| 1. |
- Badam, Tejaswi, et al.
(författare)
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CD4(+) T-cell DNA methylation changes during pregnancy significantly correlate with disease-associated methylation changes in autoimmune diseases
- 2022
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Ingår i: Epigenetics. - : Taylor & Francis Group. - 1559-2294 .- 1559-2308. ; 17:9, s. 1040-1055
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Tidskriftsartikel (refereegranskat)abstract
- Epigenetics may play a central, yet unexplored, role in the profound changes that the maternal immune system undergoes during pregnancy and could be involved in the pregnancy-induced modulation of several autoimmune diseases. We investigated changes in the methylome in isolated circulating CD4(+) T-cells in non-pregnant and pregnant women, during the 1(st) and 2(nd) trimester, using the Illumina Infinium Human Methylation 450K array, and explored how these changes were related to autoimmune diseases that are known to be affected during pregnancy. Pregnancy was associated with several hundreds of methylation differences, particularly during the 2(nd) trimester. A network-based modular approach identified several genes, e.g., CD28, FYN, VAV1 and pathways related to T-cell signalling and activation, highlighting T-cell regulation as a central component of the observed methylation alterations. The identified pregnancy module was significantly enriched for disease-associated methylation changes related to multiple sclerosis, rheumatoid arthritis and systemic lupus erythematosus. A negative correlation between pregnancy-associated methylation changes and disease-associated changes was found for multiple sclerosis and rheumatoid arthritis, diseases that are known to improve during pregnancy whereas a positive correlation was found for systemic lupus erythematosus, a disease that instead worsens during pregnancy. Thus, the directionality of the observed changes is in line with the previously observed effect of pregnancy on disease activity. Our systems medicine approach supports the importance of the methylome in immune regulation of T-cells during pregnancy. Our findings highlight the relevance of using pregnancy as a model for understanding and identifying disease-related mechanisms involved in the modulation of autoimmune diseases.
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| 2. |
- Barrenäs, Fredrik, et al.
(författare)
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Disease-Associated MRNA Expression Differences in Genes with Low DNA Methylation
- 2012
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- Although the importance of DNA methylation for mRNA expression has been shown for individualgenes in several complex diseases, such a relation has been difficult to show on a genome-wide scale.Here, we used microarrays to examine the relationship between DNA methylation and mRNAexpression in CD4+ T cells from patients with seasonal allergic rhinitis (SAR) and healthy controls.SAR is an optimal disease model because the disease process can be studied by comparing allergenchallengedCD4+ T cells obtained from patients and controls, and mimicked in Th2 polarised T cellsfrom healthy controls. The cells from patients can be analyzed to study relations between methylationand mRNA expression, while the Th2 cells can be used for functional studies. We found that DNAmethylation, but not mRNA expression clearly separated patients from controls. Similar to studies ofother complex diseases, we found no general relation between DNA methylation and mRNAexpression. However, when we took into account the absence or presence of CpG islands in thepromoters of disease associated genes an association was found: low methylation genes without CpGislands had significantly higher expression levels of disease-associated genes. This association wasconfirmed for genes whose expression levels were regulated by a transcription factor of knownrelevance for allergy, IRF4, using combined ChIP-chip and siRNA mediated silencing of IRF4expression. In summary, disease-associated increases of mRNA expression were found in lowmethylation genes without CpG islands in CD4+ T cells from patients with SAR. Further studies arewarranted to examine if a similar association is found in other complex diseases.
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| 3. |
- Bensberg, Maike, 1993-
(författare)
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DNA methylation in T cell leukaemia
- 2024
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- T cell acute lymphoblastic leukaemia (T-ALL) is a predominantly paediatric cancer that stems from malignant transformation of developing T cells. While the disease has an overall survival rate of 80%, the intense chemotherapy treatment causes severe toxicity and long-term side effects. Furthermore, the survival rate for patients in relapse is less than 25%. Consequently, there is a need for improved therapy options to reduce treatment-related side effects and improve the survival rate of relapsed patients. Targeting aberrant DNA methylation with hypomethylating agents (HMAs) has been successful in the treatment of myelodysplastic syndromes and acute myeloid leukaemia but has not been routinely used in the treatment of T-ALL, despite DNA hypomethylation being observed in T-ALL patients. In this work, we employed a comprehensive set of molecular and sequencing-based techniques to explore the possibilities of HMAs as a treatment option for T-ALL.We made the discovery that the DNA demethylating enzyme ten-eleven translocation 2, TET2, is downregulated or completely silenced in primary T-ALL. Moreover, the TET2 promoter was highly methylated in a group of patients, suggesting that TET2 itself can be silenced through DNA methylation in T-ALL. By treatment with HMAs, TET2 was demethylated in T-ALL cell lines and was one of few genes that was activated upon loss of DNA methylation, indicating that TET2 expression is regulated by DNA methylation in T-ALL cell lines. The development of a novel HMA, the DNMT1-specific inhibitor GSK-3685032, offers a tool to reveal the mechanism of action of the traditional HMAs, 5- azacytidine and decitabine, and to study the effects of acute loss of DNA methylation on cancer cells. We found that 5-azacytidine and decitabine are cytotoxic to T-ALL cells primarily by creating DNA double strand breaks. In contrast, GSK did not prompt a DNA damage response and instead reduced global DNA methylation to as little as 18% with limited cytotoxicity only occurring after levels of DNA methylation had dropped below 30%, a level of demethylation not achieved with DEC or AZA.T-ALL is more than two times more common in boys than girls and mutations in X-linked tumour suppressor genes that escape X inactivation, have been suggested as an underlying cause for the observed sex-bias. In theory, these aberrations would be more detrimental in XYmale cells than in XX-female cells due to the presence of an extra protective copy of the gene in females. We profiled DNA methylation during T cell development and created a map of sex-specific gene expression and expression from the inactive X chromosome, finding that some, but not all, suggested tumour suppressor genes in fact escape X inactivation. These results highlight the importance of profiling the healthy cells that T-ALL arises from to correctly judge the functional impact of gene dysregulation in cancer.In the last study, we aimed to investigate the role of N6-adenine methylation (6mdA) during T cell differentiation. While 6mdA is common in bacteria it is much rarer in humans. Nevertheless, 6mdA has previously been associated with several cellular processes, including cancer progression. Our study calls the presence of 6mdA in mammals into question by exposing limitations of the techniques used in its analysis. We show that contamination with bacterial DNA or 6mAcontaining RNA, nonspecific antibody binding, and low precision of third-generation sequencing techniques all hinder the detection and investigation of rare DNA modifications, such as 6mdA.Together, this work is an in-depth study of the function and the potential of DNA methylation in the biology of healthy and malignant T cells.
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| 4. |
- Bensberg, Maike, et al.
(författare)
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TET2 as a tumor suppressor and therapeutic target in T-cell acute lymphoblastic leukemia
- 2021
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Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 118:34
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Tidskriftsartikel (refereegranskat)abstract
- Pediatric T-cell acute lymphoblastic leukemia (T-ALL) is an aggres-sive malignancy resulting from overproduction of immature T-cells in the thymus and is typified by widespread alterations in DNA methyl-ation. As survival rates for relapsed T-ALL remain dismal (10 to 25%), development of targeted therapies to prevent relapse is key to improv-ing prognosis. Whereas mutations in the DNA demethylating enzyme TET2 are frequent in adult T-cell malignancies, TET2 mutations in T-ALL are rare. Here, we analyzed RNA-sequencing data of 321 primary T-ALLs, 20 T-ALL cell lines, and 25 normal human tissues, revealing that TET2 is transcriptionally repressed or silenced in 71% and 17% of T-ALL, respec-tively. Furthermore, we show that TET2 silencing is often associated with hypermethylation of the TET2 promoter in primary T-ALL. Impor-tantly, treatment with the DNA demethylating agent, 5-azacytidine (5-aza), was significantly more toxic to TET2-silenced T-ALL cells and resulted in stable re-expression of the TET2 gene. Additionally, 5-aza led to up-regulation of methylated genes and human endogenous ret-roviruses (HERVs), which was further enhanced by the addition of phys-iological levels of vitamin C, a potent enhancer of TET activity. Together, our results clearly identify 5-aza as a potential targeted therapy for TET2-silenced T-ALL.
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| 5. |
- Bruhn, Sören, et al.
(författare)
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A Generally Applicable Translational Strategy Identifies S100A4 as a Candidate Gene in Allergy
- 2014
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Ingår i: Science Translational Medicine. - : American Association for the Advancement of Science (AAAS). - 1946-6234 .- 1946-6242. ; 6:218
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Tidskriftsartikel (refereegranskat)abstract
- The identification of diagnostic markers and therapeutic candidate genes in common diseases is complicated by the involvement of thousands of genes. We hypothesized that genes co-regulated with a key gene in allergy, IL13, would form a module that could help to identify candidate genes. We identified a T helper 2 (T(H)2) cell module by small interfering RNA-mediated knockdown of 25 putative IL13-regulating transcription factors followed by expression profiling. The module contained candidate genes whose diagnostic potential was supported by clinical studies. Functional studies of human TH2 cells as well as mouse models of allergy showed that deletion of one of the genes, S100A4, resulted in decreased signs of allergy including TH2 cell activation, humoral immunity, and infiltration of effector cells. Specifically, dendritic cells required S100A4 for activating T cells. Treatment with an anti-S100A4 antibody resulted in decreased signs of allergy in the mouse model as well as in allergen-challenged T cells from allergic patients. This strategy, which may be generally applicable to complex diseases, identified and validated an important diagnostic and therapeutic candidate gene in allergy.
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| 6. |
- Doumpas, Nikolaos, et al.
(författare)
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TCF/LEF dependent and independent transcriptional regulation of Wnt/beta-catenin target genes
- 2019
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Ingår i: EMBO Journal. - : WILEY. - 0261-4189 .- 1460-2075. ; 38:2
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Tidskriftsartikel (refereegranskat)abstract
- During canonical Wnt signalling, the activity of nuclear beta-catenin is largely mediated by the TCF/LEF family of transcription factors. To challenge this view, we used the CRISPR/Cas9 genome editing approach to generate HEK 293T cell clones lacking all four TCF/LEF genes. By performing unbiased whole transcriptome sequencing analysis, we found that a subset of beta-catenin transcriptional targets did not require TCF/LEF factors for their regulation. Consistent with this finding, we observed in a genome-wide analysis that beta-catenin occupied specific genomic regions in the absence of TCF/LEF. Finally, we revealed the existence of a transcriptional activity of beta-catenin that specifically appears when TCF/LEF factors are absent, and refer to this as beta-catenin-GHOST response. Collectively, this study uncovers a previously neglected modus operandi of beta-catenin that bypasses the TCF/LEF transcription factors.
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| 7. |
- Douvlataniotis, Karolos, et al.
(författare)
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No evidence for DNA N-6-methyladenine in mammals
- 2020
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Ingår i: Science Advances. - Washington, DC, United States : American Association for the Advancement of Science (A A A S). - 2375-2548. ; 6:12
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Tidskriftsartikel (refereegranskat)abstract
- N-6-methyladenine (6mdA) is a widespread DNA modification in bacteria. More recently, 6mdA has also been characterized in mammalian DNA. However, measurements of 6mdA abundance and profiles are often very dissimilar between studies, even when performed on DNA from identical mammalian cell types. Using comprehensive bioinformatics analyses of published data and novel experimental approaches, we reveal that efforts to assay 6mdA in mammals have been severely compromised by bacterial contamination, RNA contamination, technological limitations, and antibody nonspecificity. These complications render 6mdA an exceptionally problematic DNA modification to study and have resulted in erroneous detection of 6mdA in several mammalian systems. Together, our results strongly imply that the evidence published to date is not sufficient to support the presence of 6mdA in mammals.
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| 8. |
- Gawel, Danuta, et al.
(författare)
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A validated single-cell-based strategy to identify diagnostic and therapeutic targets in complex diseases
- 2019
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Ingår i: Genome Medicine. - : Springer Science and Business Media LLC. - 1756-994X. ; 11
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Tidskriftsartikel (refereegranskat)abstract
- Background: Genomic medicine has paved the way for identifying biomarkers and therapeutically actionable targets for complex diseases, but is complicated by the involvement of thousands of variably expressed genes across multiple cell types. Single-cell RNA-sequencing study (scRNA-seq) allows the characterization of such complex changes in whole organs. Methods: The study is based on applying network tools to organize and analyze scRNA-seq data from a mouse model of arthritis and human rheumatoid arthritis, in order to find diagnostic biomarkers and therapeutic targets. Diagnostic validation studies were performed using expression profiling data and potential protein biomarkers from prospective clinical studies of 13 diseases. A candidate drug was examined by a treatment study of a mouse model of arthritis, using phenotypic, immunohistochemical, and cellular analyses as read-outs. Results: We performed the first systematic analysis of pathways, potential biomarkers, and drug targets in scRNA-seq data from a complex disease, starting with inflamed joints and lymph nodes from a mouse model of arthritis. We found the involvement of hundreds of pathways, biomarkers, and drug targets that differed greatly between cell types. Analyses of scRNA-seq and GWAS data from human rheumatoid arthritis (RA) supported a similar dispersion of pathogenic mechanisms in different cell types. Thus, systems-level approaches to prioritize biomarkers and drugs are needed. Here, we present a prioritization strategy that is based on constructing network models of disease-associated cell types and interactions using scRNA-seq data from our mouse model of arthritis, as well as human RA, which we term multicellular disease models (MCDMs). We find that the network centrality of MCDM cell types correlates with the enrichment of genes harboring genetic variants associated with RA and thus could potentially be used to prioritize cell types and genes for diagnostics and therapeutics. We validated this hypothesis in a large-scale study of patients with 13 different autoimmune, allergic, infectious, malignant, endocrine, metabolic, and cardiovascular diseases, as well as a therapeutic study of the mouse arthritis model. Conclusions: Overall, our results support that our strategy has the potential to help prioritize diagnostic and therapeutic targets in human disease.
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| 9. |
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| 10. |
- Gawel, Danuta R., 1988-
(författare)
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Identification of genes and regulators that are shared across T cell associated diseases
- 2018
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Genome-wide association studies (GWASs) of hundreds of diseases and millions of patients have led to the identification of genes that are associated with more than one disease. The aims of this PhD thesis were to a) identify a group of genes important in multiple diseases (shared disease genes), b) identify shared up-stream disease regulators, and c) determine how the same genes can be involved in the pathogenesis of different diseases. These aims have been tested on CD4+ T cells because they express the T helper cell differentiation pathway, which was the most enriched pathway in analyses of all disease associated genes identified with GWASs.Combining information about known gene-gene interactions from the protein-protein interaction (PPI) network with gene expression changes in multiple T cell associated diseases led to the identification of a group of highly interconnected genes that were miss-expressed in many of those diseases – hereafter called ‘shared disease genes’. Those genes were further enriched for inflammatory, metabolic and proliferative pathways, genetic variants identified by all GWASs, as well as mutations in cancer studies and known diagnostic and therapeutic targets. Taken together, these findings supported the relevance of the shared disease genes.Identification of the shared upstream disease regulators was addressed in the second project of this PhD thesis. The underlying hypothesis assumed that the determination of the shared upstream disease regulators is possible through a network model showing in which order genes activate each other. For that reason a transcription factor–gene regulatory network (TF-GRN) was created. The TF-GRN was based on the time-series gene expression profiling of the T helper cell type 1 (Th1), and T helper cell type 2 (Th2) differentiation from Native T-cells. Transcription factors (TFs) whose expression changed early during polarization and had many downstream predicted targets (hubs) that were enriched for disease associated single nucleotide polymorphisms (SNPs) were prioritised as the putative early disease regulators. These analyses identified three transcription factors: GATA3, MAF and MYB. Their predicted targets were validated by ChIP-Seq and siRNA mediated knockdown in primary human T-cells. CD4+ T cells isolated from seasonal allergic rhinitis (SAR) and multiple sclerosis (MS) patients in their non-symptomatic stages were analysed in order to demonstrate predictive potential of those three TFs. We found that those three TFs were differentially expressed in symptom-free stages of the two diseases, while their TF-GRN{predicted targets were differentially expressed during symptomatic disease stages. Moreover, using RNA-Seq data we identified a disease associated SNP that correlated with differential splicing of GATA3.A limitation of the above study is that it concentrated on TFs as main regulators in cells, excluding other potential regulators such as microRNAs. To this end, a microRNA{gene regulatory network (mGRN) of human CD4+ T cell differentiation was constructed. Within this network, we defined regulatory clusters (groups of microRNAs that are regulating groups of mRNAs). One regulatory cluster was differentially expressed in all of the tested diseases, and was highly enriched for GWAS SNPs. Although the microRNA processing machinery was dynamically upregulated during early T-cell activation, the majority of microRNA modules showed specialisation in later time-points.In summary this PhD thesis shows the relevance of shared genes and up-stream disease regulators. Putative mechanisms of why shared genes can be involved in pathogenesis of different diseases have also been demonstrated: a) differential gene expression in different diseases; b) alternative transcription factor splicing variants may affect different downstream gene target group; and c) SNPs might cause alternative splicing.
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