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In patients with proximal hypospadias, often no genetic cause is identified despite extensive genetic testing. Many genes involved in sex development encode transcription factors with strict timing and dosing of the gene products. We hypothesised that there might be recurrent differences in DNA methylation in boys with hypospadias and that these might differ between patients born small versus appropriate for gestational age. Genome-wide Methylated DNA sequencing (MeD-seq) was performed on 32bp LpnPI restriction enzyme fragments after RE-digestion in leucocytes from 16 XY boys with unexplained proximal hypospadias, one with an unexplained XX testicular disorder/difference of sex development (DSD) and twelve, healthy, sex- and age-matched controls. Five of seven differentially methylated regions (DMRs) between patients and XY controls were in the Long Intergenic Non-Protein Coding RNA 665 (LINC00665; CpG24525). Three patients showed hypermethylation of MAP3K1. Finally, no DMRs in XX testicular DSD associated genes were identified in the XX boy versus XX controls. In conclusion, we observed no recognizable epigenetic signature in 16 boys with XY proximal hypospadias and no difference between children born small versus appropriate for gestational age. Comparison to previous methylation studies in individuals with hypospadias did not show consistent findings, possibly due to the use of different inclusion criteria, tissues and methods.
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Metilación de ADN , Hipospadias , Humanos , Masculino , Hipospadias/genética , Proyectos Piloto , Epigénesis Genética , Islas de CpG , ARN Largo no Codificante/genética , Niño , Preescolar , Lactante , Estudio de Asociación del Genoma Completo , Estudios de Casos y ControlesRESUMEN
BACKGROUND: As new treatment options for patients with higher-risk myelodysplastic syndromes are emerging, identification of prognostic markers for hypomethylating agent (HMA) treatment and understanding mechanisms of their delayed and short-term responses are essential. Early fetal hemoglobin (HbF) induction has been suggested as a prognostic indicator for decitabine-treated patients. Although epigenetic mechanisms are assumed, responding patients' epigenomes have not been thoroughly examined. We aimed to clarify HbF kinetics and prognostic value for azacytidine treated patients, as well as the epigenetic landscape that might influence HbF re-expression and its clinical relevance. RESULTS: Serial HbF measurements by high-performance liquid chromatography (n = 20) showed induction of HbF only among responders (p = 0.030). Moreover, HbF increase immediately after the first azacytidine cycle demonstrated prognostic value for progression-free survival (PFS) (p = 0.032, HR = 0.19, CI 0.24-1.63). Changes in methylation patterns were revealed with methylated DNA genome-wide sequencing analysis (n = 7) for FOG-1, RCOR-1, ZBTB7A and genes of the NuRD-complex components. Targeted pyrosequencing methodology (n = 28) revealed a strong inverse correlation between the degree of γ-globin gene (HBG2) promoter methylation and baseline HbF levels (p = 0.003, rs = - 0.663). A potential epigenetic mechanism of HbF re-expression in azacytidine responders was enlightened by targeted methylation analysis, through hypomethylation of site -53 of HBG2 promoter (p = 0.039, rs = - 0.504), which corresponds to MBD2-NuRD binding site, and to hypermethylation of the CpG326 island of ZBTB7A (p = 0.05, rs = 0.482), a known HbF repressor. These changes were associated to blast cell clearance (pHBG2 = 0.011, rs = 0.480/pZBTB7A = 0.026, rs = 0.427) and showed prognostic value for PFS (pZBTB7A = 0.037, HR = 1.14, CI 0.34-3.8). CONCLUSIONS: Early HbF induction is featured as an accessible prognostic indicator for HMA treatment and the proposed potential epigenetic mechanism of HbF re-expression in azacytidine responders includes hypomethylation of the γ-globin gene promoter region and hypermethylation of the CpG326 island of ZBTB7A. The association of these methylation patterns with blast clearance and their prognostic value for PFS paves the way to discuss in-depth azacytidine epigenetic mechanism of action.
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Azacitidina , Metilación de ADN , Epigénesis Genética , Hemoglobina Fetal , Síndromes Mielodisplásicos , Humanos , Hemoglobina Fetal/genética , Metilación de ADN/efectos de los fármacos , Metilación de ADN/genética , Azacitidina/farmacología , Femenino , Masculino , Anciano , Epigénesis Genética/efectos de los fármacos , Epigénesis Genética/genética , Persona de Mediana Edad , Síndromes Mielodisplásicos/tratamiento farmacológico , Síndromes Mielodisplásicos/genética , Pronóstico , Anciano de 80 o más Años , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Antimetabolitos Antineoplásicos/uso terapéutico , Antimetabolitos Antineoplásicos/farmacologíaRESUMEN
BACKGROUND: Patients with advanced-stage epithelial ovarian cancer (EOC) receive treatment with a poly-ADP ribose-polymerase (PARP) inhibitor (PARPi) as maintenance therapy after surgery and chemotherapy. Unfortunately, many patients experience disease progression because of acquired therapy resistance. This study aims to characterize epigenetic and genomic changes in cell-free DNA (cfDNA) associated with PARPi resistance. MATERIALS AND METHODS: Blood was taken from 31 EOC patients receiving PARPi therapy before treatment and at disease progression during/after treatment. Resistance was defined as disease progression within 6 months after starting PARPi and was seen in fifteen patients, while sixteen patients responded for 6 to 42 months. Blood cfDNA was evaluated via Modified Fast Aneuploidy Screening Test-Sequencing System (mFast-SeqS to detect aneuploidy, via Methylated DNA Sequencing (MeD-seq) to find differentially methylated regions (DMRs), and via shallow whole-genome and -exome sequencing (shWGS, exome-seq) to define tumor fractions and mutational signatures. RESULTS: Aneuploid cfDNA was undetectable pre-treatment but observed in six patients post-treatment, in five resistant and one responding patient. Post-treatment ichorCNA analyses demonstrated in shWGS and exome-seq higher median tumor fractions in resistant (7% and 9%) than in sensitive patients (7% and 5%). SigMiner analyses detected predominantly mutational signatures linked to mismatch repair and chemotherapy. DeSeq2 analyses of MeD-seq data revealed three methylation signatures and more tumor-specific DMRs in resistant than in responding patients in both pre- and post-treatment samples (274 vs. 30 DMRs, 190 vs. 57 DMRs, Χ2-test p < 0.001). CONCLUSION: Our genome-wide Next-Generation Sequencing (NGS) analyses in PARPi-resistant patients identified epigenetic differences in blood before treatment, whereas genomic alterations were more frequently observed after progression. The epigenetic differences at baseline are especially interesting for further exploration as putative predictive biomarkers for PARPi resistance.
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Carcinoma Epitelial de Ovario , Metilación de ADN , Resistencia a Antineoplásicos , Epigénesis Genética , Neoplasias Ováricas , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Humanos , Femenino , Resistencia a Antineoplásicos/genética , Persona de Mediana Edad , Neoplasias Ováricas/genética , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/patología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Anciano , Carcinoma Epitelial de Ovario/genética , Carcinoma Epitelial de Ovario/tratamiento farmacológico , Carcinoma Epitelial de Ovario/patología , Adulto , Aneuploidia , Genómica/métodosRESUMEN
XIST (X-inactive specific transcript) long noncoding RNA (lncRNA) is responsible for X chromosome inactivation (XCI) in placental mammals, yet it accumulates on both X chromosomes in human female preimplantation embryos without triggering X chromosome silencing. The XACT (X-active coating transcript) lncRNA coaccumulates with XIST on active X chromosomes and may antagonize XIST function. Here, we used human embryonic stem cells in a naive state of pluripotency to assess the function of XIST and XACT in shaping the X chromosome chromatin and transcriptional landscapes during preimplantation development. We show that XIST triggers the deposition of polycomb-mediated repressive histone modifications and dampens the transcription of most X-linked genes in a SPEN-dependent manner, while XACT deficiency does not significantly affect XIST activity or X-linked gene expression. Our study demonstrates that XIST is functional before XCI, confirms the existence of a transient process of X chromosome dosage compensation and reveals that XCI and dampening rely on the same set of factors.
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OBJECTIVE: Methylation of plasma cell-free DNA (cfDNA) has potential as a marker of brain damage in neurodegenerative diseases such as frontotemporal dementia (FTD). Here, we study methylation of cfDNA in presymptomatic and symptomatic carriers of genetic FTD pathogenic variants, next to healthy controls. METHODS: cfDNA was isolated from cross-sectional plasma of 10 presymptomatic carriers (4 C9orf72, 4 GRN, and 2 MAPT), 10 symptomatic carriers (4 C9orf72, 4 GRN, and 2 MAPT), and 9 healthy controls. Genome-wide methylation of cfDNA was determined using a high-resolution sequencing technique (MeD-seq). Cumulative scores based on the identified differentially methylated regions (DMRs) were estimated for presymptomatic carriers (vs. controls and symptomatic carriers), and reevaluated in a validation cohort (8 presymptomatic: 3 C9orf72, 3 GRN, and 2 MAPT; 26 symptomatic: 7 C9orf72, 6 GRN, 12 MAPT, and 1 TARDBP; 13 noncarriers from genetic FTD families). RESULTS: Presymptomatic carriers showed a distinctive methylation profile compared to healthy controls and symptomatic carriers. Cumulative DMR scores in presymptomatic carriers enabled to significantly differentiate presymptomatic carriers from healthy controls (p < 0.001) and symptomatic carriers (p < 0.001). In the validation cohort, these scores differentiated presymptomatic carriers from symptomatic carriers (p ≤ 0.007) only. Transcription-start-site methylation in presymptomatic carriers, generally associated with gene downregulation, was enriched for genes involved in ubiquitin-dependent processes, while gene body methylation, generally associated with gene upregulation, was enriched for genes involved in neuronal cell processes. INTERPRETATION: A distinctive methylation profile of cfDNA characterizes the presymptomatic stage of genetic FTD, and could reflect neuronal death in this stage.
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Ácidos Nucleicos Libres de Células , Demencia Frontotemporal , Enfermedad de Pick , Humanos , Demencia Frontotemporal/patología , Proteína C9orf72/genética , Estudios Transversales , Metilación de ADN , Mutación , Enfermedad de Pick/genética , Ácidos Nucleicos Libres de Células/genéticaRESUMEN
Aberrant DNA methylation changes have been reported to be associated with carcinogenesis in cirrhotic HCC, but DNA methylation patterns for these non-cirrhotic HCC cases were not examined. Therefore, we sought to investigate DNA methylation changes on non-cirrhotic HCC using reported promising DNA methylation markers (DMMs), including HOXA1, CLEC11A, AK055957, and TSPYL5, on 146 liver tissues using quantitative methylation-specific PCR and methylated DNA sequencing. We observed a high frequency of aberrant methylation changes in the four DMMs through both techniques in non-cirrhotic HCC compared to cirrhosis, hepatitis, and benign lesions (p < 0.05), suggesting that hypermethylation of these DMMs is specific to non-cirrhotic HCC development. Also, the combination of the four DMMs exhibited 78% sensitivity at 80% specificity with an AUC of 0.85 in discriminating non-cirrhotic HCC from hepatitis and benign lesions. In addition, HOXA1 showed a higher aberrant methylation percentage in non-cirrhotic HCC compared to cirrhotic HCC (43.3% versus 13.3%, p = 0.039), which was confirmed using multivariate linear regression (p < 0.05). In summary, we identified aberrant hypermethylation changes in HOXA1, CLEC11A, AK055957, and TSPYL5 in non-cirrhotic HCC tissues compared to cirrhosis, hepatitis, and benign lesions, providing information that could be used as potentially detectable biomarkers for these unusual HCC cases in clinical practice.
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Colorectal cancer (CRC) colonoscopic surveillance is effective but burdensome. Circulating tumor DNA (ctDNA) analysis has emerged as a promising, minimally invasive tool for disease detection and management. Here, we assessed which ctDNA assay might be most suitable for a ctDNA-based CRC screening/surveillance blood test. In this prospective, proof-of-concept study, patients with colonoscopies for Lynch surveillance or the National Colorectal Cancer screening program were included between 7 July 2019 and 3 June 2022. Blood was drawn, and if advanced neoplasia (adenoma with villous component, high-grade dysplasia, ≥10 mm, or CRC) was detected, it was analyzed for chromosomal copy number variations, single nucleotide variants, and genome-wide methylation (MeD-seq). Outcomes were compared with corresponding patients' tissues and the MeD-seq results of healthy blood donors. Two Lynch carriers and eight screening program patients were included: five with CRC and five with advanced adenomas. cfDNA showed copy number variations and single nucleotide variants in one patient with CRC and liver metastases. Eight patients analyzed with MeD-seq showed clustering of Lynch-associated and sporadic microsatellite instable lesions separate from microsatellite stable lesions, as did healthy blood donors. In conclusion, whereas copy number changes and single nucleotide variants were only detected in one patient, cfDNA methylation profiles could discriminate all microsatellite instable advanced neoplasia, rendering this tool particularly promising for LS surveillance. Larger studies are warranted to validate these findings.
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Myelodysplastic syndromes (MDS) consist of a group of hematological malignancies characterized by ineffective hematopoiesis, cytogenetic abnormalities, and often a high risk of transformation to acute myeloid leukemia (AML). So far, there have been only a very limited number of studies assessing the epigenetics component contributing to the pathophysiology of these disorders, but not a single study assessing this at a genome-wide level. Here, we implemented a generic high throughput epigenomics approach, using methylated DNA sequencing (MeD-seq) of LpnPI digested fragments to identify potential epigenomic targets associated with MDS subtypes. Our results highlighted that PCDHG and ZNF gene families harbor potential epigenomic targets, which have been shown to be differentially methylated in a variety of comparisons between different MDS subtypes. Specifically, CpG islands, transcription start sites and post-transcriptional start sites within ZNF124, ZNF497 and PCDHG family are differentially methylated with fold change above 3,5. Overall, these findings highlight important aspects of the epigenomic component of MDS syndromes pathogenesis and the pharmacoepigenomic basis to the hypomethylating agents drug treatment response, while this generic high throughput whole epigenome sequencing approach could be readily implemented to other genetic diseases with a strong epigenetic component.
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Metilación de ADN , Síndromes Mielodisplásicos , Humanos , Metilación de ADN/genética , Epigenómica , Epigénesis Genética , Síndromes Mielodisplásicos/tratamiento farmacológico , Síndromes Mielodisplásicos/genética , Síndromes Mielodisplásicos/patología , Progresión de la Enfermedad , Islas de CpG/genética , Proteínas de Unión al ADN/genéticaRESUMEN
According to the current guidelines, watchful waiting (WW) is a feasible option for patients with good or intermediate prognosis renal-cell carcinoma (RCC). However, some patients rapidly progress during WW, requiring the initiation of treatment. Here, we explore whether we can identify those patients using circulating cell-free DNA (cfDNA) methylation. We first defined a panel of RCC-specific circulating methylation markers by intersecting differentially methylated regions from a publicly available dataset with known RCC methylation markers from the literature. The resulting RCC-specific methylation marker panel of 22 markers was subsequently evaluated for an association with rapid progression by methylated DNA sequencing (MeD-seq) in serum from 10 HBDs and 34 RCC patients with a good or intermediate prognosis starting WW in the IMPACT-RCC study. Patients with an elevated RCC-specific methylation score compared to HBDs had a shorter progression-free survival (PFS, p = 0.018), but not a shorter WW-time (p = 0.15). Cox proportional hazards regression showed that only the International Metastatic Renal Cell Carcinoma Database Consortium (IMDC) criteria were significantly associated with WW time (HR 2.01, p = 0.01), whereas only our RCC-specific methylation score (HR 4.45, p = 0.02) was significantly associated with PFS. The results of this study suggest that cfDNA methylation is predictive of PFS but not WW.
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Cell state changes in development and disease are controlled by gene regulatory networks, the dynamics of which are difficult to track in real time. In this study, we used an inducible DCM-RNA polymerase subunit b fusion protein which labels active genes and enhancers with a bacterial methylation mark that does not affect gene transcription and is propagated in S-phase. This DCM-RNA polymerase fusion protein enables transcribed genes and active enhancers to be tagged and then examined at later stages of development or differentiation. We apply this DCM-time machine (DCM-TM) technology to study intestinal homeostasis, revealing rapid and coordinated activation of enhancers and nearby genes during enterocyte differentiation. We provide new insights in absorptive-secretory lineage decision-making in intestinal stem cell (ISC) differentiation and show that ISCs retain a unique chromatin landscape required to maintain ISC identity and delineate future expression of differentiation-associated genes. DCM-TM has wide applicability in tracking cell states, providing new insights in the regulatory networks underlying cell state changes.
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Cromatina , Transcriptoma , Linaje de la Célula/genética , Transcriptoma/genética , Estudios Retrospectivos , Diferenciación Celular/genética , Cromatina/genética , ARN Polimerasas Dirigidas por ADN/metabolismo , Elementos de Facilitación Genéticos/genéticaRESUMEN
Induced pluripotent stem cell (iPSC)-derived kidney organoids are a potential tool for the regeneration of kidney tissue. They represent an early stage of nephrogenesis and have been shown to successfsully vascularize and mature further in vivo. However, there are concerns regarding the long-term safety and stability of iPSC derivatives. Specifically, the potential for tumorigenesis may impede the road to clinical application. To study safety and stability of kidney organoids, we analyzed their potential for malignant transformation in a teratoma assay and following long-term subcutaneous implantation in an immune-deficient mouse model. We did not detect fully functional residual iPSCs in the kidney organoids as analyzed by gene expression analysis, single-cell sequencing and immunohistochemistry. Accordingly, kidney organoids failed to form teratoma. Upon long-term subcutaneous implantation of whole organoids in immunodeficient IL2Ry-/-RAG2-/- mice, we observed tumor formation in 5 out of 103 implanted kidney organoids. These tumors were composed of WT1+CD56+ immature blastemal cells and showed histological resemblance with Wilms tumor. No genetic changes were identified that contributed to the occurrence of tumorigenic cells within the kidney organoids. However, assessment of epigenetic changes revealed a unique cluster of differentially methylated genes that were also present in undifferentiated iPSCs. We discovered that kidney organoids have the capacity to form tumors upon long-term implantation. The presence of epigenetic modifications combined with the lack of environmental cues may have caused an arrest in terminal differentiation. Our results indicate that the safe implementation of kidney organoids should exclude the presence of pro-tumorigenic methylation in kidney organoids.
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Células Madre Pluripotentes Inducidas , Teratoma , Animales , Diferenciación Celular , Células Madre Pluripotentes Inducidas/metabolismo , Riñón/patología , Ratones , Organogénesis , Organoides/metabolismo , Teratoma/patologíaRESUMEN
Uveal melanoma (UM) is an aggressive intra-ocular cancer with a strong tendency to metastasize. Metastatic UM is associated with mutations in BAP1 and SF3B1, however only little is known about the epigenetic modifications that arise in metastatic UM. In this study we aim to unravel epigenetic changes contributing to UM metastasis using a new genome-wide methylation analysis technique that covers over 50% of all CpG's. We identified aberrant methylation contributing to BAP1 and SF3B1-mediated UM metastasis. The methylation data was integrated with expression data and surveyed in matched UM metastases from the liver, skin and bone. UM metastases showed no commonly shared novel epigenetic modifications, implying that epigenetic changes contributing to metastatic spreading and colonization in distant tissues occur early in the development of UM and epigenetic changes that occur after metastasis are mainly patient-specific. Our findings reveal a plethora of epigenetic modifications in metastatic UM and its metastases, which could subsequently result in aberrant repression or activation of many tumor-related genes. This observation points towards additional layers of complexity at the level of gene expression regulation, which may explain the low mutational burden of UM.
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Melanoma/genética , Melanoma/metabolismo , Metástasis de la Neoplasia/genética , Neoplasias de la Úvea/genética , Neoplasias de la Úvea/metabolismo , Análisis Mutacional de ADN , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Humanos , Metilación , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Factores de Empalme de ARN/genética , Factores de Empalme de ARN/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Ubiquitina Tiolesterasa/genética , Ubiquitina Tiolesterasa/metabolismoRESUMEN
BACKGROUND: DNA methylation detection in liquid biopsies provides a highly promising and much needed means for real-time monitoring of disease load in advanced cancer patient care. Compared to the often-used somatic mutations, tissue- and cancer-type specific epigenetic marks affect a larger part of the cancer genome and generally have a high penetrance throughout the tumour. Here, we describe the successful application of the recently described MeD-seq assay for genome-wide DNA methylation profiling on cell-free DNA (cfDNA). The compatibility of the MeD-seq assay with different types of blood collection tubes, cfDNA input amounts, cfDNA isolation methods, and vacuum concentration of samples was evaluated using plasma from both metastatic cancer patients and healthy blood donors (HBDs). To investigate the potential value of cfDNA methylation profiling for tumour load monitoring, we profiled paired samples from 8 patients with resectable colorectal liver metastases (CRLM) before and after surgery. RESULTS: The MeD-seq assay worked on plasma-derived cfDNA from both EDTA and CellSave blood collection tubes when at least 10 ng of cfDNA was used. From the 3 evaluated cfDNA isolation methods, both the manual QIAamp Circulating Nucleic Acid Kit (Qiagen) and the semi-automated Maxwell® RSC ccfDNA Plasma Kit (Promega) were compatible with MeD-seq analysis, whereas the QiaSymphony DSP Circulating DNA Kit (Qiagen) yielded significantly fewer reads when compared to the QIAamp kit (p < 0.001). Vacuum concentration of samples before MeD-seq analysis was possible with samples in AVE buffer (QIAamp) or water, but yielded inconsistent results for samples in EDTA-containing Maxwell buffer. Principal component analysis showed that pre-surgical samples from CRLM patients were very distinct from HBDs, whereas post-surgical samples were more similar. Several described methylation markers for colorectal cancer monitoring in liquid biopsies showed differential methylation between pre-surgical CRLM samples and HBDs in our data, supporting the validity of our approach. Results for MSC, ITGA4, GRIA4, and EYA4 were validated by quantitative methylation specific PCR. CONCLUSIONS: The MeD-seq assay provides a promising new method for cfDNA methylation profiling. Potential future applications of the assay include marker discovery specifically for liquid biopsy analysis as well as direct use as a disease load monitoring tool in advanced cancer patients.
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Ácidos Nucleicos Libres de Células/análisis , Metilación de ADN/genética , Ácidos Nucleicos Libres de Células/genética , Metilación de ADN/fisiología , Estudio de Asociación del Genoma Completo/métodos , Estudio de Asociación del Genoma Completo/estadística & datos numéricos , Humanos , Biopsia Líquida/métodos , Biopsia Líquida/estadística & datos numéricos , Análisis de Secuencia de ADN/métodos , Análisis de Secuencia de ADN/estadística & datos numéricosRESUMEN
BACKGROUND: Alveolar capillary dysplasia with or without misalignment of the pulmonary veins (ACD/MPV) is a lethal congenital lung disorder associated with a variety of heterozygous genomic alterations in the FOXF1 gene or its 60 kb enhancer. Cases without a genomic alteration in the FOXF1 locus have been described as well. The mechanisms responsible for FOXF1 haploinsufficiency and the cause of ACD/MPV in patients without a genomic FOXF1 variant are poorly understood, complicating the search for potential therapeutic targets for ACD/MPV. To investigate the contribution of aberrant DNA methylation, genome wide methylation patterns of ACD/MPV lung tissues were compared with methylation patterns of control lung tissues using the recently developed technique Methylated DNA sequencing (MeD-seq). RESULTS: Eight ACD/MPV lung tissue samples and three control samples were sequenced and their mutual comparison resulted in identification of 319 differentially methylated regions (DMRs) genome wide, involving 115 protein coding genes. The potentially upregulated genes were significantly enriched in developmental signalling pathways, whereas potentially downregulated genes were mainly enriched in O-linked glycosylation. In patients with a large maternal deletion encompassing the 60 kb FOXF1 enhancer, DNA methylation patterns in this FOXF1 enhancer were not significantly different compared to controls. However, two hypermethylated regions were detected in the 60 kb FOXF1 enhancer of patients harbouring a FOXF1 point mutation. Lastly, a large hypermethylated region overlapping the first FOXF1 exon was found in one of the ACD/MPV patients without a known pathogenic FOXF1 variation. CONCLUSION: This is the first study providing genome wide methylation data on lung tissue of ACD/MPV patients. DNA methylation analyses in the FOXF1 locus excludes maternal imprinting of the 60 kb FOXF1 enhancer. Hypermethylation at the 60 kb FOXF1 enhancer might contribute to FOXF1 haploinsufficiency caused by heterozygous mutations in the FOXF1 coding region. Interestingly, DNA methylation analyses of patients without a genomic FOXF1 variant suggest that abnormal hypermethylation of exon 1 might play a role in some ACD/MPV in patients.
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Factores de Transcripción Forkhead/análisis , Pulmón/anomalías , Síndrome de Circulación Fetal Persistente/genética , Alveolos Pulmonares/anomalías , Metilación de ADN/genética , Metilación de ADN/fisiología , Femenino , Factores de Transcripción Forkhead/sangre , Estudio de Asociación del Genoma Completo/métodos , Humanos , Lactante , Recién Nacido , Pulmón/metabolismo , Masculino , Síndrome de Circulación Fetal Persistente/sangreRESUMEN
The 11 zinc finger (ZF) protein CTCF regulates topologically associating domain formation and transcription through selective binding to thousands of genomic sites. Here, we replaced endogenous CTCF in mouse embryonic stem cells with green-fluorescent-protein-tagged wild-type or mutant proteins lacking individual ZFs to identify additional determinants of CTCF positioning and function. While ZF1 and ZF8-ZF11 are not essential for cell survival, ZF8 deletion strikingly increases the DNA binding off-rate of mutant CTCF, resulting in reduced CTCF chromatin residence time. Loss of ZF8 results in widespread weakening of topologically associating domains, aberrant gene expression and increased genome-wide DNA methylation. Thus, important chromatin-templated processes rely on accurate CTCF chromatin residence time, which we propose depends on local sequence and chromatin context as well as global CTCF protein concentration.
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Factor de Unión a CCCTC/fisiología , Cromatina/metabolismo , Metilación de ADN , Regulación de la Expresión Génica , Genoma , Células Madre Pluripotentes/fisiología , Animales , Factor de Unión a CCCTC/genética , Femenino , Proteínas Fluorescentes Verdes/genética , Masculino , Ratones , Mitosis , Células Madre Embrionarias de Ratones , Mutación , Células Madre Pluripotentes/metabolismo , Factores de Tiempo , Elongación de la Transcripción GenéticaRESUMEN
INTRODUCTION: The majority of desmoid-type fibromatosis (DTF) tumors harbor a ß-catenin mutation, affecting specific codons in CTNNB1 exon 3. S45F tumors are reported to have a higher chance of recurrence after surgery and more resistance to systemic treatments compared to wild-type (WT) and T41A tumors. The aim of this pilot study was to examine the genome-wide DNA methylation profiles of S45F and T41A mutated DTF, to explain the observed differences in clinical behavior between these DTF subtypes. MATERIAL AND METHODS: Genome-wide analysis of DNA methylation was performed using MeD-seq on formalin-fixed, paraffin-embedded primary DTF samples harboring a S45F (n = 14) or a T41A (n = 15) mutation. Differentially methylated regions (DMRs) between S45F and T41A DTF were identified and used for a supervised hierarchical cluster analysis. DMRs with a fold-change ≥1.5 were considered to be differentially methylated and differences between S45F and T41A tumors were quantitatively assessed. The effect of DMRs on the expression of associated genes was assessed using an independent mRNA expression dataset. Protein-protein interactions between WT ß-catenin and mutant variants and DNA methyltransferase 1 (DNMT1) were examined by immunoprecipitation experiments. RESULTS: MeD-seq analyses indicated 354 regions that displayed differential methylation. Cluster analysis yielded no distinct clusters based on mutation, sex, tumor site or tumor size. A supervised clustering based on DMRs between small (≤34 mm) and large (>87 mm) DTF distinguished the two groups. Only ten DMRs displayed a fold change of ≥1.5 and six of them were found associated with the following genes: NLRP4, FOXK2, PERM1, CCDC6, NOC4L, and DUX4L6. The effects of DMRs on gene expression yielded a significant difference (p < 0.05) in the expression between S45F and T41A for CCDC6 and FOXK2 but not for all Affymetrix probe-sets used to detect these genes. Immunoprecipitations did not reveal an association of WT ß-catenin or mutant variants with DNMT1. CONCLUSION: This study demonstrated that S45F and T41A DTF tumors did not exhibit gross differences in DNA methylation patterns. This implies that distinct DNA methylation profiles are not the sole determinant for the divergent clinical behavior of these different DTF mutant subtypes.
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Exhaustion is a dysfunctional state of cytotoxic CD8+ T cells (CTL) observed in chronic infection and cancer. Current in vivo models of CTL exhaustion using chronic viral infections or cancer yield very few exhausted CTL, limiting the analysis that can be done on these cells. Establishing an in vitro system that rapidly induces CTL exhaustion would therefore greatly facilitate the study of this phenotype, identify the truly exhaustion-associated changes and allow the testing of novel approaches to reverse or prevent exhaustion. Here we show that repeat stimulation of purified TCR transgenic OT-I CTL with their specific peptide induces all the functional (reduced cytokine production and polyfunctionality, decreased in vivo expansion capacity) and phenotypic (increased inhibitory receptors expression and transcription factor changes) characteristics of exhaustion. Importantly, in vitro exhausted cells shared the transcriptomic characteristics of the gold standard of exhaustion, CTL from LCMV cl13 infections. Gene expression of both in vitro and in vivo exhausted CTL was distinct from T cell anergy. Using this system, we show that Tcf7 promoter DNA methylation contributes to TCF1 downregulation in exhausted CTL. Thus this novel in vitro system can be used to identify genes and signaling pathways involved in exhaustion and will facilitate the screening of reagents that prevent/reverse CTL exhaustion.
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Linfocitos T CD8-positivos/inmunología , Metilación de ADN/inmunología , Factor Nuclear 1-alfa del Hepatocito/inmunología , Coriomeningitis Linfocítica/inmunología , Virus de la Coriomeningitis Linfocítica/inmunología , Regiones Promotoras Genéticas/inmunología , Animales , Linfocitos T CD8-positivos/patología , Factor Nuclear 1-alfa del Hepatocito/genética , Coriomeningitis Linfocítica/genética , Coriomeningitis Linfocítica/patología , Virus de la Coriomeningitis Linfocítica/genética , Ratones , Ratones Transgénicos , Transducción de Señal/genética , Transducción de Señal/inmunologíaRESUMEN
Autosomal-dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease, leading to kidney failure in most patients. In approximately 85% of cases, the disease is caused by mutations in PKD1. How dysregulation of PKD1 leads to cyst formation on a molecular level is unknown. Induced pluripotent stem cells (iPSCs) are a powerful tool for in vitro modeling of genetic disorders. Here, we established ADPKD patient-specific iPSCs to study the function of PKD1 in kidney development and cyst formation in vitro. Somatic mutations are proposed to be the initiating event of cyst formation, and therefore, iPSCs were derived from cystic renal epithelial cells rather than fibroblasts. Mutation analysis of the ADPKD iPSCs revealed germline mutations in PKD1 but no additional somatic mutations in PKD1/PKD2. Although several somatic mutations in other genes implicated in ADPKD were identified in cystic renal epithelial cells, only few of these mutations were present in iPSCs, indicating a heterogeneous mutational landscape, and possibly in vitro cell selection before and during the reprogramming process. Whole-genome DNA methylation analysis indicated that iPSCs derived from renal epithelial cells maintain a kidney-specific DNA methylation memory. In addition, comparison of PKD1+/- and control iPSCs revealed differences in DNA methylation associated with the disease history. In conclusion, we generated and characterized iPSCs derived from cystic and healthy control renal epithelial cells, which can be used for in vitro modeling of kidney development in general and cystogenesis in particular.
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Células Epiteliales/patología , Células Madre Pluripotentes Inducidas/patología , Riñón/patología , Riñón Poliquístico Autosómico Dominante/patología , Línea Celular , Reprogramación Celular , Metilación de ADN/genética , Análisis Mutacional de ADN , Epigénesis Genética , Humanos , Túbulos Renales/patología , Mutación/genética , Riñón Poliquístico Autosómico Dominante/genética , Canales Catiónicos TRPP/genética , Canales Catiónicos TRPP/metabolismoRESUMEN
Approximately one-third of the patients with well-differentiated liposarcoma (WDLPS) will develop a local recurrence. Not much is known about the molecular relationship between the primary tumor and the recurrent tumor, which is important to reveal potential drivers of recurrence. Here we investigated the biology of recurrent WDLPS by comparing paired primary and recurrent WDLPS using microRNA profiling and genome-wide DNA methylation analyses. In total, 27 paired primary and recurrent WDLPS formalin-fixed and paraffin-embedded tumor samples were collected. MicroRNA expression profiles were determined using TaqMan® Low Density Array (TLDA) cards. Genome-wide DNA methylation and differentially methylated regions (DMRs) were assessed by methylated DNA sequencing (MeD-seq). A supervised cluster analysis based on differentially expressed microRNAs between paired primary and recurrent WDLPS did not reveal a clear cluster pattern separating the primary from the recurrent tumors. The clustering was also not based on tumor localization, time to recurrence, age or status of the resection margins. Changes in DNA methylation between primary and recurrent tumors were extremely variable, and no consistent DNA methylation changes were found. As a result, a supervised clustering analysis based on DMRs between primary and recurrent tumors did not show a distinct cluster pattern based on any of the features. Subgroup analysis for tumors localized in the extremity or the retroperitoneum also did not yield a clear distinction between primary and recurrent WDLPS samples. In conclusion, microRNA expression profiles and DNA methylation profiles do not distinguish between primary and recurrent WDLPS and no putative common drivers could be identified.
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Metilación de ADN/genética , Regulación Neoplásica de la Expresión Génica , Liposarcoma/genética , MicroARNs/genética , Recurrencia Local de Neoplasia/genética , Adulto , Anciano , Análisis por Conglomerados , Femenino , Heterogeneidad Genética , Humanos , Masculino , MicroARNs/metabolismo , Persona de Mediana Edad , Análisis de Componente PrincipalRESUMEN
Circadian rhythm disturbance (CRD) increases the risk of disease, e.g. metabolic syndrome, cardiovascular disease, and cancer. In the present study, we investigated later life adverse health effects triggered by repeated jet lag during gestation. Pregnant mice were subjected to a regular light-dark cycle (CTRL) or to a repeated delay (DEL) or advance (ADV) jet lag protocol. Both DEL and ADV offspring showed reduced weight gain. ADV offspring had an increased circadian period, and an altered response to a jet lag was observed in both DEL and ADV offspring. Analysis of the bones of adult male ADV offspring revealed reduced cortical bone mass and strength. Strikingly, analysis of the heart identified structural abnormalities and impaired heart function. Finally, DNA methylation analysis revealed hypermethylation of miR17-92 cluster and differential methylation within circadian clock genes, which correlated with altered gene expression. We show that developmental CRD affects the circadian system and predisposes to non-communicable disease in adult life.