Investigation of mutational signatures associated with DNMT3A deficiency
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ABSTRACT:
PROVIDER: EGAS00001002329 | EGA |
REPOSITORIES: EGA
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Project description:Mutational signatures have been shown to be attributable to specific genetic contexts, such as mutations in DNA repair genes. DNMT3A is a DNA methyltransferase that helps maintain the DNA methylation pattern in a site-specific manner and may participate in DNA repair or the stress response. We have identified an adult individual who is a germline mosaic for a DNMT3A mutation. We have obtained clonal lymphoblastoid cells (LCLs) from the subject representing both WT and mutant lines grown in the same individual for >50 years. These clones represent a unique opportunity to examine the mutational impact of the DNMT3A mutation in a well-controlled setting. Our goal is to perform WGS on whole blood, representing the pool, as well as several WT and several mutant clones, in order to investigate the contribution of DNMT3A to mutation rates and signatures. .
This dataset contains all the data available for this study on 2019-04-03.
| EGAD00001004889 | EGA
Project description:DNA methyltransferase 3A (DNMT3A) is the most frequently mutated gene in clonal hematopoiesis (CH) and serves as a key tumor suppressor in the hematopoietic system. Somatic mutations in DNMT3A arise in hematopoietic stem and progenitor cells (HSPCs) many years before malignancies develop, but how they prime cells for transformation remains unknown, partly due to our inability to compare mutant to equivalent wild-type cells before frank disease. To overcome this challenge, we derived multiple normal and DNMT3A-mutant lymphoblastoid cell clones from a germ-line mosaic individual in whom these cells co-existed for nearly six decades. Although arising early in gestation, mutant cells dominated the blood, but not other tissues. Deep sequencing revealed similar mutational burdens and mutational signatures across normal and mutant clones. In contrast, epigenetic profiling uncovered focal erosion of DNA methylation at oncogenic regulatory regions in mutant clones. These regions overlapped with those sensitive to DNMT3A loss after DNMT3A ablation in HSPCs and with those present in leukemia samples. These results suggest that DNMT3A is responsible for maintaining a conserved DNA methylation pattern that is maintained for decades and across cell types and states. Erosion of this pattern confers a distinct competitive advantage, increasing the likelihood of malignant transformation
2020-07-15 | GSE150515 | GEO
Project description:DNA double strand breaks (DSBs) are a major source of mutations. Both non-homologous-end-joining (NHEJ) and microhomology-mediated-end-joining (MMEJ) DSB repair pathways are error prone and produce deletions, which can lead to cancer. DSBs also lead to epigenetic changes, including demethylation, which is involved in carcinogenesis. Of specific interest is the MMEJ repair pathway, as it requires methylation restoration around the break, as a result of the resection and formation of single stranded (ssDNA) intermediates. While, methylation patterns after homologous recombination (HR) have been partially studied, the methylation status after MMEJ and NHEJ remains poorly reported, and can be relevant for cancer. To study methylation patterns around DSB after NHEJ and MMEJ repair, we used targeted bisulfite-sequencing (BS-seq) to quantify methylation of dozens of single cell clones after induction of DSB by CRISPR. Each single cell clone was classified according to the sequence signature to a specific repair mechanism: NHEJ or MMEJ. Comparison of single cell clones after DSB to control cells, without DSB, demonstrated correct restoration of the methylation levels. No difference in methylation patterns was noticed when comparing NHEJ to MMEJ. Methylation levels in gene body, highly methylated CpGs (n=61, 4000 base pairs around DSB) and in low methylation CpGs (n=19), remained stable after both MMEJ and NHEJ. Gene body methylation persisted even on the background of DNMT3A R882C mutation, the most prevalent preleukemic mutation, in which the de novo methylation machinery is compromised. An exception observed in a single CpG site (ASXL1 995) which demonstrated elevated methylation rate after DSB repair only in the presence of WT DNMT3A. In summary, DNA methylation restoration demonstrated high fidelity after DSB both in methylated and unmethylated gene body, even in cases where DNA resections and deletions occurred.
2022-09-11 | E-MTAB-12037 | biostudies-arrayexpress
Project description:DNA double strand breaks (DSBs) are a major source of mutations. Both non-homologous-end-joining (NHEJ) and microhomology-mediated-end-joining (MMEJ) DSB repair pathways are error prone and produce deletions, which can lead to cancer. DSBs also lead to epigenetic changes, including demethylation, which is involved in carcinogenesis. Of specific interest is the MMEJ repair pathway, as it requires methylation restoration around the break, as a result of the resection and formation of single stranded (ssDNA) intermediates. While, methylation patterns after homologous recombination (HR) have been partially studied, the methylation status after MMEJ and NHEJ remains poorly reported, and can be relevant for cancer. To study methylation patterns around DSB after NHEJ and MMEJ repair, we used targeted bisulfite-sequencing (BS-seq) to quantify methylation of dozens of single cell clones after induction of DSB by CRISPR. Each single cell clone was classified according to the sequence signature to a specific repair mechanism: NHEJ or MMEJ. Comparison of single cell clones after DSB to control cells, without DSB, demonstrated correct restoration of the methylation levels. No difference in methylation patterns was noticed when comparing NHEJ to MMEJ. Methylation levels in gene body, highly methylated CpGs (n=61, 4000 base pairs around DSB) and in low methylation CpGs (n=19), remained stable after both MMEJ and NHEJ. Gene body methylation persisted even on the background of DNMT3A R882C mutation, the most prevalent preleukemic mutation, in which the de novo methylation machinery is compromised. An exception observed in a single CpG site (ASXL1 995) which demonstrated elevated methylation rate after DSB repair only in the presence of WT DNMT3A. In summary, DNA methylation restoration demonstrated high fidelity after DSB both in methylated and unmethylated gene body, even in cases where DNA resections and deletions occurred.
2022-08-25 | E-MTAB-12061 | biostudies-arrayexpress
Project description:Purpose: DNA methyltransferase 3A (DNMT3A) mediates de novo DNA methylation. Mutations in DNMT3A are associated with hematological malignancies, most frequently acute myeloid leukemia. DNMT3A mutations are hypothesized to establish a pre-leukemic state, rendering cells vulnerable to secondary oncogenic mutations and malignant transformation. However, the mechanisms by which DNMT3A mutations contribute to leukemogenesis are not well-defined. Methods: Methylated DNA immunoprecipitation (MeDIP) was used to detect regional differences in DNA methylation between DNMT3A WT and MT clones. DNA extraction of WTblk, WT1, MT1, MT2, and MT4 cell lines was performed as previously described. 5-mC profiling, fragmentation, library preparation and sequencing were performed by Otogenetics (Atlanta, GA, USA). In brief, paired-end libraries were generated using the Illumina TruSeq DNA sample preparation kit. Illumina HiSeq2500 was used for sequencing with a paired-end sequencing length of 100-125 bp and approximately 40 million reads per sample. Results: We attempted to identify differentially methylated genes based on DNMT3A mutation status and found very few regions of differential methylation between WT and MT cell lines (Figure 3C), which did not exceed the number of regions expected to exhibit differential methylation by chance alone. These data suggest that regional methylation patterns do not differ between DNMT3A MT and WT cell lines in our gene-edited K562 cells. Conclusions: CRISPR/Cas9-mediated DNMT3A-mutated K562 cells may be used to model effects of DNMT3A mutations in human cells. Our findings implicate aberrant splicing and induction of genomic instability as potential mechanisms by which DNMT3A mutations might predispose to malignancy.
2018-01-09 | GSE96577 | GEO
Project description:Despite the impact of DNMT3A mutation in acute myeloid leukemia has been emphasized, the precise molecular mechanisms in leukemogenesis are largely unknown. Here we show that, in murine transplantation experiments, recipients transplanted with DNMT3A mutant-transduced cells exhibit aberrant hematopoietic stem cell (HSC) accumulation. Differentiation-associated genes are down-regulated without accompanying changes in methylation status of their promoter-associated CpG islands in DNMT3A mutant-transduced stem/progenitor cells. DNMT3A mutant also promotes monoblastic transformation in vitro in combination with HOXA9. Molecularly, DNMT3A mutant interacts with polycomb repressive complex 1 (PRC1), leading to transcriptional silencing of PU.1. Suppression of PRC1 impairs aberrant HSC accumulation and monoblastic transformation. Taken together, our results highlight the functional role of DNMT3A mutation, forming the basis for leukemia development. GFP-labeled empty vector, DNMT3A wild-type (WT), R882H-transduced LSK cells derived from transplanted mice were utilized for compared the expression profiles (3 sorted empty vector-transduced LSK cells, 3 sorted DNMT3A WT-transduced LSK cells, and 3 sorted DNMT3A R882H-transduced LSK cells. Total RNA was extracted by TaKaRa NucleoSpin RNA XS according to the manufacturerâs protocol. Amplification and biotin labeling of fragmented cDNA was carried out from 3.67 ng of total RNA by using NuGen Ovation Pico WTA System V2 (NuGEN) and SureTag Complete DNA Labeling Kit (Agilent). Each 2 μg of cDNA were hybridized to the Agilent SurePrint G3 Mouse Gene Expression 8x60K (Agilent) using Gene Expression Hybridization Kit (Agilent). After scanning, the signal intensity for each feature was measured by Agilent Feature Extraction (Agilent).
2016-02-10 | E-GEOD-55262 | biostudies-arrayexpress
Project description:DNA Methyltransferase 3A (DNMT3A) is frequently mutated in various hematopoietic malignancies; however, the underlying oncogenic mechanisms remain elusive. Here, we report that DNMT3A mutational âhotspotâ at Arg882 (DNMT3A-R882H) cooperates with constitutively activated RAS in transforming murine hematopoietic stem/progenitor cells (HSPCs) ex vivo and inducing acute leukemias in vivo. DNMT3A-R882H potentiates aberrant transactivation of âstemnessâ gene expression programs, notably transcription factors Meis1, Hox-A, Mn1 and Mycn. Mechanistically, R882-mutated DNMT3A directly binds to cis-regulatory elements of these genes and induces focal CpG hypomethylation reminiscent of what was seen in human leukemias bearing DNMT3A R882 mutation. Furthermore, DNMT3A-R882H induced DNA hypomethylation facilitates gene enhancer/promoter activation and recruitment of Dot1l-associated transcription elongation machineries. Inactivation of Dot1l represses DNMT3AR882H-mediated stem cell gene dysregulation and acute leukemogenicity. In this dataset, we provided enhanced Reduced Representation Bisulfite Sequencing (eRRBS) DNA methylome profiling data showing effect of DNMT3A R882H mutation or WT expression on hematopoietic stem/progenitor cells with NRAS G12D co-transduction. eRRBBs DNA methylome analysis of Lin- enriched hematopoietic stem/progenitor cells with retroviral infection of NRAS G12D alone (EV-RAS), DNMT3A R882H with NRAS G12D (RH-RAS) or DNMT3A WT with NRAS G12D (WT-RAS) at day 16 post-transduction.
2016-06-23 | E-GEOD-71473 | biostudies-arrayexpress
Project description:Peripheral T-cell lymphomas (PTCLs) are heterogenous T-cell neoplasms often associated with epigenetic dysregulation. We investigated de novo DNA methyltransferase 3A (DNMT3A) mutations in common PTCL entities, including angioimmunoblastic T-cell lymphoma and novel molecular subtypes identified within PTCL-not otherwise specified (PTCL-NOS) designated as PTCL-GATA3 and PTCL-TBX21. DNMT3A-mutated PTCL-TBX21 cases showed inferior overall-survival, with DNMT3A mutated residues skewed towards the methyltransferase domain and dimerization motif (S881–R887). Transcriptional profiling demonstrated significant enrichment of activated CD8+ T-cell cytotoxic gene signatures in the DNMT3A-mutant PTCL-TBX21 cases, which was further validated using immunohistochemistry. Genome-wide methylation analysis of DNMT3A-mutant versus wild-type PTCL-TBX21 cases demonstrated hypomethylation in target genes regulating IFN-g, TCR signaling and EOMES, a master transcriptional regulator of cytotoxic effector cells. Similar findings were observed in a murine model of PTCL with Dnmt3a loss (in-vivo) and further validated in-vitro by ectopic expression of DNMT3A-mutants (DNMT3A-R882, -Q886, -V716, versus WT) in CD8+T-cell line, resulting in T-cell activation and EOMES upregulation. Furthermore, stable, ectopic expression of the DNMT3A-mutants in primary CD3+ T-cell cultures resulted in the preferential outgrowth of CD8+ T-cells with DNMT3AR882H mutation. Single-cell-RNA-seq analysis of CD3+ T-cells revealed differential CD8+ T-cell subset polarization, mirroring findings in DNMT3A-mutated PTCL-TBX21 and validating the cytotoxic and T-cell memory transcriptional programs associated with the DNMT3AR882H mutation. Our findings indicate that DNMT3A mutations define a cytotoxic subset in PTCL-TBX21 with prognostic significance, thus may further refine pathological heterogeneity in PTCL-NOS and suggest alternative treatment strategies for this subset.
2022-05-29 | GSE204876 | GEO
Project description:Peripheral T-cell lymphomas (PTCLs) are heterogenous T-cell neoplasms often associated with epigenetic dysregulation. We investigated de novo DNA methyltransferase 3A (DNMT3A) mutations in common PTCL entities, including angioimmunoblastic T-cell lymphoma and novel molecular subtypes identified within PTCL-not otherwise specified (PTCL-NOS) designated as PTCL-GATA3 and PTCL-TBX21. DNMT3A-mutated PTCL-TBX21 cases showed inferior overall-survival, with DNMT3A mutated residues skewed towards the methyltransferase domain and dimerization motif (S881–R887). Transcriptional profiling demonstrated significant enrichment of activated CD8+ T-cell cytotoxic gene signatures in the DNMT3A-mutant PTCL-TBX21 cases, which was further validated using immunohistochemistry. Genome-wide methylation analysis of DNMT3A-mutant versus wild-type PTCL-TBX21 cases demonstrated hypomethylation in target genes regulating IFN-g, TCR signaling and EOMES, a master transcriptional regulator of cytotoxic effector cells. Similar findings were observed in a murine model of PTCL with Dnmt3a loss (in-vivo) and further validated in-vitro by ectopic expression of DNMT3A-mutants (DNMT3A-R882, -Q886, -V716, versus WT) in CD8+T-cell line, resulting in T-cell activation and EOMES upregulation. Furthermore, stable, ectopic expression of the DNMT3A-mutants in primary CD3+ T-cell cultures resulted in the preferential outgrowth of CD8+ T-cells with DNMT3AR882H mutation. Single-cell-RNA-seq analysis of CD3+ T-cells revealed differential CD8+ T-cell subset polarization, mirroring findings in DNMT3A-mutated PTCL-TBX21 and validating the cytotoxic and T-cell memory transcriptional programs associated with the DNMT3AR882H mutation. Our findings indicate that DNMT3A mutations define a cytotoxic subset in PTCL-TBX21 with prognostic significance, thus may further refine pathological heterogeneity in PTCL-NOS and suggest alternative treatment strategies for this subset.
2022-05-29 | GSE204875 | GEO
Project description:DNA methyltransferase 3A (DNMT3A) gene is mutated in various myeloid neoplasms including acute myeloid leukemia (AML), especially at the Arg882 and associated with inferior outcomes. Despite the current progress of functional role of DNMT3A mutations, the molecular pathogenesis of myeloid malignancies remains poorly understood. The mechanisms of AML transformation and functional role of DNMT3A mutations through its target genes in the leukemogenesis remain to be explored. Here we wished to perform the differential gene expression profile in U937 cells over-expressed with DNMT3A-Arg882His/Cys (R882H/C) mutations including DNMT3A-WT and vector. Results: Gene expression profiling analysis revealed aberrant expression of several cell-cycle and apoptosis-related genes in U937 cells transduced with mutant DNMT3A compared to WT- or vector control.
2017-12-01 | GSE90931 | GEO