| 1. |
- Gawel, Danuta R., 1988-
(författare)
-
Identification of genes and regulators that are shared across T cell associated diseases
- 2018
-
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.
|
|
| 2. |
- Lentini, Antonio, 1990-
(författare)
-
Dynamic regulation of DNA methylation in human T-cell biology
- 2019
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- T helper cells play a central role in orchestrating immune responses in humans. Upon encountering a foreign antigen, T helper cells are activated followed by a differentiation process where the cells are specialised to help combating the infection. Dysregulation of T helper cell activation, differentiation and function has been implicated in numerous diseases, including autoimmunity and cancer. Whereas gene-regulatory networks help drive T-cell differentiation, acquisition of stable cell states require heritable epigenetic signals, such as DNA methylation. Indeed, the establishment of DNA methylation patterns is a key part of appropriate T-cell differentiation but how this is regulated over time remains unknown. Methylation can be directly attached to cytosine residues in DNA to form 5-methylcytosine (5mC) but the removal of DNA methylation requires multiple enzymatic reactions, commonly initiated by the conversion into 5-hydroxymethylcytosine (5hmC), thus creating a highly complex regulatory system. This thesis aimed to investigate how DNA methylation is dynamically regulated during T-cell differentiation.To this end, we employed large-scale profiling techniques combining gene expression as well as genome-wide 5mC and 5hmC measurements to construct a time-series model of epigenetic regulation of differentiation. This revealed that early T-cell activation was accompanied by extensive genome-wide deposition of 5hmC which resulted in demethylation upon proliferation. Early DNA methylation remodelling through 5hmC was not only indicative of demethylation events during T-cell differentiation but also marked changes persisting longterm in memory T-cell subsets. These results suggest that priming of epigenetic landscapes in T-cells is initiated during early activation events, preceding any establishment of a stable lineage, which are then maintained throughout the cells lifespan. The regions undergoing remodelling were also highly enriched for genetic variants in autoimmune diseases which we show to be functional through disruption of protein binding. These variants could potentially disrupt gene-regulatory networks and the establishment of epigenetic priming, highlighting the complex interplay between genetic and epigenetic layers. In the course of this work, we discovered that a commonly used technique to study genome-wide DNA modifications, DNA immunoprecipitation (DIP)-seq, had a false discovery rate between 50-99% depending on the modification and cell type being assayed. This represented inherent technical errors related to the use of antibodies resulting in off-target binding of repetitive sequences lacking any DNA modifications. These sequences are common in mammalian genomes making robust detection of rare DNA modifications very difficult due to the high background signals. However, offtarget binding could easily be controlled for using a non-specific antibody control which greatly improved data quality and biological insight of the data. Although future studies are advised to use alternative methods where available, error correction is an acceptable alternative which will help fuel new discoveries through the removal of extensive background signals.Taken together, this thesis shows how integrative use of high-resolution epigenomic data can be used to study complex biological systems over time as well as how these techniques can be systematically characterised to identify and correct errors resulting in improved detection.
|
|
| 3. |
- Rundquist, Olof, 1991-
(författare)
-
Multi-omic time-series analysis of T-cells as a model for identification of biomarkers, treatments and upstream disease regulators
- 2021
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- CD4+ T-cell function and their process of differentiation is a central piece of the puzzle in a multitude of diseases. CD4+ T-cells are part of the adaptive immune system and function by directing other immune cells to the site of infection and instructing B-cells to produce antibodies, among many other functions. CD4+ T-cells may differentiate into several different sub-types, such as T-helper 1, 2 and 17, with differing functions within the immune system. T-helper 1 (Th1) cells are most closely associated with the elimination of viral infections but are also associated with autoimmune diseases such as multiple sclerosis (MS) and rheumatoid arthritis (RA). T-cells develop in the thymus first as double-negative T-cells, that express neither CD4 nor CD8, going through multiple development stages before becoming double-positive T-cell that express both CD4 and CD8, before eventually giving rise to single positive CD4+ and CD8+ T-cells. This process of development is under tight control and if this control fails, cancer may result. Once CD4+ T-cells are fully developed, they may specialize as outlined above and if said process is not properly controlled, autoimmunity may result. As such, the proper understanding of these control mechanisms is of great importance for the understanding of diseases of the immune system and the discovery of biomarkers and treatments against said diseases. These control processes are often studied in a singular fashion using one omic technique, e.g., RNA sequencing (RNA-seq), with the assumption that a signal in one omic layer will be reflected in another. Recent studies attempting to integrate multiple omics have however cast doubt on this and it is becoming increasingly apparent that to gain a complete understanding of a system, the system needs to be studied at multiple levels of regulation, i.e., multiple omics.The aim of this thesis was to use multi-omics to investigate the development and differentiation process of CD4+ T-helper cells and relate it to disease mechanisms. To start, we studied T-cell development through the model of T-cell acute lymphoblastic leukaemia (T-ALL). More specifically, we studied the TET2 gene and investigated its importance in T-ALL for treatment susceptibility and mechanism in vitro. TET2 is a demethylase and functions through the removal of cytosine methylation on the DNA, a marker of gene silencing. Through treatment with decitabine, an inhibitor of DNA-methylation, and Vitamin C, a co-factor for TET2, we showed that TET2 deficient cancer cell lines were more vulnerable to treatment targeting DNA methylation and investigated the mechanistic effects of said treatment by RNA sequencing. We then moved on to study primary human naïve CD4+ T-cells and their differentiation into Th1-cells. First, we focused on T-cell activation and its importance to MS to understand the role of T-cells in mediating the lowered disease activity usually observed during pregnancy in MS. This showed that the major pregnancy hormone progesterone significantly dampens T-cell activation, providing a possible explanation for the beneficial effects of pregnancy on MS. Then, using ATAC sequencing (ATAC-seq), RNA-seq and proteomics we studied Th1-differentiation as a time series to elucidate regulatory events throughout the differentiation process and to study their implications for MS with the inclusion of progesterone treatment. The integration of several omic techniques presents unique challenges as one does not necessarily directly translate to the other. As such, we first focused on the integration of RNA-seq and proteomics by designing a model for the prediction of protein abundance from RNA-seq and validated it through biomarker discovery. Next, we focused on the integration of ATAC-seq and RNA-seq using correlation between time series of the two techniques. This thesis provides a thorough investigation of Th1-cell differentiation and its potential involvement in disease. Time series datasets were produced to study gene regulation (ATAC-seq), gene expression (RNA-seq) and protein expression (mass spectrometry) and the work focused on their integration. This profoundly showed that through combining multiple omic techniques it was possible to gain new insights that were not possible to discover with one or the other. Multi-omic analyses are becoming more and more common in medicine today as their power to produce new insight into the complexity of complex diseases is being increasingly recognized. As such, this work forms an important foundation for future discovery of biomarkers and treatments in such diseases.
|
|
| 4. |
- Bensberg, Maike, 1993-
(författare)
-
DNA methylation in T cell leukaemia
- 2024
-
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.
|
|
| 5. |
- Faustini, Elena, 1992-
(författare)
-
Nuclear architecture, chromatin dynamics and DSB repair: characterization of new factors that regulate genome integrity
- 2023
-
Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cells are continuously exposed to DNA damaging agents that cause different types of lesions. Double strand breaks (DSBs), where both strands of DNA are broken, are the most toxic lesions. To repair DSBs and ensure genome stability and cell survival, mammalian cells evolved two main pathways, Homologous recombination (HR) and classical Nonhomologous end joining (c-NHEJ). Failure in these pathways triggers genome instability, which correlates with tumorigenesis and cancer progression, but can also contribute to cancer treatment when properly exploited. At the same time, the end of our chromosomes, the telomeres, should be protected from being sensed as DSBs and aberrantly repaired. In fact, failure in telomere protection due to short or dysfunctional telomeres can cause chromosome fusions, genome instability and, in some cases, contribute to tumorigenesis. To this end, telomeres are protected by the shelterin complex in all cell types and elongated by telomerase in germline and somatic stem cells. This thesis aims to find new factors involved in the DNA damage response (DDR) that could be used as markers for cancer diagnosis and/or targets in cancer therapy. In paper I, we explore the effect of two novel variants in the H/ACA RNA binding complex component NHP2 identified in a patient with Aplastic anemia, gastric cancer, and signs of premature aging. H/ACA RNA binding complex is essential for the stability and maturation of both ribosomal RNA and the telomerase RNA component hTR. By in silico and in cells analysis, we found that both mutations reduce the affinity of NHP2 for the other components of the H/ACA RNA binding complex due to the misplacement of the N-terminus, affecting protein stability. Furthermore, these variants cause reduced telomerase activity by failing to preserve hTR. However, they do not affect the DDR. In papers II and III, we investigated the role of chromatin mobility in the maintenance of genome stability. In fact, the mobility of DSBs can cause translocations, one of the main hallmarks of cancer initiation and progression, but is also one of the mechanisms responsible for the efficacy of therapies based on PARP inhibition against breast and ovarian cancer defective in HR. In paper II, we describe a method for consistent and unbiased quantification of DSB mobility and nuclear deformations in the presence of multiple DSBs by fluorescent live-cell imaging. This method can be used with any fluorescent-tagged proteins binding specific genome regions, such as telomeres. In paper III Section A, we analyzed DSB mobility and mis-repair in the context of nuclear envelope (NE) alterations as Lamin A/C deletion or reduction in sphingolipids synthesis. In fact, previous studies have shown that different components of the NE are involved in promoting and/or counteracting such mobility, but the role of the NE itself was not yet explored. By combining genetic modifications and/or chemical inhibition with microscopy techniques, we found that the presence of invaginations significantly increases DSB mobility and mis-repair after treatment with PARP inhibitor in the absence of BRCA1, suggesting that the visualization of the NE could be used as marker for predicting the cancer therapy outcome and that, in the long run, the NE structure could be used as target in cancer therapy. In paper III Section B, we demonstrated that cells subjected to mechanical shear stress and/or to an agonist of the mechanosensor channel Piezo1 reduced DSB mobility and mis-repair. This effect is associated with chromatin decompaction, as observed by electron microscopy and ATAC sequencing, and reduced telomere mobility, suggesting that the shear stress-mediated activation of Piezo1 promotes the chromatin opening and that chromatin compaction can influence DSB mobility and repair. Since cancer cells are exposed to mechanical stress during metastasis, our work suggests that cotreatment with drugs closing the chromatin and/or inhibiting Piezo1 could increase the sensitivity of cancer cells to chemotherapy.
|
|
| 6. |
- Hackett, Jamie A., et al.
(författare)
-
Promoter DNA methylation couples genome-defence mechanisms to epigenetic reprogramming in the mouse germline
- 2012
-
Ingår i: Development. - : The Company of Biologists. - 0950-1991 .- 1477-9129. ; 139:19, s. 3623-3632
-
Tidskriftsartikel (refereegranskat)abstract
- Mouse primordial germ cells (PGCs) erase global DNA methylation (5mC) as part of the comprehensive epigenetic reprogramming that occurs during PGC development. 5mC plays an important role in maintaining stable gene silencing and repression of transposable elements (TE) but it is not clear how the extensive loss of DNA methylation impacts on gene expression and TE repression in developing PGCs. Using a novel epigenetic disruption and recovery screen and genetic analyses, we identified a core set of germline-specific genes that are dependent exclusively on promoter DNA methylation for initiation and maintenance of developmental silencing. These gene promoters appear to possess a specialised chromatin environment that does not acquire any of the repressive H3K27me3, H3K9me2, H3K9me3 or H4K20me3 histone modifications when silenced by DNA methylation. Intriguingly, this methylation-dependent subset is highly enriched in genes with roles in suppressing TE activity in germ cells. We show that the mechanism for developmental regulation of the germline genome-defence genes involves DNMT3B-dependent de novo DNA methylation. These genes are then activated by lineage-specific promoter demethylation during distinct global epigenetic reprogramming events in migratory (~E8.5) and post-migratory (E10.5-11.5) PGCs. We propose that genes involved in genome defence are developmentally regulated primarily by promoter DNA methylation as a sensory mechanism that is coupled to the potential for TE activation during global 5mC erasure, thereby acting as a failsafe to ensure TE suppression and maintain genomic integrity in the germline.
|
|
| 7. |
- Lentini, Antonio, et al.
(författare)
-
Analyzing DNA-Immunoprecipitation Sequencing Data
- 2021
-
Ingår i: DNA Modifications. - New York, NY : Humana Press. - 9781071608753 - 9781071608760 ; 2198, s. 431-439
-
Bokkapitel (refereegranskat)abstract
- Genome-wide profiling of DNA modifications has advanced our understanding of epigenetics in mammalian biology. Whereas several different methods for profiling DNA modifications have been developed over the last decade, DNA-immunoprecipitation coupled with high-throughput sequencing (DIP-seq) has proven a particularly adaptable and cost-effective approach. DIP-seq was especially valuable in initial studies of the more recently discovered DNA modifications, 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine. As an enrichment-based profiling method, analysis of DIP-seq data poses several unique, and often unappreciated bioinformatics challenges, which if unmet, can profoundly affect the results and conclusions drawn from the data. Here, we outline key considerations in both the design of DIP-seq assays and analysis of DIP-seq data to ensure the accuracy and reproducibility of DIP-seq based studies.
|
|
| 8. |
- Lentini, Antonio, et al.
(författare)
-
Mapping DNA Methylation in Mammals: The State of the Art
- 2021
-
Ingår i: DNA Modifications. - New York, NY : Humana Press. - 9781071608753 - 9781071608760 ; 2198, s. 37-50
-
Bokkapitel (refereegranskat)abstract
- A complete understanding of the dynamics and function of cytosine modifications in mammalian biology is lacking. Central to achieving this understanding is the availability of techniques that permit sensitive and specific genome-wide mapping of DNA modifications in mammalian DNA. The last decade has seen the development of a vast arsenal of novel profiling approaches enabling epigeneticists to tackle research questions that were previously out of reach. Here, we review the techniques currently available for profiling DNA modifications in mammals, discuss their strengths and weaknesses, and speculate on the future direction of DNA modification profiling technologies.
|
|
| 9. |
- Nestor, Colm, 1977-, et al.
(författare)
-
Correlation of inter-locus polyglutamine toxicity with CAG•CTG triplet repeat expandability and flanking genomic DNA GC content
- 2011
-
Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 6:12
-
Tidskriftsartikel (refereegranskat)abstract
- Dynamic expansions of toxic polyglutamine (polyQ)-encoding CAG repeats in ubiquitously expressed, but otherwise unrelated, genes cause a number of late-onset progressive neurodegenerative disorders, including Huntington disease and the spinocerebellar ataxias. As polyQ toxicity in these disorders increases with repeat length, the intergenerational expansion of unstable CAG repeats leads to anticipation, an earlier age-at-onset in successive generations. Crucially, disease associated alleles are also somatically unstable and continue to expand throughout the lifetime of the individual. Interestingly, the inherited polyQ length mediating a specific age-at-onset of symptoms varies markedly between disorders. It is widely assumed that these inter-locus differences in polyQ toxicity are mediated by protein context effects. Previously, we demonstrated that the tendency of expanded CAG•CTG repeats to undergo further intergenerational expansion (their 'expandability') also differs between disorders and these effects are strongly correlated with the GC content of the genomic flanking DNA. Here we show that the inter-locus toxicity of the expanded polyQ tracts of these disorders also correlates with both the expandability of the underlying CAG repeat and the GC content of the genomic DNA flanking sequences. Inter-locus polyQ toxicity does not correlate with properties of the mRNA or protein sequences, with polyQ location within the gene or protein, or steady state transcript levels in the brain. These data suggest that the observed inter-locus differences in polyQ toxicity are not mediated solely by protein context effects, but that genomic context is also important, an effect that may be mediated by modifying the rate at which somatic expansion of the DNA delivers proteins to their cytotoxic state.
|
|
| 10. |
- Nestor, Colm, 1977-, et al.
(författare)
-
Enzymatic approaches and bisulfite sequencing cannot distinguish between 5-methylcytosine and 5-hydroxymethylcytosine in DNA
- 2010
-
Ingår i: BioTechniques. - : Informa Healthcare. - 0736-6205 .- 1940-9818. ; 48:4, s. 317-319
-
Tidskriftsartikel (refereegranskat)abstract
- DNA cytosine methylation (5mC) is highly abundant in mammalian cells and is associated with transcriptional repression. Recently, hydroxymethylcytosine (hmC) has been detected at high levels in certain human cell types; however, its roles are unknown. Due to the structural similarity between 5mC and hmC, it is unclear whether 5mC analyses can discriminate between these nucleotides. Here we show that 5mC and hmC are experimentally indistinguishable using established 5mC mapping methods, thereby implying that existing 5mC data sets will require careful re-evaluation in the context of the possible presence of hmC. Potential differential enrichment of 5mC and hmC DNA sequences may be facilitated using a 5mC monoclonal antibody.
|
|
| 11. |
- Nestor, Colm, 1977-, et al.
(författare)
-
Tissue type is a major modifier of the 5-hydroxymethylcytosine content of human genes
- 2012
-
Ingår i: Genome Research. - : Cold Spring Harbor Laboratory Press (CSHL). - 1088-9051 .- 1549-5469. ; 22:3, s. 467-477
-
Tidskriftsartikel (refereegranskat)abstract
- The discovery of substantial amounts of 5-hydroxymethylcytosine (5hmC), formed by the oxidation of 5-methylcytosine (5mC), in various mouse tissues and human embryonic stem (ES) cells has necessitated a reevaluation of our knowledge of 5mC/5hmC patterns and functions in mammalian cells. Here, we investigate the tissue specificity of both the global levels and locus-specific distribution of 5hmC in several human tissues and cell lines. We find that global 5hmC content of normal human tissues is highly variable, does not correlate with global 5mC content, and decreases rapidly as cells from normal tissue adapt to cell culture. Using tiling microarrays to map 5hmC levels in DNA from normal human tissues, we find that 5hmC patterns are tissue specific; unsupervised hierarchical clustering based solely on 5hmC patterns groups independent biological samples by tissue type. Moreover, in agreement with previous studies, we find 5hmC associated primarily, but not exclusively, with the body of transcribed genes, and that within these genes 5hmC levels are positively correlated with transcription levels. However, using quantitative 5hmC-qPCR, we find that the absolute levels of 5hmC for any given gene are primarily determined by tissue type, gene expression having a secondary influence on 5hmC levels. That is, a gene transcribed at a similar level in several different tissues may have vastly different levels of 5hmC (>20-fold) dependent on tissue type. Our findings highlight tissue type as a major modifier of 5hmC levels in expressed genes and emphasize the importance of using quantitative analyses in the study of 5hmC levels.
|
|
| 12. |
- Reddington, James P, et al.
(författare)
-
Redistribution of H3K27me3 upon DNA hypomethylation results in de-repression of Polycomb target genes
- 2013
-
Ingår i: Genome Biology. - : Springer Science and Business Media LLC. - 1465-6906 .- 1474-760X. ; 14:3, s. R25-
-
Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: DNA methylation and the Polycomb repression system are epigenetic mechanisms that play important roles in maintaining transcriptional repression. Recent evidence suggests that DNA methylation can attenuate the binding of Polycomb protein components to chromatin and thus plays a role in determining their genomic targeting. However, whether this role of DNA methylation is important in the context of transcriptional regulation is unclear.RESULTS: By genome-wide mapping of the Polycomb Repressive Complex 2-signature histone mark, H3K27me3, in severely DNA hypomethylated mouse somatic cells, we show that hypomethylation leads to widespread H3K27me3 redistribution, in a manner that reflects the local DNA methylation status in wild-type cells. Unexpectedly, we observe striking loss of H3K27me3 and Polycomb Repressive Complex 2 from Polycomb target gene promoters in DNA hypomethylated cells, including Hox gene clusters. Importantly, we show that many of these genes become ectopically expressed in DNA hypomethylated cells, consistent with loss of Polycomb-mediated repression.CONCLUSIONS: An intact DNA methylome is required for appropriate Polycomb-mediated gene repression by constraining Polycomb Repressive Complex 2 targeting. These observations identify a previously unappreciated role for DNA methylation in gene regulation and therefore influence our understanding of how this epigenetic mechanism contributes to normal development and disease.
|
|
| 13. |
- Sproul, Duncan, et al.
(författare)
-
Tissue of origin determines cancer-associated CpG island promoter hypermethylation patterns
- 2012
-
Ingår i: Genome Biology. - : BioMed Central. - 1465-6906 .- 1474-760X. ; 13:10
-
Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Aberrant CpG island promoter DNA hypermethylation is frequently observed in cancer and is believed to contribute to tumor progression by silencing the expression of tumor suppressor genes. Previously, we observed that promoter hypermethylation in breast cancer reflects cell lineage rather than tumor progression and occurs at genes that are already repressed in a lineage-specific manner. To investigate the generality of our observation we analyzed the methylation profiles of 1,154 cancers from 7 different tissue types.RESULTS:We find that 1,009 genes are prone to hypermethylation in these 7 types of cancer. Nearly half of these genes varied in their susceptibility to hypermethylation between different cancer types. We show that the expression status of hypermethylation prone genes in the originator tissue determines their propensity to become hypermethylated in cancer; specifically, genes that are normally repressed in a tissue are prone to hypermethylation in cancers derived from that tissue. We also show that the promoter regions of hypermethylation-prone genes are depleted of repetitive elements and that DNA sequence around the same promoters is evolutionarily conserved. We propose that these two characteristics reflect tissue-specific gene promoter architecture regulating the expression of these hypermethylation prone genes in normal tissues.CONCLUSIONS:As aberrantly hypermethylated genes are already repressed in pre-cancerous tissue, we suggest that their hypermethylation does not directly contribute to cancer development via silencing. Instead aberrant hypermethylation reflects developmental history and the perturbation of epigenetic mechanisms maintaining these repressed promoters in a hypomethylated state in normal cells.
|
|
| 14. |
- Sproul, Duncan, et al.
(författare)
-
Transcriptionally repressed genes become aberrantly methylated and distinguish tumors of different lineages in breast cancer
- 2011
-
Ingår i: Proceedings of the National Academy of Sciences of the United States of America. - : National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 108:11, s. 4364-4369
-
Tidskriftsartikel (refereegranskat)abstract
- Aberrant promoter hypermethylation is frequently observed in cancer. The potential for this mechanism to contribute to tumor development depends on whether the genes affected are repressed because of their methylation. Many aberrantly methylated genes play important roles in development and are bivalently marked in ES cells, suggesting that their aberrant methylation may reflect developmental processes. We investigated this possibility by analyzing promoter methylation in 19 breast cancer cell lines and 47 primary breast tumors. In cell lines, we defined 120 genes that were significantly repressed in association with methylation (SRAM). These genes allowed the unsupervised segregation of cell lines into epithelial (EPCAM+ve) and mesenchymal (EPCAM-ve) lineages. However, the methylated genes were already repressed in normal cells of the same lineage, and >90% could not be derepressed by treatment with 5-aza-2'-deoxycytidine. The tumor suppressor genes APC and CDH1 were among those methylated in a lineage-specific fashion. As predicted by the epithelial nature of most breast tumors, SRAM genes that were methylated in epithelial cell lines were frequently aberrantly methylated in primary tumors, as were genes specifically repressed in normal epithelial cells. An SRAM gene expression signature also correctly identified the rare claudin-low and metaplastic tumors as having mesenchymal characteristics. Our findings implicate aberrant DNA methylation as a marker of cell lineage rather than tumor progression and suggest that, in most cases, it does not cause the repression with which it is associated.
|
|
| 15. |
- Thomson, John P., et al.
(författare)
-
5-Hydroxymethylcytosine Profiling in Human DNA
- 2017
-
Ingår i: Population Epigenetics. - New York, NY : Humana Press. - 9781493969012 - 9781493969036 ; , s. 89-98
-
Bokkapitel (refereegranskat)abstract
- Since its "re-discovery" in 2009, there has been significant interest in defining the genome-wide distribution of DNA marked by 5-hydroxymethylation at cytosine bases (5hmC). In recent years, technological advances have resulted in a multitude of unique strategies to map 5hmC across the human genome. Here we discuss the wide range of approaches available to map this modification and describe in detail the affinity based methods which result in the enrichment of 5hmC marked DNA for downstream analysis.
|
|
| 16. |
- Thomson, John P, et al.
(författare)
-
Non-genotoxic carcinogen exposure induces defined changes in the 5-hydroxymethylome
- 2012
-
Ingår i: Genome Biology. - : BioMed Central. - 1465-6906 .- 1474-760X. ; 13:10
-
Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Induction and promotion of liver cancer by exposure to non-genotoxic carcinogens coincides with epigenetic perturbations, including specific changes in DNA methylation. Here we investigate the genome-wide dynamics of 5-hydroxymethylcytosine (5hmC) as a likely intermediate of 5-methylcytosine (5mC) demethylation in a DNA methylation reprogramming pathway. We use a rodent model of non-genotoxic carcinogen exposure using the drug phenobarbital.RESULTS:Exposure to phenobarbital results in dynamic and reciprocal changes to the 5mC/5hmC patterns over the promoter regions of a cohort of genes that are transcriptionally upregulated. This reprogramming of 5mC/5hmC coincides with characteristic changes in the histone marks H3K4me2, H3K27me3 and H3K36me3. Quantitative analysis of phenobarbital-induced genes that are involved in xenobiotic metabolism reveals that both DNA modifications are lost at the transcription start site, while there is a reciprocal relationship between increasing levels of 5hmC and loss of 5mC at regions immediately adjacent to core promoters.CONCLUSIONS:Collectively, these experiments support the hypothesis that 5hmC is a potential intermediate in a demethylation pathway and reveal precise perturbations of the mouse liver DNA methylome and hydroxymethylome upon exposure to a rodent hepatocarcinogen.
|
|
| 17. |
- Whitelaw, Nadia C, et al.
(författare)
-
Reduced levels of two modifiers of epigenetic gene silencing, Dnmt3a and Trim28, cause increased phenotypic noise
- 2010
-
Ingår i: Genome Biology. - : BioMed Central. - 1465-6906 .- 1474-760X. ; 11:11
-
Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Inbred individuals reared in controlled environments display considerable variance in many complex traits but the underlying cause of this intangible variation has been an enigma. Here we show that two modifiers of epigenetic gene silencing play a critical role in the process.RESULTS: Inbred mice heterozygous for a null mutation in DNA methyltransferase 3a (Dnmt3a) or tripartite motif protein 28 (Trim28) show greater coefficients of variance in body weight than their wild-type littermates. Trim28 mutants additionally develop metabolic syndrome and abnormal behavior with incomplete penetrance. Genome-wide gene expression analyses identified 284 significantly dysregulated genes in Trim28 heterozygote mutants compared to wild-type mice, with Mas1, which encodes a G-protein coupled receptor implicated in lipid metabolism, showing the greatest average change in expression (7.8-fold higher in mutants). This gene also showed highly variable expression between mutant individuals.CONCLUSIONS: These studies provide a molecular explanation of developmental noise in whole organisms and suggest that faithful epigenetic control of transcription is central to suppressing deleterious levels of phenotypic variation. These findings have broad implications for understanding the mechanisms underlying sporadic and complex disease in humans.
|
|
