RESUMEN
Gene expression is controlled in part by post-translational modifications of core histones. Methylation of lysine 4 of histone H3 (H3K4), associated with open chromatin and gene transcription, is catalyzed by type 2 lysine methyltransferase complexes that require WDR5, RBBP5, ASH2L and DPY30 as core subunits. Ash2l is essential during embryogenesis and for maintaining adult tissues. To expand on the mechanistic understanding of Ash2l, we generated mouse embryo fibroblasts (MEFs) with conditional Ash2l alleles. Upon loss of Ash2l, methylation of H3K4 and gene expression were downregulated, which correlated with inhibition of proliferation and cell cycle progression. Moreover, we observed induction of senescence concomitant with a set of downregulated signature genes but independent of SASP. Many of the signature genes are FoxM1 responsive. Indeed, exogenous FOXM1 was sufficient to delay senescence. Thus, although the loss of Ash2l in MEFs has broad and complex consequences, a distinct set of downregulated genes promotes senescence.
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Proteínas de Unión al ADN , Proteína de la Leucemia Mieloide-Linfoide , Animales , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/genética , Histonas/metabolismo , Lisina/metabolismo , Ratones , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: Massive amounts of data are produced by combining next-generation sequencing with complex biochemistry techniques to characterize regulatory genomics profiles, such as protein-DNA interaction and chromatin accessibility. Interpretation of such high-throughput data typically requires different computation methods. However, existing tools are usually developed for a specific task, which makes it challenging to analyze the data in an integrative manner. RESULTS: We here describe the Regulatory Genomics Toolbox (RGT), a computational library for the integrative analysis of regulatory genomics data. RGT provides different functionalities to handle genomic signals and regions. Based on that, we developed several tools to perform distinct downstream analyses, including the prediction of transcription factor binding sites using ATAC-seq data, identification of differential peaks from ChIP-seq data, and detection of triple helix mediated RNA and DNA interactions, visualization, and finding an association between distinct regulatory factors. CONCLUSION: We present here RGT; a framework to facilitate the customization of computational methods to analyze genomic data for specific regulatory genomics problems. RGT is a comprehensive and flexible Python package for analyzing high throughput regulatory genomics data and is available at: https://github.com/CostaLab/reg-gen . The documentation is available at: https://reg-gen.readthedocs.io.
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Cromatina , Genómica , Secuenciación de Inmunoprecipitación de Cromatina , Documentación , Biblioteca de GenesRESUMEN
BACKGROUND: DNA methylation is involved in the epigenetic regulation of gene expression during developmental processes and is primarily established by the DNA methyltransferase 3A (DNMT3A) and 3B (DNMT3B). DNMT3A is one of the most frequently mutated genes in clonal hematopoiesis and leukemia, indicating that it plays a crucial role for hematopoietic differentiation. However, the functional relevance of Dnmt3a for hematopoietic differentiation and hematological malignancies has mostly been analyzed in mice, with the specific role for human hematopoiesis remaining elusive. In this study, we therefore investigated if DNMT3A is essential for hematopoietic differentiation of human induced pluripotent stem cells (iPSCs). RESULTS: We generated iPSC lines with knockout of either exon 2, 19, or 23 and analyzed the impact of different DNMT3A exon knockouts on directed differentiation toward mesenchymal and hematopoietic lineages. Exon 19-/- and 23-/- lines displayed an almost entire absence of de novo DNA methylation during mesenchymal and hematopoietic differentiation. Yet, differentiation efficiency was only slightly reduced in exon 19-/- and rather increased in exon 23-/- lines, while there was no significant impact on gene expression in hematopoietic progenitors (iHPCs). Notably, DNMT3A-/- iHPCs recapitulate some DNA methylation patterns of acute myeloid leukemia (AML) with DNMT3A mutations. Furthermore, multicolor genetic barcoding revealed growth advantage of exon 23-/- iHPCs in a syngeneic competitive differentiation assay. CONCLUSIONS: Our results demonstrate that iPSCs with homozygous knockout of different exons of DNMT3A remain capable of mesenchymal and hematopoietic differentiation-and exon 23-/- iHPCs even gained growth advantage-despite loss of almost the entire de novo DNA methylation. Partial recapitulation of DNA methylation patterns of AML with DNMT3A mutations by our DNMT3A knockout iHPCs indicates that our model system can help to elucidate mechanisms of clonal hematopoiesis.
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Células Madre Pluripotentes Inducidas , Leucemia Mieloide Aguda , Animales , Diferenciación Celular/genética , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN , ADN Metiltransferasa 3A , Epigénesis Genética , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , RatonesRESUMEN
We evaluated the suitability of 2% human platelet lysate medium (2%HPL) as a replacement for 2% fetal bovine serum medium (2%FBS) for the xeno-free organ culture of human donor corneas. A total of 32 corneas from 16 human donors were cultured in 2%FBS for 3 days (TP1), then evaluated using phase contrast microscopy (endothelial cell density (ECD) and cell morphology). Following an additional 25-day culture period (TP2) in either 2%FBS or 2%HPL, the pairs were again compared using microscopy; then stroma and Descemet membrane/endothelium (DmE) were processed for next generation sequencing (NGS). At TP2 the ECD was higher in the 2%HPL group (2179 ± 288 cells/mm2) compared to 2%FBS (2113 ± 331 cells/mm2; p = 0.03), and endothelial cell loss was lower (ECL HPL = -0.7% vs. FBS = -3.8%; p = 0.01). There were no significant differences in cell morphology between TP1 and 2, or between 2%HPL and 2%FBS. NGS showed the differential expression of 1644 genes in endothelial cells and 217 genes in stromal cells. It was found that 2%HPL led to the upregulation of cytoprotective, anti-inflammatory and anti-fibrotic genes (HMOX1, SERPINE1, ANGPTL4, LEFTY2, GADD45B, PLIN2, PTX3, GFRA1/2), and the downregulation of pro-inflammatory/apoptotic genes (e.g., CXCL14, SIK1B, PLK5, PPP2R3B, FABP5, MAL, GATA3). 2%HPL is a suitable xeno-free substitution for 2%FBS in human cornea organ culture, inducing less ECL and producing potentially beneficial alterations in gene expression.
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Técnicas de Cultivo de Célula , Células Endoteliales , Humanos , Proliferación Celular , Plaquetas/metabolismo , Células Cultivadas , Córnea , Medios de Cultivo/farmacología , Diferenciación Celular , Proteínas de Unión a Ácidos Grasos/metabolismoRESUMEN
RNA can directly bind to purine-rich DNA via Hoogsteen base pairing, forming a DNA:RNA triple helical structure that anchors the RNA to specific sequences and allows guiding of transcription regulators to distinct genomic loci. To unravel the prevalence of DNA:RNA triplexes in living cells, we have established a fast and cost-effective method that allows genome-wide mapping of DNA:RNA triplex interactions. In contrast to previous approaches applied for the identification of chromatin-associated RNAs, this method uses protein-free nucleic acids isolated from chromatin. High-throughput sequencing and computational analysis of DNA-associated RNA revealed a large set of RNAs which originate from non-coding and coding loci, including super-enhancers and repeat elements. Combined analysis of DNA-associated RNA and RNA-associated DNA identified genomic DNA:RNA triplex structures. The results suggest that triplex formation is a general mechanism of RNA-mediated target-site recognition, which has major impact on biological functions.
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ADN/química , ADN/aislamiento & purificación , Conformación de Ácido Nucleico , ARN/química , ARN/aislamiento & purificación , Emparejamiento Base , Secuencia de Bases , Sitios de Unión , Cromatina/genética , Cromatina/metabolismo , Mapeo Cromosómico , ADN/genética , Elementos de Facilitación Genéticos/genética , Células HeLa , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Purinas/química , Purinas/metabolismo , ARN/genética , ARN Largo no Codificante/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Reproducibilidad de los Resultados , Factores de Transcripción/metabolismoRESUMEN
Long non-coding RNAs (lncRNAs) can act as scaffolds that promote the interaction of proteins, RNA, and DNA. There is increasing evidence of sequence-specific interactions of lncRNAs with DNA via triple-helix (triplex) formation. This process allows lncRNAs to recruit protein complexes to specific genomic regions and regulate gene expression. Here we propose a computational method called Triplex Domain Finder (TDF) to detect triplexes and characterize DNA-binding domains and DNA targets statistically. Case studies showed that this approach can detect the known domains of lncRNAs Fendrr, HOTAIR and MEG3. Moreover, we validated a novel DNA-binding domain in MEG3 by a genome-wide sequencing method. We used TDF to perform a systematic analysis of the triplex-forming potential of lncRNAs relevant to human cardiac differentiation. We demonstrated that the lncRNA with the highest triplex-forming potential, GATA6-AS, forms triple helices in the promoter of genes relevant to cardiac development. Moreover, down-regulation of GATA6-AS impairs GATA6 expression and cardiac development. These data indicate the unique ability of our computational tool to identify novel triplex-forming lncRNAs and their target genes.
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Biología Computacional/métodos , ADN/metabolismo , ARN Largo no Codificante/química , ARN Largo no Codificante/metabolismo , Algoritmos , Secuencia de Bases , Sitios de Unión/genética , ADN/química , Expresión Génica , Humanos , Conformación de Ácido Nucleico , Unión Proteica , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: Age-associated DNA methylation changes provide a promising biomarker for the aging process. While genome-wide DNA methylation profiles enable robust age-predictors by integration of many age-associated CG dinucleotides (CpGs), there are various alternative approaches for targeted measurements at specific CpGs that better support standardized and cost-effective high-throughput analysis. RESULTS: In this study, we utilized 4647 Illumina BeadChip profiles of blood to select CpG sites that facilitate reliable age-predictions based on pyrosequencing. We demonstrate that the precision of DNA methylation measurements can be further increased with droplet digital PCR (ddPCR). In comparison, bisulfite barcoded amplicon sequencing (BBA-seq) gave slightly lower correlation between chronological age and DNA methylation at individual CpGs, while the age-predictions were overall relatively accurate. Furthermore, BBA-seq data revealed that the correlation of methylation levels with age at neighboring CpG sites follows a bell-shaped curve, often associated with a CTCF binding site. We demonstrate that within individual BBA-seq reads the DNA methylation at neighboring CpGs is not coherently modified, but reveals a stochastic pattern. Based on this, we have developed a new approach for epigenetic age predictions based on the binary sequel of methylated and non-methylated sites in individual reads, which reflects heterogeneity in epigenetic aging within a sample. CONCLUSION: Targeted DNA methylation analysis at few age-associated CpGs by pyrosequencing, BBA-seq, and particularly ddPCR enables high precision of epigenetic age-predictions. Furthermore, we demonstrate that the stochastic evolution of age-associated DNA methylation patterns in BBA-seq data enables epigenetic clocks for individual DNA strands.
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Envejecimiento/genética , Metilación de ADN , Epigénesis Genética/fisiología , Epigenómica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento , Sangre/metabolismo , Marcadores Genéticos , HumanosRESUMEN
There is a growing perception that long non-coding RNAs (lncRNAs) modulate cellular function. In this study, we analyzed the role of the lncRNA HOTAIR in mesenchymal stem cells (MSCs) with particular focus on senescence-associated changes in gene expression and DNA-methylation (DNAm). HOTAIR binding sites were enriched at genomic regions that become hypermethylated with increasing cell culture passage. Overexpression and knockdown of HOTAIR inhibited or stimulated adipogenic differentiation of MSCs, respectively. Modification of HOTAIR expression evoked only very moderate effects on gene expression, particularly of polycomb group target genes. Furthermore, overexpression and knockdown of HOTAIR resulted in DNAm changes at HOTAIR binding sites. Five potential triple helix forming domains were predicted within the HOTAIR sequence based on reverse Hoogsteen hydrogen bonds. Notably, the predicted triple helix target sites for these HOTAIR domains were also enriched in differentially expressed genes and close to DNAm changes upon modulation of HOTAIR Electrophoretic mobility shift assays provided further evidence that HOTAIR domains form RNA-DNA-DNA triplexes with predicted target sites. Our results demonstrate that HOTAIR impacts on differentiation of MSCs and that it is associated with senescence-associated DNAm. Targeting of epigenetic modifiers to relevant loci in the genome may involve triple helix formation with HOTAIR.
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Células Madre Mesenquimatosas/fisiología , ARN Largo no Codificante/fisiología , Secuencia de Bases , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Senescencia Celular , Metilación de ADN , Epigénesis Genética , Expresión Génica , Humanos , Conformación de Ácido Nucleico , Unión Proteica , ARN Largo no Codificante/químicaRESUMEN
OBJECTIVE: The generation of acinar and ductal cells from human pluripotent stem cells (PSCs) is a poorly studied process, although various diseases arise from this compartment. DESIGN: We designed a straightforward approach to direct human PSCs towards pancreatic organoids resembling acinar and ductal progeny. RESULTS: Extensive phenotyping of the organoids not only shows the appropriate marker profile but also ultrastructural, global gene expression and functional hallmarks of the human pancreas in the dish. Upon orthotopic transplantation into immunodeficient mice, these organoids form normal pancreatic ducts and acinar tissue resembling fetal human pancreas without evidence of tumour formation or transformation. Finally, we implemented this unique phenotyping tool as a model to study the pancreatic facets of cystic fibrosis (CF). For the first time, we provide evidence that in vitro, but also in our xenograft transplantation assay, pancreatic commitment occurs generally unhindered in CF. Importantly, cystic fibrosis transmembrane conductance regulator (CFTR) activation in mutated pancreatic organoids not only mirrors the CF phenotype in functional assays but also at a global expression level. We also conducted a scalable proof-of-concept screen in CF pancreatic organoids using a set of CFTR correctors and activators, and established an mRNA-mediated gene therapy approach in CF organoids. CONCLUSIONS: Taken together, our platform provides novel opportunities to model pancreatic disease and development, screen for disease-rescuing agents and to test therapeutic procedures.
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Fibrosis Quística/terapia , Modelos Animales de Enfermedad , Organoides/crecimiento & desarrollo , Organoides/trasplante , Páncreas/citología , ARN Mensajero/uso terapéutico , Células Acinares/citología , Animales , Fibrosis Quística/genética , Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/genética , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Perfilación de la Expresión Génica , Terapia Genética , Humanos , Ratones , Organoides/citología , Organoides/metabolismo , Páncreas/crecimiento & desarrollo , Páncreas/metabolismo , Conductos Pancreáticos/citología , Fenotipo , Células Madre PluripotentesRESUMEN
Visual processing depends on sensitive and balanced synaptic neurotransmission. Extracellular matrix proteins in the environment of cells are key modulators in synaptogenesis and synaptic plasticity. In the present study, we provide evidence that the combined loss of the four extracellular matrix components, brevican, neurocan, tenascin-C, and tenascin-R, in quadruple knockout mice leads to severe retinal dysfunction and diminished visual motion processing in vivo. Remarkably, impaired visual motion processing was accompanied by a developmental loss of cholinergic direction-selective starburst amacrine cells. Additionally, we noted imbalance of inhibitory and excitatory synaptic signaling in the quadruple knockout retina. Collectively, the study offers insights into the functional importance of four key extracellular matrix proteins for retinal function, visual motion processing, and synaptic signaling.
RESUMEN
PURPOSE: We evaluated the suitability of 2% human platelet lysate (2%HPL) to replace 2% fetal bovine serum containing medium (2%FBS) for the xeno-free organ culture of human donor corneas. METHODS: 32 human corneas unsuitable for transplantation from 16 human donors (age 69.3±15.7years) were collected 38.5±17.1 hours after death. They were first cultured in 2%FBS containing medium for 3 days (time point TP1), then evaluated by phase contrast microscopy (endothelial cell density (ECD) and cell morphology. Following an additional 25-days culture period (time point TP2) in either 2%FBS or 2%HPL medium the pairs were again compared by phase contrast microscopy (ECD and morphology), stroma and Descemet membrane/endothelium (DmE) were processed for next generation sequencing (NGS). RESULTS: ECD did not differ between the 2%HPL and 2%FBS group at TP1 (p=0.87). At TP2 the ECD was higher in the 2%HPL group (2179±288cells/mm2) compared to 2%FBS (2113±331cells/mm2; p=0.03), and endothelial cell loss was lower (ECL hPL=-0.7% vs. FBS=-3.8%; p=0.01). There were no significant differences in cell morphology, neither between TP1 and 2 nor between 2%HPL and 2%FBS. NGS showed the differential expression of 1644 genes in endothelial and 217 genes in stromal cells. 2%HPL led to the upregulation of cytoprotective, anti-inflammatory and anti-fibrotic genes (e.g. HMOX1, SERPINE1, ANGPTL4, LEFTY2, GADD45B, PLIN2, PTX3, GFRA1/2) and the downregulation of pro-inflammatory/apoptotic genes (e.g. CXCL14, SIK1B, PLK5, PPP2R3B, SLURP1, FABP5, MAL, GATA3). CONCLUSION: 2%HPL is a suitable xeno-free substitution for 2%FBS in human cornea organ culture, inducing less ECL and potentially beneficial alterations in gene expression.
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Córnea , Donantes de Tejidos , Humanos , Persona de Mediana Edad , Anciano , Anciano de 80 o más Años , Regulación hacia Abajo , Células Endoteliales , Secuenciación de Nucleótidos de Alto Rendimiento , Antígenos Ly , Activador de Plasminógeno de Tipo Uroquinasa , Proteínas de Unión a Ácidos GrasosRESUMEN
Transcription factors play a determining role in lineage commitment and cell differentiation. Interferon regulatory factor 8 (IRF8) is a lineage determining transcription factor in hematopoiesis and master regulator of dendritic cells (DC), an important immune cell for immunity and tolerance. IRF8 is prominently upregulated in DC development by autoactivation and controls both DC differentiation and function. However, it is unclear how Irf8 autoactivation is controlled and eventually limited. Here, we identified a novel long non-coding RNA transcribed from the +32 kb enhancer downstream of Irf8 transcription start site and expressed specifically in mouse plasmacytoid DC (pDC), referred to as lncIrf8. The lncIrf8 locus interacts with the lrf8 promoter and shows differential epigenetic signatures in pDC versus classical DC type 1 (cDC1). Interestingly, a sequence element of the lncIrf8 promoter, but not lncIrf8 itself, is crucial for mouse pDC and cDC1 differentiation, and this sequence element confers feedback inhibition of Irf8 expression. Taken together, in DC development Irf8 autoactivation is first initiated by flanking enhancers and then second controlled by feedback inhibition through the lncIrf8 promoter element in the +32 kb enhancer. Our work reveals a previously unrecognized negative feedback loop of Irf8 that orchestrates its own expression and thereby controls DC differentiation.
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ARN Largo no Codificante , Ratones , Animales , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Retroalimentación , Factores Reguladores del Interferón/metabolismo , Diferenciación Celular/fisiología , Elementos de Facilitación Genéticos , Células DendríticasRESUMEN
Long noncoding RNAs (lncRNAs) regulate the expression of protein-coding genes and have been shown to play important roles in inflammatory skin diseases. However, we still have limited understanding of the functional impact of lncRNAs in skin, partly due to their tissue specificity and lower expression levels compared with protein-coding genes. We compiled a comprehensive list of 18,517 lncRNAs from different sources and studied their expression profiles in 834 RNA-Seq samples from multiple inflammatory skin conditions and cytokine-stimulated keratinocytes. Applying a balanced random forest to predict involvement in biological functions, we achieved a median AUROC of 0.79 in 10-fold cross-validation, identifying significant DNA binding domains (DBDs) for 39 lncRNAs. G18244, a skin-expressing lncRNA predicted for IL-4/IL-13 signaling in keratinocytes, was highly correlated in expression with F13A1, a protein-coding gene involved in macrophage regulation, and we further identified a significant DBD in F13A1 for G18244. Reflecting clinical implications, AC090198.1 (predicted for IL-17 pathway) and AC005332.6 (predicted for IFN-γ pathway) had significant negative correlation with the SCORAD metric for atopic dermatitis. We also utilized single-cell RNA and spatial sequencing data to validate cell type specificity. Our research demonstrates lncRNAs have important immunological roles and can help prioritize their impact on inflammatory skin diseases.
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ARN Largo no Codificante , Enfermedades de la Piel , Humanos , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Piel/metabolismo , Enfermedades de la Piel/genéticaRESUMEN
Assessment of measurable residual disease (MRD) upon treatment of acute myeloid leukemia (AML) remains challenging. It is usually addressed by highly sensitive PCR- or sequencing-based screening of specific mutations, or by multiparametric flow cytometry. However, not all patients have suitable mutations and heterogeneity of surface markers hampers standardization in clinical routine. In this study, we propose an alternative approach to estimate MRD based on AML-associated DNA methylation (DNAm) patterns. We identified four CG dinucleotides (CpGs) that commonly reveal aberrant DNAm in AML and their combination could reliably discern healthy and AML samples. Interestingly, bisulfite amplicon sequencing demonstrated that aberrant DNAm patterns were symmetric on both alleles, indicating that there is epigenetic crosstalk between homologous chromosomes. We trained shallow-learning and deep-learning algorithms to identify anomalous DNAm patterns. The method was then tested on follow-up samples with and without MRD. Notably, even samples that were classified as MRD negative often revealed higher anomaly ratios than healthy controls, which may reflect clonal hematopoiesis. Our results demonstrate that targeted DNAm analysis facilitates reliable discrimination of malignant and healthy samples. However, since healthy samples also comprise few abnormal-classified DNAm reads the approach does not yet reliably discriminate MRD positive and negative samples.
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Biomarcadores de Tumor/genética , Metilación de ADN , Regulación Leucémica de la Expresión Génica , Leucemia Mieloide Aguda/patología , Mutación , Recurrencia Local de Neoplasia/patología , Neoplasia Residual/patología , Humanos , Leucemia Mieloide Aguda/genética , Recurrencia Local de Neoplasia/genética , Neoplasia Residual/genética , Pronóstico , Tasa de SupervivenciaRESUMEN
Changes in gene expression programs are intimately linked to cell fate decisions. Post-translational modifications of core histones contribute to control gene expression. Methylation of lysine 4 of histone H3 (H3K4) correlates with active promoters and gene transcription. This modification is catalyzed by KMT2 methyltransferases, which require interaction with 4 core subunits, WDR5, RBBP5, ASH2L and DPY30, for catalytic activity. Ash2l is necessary for organismal development and for tissue homeostasis. In mouse embryo fibroblasts (MEFs), Ash2l loss results in gene repression, provoking a senescence phenotype. We now find that upon knockout of Ash2l both H3K4 mono- and tri-methylation (H3K4me1 and me3, respectively) were deregulated. In particular, loss of H3K4me3 at promoters correlated with gene repression, especially at CpG island promoters. Ash2l loss resulted in increased loading of histone H3 and reduced chromatin accessibility at promoters, accompanied by an increase of repressing and a decrease of activating histone marks. Moreover, we observed altered binding of CTCF upon Ash2l loss. Lost and gained binding was noticed at promoter-associated and intergenic sites, respectively. Thus, Ash2l loss and reduction of H3K4me3 correlate with altered chromatin accessibility and transcription factor binding. These findings contribute to a more detailed understanding of mechanistic consequences of H3K4me3 loss and associated repression of gene transcription and thus of the observed cellular consequences.
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Cromatina , Histonas , Animales , Ratones , Cromatina/genética , Proteínas de Unión al ADN/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismoRESUMEN
DNA:DNA:RNA triplexes that are formed through Hoogsteen base-pairing of the RNA in the major groove of the DNA duplex have been observed in vitro, but the extent to which these interactions occur in cells and how they impact cellular functions remains elusive. Using a combination of bioinformatic techniques, RNA/DNA pulldown and biophysical studies, we set out to identify functionally important DNA:DNA:RNA triplex-forming long non-coding RNAs (lncRNA) in human endothelial cells. The lncRNA HIF1α-AS1 was retrieved as a top hit. Endogenous HIF1α-AS1 reduces the expression of numerous genes, including EPH Receptor A2 and Adrenomedullin through DNA:DNA:RNA triplex formation by acting as an adapter for the repressive human silencing hub complex (HUSH). Moreover, the oxygen-sensitive HIF1α-AS1 is down-regulated in pulmonary hypertension and loss-of-function approaches not only result in gene de-repression but also enhance angiogenic capacity. As exemplified here with HIF1α-AS1, DNA:DNA:RNA triplex formation is a functionally important mechanism of trans-acting gene expression control.
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ARN Largo no Codificante , Humanos , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Células Endoteliales/metabolismo , ADN/genética , ADN/metabolismo , Emparejamiento Base , Oligonucleótidos , Regulación Neoplásica de la Expresión GénicaRESUMEN
Culture expansion of primary cells evokes highly reproducible DNA methylation (DNAm) changes. We have identified CG dinucleotides (CpGs) that become continuously hyper- or hypomethylated during long-term culture of mesenchymal stem cells (MSCs) and other cell types. Bisulfite barcoded amplicon sequencing (BBA-seq) demonstrated that DNAm patterns of neighboring CpGs become more complex without evidence of continuous pattern development and without association to oligoclonal subpopulations. Circularized chromatin conformation capture (4C) revealed reproducible changes in nuclear organization between early and late passages, while there was no enriched interaction with other genomic regions that also harbor culture-associated DNAm changes. Chromatin immunoprecipitation of CTCF did not show significant differences during long-term culture of MSCs, however culture-associated hypermethylation was enriched at CTCF binding sites and hypomethylated CpGs were devoid of CTCF. Taken together, our results support the notion that DNAm changes during culture-expansion are not directly regulated by a targeted mechanism but rather resemble epigenetic drift.
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Factor de Unión a CCCTC/genética , Cromatina/metabolismo , Metilación de ADN , Epigénesis Genética , Flujo Genético , Células Madre Mesenquimatosas/metabolismo , Envejecimiento , Células Cultivadas , Cromatina/genética , Islas de CpG , Humanos , Técnicas In Vitro , Células Madre Mesenquimatosas/citologíaRESUMEN
BACKGROUND: Dyskeratosis congenita (DKC) and idiopathic aplastic anemia (AA) are bone marrow failure syndromes that share characteristics of premature aging with severe telomere attrition. Aging is also reflected by DNA methylation changes, which can be utilized to predict donor age. There is evidence that such epigenetic age predictions are accelerated in premature aging syndromes, but it is yet unclear how this is related to telomere length. DNA methylation analysis may support diagnosis of DKC and AA, which still remains a challenge for these rare diseases. RESULTS: In this study, we analyzed blood samples of 70 AA and 18 DKC patients to demonstrate that their epigenetic age predictions are overall increased, albeit not directly correlated with telomere length. Aberrant DNA methylation was observed in the gene PRDM8 in DKC and AA as well as in other diseases with premature aging phenotype, such as Down syndrome and Hutchinson-Gilford-Progeria syndrome. Aberrant DNA methylation patterns were particularly found within subsets of cell populations in DKC and AA samples as measured with barcoded bisulfite amplicon sequencing (BBA-seq). To gain insight into the functional relevance of PRDM8, we used CRISPR/Cas9 technology to generate induced pluripotent stem cells (iPSCs) with heterozygous and homozygous knockout. Loss of PRDM8 impaired hematopoietic and neuronal differentiation of iPSCs, even in the heterozygous knockout clone, but it did not impact on epigenetic age. CONCLUSION: Taken together, our results demonstrate that epigenetic aging is accelerated in DKC and AA, independent from telomere attrition. Furthermore, aberrant DNA methylation in PRDM8 provides another biomarker for bone marrow failure syndromes and modulation of this gene in cellular subsets may be related to the hematopoietic and neuronal phenotypes observed in premature aging syndromes.
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Anemia Aplásica/sangre , Anemia Aplásica/genética , Metilación de ADN/genética , Proteínas de Unión al ADN/sangre , Proteínas de Unión al ADN/genética , Disqueratosis Congénita/sangre , Disqueratosis Congénita/genética , Histona Metiltransferasas/sangre , Histona Metiltransferasas/genética , Femenino , Células Madre Hematopoyéticas/metabolismo , Humanos , Masculino , Neuronas/metabolismo , Fenotipo , Telómero/metabolismoRESUMEN
Long non-coding RNAs (lncRNAs) contribute to cardiac (patho)physiology. Aging is the major risk factor for cardiovascular disease with cardiomyocyte apoptosis as one underlying cause. Here, we report the identification of the aging-regulated lncRNA Sarrah (ENSMUST00000140003) that is anti-apoptotic in cardiomyocytes. Importantly, loss of SARRAH (OXCT1-AS1) in human engineered heart tissue results in impaired contractile force development. SARRAH directly binds to the promoters of genes downregulated after SARRAH silencing via RNA-DNA triple helix formation and cardiomyocytes lacking the triple helix forming domain of Sarrah show an increase in apoptosis. One of the direct SARRAH targets is NRF2, and restoration of NRF2 levels after SARRAH silencing partially rescues the reduction in cell viability. Overexpression of Sarrah in mice shows better recovery of cardiac contractile function after AMI compared to control mice. In summary, we identified the anti-apoptotic evolutionary conserved lncRNA Sarrah, which is downregulated by aging, as a regulator of cardiomyocyte survival.
Asunto(s)
Apoptosis , Infarto del Miocardio/genética , Miocitos Cardíacos/citología , ARN Largo no Codificante/genética , Envejecimiento , Animales , Proteínas Portadoras/genética , Supervivencia Celular , Coenzima A Transferasas/genética , Modelos Animales de Enfermedad , Silenciador del Gen , Humanos , Proteínas con Dominio LIM/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Factor 2 Relacionado con NF-E2/genética , ARN sin Sentido/genética , ARN Interferente Pequeño/genética , Factores de Transcripción p300-CBP/genéticaRESUMEN
Post-translational modifications of core histones participate in controlling the expression of genes. Methylation of lysine 4 of histone H3 (H3K4), together with acetylation of H3K27, is closely associated with open chromatin and gene transcription. H3K4 methylation is catalyzed by KMT2 lysine methyltransferases that include the mixed-lineage leukemia 1-4 (MLL1-4) and SET1A and B enzymes. For efficient catalysis, all six require a core complex of four proteins, WDR5, RBBP5, ASH2L, and DPY30. We report that targeted disruption of Ash2l in the murine hematopoietic system results in the death of the mice due to a rapid loss of mature hematopoietic cells. However, lin-Sca1+Kit+ (LSK) cells, which are highly enriched in hematopoietic stem and multi-potent progenitor cells, accumulated in the bone marrow. The loss of Ash2l resulted in global reduction of H3K4 methylation and deregulated gene expression, including down-regulation of many mitosis-associated genes. As a consequence, LSK cells accumulated in the G2-phase of the cell cycle and were unable to proliferate and differentiate. In conclusion, Ash2l is essential for balanced gene expression and for hematopoietic stem and multi-potent progenitor cell physiology.