/genética , Células Madre Embrionarias/citología , Genes del Desarrollo , Glicosilación , Ratones , N-Acetilglucosaminiltransferasas/antagonistas & inhibidores , Células Madre Pluripotentes/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas/genéticaRESUMEN
Polycomb group (PcG) proteins play important roles in repressing lineage-specific genes and maintaining the undifferentiated state of mouse embryonic stem cells (mESCs). However, how PcG proteins are recruited to their target genes is largely unknown. Here, we show that the H3K36-specific histone demethylase Kdm2b is highly expressed in mESCs and regulated by the pluripotent factors Oct4 and Sox2 directly. Depletion of Kdm2b in mESCs causes de-repression of lineage-specific genes and induces early differentiation. The function of Kdm2b depends on its CxxC-ZF domain, which mediates its genome-wide binding to CpG islands (CGIs). Kdm2b interacts with the core components of polycomb repressive complex 1 (PRC1) and recruits the complex to the CGIs of early lineage-specific genes. Thus, our study not only reveals an Oct4-Sox2-Kdm2b-PRC1-CGI regulatory axis and its function in maintaining the undifferentiated state of mESCs, but also demonstrates a critical function of Kdm2b in recruiting PRC1 to the CGIs of lineage-specific genes to repress their expression.
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Diferenciación Celular , Islas de CpG/genética , Células Madre Embrionarias/fisiología , Proteínas F-Box/metabolismo , Regulación del Desarrollo de la Expresión Génica , Genes del Desarrollo , Histona Demetilasas con Dominio de Jumonji/metabolismo , Complejo Represivo Polycomb 1/metabolismo , Animales , Secuencia de Bases , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Western Blotting , Linaje de la Célula , Proliferación Celular , Células Cultivadas , Inmunoprecipitación de Cromatina , Metilación de ADN , Ensayo de Cambio de Movilidad Electroforética , Células Madre Embrionarias/citología , Proteínas F-Box/antagonistas & inhibidores , Proteínas F-Box/genética , Técnica del Anticuerpo Fluorescente , Perfilación de la Expresión Génica , Genoma , Inmunoprecipitación , Histona Demetilasas con Dominio de Jumonji/antagonistas & inhibidores , Histona Demetilasas con Dominio de Jumonji/genética , Luciferasas/metabolismo , Ratones , Datos de Secuencia Molecular , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/fisiología , Complejo Represivo Polycomb 1/genética , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción SOXB1/genética , Factores de Transcripción SOXB1/metabolismo , Homología de Secuencia de Ácido Nucleico , TransfecciónRESUMEN
Embryonic stem (ES) cells exhibit general characteristics of open chromatin, a state that may be necessary for ES cells to efficiently self-renew while remaining poised for differentiation. Histone H3K4 and H3K9 trimethylation associate as a general rule, with open and silenced chromatin, respectively, for ES cell pluripotency maintenance. However, how histone modifications are regulated to maintain open chromatin in ES cells remains largely unknown. Here, we demonstrate that trithorax protein Ash2l, homologue of the Drosophila Ash2 (absent, small, homeotic-2) protein, is a key regulator of open chromatin in ES cells. Consistent with Ash2l being a core subunit of mixed lineage leukemia methyltransferase complex, RNAi knockdown of Ash2l was sufficient to reduce H3K4 methylation levels and drive ES cells to a silenced chromatin state with high H3K9 trimethylation. Genome-wide ChIP-seq analysis indicated that Ash2l is recruited to target loci through two distinct modes and enriched at a family of genes implicated in open chromatin regulation, including chromatin remodeler Cdh7, transcription factor c-Myc, and H3K9 demethylase Kdm4c. Our results underscore the importance of Ash2l in open chromatin regulation and provide insight into how the open chromatin landscape is maintained in ES cells.
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Cromatina/química , Proteínas de Unión al ADN/fisiología , Células Madre Embrionarias/citología , Regulación del Desarrollo de la Expresión Génica , Proteínas Nucleares/fisiología , Factores de Transcripción/fisiología , Animales , Diferenciación Celular , Cromatina/metabolismo , Inmunoprecipitación de Cromatina , Proteínas de Unión al ADN/metabolismo , Genoma , Histonas/metabolismo , Metilación , Ratones , Microscopía Fluorescente/métodos , Modelos Biológicos , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Proteínas Nucleares/metabolismo , Células Madre Pluripotentes/citología , Interferencia de ARN , Factores de Transcripción/metabolismoRESUMEN
In mammals, maintenance of the linear chromosome ends (or telomeres) involves faithful replication of genetic materials and protection against DNA damage signals, to ensure genome stability and integrity. These tasks are carried out by the telomerase holoenzyme and a unique nucleoprotein structure in which an array of telomere-associated proteins bind to telomeric DNA to form special protein/DNA complexes. The telomerase complex, which is comprised of telomeric reverse transcriptase (TERT), telomeric RNA component (TERC), and other assistant factors, is responsible for adding telomeric repeats to the ends of chromosomes. Without proper telomere maintenance, telomere length will shorten with successive round of DNA replication due to the so-called end replication problem. Aberrant regulation of telomeric proteins and/or telomerase may lead to abnormalities that can result in diseases such as dyskeratosis congenita (DC) and cancers. Understanding the mechanisms that regulate telomere homeostasis and the factors that contribute to telomere dysfunction should aid us in developing diagnostic and therapeutic tools for these diseases.
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Telómero/química , Telómero/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , ADN/química , ADN/metabolismo , G-Cuádruplex , Humanos , Unión Proteica , Telómero/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
While breast cancer mortality rate has seen a steady decline in the last few decades, advances in better treatment and diagnostic tools remain important as we come into the age of personalized therapy. In this report, we describe our studies of SGK3's role in breast cancer. SGK3 (also known as CISK) is a member of the AGC family of kinases. Our previous work indicates that SGK3 functions downstream of the PI 3-kinase cascade and shares molecular and biochemical similarities with Akt. Here, we show that SGK3 expression is linked to estrogen receptor (ER) both in breast caner cell lines and in primary tumor samples. Our analysis also indicated a positive correlation between SGK3 expression and tumor prognosis. Importantly, our immunochemistry analysis of human tumor samples established a clinical link between SGK3 expression and ER+ tumors. These findings implicate SGK3 as an additional component to a complex and heterogeneous disease, and point to the potential benefits of incorporating SGK3 into the process of breast cancer diagnosis and treatment.
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Neoplasias de la Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Estrógenos/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Carcinoma Ductal de Mama/mortalidad , Carcinoma Ductal de Mama/patología , Línea Celular Tumoral , Supervivencia sin Enfermedad , Femenino , Expresión Génica , Humanos , Modelos Logísticos , Persona de Mediana Edad , Análisis Multivariante , Pronóstico , Modelos de Riesgos Proporcionales , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
Short repetitive sequences are common in the human genome, and many fall within transcription units. We have previously shown that transcription through CAG repeat tracts destabilizes them in a way that depends on transcription-coupled nucleotide excision repair and mismatch repair. Recent observations that antisense transcription accompanies sense transcription in many human genes led us to test the effects of antisense transcription on triplet repeat instability in human cells. Here, we report that simultaneous sense and antisense transcription (convergent transcription) initiated from two inducible promoters flanking a CAG95 tract in a nonessential gene enhances repeat instability synergistically, arrests the cell cycle, and causes massive cell death via apoptosis. Using chemical inhibitors and small interfering RNA (siRNA) knockdowns, we identified the ATR (ataxia-telangiectasia mutated [ATM] and Rad3 related) signaling pathway as a key mediator of this cellular response. RNA polymerase II, replication protein A (RPA), and components of the ATR signaling pathway accumulate at convergently transcribed repeat tracts, accompanied by phosphorylation of ATR, CHK1, and p53. Cell death depends on simultaneous sense and antisense transcription and is proportional to their relative levels, it requires the presence of the repeat tract, and it occurs in both proliferating and nonproliferating cells. Convergent transcription through a CAG repeat represents a novel mechanism for triggering a cellular stress response, one that is initiated by events at a single locus in the genome and resembles the response to DNA damage.
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Apoptosis/genética , Secuencias Repetitivas de Ácidos Nucleicos , Transcripción Genética , Animales , Elementos sin Sentido (Genética) , Proteínas de la Ataxia Telangiectasia Mutada , Inhibidores de Caspasas , Caspasas/metabolismo , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Regiones Promotoras Genéticas , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal/genética , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
The telosome/shelterin, a six-protein complex formed by TRF1, TRF2, RAP1, TIN2, POT1, and TPP1, functions as the core of the telomere interactome, acting as the molecular platform for the assembly of higher order complexes and coordinating cross-talks between various protein subcomplexes. Within the telosome, there are two oligonucleotide- or oligosaccharide-binding (OB) fold-containing proteins, TPP1 and POT1. They can form heterodimers that bind to the telomeric single-stranded DNA, an activity that is central for telomere end capping and telomerase recruitment. Through proteomic analyses, we found that in addition to POT1, TPP1 can associate with another OB fold-containing protein, OBFC1/AAF44. The yeast homolog of OBFC1 is Stn1, which plays a critical role in telomere regulation. We show here that OBFC1/AAF44 can localize to telomeres in human cells and bind to telomeric single-stranded DNA in vitro. Furthermore, overexpression of an OBFC1 mutant resulted in elongated telomeres in human cells, implicating OBFC1/AAF4 in telomere length regulation. Taken together, our studies suggest that OBFC1/AAF44 represents a new player in the telomere interactome for telomere maintenance.