scholarly journals VHL inactivation without hypoxia is sufficient to achieve genome hypermethylation

2016 ◽  
Author(s):  
Artem V. Artemov ◽  
Nadezhda Zhigalova ◽  
Svetlana Zhenilo ◽  
Alexander M. Mazur ◽  
Egor B. Prokhortchouk
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Binding Sites ◽  
Promoter Hypermethylation ◽  
Gene Promoters ◽  
Dna Hypermethylation ◽  
Hypoxic Conditions ◽  
Transcription Regulators

AbstractVHL inactivation is a key oncogenic event for renal carcinomas. In normoxia, VHL suppresses HIF1a-mediated response to hypoxia. It has previously been shown that hypoxic conditions inhibit TET-dependent hydroxymethylation of cytosines and cause DNA hypermethylation at gene promoters. In this work, we performed VHL inactivation by CRISPR/Cas9 and studied its effects on gene expression and DNA methylation. We showed that even without hypoxia, VHL inactivation leads to hypermethylation of the genome which mainly occurred in AP-1 and TRIM28 binding sites. We also observed promoter hypermethylation of several transcription regulators associated with decreased gene expression.

scholarly journals CRISPR/Cas9-Targeted De Novo DNA Methylation Is Maintained and Impacts the Colony Forming Potential of Human Hematopoietic CD34+ Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2517-2517
Author(s):  
Emily A Saunderson ◽  
Kevin Rouault-Pierre ◽  
John G. Gribben ◽  
Gabriella Ficz
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Research Funding ◽  
De Novo ◽  
Gene Promoter ◽  
Promoter Hypermethylation ◽  
Gene Promoters ◽  
Dna Hypermethylation ◽  
Human Cd34 ◽  
Cd34 Cells

Introduction The epigenome is significantly perturbed in hematological malignancies with global DNA hypomethylation and localized hypermethylation of gene promoter CpG islands. Whether specific gene promoter hypermethylation can contribute to the clonal expansion of hematopoietic stem and progenitor cells (HSPCs) in humans by affecting HSPC biology, independently of genetic mutations, has not previously been investigated due to the lack of appropriate tools. We show for the first time that it is possible to target de novo DNA methylation using CRISPR/Cas9 in human CD34+ cells isolated from cord blood (CB). DNA methylation targeted to key cell cycle control gene promoters, INK4b (p15) and ARF (p14), is permanently maintained after dCas9 3A3L degradation and inherited as cells differentiate; inhibiting gene expression and affecting the colony forming potential of CD34+ cells. This demonstrates that specific DNA hypermethylation events can permanently change HSPC biology and impact differentiation, potentially contributing to pre-malignant processes. Methods Human CD34+ HSPCs were isolated from human CB and maintained in liquid culture for 24 hours before nucleofection with mRNA encoding an adapted form of CRISPR/Cas9 which has no nuclease activity (dCas9) and is fused to the catalytic domain of DNA methyltransferase 3A (DNMT3A) and 3L (3A3L). The nucleofection cocktail contained dCas9 3A3L or dCas9 3A3L-mut (lacks methyltransferase activity) and 1 to 3 guide RNAs to target DNA methylation to combinations of the INK4a-ARF-INK4b locus. Cells were then seeded into methylcellulose for a primary colony forming assay (CFU). Colonies were scored after 14 days and cells were either harvested and pooled or individual colonies were picked for single-colony molecular analyses. The DNA was extracted and methylation at the INK4a-ARF-INK4b promoters was quantified using targeted bisulfite sequencing; target gene expression was measured using qPCR. The remaining cells from the primary CFU were re-plated a second (secondary CFU) and third (tertiary CFU) time and colonies were again scored after 14 days. Results and Conclusions Targeting DNA methylation to the INK4a-ARF-INK4b locus or INK4b individually in human CD34+ cells resulted in maintenance of hypermethylation at ARF and/or INK4b gene promoters in individual BFU-E (burst-forming unit-erythroid) and CFU-GM (granulocyte, macrophage) colonies as measured by single-colony targeted bisulfite sequencing after the primary CFU; causing heritable repression of INK4b gene expression in the differentiated cells. Some CpGs were up to 90% methylated, indicating that DNA methylation added at these gene promoters is highly stable as cells differentiate. Hypermethylation of ARF and INK4b was found in some colonies even after the tertiary CFU, demonstrating long-term maintenance of promoter hypermethylation. Unexpectedly, no DNA hypermethylation was detected at INK4a in differentiated cells, but whether this is the case for all subpopulations of HSPCs (i.e. HSCs or lymphoid progenitors) is under investigation. Hypermethylation of INK4b and ARF increased the colony forming potential of CD34+ cells in primary, secondary and tertiary CFUs, compared to the control. Conversely, methylation targeted to INK4b alone did not significantly affect the number of colonies in the first CFU, and decreased the number of colonies in the secondary CFU. This suggests a complex interplay between key cell cycle regulators ARF and INK4b in CD34+ cells and during differentiation which can be disrupted by DNA hypermethylation and gene repression. These findings demonstrate the novel insights we can gain by using CRISPR/Cas9 tools to target DNA methylation and these investigations will reveal how gene promoter hypermethylation can impact HSPC function. Furthermore, studying this locus may uncover an important role for DNA hypermethylation in the development of myeloid malignancies, since INK4b is frequently hypermethylated, but rarely mutated, in myeloid dysplastic/proliferative neoplasms and acute myeloid leukemia. Disclosures Gribben: Janssen: Consultancy, Honoraria, Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria, Research Funding; Acerta/Astra Zeneca: Consultancy, Honoraria, Research Funding.

scholarly journals Deletion of Tet proteins results in quantitative disparities during ESC differentiation partially attributable to alterations in gene expression

2019 ◽  
Author(s):  
Michael J Reimer ◽  
Kirthi Pulakanti ◽  
Linzheng Shi ◽  
Alex Abel ◽  
Mingyu Liang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Critical Role ◽  
Embryonic Stem ◽  
Dna Demethylation ◽  
Intermediate Phenotype ◽  
Gene Promoters ◽  
Dna Hypermethylation ◽  
Tet Proteins ◽  
Tet Protein

Abstract Background: The Tet protein family (Tet1, Tet2, and Tet3) regulate DNA methylation through conversion of 5-methylcytosine to 5-hydroxymethylcytosine which can ultimately result in DNA demethylation and play a critical role during early mammalian development and pluripotency¬. While multiple groups have generated knockouts combining loss of different Tet proteins in murine embryonic stem cells (ESCs), differences in genetic background and approaches has made it difficult to directly compare results and discern the direct mechanism by which Tet proteins regulate the transcriptome. To address this concern, we utilized genomic editing in an isogenic pluripotent background which permitted a quantitative, flow-cytometry based measurement of pluripotency in combination with genome-wide assessment of gene expression and DNA methylation changes. Our ultimate goal was to generate a resource of large-scale datasets to permit hypothesis-generating experiments. Results: We demonstrate a quantitative disparity in the differentiation ability among Tet protein deletions, with Tet2 single knockout exhibiting the most severe defect, while loss of Tet1 ¬alone or combinations of Tet genes showed a quantitatively intermediate phenotype. Using a combination of transcriptomic and epigenomic approaches we demonstrate an increase in DNA hypermethylation and a divergence of transcriptional profiles in pluripotency among Tet deletions, with loss of Tet2 having the most profound effect in undifferentiated ESCs. Conclusions: We conclude that loss of Tet2 has the most dramatic effect both on the phenotype of ESCs and the transcriptome compared to other genotypes. While loss of Tet proteins increased DNA hypermethylation, especially in gene promoters, these changes in DNA methylation did not correlate with gene expression changes. Thus, while loss of different Tet proteins alters DNA methylation, this change does not appear to be directly responsible for transcriptome changes. Thus, loss of Tet proteins likely regulates the transcriptome epigenetically both through altering 5mC but also through additional mechanisms. Nonetheless, the transcriptome changes in pluripotent Tet2-/- ESCs compared to wild-type implies that the disparities in differentiation can be partially attributed to baseline alterations in gene expression.

scholarly journals Deletion of Tet proteins results in quantitative disparities during ESC differentiation partially attributable to differences in gene expression

2019 ◽  
Author(s):  
Michael J Reimer ◽  
Kirthi Pulakanti ◽  
Linzheng Shi ◽  
Alex Abel ◽  
Mingyu Liang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Critical Role ◽  
Embryonic Stem ◽  
Dna Demethylation ◽  
Intermediate Phenotype ◽  
Gene Promoters ◽  
Dna Hypermethylation ◽  
Tet Proteins ◽  
Tet Protein

Abstract Background: The Tet protein family (Tet1, Tet2, and Tet3) regulate DNA methylation through conversion of 5-methylcytosine to 5-hydroxymethylcytosine which can ultimately result in DNA demethylation and play a critical role during early mammalian development and pluripotency¬. While multiple groups have generated knockouts combining loss of different Tet proteins in murine embryonic stem cells (ESCs), differences in genetic background and approaches has made it difficult to directly compare results and discern the direct mechanism by which Tet proteins regulate the transcriptome. To address this concern, we utilized genomic editing in an isogenic pluripotent background which permitted a quantitative, flow-cytometry based measurement of pluripotency in combination with genome-wide assessment of gene expression and DNA methylation changes. Our ultimate goal was to generate a resource of large-scale datasets to permit hypothesis-generating experiments. Results: We demonstrate a quantitative disparity in the differentiation ability among Tet protein deletions, with Tet2 single knockout exhibiting the most severe defect, while loss of Tet1 ¬alone or combinations of Tet genes showed a quantitatively intermediate phenotype. Using a combination of transcriptomic and epigenomic approaches we demonstrate an increase in DNA hypermethylation and a divergence of transcriptional profiles in pluripotency among Tet deletions, with loss of Tet2 having the most profound effect in undifferentiated ESCs. Conclusions: We conclude that loss of Tet2 has the most dramatic effect both on the phenotype of ESCs and the transcriptome compared to other genotypes. While loss of Tet proteins increased DNA hypermethylation, especially in gene promoters, these changes in DNA methylation did not correlate with gene expression changes. Thus, while loss of different Tet proteins alters DNA methylation, this change does not appear to be directly responsible for transcriptome changes. Thus, loss of Tet proteins likely regulates the transcriptome epigenetically both through altering 5mC but also through additional mechanisms. Nonetheless, the transcriptome changes in pluripotent Tet2-/- ESCs compared to wild-type implies that the disparities in differentiation can be partially attributed to baseline alterations in gene expression.

scholarly journals Deletion of Tet proteins results in quantitative disparities during ESC differentiation partially attributable to alterations in gene expression

2019 ◽  
Author(s):  
Michael J Reimer ◽  
Kirthi Pulakanti ◽  
Linzheng Shi ◽  
Alex Abel ◽  
Mingyu Liang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Critical Role ◽  
Embryonic Stem ◽  
Dna Demethylation ◽  
Intermediate Phenotype ◽  
Gene Promoters ◽  
Dna Hypermethylation ◽  
Tet Proteins ◽  
Tet Protein

Abstract Background: The Tet protein family (Tet1, Tet2, and Tet3) regulate DNA methylation through conversion of 5-methylcytosine to 5-hydroxymethylcytosine which can ultimately result in DNA demethylation and play a critical role during early mammalian development and pluripotency¬. While multiple groups have generated knockouts combining loss of different Tet proteins in murine embryonic stem cells (ESCs), differences in genetic background and approaches has made it difficult to directly compare results and discern the direct mechanism by which Tet proteins regulate the transcriptome. To address this concern, we utilized genomic editing in an isogenic pluripotent background which permitted a quantitative, flow-cytometry based measurement of pluripotency in combination with genome-wide assessment of gene expression and DNA methylation changes. Our ultimate goal was to generate a resource of large-scale datasets to permit hypothesis-generating experiments. Results: We demonstrate a quantitative disparity in the differentiation ability among Tet protein deletions, with Tet2 single knockout exhibiting the most severe defect, while loss of Tet1 ¬alone or combinations of Tet genes showed a quantitatively intermediate phenotype. Using a combination of transcriptomic and epigenomic approaches we demonstrate an increase in DNA hypermethylation and a divergence of transcriptional profiles in pluripotency among Tet deletions, with loss of Tet2 having the most profound effect in undifferentiated ESCs. Conclusions: We conclude that loss of Tet2 has the most dramatic effect both on the phenotype of ESCs and the transcriptome compared to other genotypes. While loss of Tet proteins increased DNA hypermethylation, especially in gene promoters, these changes in DNA methylation did not correlate with gene expression changes. Thus, while loss of different Tet proteins alters DNA methylation, this change does not appear to be directly responsible for transcriptome changes. Thus, loss of Tet proteins likely regulates the transcriptome epigenetically both through altering 5mC but also through additional mechanisms. Nonetheless, the transcriptome changes in pluripotent Tet2-/- ESCs compared to wild-type implies that the disparities in differentiation can be partially attributed to baseline alterations in gene expression.

Abstract 40: Disturbed Flow Alters Genomewide DNA Methylation Patterns, Regulating Endothelial Gene Expression and Atherosclerosis

2014 ◽  
Vol 34 (suppl_1) ◽  
Author(s):  
Jessilyn Dunn ◽  
Haiwei Qiu ◽  
Soyeon Kim ◽  
Daudi Jjingo ◽  
Ryan Hoffman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Dna Methyltransferase ◽  
Methylation Status ◽  
Western Diet ◽  
Dependent Manner ◽  
Gene Promoters ◽  
Genome Wide ◽  
Methylation Patterns ◽  
Endothelial Gene Expression

Atherosclerosis preferentially occurs in arterial regions of disturbed blood flow (d-flow), which alters gene expression, endothelial function, and atherosclerosis. Here, we show that d-flow regulates genome-wide DNA methylation patterns in a DNA methyltransferase (DNMT)-dependent manner. We found that d-flow induced expression of DNMT1, but not DNMT3a or DNMT3b, in mouse arterial endothelium in vivo and in cultured endothelial cells by oscillatory shear (OS) compared to unidirectional laminar shear in vitro. The DNMT inhibitor 5-Aza-2’deoxycytidine (5Aza) or DNMT1 siRNA significantly reduced OS-induced endothelial inflammation. Moreover, 5Aza reduced lesion formation in two atherosclerosis models using ApoE-/- mice (western diet for 3 months and the partial carotid ligation model with western diet for 3 weeks). To identify the 5Aza mechanisms, we conducted two genome-wide studies: reduced representation bisulfite sequencing (RRBS) and transcript microarray using endothelial-enriched gDNA and RNA, respectively, obtained from the partially-ligated left common carotid artery (LCA exposed to d-flow) and the right contralateral control (RCA exposed to s-flow) of mice treated with 5Aza or vehicle. D-flow induced DNA hypermethylation in 421 gene promoters, which was significantly prevented by 5Aza in 335 genes. Systems biological analyses using the RRBS and the transcriptome data revealed 11 mechanosensitive genes whose promoters were hypermethylated by d-flow but rescued by 5Aza treatment. Of those, five genes contain hypermethylated cAMP-response-elements in their promoters, including the transcription factors HoxA5 and Klf3. Their methylation status could serve as a mechanosensitive master switch in endothelial gene expression. Our results demonstrate that d-flow controls epigenomic DNA methylation patterns in a DNMT-dependent manner, which in turn alters endothelial gene expression and induces atherosclerosis.

scholarly journals Integrated Analysis of Global DNA Methylation and Transcription Reveals a Leukemia Subtype with Extreme Hypermethylation Associated with Silencing of Regulators of Differentiation

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2608-2608
Author(s):  
Claudia Gebhard ◽  
Roger Mulet-Lazaro ◽  
Lucia Schwarzfischer ◽  
Dagmar Glatz ◽  
Margit Nuetzel ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Promoter Methylation ◽  
Research Funding ◽  
Promoter Hypermethylation ◽  
Ctcf Binding ◽  
P Value ◽  
Global Dna Methylation ◽  
Employment Equity ◽  
Equity Ownership

Abstract Acute myeloid leukemia (AML) represents a highly heterogeneous myeloid stem cell disorder classified based on various genetic defects. Besides genetic alterations, epigenetic changes are recognized as an additional mechanism contributing to leukemogenesis, but insight into the latter process remains minor. Using a combination of Methyl-CpG-Immunoprecipitation (MCIp-chip) and MALDI-TOF analysis of bisulfite-treated DNA in a cohort of 196 AML patients we previously demonstrated that (cyto)genetically defined AML subtypes, including CBFB-MYH11, AML-ETO, NPM1-mut, CEBPA-mut or IDH1/2-mut subtypes, express specific DNA-methylation profiles (Gebhard et al, Leukemia, 2018). A fraction of AML patients (5/196) displayed a unique abnormal hypermethylation profile that was completely distinct from any other AML subtype. These patients present immature leukemia (FAB M0, M1) with various chromosomal aberrations but very few mutations (e.g. no IDH1/2, KRAS, DNMT3A) that might explain the CpG island methylator phenotype (CIMP) phenotype. The CIMP patients showed high resemblance with a recently reported CEBPA methylated subgroup (Wouters et al, 2007 and Figueroa et al, 2009), which we confirmed by MCIp-chip and MALDI-TOF analysis. To explore the whole range of epigenetic alterations in the CIMP-AML patients we performed in-depth global DNA methylation and gene expression analyses (MCIp-seq and RNA-seq) in 45 AML and 12 CIMP patients from both studies. Principle component analysis and t-distributed stochastic neighbor embedding (t-SNE) revealed that CIMP patients express a unique DNA-methylation and gene-expression signature that separated them from all other AMLs. We could discriminate promoter methylation from non-promoter methylation by selecting MCIp-seq peaks within 3kb around TSS. Promoter hypermethylation was highly associated with repression of genes (PCC = -0.053, p-value = 0.00075). Hypermethylation of non-promoter regions was more strongly associated with upregulation of genes (PCC = 0.046, p-value = 4.613e-06). Interestingly, differentially methylated regions also showed a positive association with myeloid lineage CTCF binding sites (27% vs 18% expected, p-value < 2.2e-16 in a chi-square test of independence). Methylation of CTCF sites causes loss of CTCF binding, which has been reported to disrupt boundaries between so-called topologically associated domains (TADs), allowing enhancers located in a particular TAD to become accessible to genes in adjacent TADs and affect their transcription. Whether this is the case is under investigation. In this study we particularly focused on the role of hypermethylation of promoters in CIMP-AMLs. Promoters of many transcriptional regulators that are involved in the differentiation of myeloid lineages of which several are frequently mutated in AML were hypermethylated and repressed, including CEBPA, CEBPD, IRF8, GATA2, KLF4, MITF or MAFB. Notably, HMGA2, a critical regulator of myeloid progenitor expansion, exhibited the largest degree of CIMP promoter hypermethylation compared to the other AMLs, accompanied by a reduction in gene expression. Moreover, multiple members of the HOXB family and KLF1 (erythroid differentiation) were methylated and repressed as well. In addition, these patients frequently showed hypermethylation of many chromatin factors (e.g. LMNA, CHD7 or TET2). Hypermethylation of the TET2 promoter could result in a loss of maintenance DNA demethylation and therefore successive hypermethylation at CpG islands. We carried out regulome-capture-bisulfite sequencing on CIMP-AMLs compared to other AML samples and normal blood cell controls and confirmed methylation of the same transcription and chromatin factor promoters. We conclude that these leukemias represent very primitive HSCPs which are blocked in differentiation into multiple hematopoietic lineages, due to the absence of regulators of these lineages. Although the underlying cause for the extreme hypermethylation signature is still subject to ongoing studies, the consequence of promoter hypermethylation is silencing of key lineage regulators causing the differentiation arrest in these cells. We argue that these patients may particularly benefit from therapies that revert DNA methylation. Disclosures Ehninger: Cellex Gesellschaft fuer Zellgewinnung mbH: Employment, Equity Ownership; GEMoaB Monoclonals GmbH: Employment, Equity Ownership; Bayer: Research Funding. Thiede:AgenDix: Other: Ownership; Novartis: Honoraria, Research Funding.

scholarly journals DNA Hypermethylation of a panel of genes as an urinary biomarker for bladder cancer diagnosis

2021 ◽  
Author(s):  
Petros Georgopoulos ◽  
Maria Papaioannou ◽  
Soultana Markopoulou ◽  
Aikaterini Fragou ◽  
George Kouvatseas ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Bladder Cancer ◽  
Smoking Status ◽  
Gene Promoter ◽  
Control Group ◽  
Gene Promoters ◽  
Dna Hypermethylation ◽  
Diagnostic Potential ◽  
Specificity And Sensitivity ◽  
Promoter Dna

Abstract PurposeThe aim of this study was to explore the diagnostic potential of a panel of five hypermethylated gene promoters in bladder cancer. Individuals with primary BCa and control individuals matching the gender, age and smoking status of the cancer patients were recruited. DNA methylation was assessed for the gene promoters of RASSF1, RARβ, DAPK, hTERT and APC in urine samples collected by spontaneous urination. Fifty patients and 35 healthy controls were recruited, with average age of 70.26 years and average smoking status of 44.78 pack-years. In the BCa group, DNA methylation was detected in 27(61.4%) samples. RASSF1 was methylated in 52.2% of samples. Only 3(13.6%) samples from the control group were methylated, all in the RASSF1 gene promoter. The specificity and sensitivity of this panel of genes to diagnose BCa was 86% and 61% respectively. The RASSF1 gene could diagnose BCa with specificity 86.4% and sensitivity 52.3%. Promoter DNA methylation of this panel of five genes could be further investigated as urine biomarker for the diagnosis of BCa. The RASSF1 could be a single candidate biomarker for predicting BCa patients versus controls. Studies are required in order to develop a geographically adjusted diagnostic biomarker for BCa.Trial registration: ACTRN12620000258954

scholarly journals Reversible DNA Hypermethylation of the Interleukin-15 (IL-15) Promoter Induces IL-15 Expression, Drives the Pathogenesis of T-Cell Large Granular Lymphocytic Leukemia and Provides a Potential Therapeutic Approach Using 5-Azacitidine

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3776-3776
Author(s):  
Jonathan E Brammer ◽  
Amy E Boles ◽  
Anthony Mansour ◽  
Aharon G. Freud ◽  
Monique Mathé-Allainmat ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
T Cell ◽  
Research Funding ◽  
Marked Decrease ◽  
Lymphocytic Leukemia ◽  
Dna Hypermethylation ◽  
Reduced Representation ◽  
Primary Driver ◽  
Interleukin 15

Background and Rationale: T-cell large granular lymphocytic leukemia (T-LGLL) is an incurable clonal proliferation of CD8+ memory T-cells that leads to profound neutropenia and anemia with limited treatment options. The primary driver of T-LGLL is overexpression of interleukin-15 (IL-15), a gamma-chain cytokine. Previously, we have demonstrated that mice overexpressing IL-15 develop DNA hypermethylation and chromosomal instability that leads to the spontaneous development of LGLL (Mishra et al. Cancer Cell 2012). Further, the IL-15 promoter is known to be hypermethylated in cutaneous T-cell lymphoma (CTCL), another IL-15 driven malignancy (Mishra et al. Cancer Discovery 2016). In CTCL patients, the counterintuitive increase in IL-15 mRNA was due to hypermethylation of its promoter at the repressor binding sequences in the IL-15 gene. However, the methylation status of the IL-15 promoter in T-LGLL patients remains unknown. Concept: We hypothesize that the IL-15 promoter is hypermethylated in patients with T-LGLL, leading to aberrant overexpression of IL-15 and that this hypermethylation is a critical event in the leukemogenesis of T-LGLL. If true, demethylation of the IL-15 promoter with a resultant decrease in IL-15 transcripts should lead to apoptosis of T-LGLL cells. Hypomethylation of the IL-15 promoter, therefore, may provide a novel therapeutic approach to inhibiting IL-15, the primary driver of T-LGLL. Results: CD3+/CD8+/CD5-/dim T-cells were purified from peripheral blood of LGLL patient (n=3) and normal donor (ND) (n=3) by flow cytometry sorting. We analyzed DNA methylation and gene expression profiling using reduced representation bisulfite and RNA sequencing. With bioinformatics analysis, we determined differential methylation (1-way ANOVA P= 0.0178) and expression (1-way ANOVA P =0.0059). These data sets revealed significant differential hypermethylation of gene promoters in leukemic samples, compared to controls (Figure 1A). Reduced representation bisulfite sequencing that can identify differentially methylated regions at single base-pair resolutions demonstrated an increase in DNA methylation of the IL-15 promoter in patient samples over controls. To determine the functional significance of this finding, we treated the MOTN-1 T-LGLL cell line in vitro with the hypomethylating agent, 5-azacytidine (5-aza) at concentrations of 0.5 uM, 1 uM, 2.5 uM, and 5 uM. At 24 and 48 hours, a marked decrease in the viability of T-LGLL cells was observed, from 100% to 49.50%, p=0.037; particularly at higher concentrations of 5-aza (100% to 27% +11.30%, p=0.0030). Next, we sought to determine whether 5-aza induced hypomethylation of the IL-15 promoter. IL-15 gene expression in MOTN-1 T-LGLL cells treated with 5-aza was measured in comparison to control treated MOTN-1 cells. A marked decrease in IL-15 expression was observed at all concentrations of 5-aza compared to control (Figure 1B, p=0.0001). These results confirm that 5-aza leads to decreased transcription of the IL-15 gene, possibly due to hypomethylation of the IL-15 promoter. Finally, to determine whether a decrease in IL-15 alone was the cause of increased apoptosis of T-LGLL cells, we exposed MOTN-1 cells to a novel IL-15 inhibitor, IBI-15, and compared cell viability against MOTN-1 cells exposed to an inactive control, IBI-40. Even more profound decrease in cell viability was observed utilizing IBI-15 that targets the binding of IL-15 to its receptor (Figure 1C). Together, these data suggest that hypermethylation of the IL-15 promoter is critical to the pathogenesis of T-LGLL, and that treatment with 5-aza is sufficient to induce hypomethylation of the IL-15 promoter, decrease IL-15 transcription, and induce apoptosis in T-LGLL cells. Conclusions: Hypermethylation of the IL-15 promoter, with subsequent increase in IL-15, is critical to the pathogenesis of T-LGLL. Inhibition of the IL-15 promoter hypermethylation by 5-aza leads to down-regulation of the IL-15 gene transcript, which is sufficient to induce apoptosis of T-LGLL cells. These data suggest that 5-aza induced hypomethylation may be a novel method to induce IL-15 inhibition and a potentially efficacious clinical strategy against T-LGLL. Disclosures Brammer: Bioniz Therapeutics, Inc.: Research Funding; Viracta Therapeutics, Inc.: Research Funding; Verastem, Inc: Research Funding. Porcu:Daiichi: Research Funding; BeiGene: Other: Scientific Board, Research Funding; Spectrum: Consultancy; Viracta: Honoraria, Other: Scientific Board, Research Funding; Innate Pharma: Honoraria, Other: Scientific Board, Research Funding; Kyowa: Honoraria, Other: Scientific Board, Research Funding; ADCT: Research Funding; Incyte: Research Funding. OffLabel Disclosure: IBI-15 IBI-40 IL-15 inhibitor

scholarly journals DNA hypermethylation associated with upregulated gene expression in prostate cancer demonstrates the diversity of epigenetic regulation

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Ieva Rauluseviciute ◽  
Finn Drabløs ◽  
Morten Beck Rye
Keyword(s):  
Gene Expression ◽  
Prostate Cancer ◽  
Dna Methylation ◽  
Epigenetic Regulation ◽  
Normal Tissue ◽  
Expression Profiles ◽  
Expression Data ◽  
Dna Hypermethylation ◽  
Genome Wide ◽  
A Genome

Abstract Background Prostate cancer (PCa) has the highest incidence rates of cancers in men in western countries. Unlike several other types of cancer, PCa has few genetic drivers, which has led researchers to look for additional epigenetic and transcriptomic contributors to PCa development and progression. Especially datasets on DNA methylation, the most commonly studied epigenetic marker, have recently been measured and analysed in several PCa patient cohorts. DNA methylation is most commonly associated with downregulation of gene expression. However, positive associations of DNA methylation to gene expression have also been reported, suggesting a more diverse mechanism of epigenetic regulation. Such additional complexity could have important implications for understanding prostate cancer development but has not been studied at a genome-wide scale. Results In this study, we have compared three sets of genome-wide single-site DNA methylation data from 870 PCa and normal tissue samples with multi-cohort gene expression data from 1117 samples, including 532 samples where DNA methylation and gene expression have been measured on the exact same samples. Genes were classified according to their corresponding methylation and expression profiles. A large group of hypermethylated genes was robustly associated with increased gene expression (UPUP group) in all three methylation datasets. These genes demonstrated distinct patterns of correlation between DNA methylation and gene expression compared to the genes showing the canonical negative association between methylation and expression (UPDOWN group). This indicates a more diversified role of DNA methylation in regulating gene expression than previously appreciated. Moreover, UPUP and UPDOWN genes were associated with different compartments — UPUP genes were related to the structures in nucleus, while UPDOWN genes were linked to extracellular features. Conclusion We identified a robust association between hypermethylation and upregulation of gene expression when comparing samples from prostate cancer and normal tissue. These results challenge the classical view where DNA methylation is always associated with suppression of gene expression, which underlines the importance of considering corresponding expression data when assessing the downstream regulatory effect of DNA methylation.

Global Epigenomic Profiling Demonstrates That Myelodysplasia Is Characterized by a Distinct Epigenetic Signature with Aberrant DNA Methylation Changes Involving Various Malignant and Hematopoietic Pathways.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2436-2436
Author(s):  
L. Zhou ◽  
J. Opalinska ◽  
D. Sohal ◽  
R. Thompson ◽  
Y. Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
Expression Analysis ◽  
Peripheral Blood ◽  
Gene Expression Analysis ◽  
Whole Genome ◽  
Gene Promoters ◽  
Global Methylation ◽  
Peripheral Blood Leucocyte ◽  
Peripheral Blood Leucocytes

Abstract Myelodysplasia (MDS) is a clonal hematopoietic disorder that leads to ineffective hematopoiesis and peripheral cytopenias. DNMT inhibitors such as azacytidine have led to clinical responses in patients, though the genes affected by epigenetic alterations are not well known. Whole genome DNA methylation was analyzed by a recently described novel method, The HELP assay (HpaII tiny fragment Enrichment by Ligation-mediated PCR; Khulan et al, Genome Res. 2006 Aug;16(8)) that uses differential methylation specific restriction digestion by HpaII and MspI followed by amplification, two color labeling and cohybridization to quantitatively determine individual promoter island methylation. A whole genome human promoter array (Nimblegen) was used to determine the level of methylation of 25626 gene promoters by calculating HpaII/MspI cut fragment intensity ratio. Peripheral blood leucocytes from 13 patients with MDS were compared to 9 age matched normal and anemic controls. Gene expression analysis was performed using 37K oligo maskless arrays on cDNA obtained from the same samples. Analysis showed that whole genome methylation profiling has greater discriminatory power in separating clusters of MDS samples from normal and anemic controls when compared to gene expression analysis. Unsupervised clustering based on epigenetic profiling demonstrated that only two cases of early MDS clustered with normals as compared to absolutely no separation between MDS and normals with clustering based on gene expression patterns. A high correlation (r=0.88–0.96) was observed between global methylation profiles of matched sets of bone marrow and peripheral blood leucocyte samples from selected patients demonstrating that peripheral blood leucocytes can be a valid surrogate for epigenomic analysis. Further analysis showed that genes consistently aberrantly methylated in MDS included Syk kinase, HOXB3, several histone acetyltranferases and others. Functional analysis by Ingenuity showed that cancer and cell signaling pathways were the most affected by epigenetic silencing. Most interestingly, a large proportion of gene promoters were also aberrantly hypomethylated. These included genes from Ras oncogene family, the CDC42 GTPase, various methyl binding proteins and other proteins mainly encoding for cancer and hematopoiesis functional pathways, thus biologically validating our analysis. Therefore, our data demonstrates that MDS is characterized by distinct epigenetic aberrations that are preserved in peripheral blood leucocytes. These can be the basis of future studies on pathogenesis and diagnosis for this disease and can potentially uncover a new set of therapeutic gene targets.

Sign in / Sign up

Export Citation Format

Share Document