Project description:DNA methylation profiling of 48 samples to determine a DNA methylation signature specific to leukaemic bone marrow samples. Matching Leukaemia bone marrow and day 28 remission bone marrow or post induction follow up (follow up up to 2 years post induction) genomic DNA were extracted from archived microscope smear slides from 11 children diagnosed with TEL/AML positive Acute Lymphoblastic Leukaemia (ALL). Unmatched bone marrow samples from an additional 8 children were also analysed. Primary tissue control samples from CD34+ and CD19+ bone marrow from adult and childhood samples as well as model cell lines (cancer and non cancerous) were compared to primary diseased samples. Disease specific DNA methylation changes were identified from these results and then verified using SEQUENOM EpiTYPER.
Project description:DNA methylation profiling of 48 samples to determine a DNA methylation signature specific to leukaemic bone marrow samples. Matching Leukaemia bone marrow and day 28 remission bone marrow or post induction follow up (follow up up to 2 years post induction) genomic DNA were extracted from archived microscope smear slides from 11 children diagnosed with TEL/AML positive Acute Lymphoblastic Leukaemia (ALL). Unmatched bone marrow samples from an additional 8 children were also analysed. Primary tissue control samples from CD34+ and CD19+ bone marrow from adult and childhood samples as well as model cell lines (cancer and non cancerous) were compared to primary diseased samples. Disease specific DNA methylation changes were identified from these results and then verified using SEQUENOM EpiTYPER. A total of 48 samples were analysed by Infinium DNA methylation analysis. As per manufacturer's protocols. Replicates were performed on one primary tumour sample and one cell line sample.
Project description:The Illumina Human Methylation EPIC array was used to assess methylation status at initial diagnosis in bone marrow or peripheral blood specimens from children with acute myeloid leukemia.
Project description:The origin of aberrant DNA methylation in cancer remains largely unknown. In this study, we elucidated the DNA methylome in primary Acute Promyelocytic Leukemia (APL) and the role of PML-RARa in establishing these patterns. APL patients showed increased genome-wide DNA methylation with higher variability than healthy CD34+ cells, promyelocytes and remission bone marrow. A core set of differentially methylated regions in APL was identified. Age at diagnosis, Sanz score and Flt3-mutation status characterized methylation subtypes. Transcription factor binding sites, e.g. c-myc binding sites were associated with low methylation. SUZ12 and REST binding sites identified in embryonic stem cells were, however, preferentially DNA hypermethylated in APL. Unexpectedly, PML-RARa binding sites were also protected from aberrant DNA methylation in APL. In line, myeloid cells from pre-leukemic PML-RARa knock-in mice did not show altered DNA methylation and expression of PML-RARa in hematopoietic progenitor cells prevented differentiation without affecting DNA methylation. ATRA treatment of APL blasts did also not result in DNA methylation changes. These results suggest that aberrant DNA methylation is associated with leukemia phenotype but not required for PML-RARa-mediated initiation of leukemogenesis. We used Reduced Representation Bisulfite Sequencing (RRBS) to determine the genome-wide methylation signature of 18 primary APL patient samples. We then compared the APL methylation signature with methylation patterns found in CD34+ progenitor cells (n=4), promyelocytes (n=4) and remission bone marrow samples (n=8). Differentially methylated regions found in all three comparisons (APL vs. all three control specimens) were then further analyzed for genomic localization, variability and association with clinical parameters. Finally, the relationship between differentially methylated regions in APL and specific transcription factor binding sites was analyzed. For this purpose, ChiP-Sequencing of SUZ12 and REST was performed in primary APL patient blasts. To further determine the contribution of the leukemogenic transcription factor PML-RARa to methylation in APL, we also performed RRBS in pre-leukemic PML-RARa knock-in mice and hematopoetic progenitor cells retrovirally transduced with PML-RARa.
Project description:An increasing body of work reveals aberrant hypermethylation of genes occurring in and potentially contributing to the pathogenesis of myeloid malignancies. Several of these diseases, such as myelodysplastic syndromes (MDS), are responsive to DNA methyltransferase inhibitors. In order to determine the extent of promoter hypermethylation in such tumors we compared the distribution of DNA methylation of 14,000 promoters in MDS and secondary AML patients enrolled in a phase I trial of 5-azacytidine and the histone deacetylase inhibitor entinostat against de novo AML patients and normal CD34+ bone marrow cells. The MDS and secondary AML patients displayed more extensive aberrant DNA methylation involving thousands of genes than did the normal CD34+ bone marrow cells or de novo AML blasts. Aberrant methylation in MDS and secondary AML tended to affect particular chromosomal regions, occurred more frequently in Alu poor genes, and included prominent involvement of genes involved in the WNT and MAPK signaling pathways. DNA methylation was also measured at days 15 and 29 after the first treatment cycle. DNA methylation was reversed at day 15 in a uniform manner throughout the genome, and this effect persisted through day 29, even without continuous administration of the study drugs. Keywords: DNA methylation profiling Direct comparison of DNA methylation in bone marrow samples from patients with Myelodysplastic syndrome or secondary Acute Myeloid Leukemia (AML) at baseline and after in vivo treatment with 5-azacytidine + etinostat. A comparison to de novo normal karyotype AML was also performed. Two control groups were included: one consisting of 8 CD34+ bone marrow samples from healthy donors and a second one consisting of matched CD34+ and CD34- fractions from the bone marrows of 4 healthy donors.
Project description:B cells provide humoral immunity by differentiating into antibody-secreting plasma cells, a process that requires cell division and is linked to DNA hypomethylation and gene regulation. Conversely, accumulation of DNA methylation in B cell differentiation is less apparent. To determine the role of de novo DNA methylation in B cell differentiation, the de novo DNA methyltransferases, Dnmt3a and Dnmt3b, were deleted in B cells resulting in phenotypically normal B cell development in the bone marrow, spleen and lymph nodes. However, upon immunologic challenge, mice deficient for Dnmt3a and Dnmt3b (Dnmt3-deficient) accumulated more antigen-specific B cells and bone marrow chimeras showed this was cell-autonomous. Additionally, a five-fold increase in splenic and bone marrow plasma cells was observed. Molecular analysis revealed that Dnmt3-deficient bone marrow plasma cells failed to repress gene expression to the same level as their Dnmt3ab-sufficient counterparts. This was coupled with a failure of Dnmt3-deficient germinal center B cells and plasma cells to gain and/or maintain DNA methylation at several thousand loci that were clustered in enhancers of genes that function in B cell activation and homing. Analysis of chromatin accessibility showed Dnmt3-deficient plasma cells had increased accessibility at several genes involved in hematopoiesis and B cell differentiation. These data show that de novo DNA methylation limits B cell activation, proliferation and differentiation, and support a model whereby DNA methylation represses the aberrant transcription of genes silenced in B cell differentiation to maintain plasma cell homeostasis.
Project description:B cells provide humoral immunity by differentiating into antibody-secreting plasma cells, a process that requires cell division and is linked to DNA hypomethylation and gene regulation. Conversely, accumulation of DNA methylation in B cell differentiation is less apparent. To determine the role of de novo DNA methylation in B cell differentiation, the de novo DNA methyltransferases, Dnmt3a and Dnmt3b, were deleted in B cells resulting in phenotypically normal B cell development in the bone marrow, spleen and lymph nodes. However, upon immunologic challenge, mice deficient for Dnmt3a and Dnmt3b (Dnmt3-deficient) accumulated more antigen-specific B cells and bone marrow chimeras showed this was cell-autonomous. Additionally, a five-fold increase in splenic and bone marrow plasma cells was observed. Molecular analysis revealed that Dnmt3-deficient bone marrow plasma cells failed to repress gene expression to the same level as their Dnmt3ab-sufficient counterparts. This was coupled with a failure of Dnmt3-deficient germinal center B cells and plasma cells to gain and/or maintain DNA methylation at several thousand loci that were clustered in enhancers of genes that function in B cell activation and homing. Analysis of chromatin accessibility showed Dnmt3-deficient plasma cells had increased accessibility at several genes involved in hematopoiesis and B cell differentiation. These data show that de novo DNA methylation limits B cell activation, proliferation and differentiation, and support a model whereby DNA methylation represses the aberrant transcription of genes silenced in B cell differentiation to maintain plasma cell homeostasis.
Project description:B cells provide humoral immunity by differentiating into antibody-secreting plasma cells, a process that requires cell division and is linked to DNA hypomethylation and gene regulation. Conversely, accumulation of DNA methylation in B cell differentiation is less apparent. To determine the role of de novo DNA methylation in B cell differentiation, the de novo DNA methyltransferases, Dnmt3a and Dnmt3b, were deleted in B cells resulting in phenotypically normal B cell development in the bone marrow, spleen and lymph nodes. However, upon immunologic challenge, mice deficient for Dnmt3a and Dnmt3b (Dnmt3-deficient) accumulated more antigen-specific B cells and bone marrow chimeras showed this was cell-autonomous. Additionally, a five-fold increase in splenic and bone marrow plasma cells was observed. Molecular analysis revealed that Dnmt3-deficient bone marrow plasma cells failed to repress gene expression to the same level as their Dnmt3ab-sufficient counterparts. This was coupled with a failure of Dnmt3-deficient germinal center B cells and plasma cells to gain and/or maintain DNA methylation at several thousand loci that were clustered in enhancers of genes that function in B cell activation and homing. Analysis of chromatin accessibility showed Dnmt3-deficient plasma cells had increased accessibility at several genes involved in hematopoiesis and B cell differentiation. These data show that de novo DNA methylation limits B cell activation, proliferation and differentiation, and support a model whereby DNA methylation represses the aberrant transcription of genes silenced in B cell differentiation to maintain plasma cell homeostasis.
Project description:TET2 is a close relative of TET1, an enzyme that converts 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in DNA. The gene encoding TET2 resides at chromosome 4q24, in a region showing recurrent microdeletions and copy-neutral loss of heterozygosity (CN-LOH) in patients with diverse myeloid malignancies. Somatic TET2 mutations are frequently observed in myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), MDS/MPN overlap syndromes including chronic myelomonocytic leukaemia (CMML), acute myeloid leukaemias (AML) and secondary AML (sAML). We show here that TET2 mutations associated with myeloid malignancies compromise catalytic activity. Bone marrow samples from patients with TET2 mutations displayed uniformly low levels of 5hmC in genomic DNA compared to bone marrow samples from healthy controls. Moreover, small hairpin RNA (shRNA)-mediated depletion of Tet2 in mouse haematopoietic precursors skewed their differentiation towards monocyte/macrophage lineages in culture. There was no significant difference in DNA methylation between bone marrow samples from patients with high 5hmC versus healthy controls, but samples from patients with low 5hmC showed hypomethylation relative to controls at the majority of differentially methylated CpG sites. Our results demonstrate that Tet2 is important for normal myelopoiesis, and suggest that disruption of TET2 enzymatic activity favours myeloid tumorigenesis. Measurement of 5hmC levels in myeloid malignancies may prove valuable as a diagnostic and prognostic tool, to tailor therapies and assess responses to anticancer drugs. Genome wide DNA methylation profiling of patients samples with various myeloid malignancies and diffrential levels of 5hmC.The Illumina Infinium 27k Human DNA methylation Beadchip v1.2 was used to obtain DNA methylation profiles across approximately 27,000 CpGs in bone marrow samples and occasionally peripheral blood samples. Samples included 28 control healthy bone marrows, 29 patients samples with low 5hmC levels (7 patients with wild-type TET2 and 22 mutant TET2) and 24 with high levels of 5hmC (22 with wild-type TET2 and 2 mutant TET2). Bisulphite converted DNA from 81 samples was hybridised to the Illumina Infinium 27k Human Methylation Beadchip v1.2
Project description:Here we used Illumina NGS for high-throughput profiling of the DNA methylome in two human colon cancer derived cell lines, two human normal bone marrow CD34+ controls and in five human Acutre Myeloid Leukeima patient samples. These data can be used to determine the CpG cytosine methylation pattern at base pair resolution in each sample and to determine differentially methylated cytosines and regions between samples Reduced Representation Bisulfite Sequencing (RRBS) and Extended Reduced Representation Bisulfite Sequencing (ERRBS) on genomic DNA. We used colon cancer cell lines (two) to establish reproducbility and range of assay sensitivity. We used Acute Myeloid Leukemia patient samples and CD34+ bone marrow cells as controls to determine the methylome pattern in the patient samples