Role of MBD2 in gene regulation and tumorigenesis

2005 ◽  
Vol 33 (6) ◽  
pp. 1537-1540 ◽  
Author(s):  
J. Berger ◽  
A. Bird
Keyword(s):  
Dna Methyltransferase ◽  
Cpg Island ◽  
Gene Dosage ◽  
Cpg Islands ◽  
Transcriptional Repressor ◽  
Epigenetic Mechanism ◽  
Tumour Suppressor Genes ◽  
Dependent Manner ◽  
Cpg Dinucleotides ◽  
Mbd Protein

DNA methylation is an epigenetic mechanism involved in transcriptional silencing of imprinted genes, genes located on the inactive X chromosome, and a number of tumour suppressor genes in cancer. MBD (methyl-CpG-binding domain) proteins selectively bind to methylated DNA and recruit chromatin remodelling and transcriptional repressor complexes, thereby establishing a repressive chromatin state. MBD2, a member of the MBD protein family, binds to methylated promoter CpG islands (clusters of high-density CpG dinucleotides) and acts as a methylation-dependent transcriptional repressor. Previous work has demonstrated that decreased CpG island methylation in mice lacking the DNA methyltransferase DNMT1 is associated with impaired tumorigenesis when crossed on the tumour-susceptible ApcMin/+ background. Mbd2 deficiency also dramatically reduces adenoma burden and extends life span in a gene dosage-dependent manner in this mouse model. Mbd2 is therefore essential for tumorigenesis in the murine intestine, although it is dispensable for the viability of the host animals. These findings validate MBD2 as a potential target for therapeutic intervention in colorectal cancer.

scholarly journals Aldosterone reprograms promoter methylation to regulate αENaC transcription in the collecting cuct

2013 ◽  
Vol 305 (7) ◽  
pp. F1006-F1013 ◽  
Author(s):  
Zhiyuan Yu ◽  
Qun Kong ◽  
Bruce C. Kone
Keyword(s):  
Promoter Methylation ◽  
Dna Methyltransferase ◽  
Cpg Island ◽  
Collecting Duct ◽  
Epigenetic Mechanism ◽  
Sequencing Analysis ◽  
Control Of Gene Expression ◽  
Bisulfite Treatment ◽  
High Level ◽  
Interfering Rna

Aldosterone increases tubular Na+ absorption largely by increasing α-epithelial Na+ channel (αENaC) transcription in collecting duct principal cells. How aldosterone reprograms basal αENaC transcription to high-level activity in the collecting duct is incompletely understood. Promoter methylation, a covalent but reversible epigenetic process, has been implicated in the control of gene expression in health and disease. We investigated the role of promoter methylation/demethylation in the epigenetic control of basal and aldosterone-stimulated αENaC transcription in mIMCD3 collecting duct cells. Bisulfite treatment and sequencing analysis after treatment of the cells with the DNA methyltransferase (DNMT) inhibitor 5-aza-2′-deoxycytidine (5-Aza-CdR) identified clusters of methylated cytosines in a CpG island near the transcription start site of the αENaC promoter. 5-Aza-CdR treatment or small interfering RNA-mediated knockdown of DNMT3b or methyl-CpG-binding domain protein (MBD)-4 derepressed basal αENaC transcription, indicating that promoter methylation suppresses basal αENaC transcription. Aldosterone triggered a time-dependent decrease in 5mC and DNMT3b and a concurrent enrichment in 5-hydroxymethylcytosine (5hmC) and ten-eleven translocation (Tet)2 at the αENaC promoter, consistent with active demethylation. 5-Aza-CdR mimicked aldosterone by enhancing Sp1 binding to the αENaC promoter. We conclude that DNMT3b- and MBD4-dependent methylation of the αENaC promoter limits basal αENaC transcription, in part by limiting Sp1 binding and trans-activation. Aldosterone stimulates the dispersal of DNMT3b and recruitment of Tet2 to demethylate the αENaC promoter to induce αENaC transcription. These results disclose a novel epigenetic mechanism for the control of basal and aldosterone-induced αENaC transcription that adds to previously described epigenetic controls exerted by histone modifications.

CD40-induced transcriptional activation of vascular endothelial growth factor involves a 68-bp region of the promoter containing a CpG island

2004 ◽  
Vol 287 (3) ◽  
pp. F512-F520 ◽  
Author(s):  
Peter H. Lapchak ◽  
Michael Melter ◽  
Soumitro Pal ◽  
Jesse A. Flaxenburg ◽  
Christopher Geehan ◽  
...  
Keyword(s):  
Vascular Endothelial Growth Factor ◽  
Growth Factor ◽  
Transcriptional Activation ◽  
Cpg Island ◽  
Transcriptional Repressor ◽  
Renal Physiology ◽  
Luciferase Reporter ◽  
Vascular Endothelial ◽  
Cpg Dinucleotides ◽  
Endothelial Growth Factor

Vascular endothelial growth factor (VEGF) is produced by several cell types in the kidney, and its expression is tightly regulated for the maintenance of normal renal physiology. Increases or decreases in its expression are associated with proteinuria and renal disease. Recently, we found that the expression of VEGF is markedly induced following interactions between CD40 ligand (CD40L) and CD40. Here, endothelial cells (EC) or Jurkat T cell lines were transiently transfected with luciferase reporter constructs under the control of the human VEGF promoter and were treated with human soluble CD40L (sCD40L). We identified a CD40-responsive 68-bp region (bp −50 to +18) of the promoter and 43 bp within this region (bp −25 to +18) that have 97% homology to a sequence of CpG dinucleotides. A computerized search revealed that the CpG region has putative binding domains for the transcriptional repressor protein methyl CpG binding protein-2 (MeCP2). In EMSA, we found that the 43-bp methylated sequence formed four complex(es) with nuclear extracts from untreated EC and reduced binding of at least one complex when nuclear lysates from sCD40L-activated EC (30 min) were used. Supershift analysis using anti-MeCP2 demonstrated that most of the complex(es) in both untreated and sCD40L-activated EC involved interactions between the 43-bp DNA and MeCP2. In addition, we found that other CpG binding proteins may also interact with this region of the promoter. Taken together, this is the first demonstration that CpG binding transcriptional repressor proteins including MeCP2 may be of importance in VEGF biology.

scholarly journals Demethylation of somatic and testis-specific histone H2A and H2B genes in F9 embryonal carcinoma cells

1993 ◽  
Vol 13 (9) ◽  
pp. 5538-5548
Author(s):  
Y C Choi ◽  
C B Chae
Keyword(s):  
Embryonal Carcinoma ◽  
Cpg Island ◽  
Cpg Islands ◽  
High Density ◽  
Carcinoma Cells ◽  
Embryonal Carcinoma Cells ◽  
Cpg Dinucleotides ◽  
F9 Embryonal Carcinoma Cells ◽  
Methylation Patterns

In contrast to many other genes containing a CpG island, the testis-specific H2B (TH2B) histone gene exhibits tissue-specific methylation patterns in correlation with gene activity. Characterization of the methylation patterns within a 20-kb segment containing the TH2A and TH2B genes in comparison with that in a somatic histone cluster revealed that: (i) the germ cell-specific unmethylated domain of the TH2A and TH2B genes is defined as a small region surrounding the CpG islands of the TH2A and TH2B genes and (ii) somatic histone genes are unmethylated in both liver and germ cells, like other genes containing CpG islands, whereas flanking sequences are methylated. Transfection of in vitro-methylated TH2B, somatic H2B, and mouse metallothionein I constructs into F9 embryonal carcinoma cells revealed that the CpG islands of the TH2A and TH2B genes were demethylated like those of the somatic H2A and H2B genes and the metallothionein I gene. The demethylation of those CpG islands became significantly inefficient at a high number of integrated copies and a high density of methylated CpG dinucleotides. In contrast, three sites in the somatic histone cluster, of which two sites are located in the long terminal repeat of an endogenous retrovirus-like sequence, were efficiently demethylated even at a high copy number and a high density of methylated CpG dinucleotides. These results suggest two possible mechanisms for demethylation in F9 cells and methylation of CpG islands of the TH2A and TH2B genes at the postblastula stage during embryogenesis.

scholarly journals Demethylation of somatic and testis-specific histone H2A and H2B genes in F9 embryonal carcinoma cells.

1993 ◽  
Vol 13 (9) ◽  
pp. 5538-5548 ◽  
Author(s):  
Y C Choi ◽  
C B Chae
Keyword(s):  
Embryonal Carcinoma ◽  
Cpg Island ◽  
Cpg Islands ◽  
High Density ◽  
Carcinoma Cells ◽  
Embryonal Carcinoma Cells ◽  
Cpg Dinucleotides ◽  
F9 Embryonal Carcinoma Cells ◽  
Methylation Patterns

In contrast to many other genes containing a CpG island, the testis-specific H2B (TH2B) histone gene exhibits tissue-specific methylation patterns in correlation with gene activity. Characterization of the methylation patterns within a 20-kb segment containing the TH2A and TH2B genes in comparison with that in a somatic histone cluster revealed that: (i) the germ cell-specific unmethylated domain of the TH2A and TH2B genes is defined as a small region surrounding the CpG islands of the TH2A and TH2B genes and (ii) somatic histone genes are unmethylated in both liver and germ cells, like other genes containing CpG islands, whereas flanking sequences are methylated. Transfection of in vitro-methylated TH2B, somatic H2B, and mouse metallothionein I constructs into F9 embryonal carcinoma cells revealed that the CpG islands of the TH2A and TH2B genes were demethylated like those of the somatic H2A and H2B genes and the metallothionein I gene. The demethylation of those CpG islands became significantly inefficient at a high number of integrated copies and a high density of methylated CpG dinucleotides. In contrast, three sites in the somatic histone cluster, of which two sites are located in the long terminal repeat of an endogenous retrovirus-like sequence, were efficiently demethylated even at a high copy number and a high density of methylated CpG dinucleotides. These results suggest two possible mechanisms for demethylation in F9 cells and methylation of CpG islands of the TH2A and TH2B genes at the postblastula stage during embryogenesis.

Pyrosequencing Analysis of the Methylation Status of the CpG Island in the Retinoic Acid Receptor-β2 (RAR-β2) Gene Promoter.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4297-4297
Author(s):  
Da-Cheng Zhou ◽  
David Reynolds ◽  
Robert E. Gallagher
Keyword(s):  
Retinoic Acid ◽  
Cell Lines ◽  
Cpg Island ◽  
Retinoic Acid Receptor ◽  
Methylation Status ◽  
Cpg Islands ◽  
Leukemia Cell ◽  
Gene Promoter ◽  
Cpg Dinucleotides ◽  
Retinoic Acid Receptor Β2

Abstract CpG islands are associated with the 5′-ends of most housekeeping genes and many regulated genes. We have hypothesized that the methylation status of CpG islands in the promoter region of all-trans retinoic acid (ATRA) target genes such as retinoic acid receptor-β2 (RAR-β2) may be related to ATRA resistance and relapse of acute promyelocytic leukemia (APL). In the present study, we developed a highly quantitative method to assess the degree of DNA methylation at specific sites using PyrosequencingTM technology (Biotage, Uppsala, Sweden). This method is more quantitative than methylation-specific PCR, and is as accurate as but simpler and more robust than combined bisulfite restriction analysis (COBRA) or direct sequencing of plasmid clones of PCR products. We used this method to study 14 CpG dinucleotides in the CpG island of the RAR-β2 promoter. In reconstruction experiments in which 100% methylated and 100% unmethylated DNAs were admixed in different proportions (100:0; 80:20, 60:40, etc), a straightline graph was obtained over the entire range from 0 – 100% for each of the 14 CpG dinucleotides (r2 > 0.98). The results were highly reproducible and the variation between the results obtained from repetitive pyrosequencing of the same DNA was very low (S.D.<2%). Also the standard deviation between measurements of different PCR-amplified, bisulfite-converted DNAs prepared in separate experiments was <5%. We then used this method to measure the methylation level of the CpG island of the RAR-β2 promoter in several leukemia cell lines. Of 3 APL cell lines, the two with PML-RARα mutations, i.e., UF-1 and AP-1060, had higher overall methylation, compared to the NB4 cell line with non-mutant PML-RARα (mean ± SD = 52 ± 25% and 55 ± 21%, versus 43 ± 20%; p = 0.04 and 0.08, respectively; SD calculated from the variation across the 14 CpG dinucleotides for each source). Two myeloid leukemia cell lines with predominantly erythroid lineage characteristics, K562 and TF-1, had much lower levels of RAR-β2 methylation (2.6 ± 0.9% and 8.9 ± 3.2%, respectively). In the AP-1060 culture system, recently developed in our lab, there was little difference in methylation status between the patient bone marrow source and an intermediate, non-immortalized cell strain AP-1060S (27 ± 13% vs. 31 ± 25%). Further, there was no difference between lower and higher passage generations of AP-1060S (31 ± 25% vs. 30 ± 26%), which had markedly different replicative potential, indicating that replicative senescence at higher AP-1060 passages was not associated with altered methylation of the RAR-β2 gene promoter. However, the established, immortalized AP-1060 cell line had significantly greater methylation (52 ± 25%) than either the bone marrow source or AP-1060S (p <0.0001 and p = 0.0002, respectively), consistent with published reports of increased promoter methylation of cell lines. In conclusion, pyrosequencing is a high throughput method with great quantitative strength, and can be used for accurate and consistent analysis of methylation status in large numbers of samples.

Effect of 5-Aza-2′-Deoxycytidine in High-Risk Myelodysplasia Cells in Vitro.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4707-4707 ◽  
Author(s):  
Hongyan Tong ◽  
Maofang Lin ◽  
Jie Jin
Keyword(s):  
High Risk ◽  
Dna Methyltransferase ◽  
Mrna Level ◽  
Cpg Islands ◽  
Antineoplastic Agent ◽  
Leukemic Cells ◽  
Refractory Anemia ◽  
Dependent Manner ◽  
Antitumor Effects

Abstract 5-Aza-2′-deoxycytidine (5-Aza-CdR; Decitabine) is an active antineoplastic agent in patients with leukemia. Due to its unique mechanism of action of demethylating DNA, 5-Aza-CdR has the potential to activate tumor suppressor and differentiation genes that have been accidentally silenced by DNA methylation in leukemic cells. Since5-Aza-CdR can induce clinical remissions in patients with high-risk myelodysplastic syndromes (MDS), we therefore attempt to investigate the mechanisms of the effect of 5-Aza-CdR by using MUTZ-1 cells in vitro, which are confirmed as a high-risk myelodysplasia Cell line that derived from a MDS patient (FAB subtype refractory anemia with excess of blasts). Our results indicated that 5-Aza-CdR showed inhibition of the growth of MUTZ-1 cells. The IC50 values of 24 hours, 48 hours and 72 hours were 6.75mmol/L, 2.82mmol/L and 5.45mol/L respectively. Characteristic changes of apoptosis emerged in MUTZ-1 cells after being exposed to 5-Aza-CdR in the different concentration from 0.8 mmol/L to 3.2 mmol/L, and the positive cells of Annexin VFITC on the membrane of MUTZ-1 cells were analyzed by flow cytometry. The percentage of apoptosis cells treated by 5-Aza-CdR at a dose of 3.2mmol/L for 48 hours was higher than that in untreated cells (16.7%±5.3% vs 2.9%±1.2%, p<0.05). The CpG islands in the promoter regions of p15INK4B gene were found to be hypermethylated in MUTZ-1 cells, resulting in a significant reduced expression of p15INK4B gene in the cells. However, the expression of p15INK4B gene was recovered and the methylation level of p15INK4B gene was also decreased (densitometry readings: 253.994±26.536 vs 369.641±28.915, p=0.003), after the cells exposed to 5-Aza-CdR at a dose of 3.2 mmol/L for 48 hours. Furthermore, 5-Aza-CdR could significantly down-regulate the expressions of DNA methyltransferase genes DNMT3A, which encodes a deno methyltransferase, at mRNA level in a dose dependent manner(r=−0.879,p=0.03). However, it had no effects on DNMT3B gene and DNMT1 gene that codes a maintenance methylase. The expression level of DNMT3A mRNA in MUTZ-1 cells treated with 5-Aza-CdR at a concentration 3.2 mmol/L for 48 hours was lower than that in untreated cells (densitometry readings:0. 385±0.086 vs 0.654±0.074 p<0.05). These observations suggest that 5-Aza-CdR can inhibit the growth and induce the apoptosis of MUTZ-1 cells within the range of concentration from 0.8 mmol/L to 3.2 mmol/L, which might be one of the mechanisms of antitumor effects of 5-Aza-CdR. The drug can activate the expression of p15INK4B gene in MUTZ-1 cells by demethylation of the p15INK4B gene through inhibiting the expression of DNMT3A gene. That might be the mechanisms of 5-Aza-CdR in the treatments of MDS.

scholarly journals Lsh Is Essential for Maintaining Global DNA Methylation Levels in Amphibia and Fish and Interacts Directly with Dnmt1

2015 ◽  
Vol 2015 ◽  
pp. 1-12 ◽  
Author(s):  
Donncha S. Dunican ◽  
Sari Pennings ◽  
Richard R. Meehan
Keyword(s):  
Dna Methylation ◽  
Dna Methyltransferase ◽  
Repetitive Sequences ◽  
Global Dna Methylation ◽  
Dependent Manner ◽  
Gene Promoters ◽  
Cpg Dinucleotides ◽  
Mouse Cells ◽  
Eukaryotic Genomes

Eukaryotic genomes are methylated at cytosine bases in the context of CpG dinucleotides, a pattern which is maintained through cell division by the DNA methyltransferase Dnmt1. Dramatic methylation losses are observed in plant and mouse cells lacking Lsh (lymphoid specific helicase), predominantly at repetitive sequences and gene promoters. However, the mechanism by which Lsh contributes to the maintenance of DNA methylation is unknown. Here we show that DNA methylation is lost in Lsh depleted frog and fish embryos, both of which exhibit developmental delay. Additionally, we show that both Lsh and Dnmt1 are associated with chromatin and that Lsh knockdown leads to a decreased Dnmt1-chromatin association. Coimmunoprecipitation experiments reveal that Lsh and Dnmt1 are found in the same protein complex, and pulldowns show this interaction is direct. Our data indicate that Lsh is usually diffuse in the nucleus but can be recruited to heterochromatin in a HP1α-dependent manner. These data together (a) show that the role of Lsh in DNA methylation is conserved in plants, amphibian, fish, and mice and (b) support a model in which Lsh contributes to Dnmt1 binding to chromatin, explaining how its loss can potentially lead to perturbations in DNA methylation maintenance.

scholarly journals Differential regulation of DNA methylation versus histone acetylation in cardiomyocytes during HHcy in vitro and in vivo: an epigenetic mechanism

2014 ◽  
Vol 46 (7) ◽  
pp. 245-255 ◽  
Author(s):  
Pankaj Chaturvedi ◽  
Anuradha Kalani ◽  
Srikanth Givvimani ◽  
Pradip Kumar Kamat ◽  
Anastasia Familtseva ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Histone Acetylation ◽  
Dna Methyltransferase ◽  
Epigenetic Mechanism ◽  
Dependent Manner ◽  
Histone Deacetylase 1 ◽  
H3k9 Acetylation ◽  
Dose Dependent

The mechanisms of homocysteine-mediated cardiac threats are poorly understood. Homocysteine, being the precursor to S-adenosyl methionine (a methyl donor) through methionine, is indirectly involved in methylation phenomena for DNA, RNA, and protein. We reported previously that cardiac-specific deletion of N-methyl-d-aspartate receptor-1 (NMDAR1) ameliorates homocysteine-posed cardiac threats, and in this study, we aim to explore the role of NMDAR1 in epigenetic mechanisms of heart failure, using cardiomyocytes during hyperhomocysteinemia (HHcy). High homocysteine levels activate NMDAR1, which consequently leads to abnormal DNA methylation vs. histone acetylation through modulation of DNA methyltransferase 1 (DNMT1), HDAC1, miRNAs, and MMP9 in cardiomyocytes. HL-1 cardiomyocytes cultured in Claycomb media were treated with 100 μM homocysteine in a dose-dependent manner. NMDAR1 antagonist (MK801) was added in the absence and presence of homocysteine at 10 μM in a dose-dependent manner. The expression of DNMT1, histone deacetylase 1 (HDAC1), NMDAR1, microRNA (miR)-133a, and miR-499 was assessed by real-time PCR as well as Western blotting. Methylation and acetylation levels were determined by checking 5′-methylcytosine DNA methylation and chromatin immunoprecipitation. Hyperhomocysteinemic mouse models (CBS+/−) were used to confirm the results in vivo. In HHcy, the expression of NMDAR1, DNMT1, and matrix metalloproteinase 9 increased with increase in H3K9 acetylation, while HDAC1, miR-133a, and miR-499 decreased in cardiomyocytes. Similar results were obtained in heart tissue of CBS+/− mouse. High homocysteine levels instigate cardiovascular remodeling through NMDAR1, miR-133a, miR-499, and DNMT1. A decrease in HDAC1 and an increase in H3K9 acetylation and DNA methylation are suggestive of chromatin remodeling in HHcy.

scholarly journals SAT-LB34 Repressive Epigenetic Programs Reinforce Steroidogenic Differentiation and Wnt/β-Catenin Signaling in Aggressive Adrenocortical Carcinoma

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Dipika R Mohan ◽  
Isabella Finco ◽  
Christopher Ryan LaPensee ◽  
Juilee Rege ◽  
Tobias Else ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Dna Methylation ◽  
Adrenocortical Carcinoma ◽  
Dna Methyltransferase ◽  
Cpg Island ◽  
Cpg Islands ◽  
Embryonic Stem ◽  
High Status ◽  
Tissue Specific ◽  
Genome Wide

Abstract Adrenocortical carcinoma (ACC) is a rare, aggressive cancer. Up to 75% of patients develop incurable metastatic disease, highlighting an urgent need for novel medical therapies. We recently identified a rapidly progressive ACC subtype characterized by CpG island hypermethylation (CIMP-high), sustained Wnt/β-catenin signaling, steroidogenic differentiation, and cell cycle activation. CIMP-high status alone accounts for 40% of ACC, but predicts 70% of recurrences and &gt;50% of deaths. Intriguingly, hypermethylated CpG islands in CIMP-high ACC are unmethylated in fetal and adult adrenal cortex, suggesting DNA methylation is supported by cancer-specific mechanisms. We therefore sought to investigate how aberrant epigenetic programming contributes to ACC biology. In embryonic stem cells, the Polycomb repressive complex 2 (PRC2) represses differentiation programs through EZH2-mediated histone H3 lysine 27 trimethylation (H3K27me3) deposition in promoter CpG islands free of DNA methylation. Gain or loss of EZH2/PRC2 function prevails in a variety of human cancers, enabling proliferation in a tissue-specific manner. Here, we identify that CIMP-high ACC exhibit high expression of EZH2/H3K27me3, but paradoxically bear DNA hypermethylation in annotated PRC2 target regions. To determine if DNA methylation of PRC2 targets disrupts or is controlled by EZH2, we characterized EZH2’s role in CIMP-high ACC cell line NCI-H295R at baseline and in response to EZH2 inhibition (EZH2i). EZH2-directed IP-MS revealed EZH2 interacts with PRC2 members and DNA methylation-sensitive accessory proteins, but no DNA methyltransferase machinery. ChIP-seq revealed EZH2 and H3K27me3 colocalize in repressive domains genome-wide, but DNA methylation and H3K27me3 are mutually exclusive. EZH2i induced H3K27 demethylation and loss of viability, but with no effect on CIMP-high DNA methylation. These data suggest PRC2 target DNA methylation in CIMP-high ACC is maintained independently of EZH2, enabling EZH2/PRC2 to coordinate alternative programs required for cell survival. We then measured the consequences of EZH2i on the NCI-H295R transcriptome (RNA-seq), EZH2/H3K27me3 deposition genome-wide (ChIP-seq), and chromatin accessibility landscape (ATAC-seq). EZH2i led to global downregulation of cell cycle, Wnt/β-catenin transcriptional programming, and steroidogenic differentiation, partially explained by EZH2i-induced offloading of EZH2 from H3K27me3 domains to accessible promoters genome-wide. Taken together, our studies illustrate how aberrant CpG island hypermethylation in CIMP-high ACC participates in a targetable repressive epigenetic cascade that reinforces oncogenic adrenocortical transcriptional programs. Ultimately, we hope to illuminate novel strategies for tissue-specific disruption of the aberrant epigenetic wiring that defines CIMP-high ACC.

Identification of 41 Novel Promoter-Associated CpG Islands Methylated in Leukemias.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1126-1126 ◽  
Author(s):  
Jaroslav Jelinek ◽  
Rajan Mannari ◽  
Jean-Pierre Issa
Keyword(s):  
Gene Silencing ◽  
Cpg Island ◽  
Methylation Status ◽  
Cpg Islands ◽  
Response To Treatment ◽  
Published Data ◽  
Promoter Regions ◽  
Cpg Dinucleotides ◽  
A Genome ◽  
Methylation Profiling

Abstract DNA methylation within promoter-associated CpG islands is a well-recognized mechanism of gene silencing and plays an important role in the development of malignancies. CpG dinucleotides in human DNA are methylated at 5′-cytosine with the exception of areas with dense concentration of CpGs (CpG islands) located in gene promoter regions. In cancer cells, methylation of CpG islands in promoter regions of tumor suppressor genes is a frequent epigenetic change with a gene-silencing effect analogous to inactivating mutations. Methylation profiling can identify biologically and clinically distinct tumor subgroups by mapping the methylation status of multiple genes, and reports in AML and ALL suggest associations between methylation and poor prognosis. Identification of methylated CpG islands can shed new light on the biology of leukemia. We used Methylated CpG Island Amplification coupled with Representative Difference Analysis (MCA-RDA) as a genome-wide screen for promoter-associated CpG islands methylated in leukemic and/or myeloproliferative cell lines and primary malignant cells, but unmethylated in blood cells from normal controls. We identified 51 unique promoter-associated CpG islands in 321 sequenced clones recovered by MCA-RDA. Forty-one CpG islands belonged to known genes, and 10 to annotated mRNAs. Of the genes with known function, 8 are involved in signaling, 7 in transcription, 3 in dephosphorylation, 2 in oxido-reductive processes, 2 in NO synthesis, 2 in adhesion, 2 in solute transport, and 2 in DNA replication. Seven out of the 51 genes were previously reported as methylated in cancer or leukemia (CDH13, HLA-B, HLA-C, PGR, SCGB3A1, SLC26A4, TERT), thus validating the MCA-RDA approach. Of the 41 new hypermethylated CpG islands recovered, 20 corresponded to genes of known function. Published data infer an association with cancer for 10 of these genes (CTDSPL, ECGF1, EDG4, FOXD2, NOR1, NOS3, OLIG2, SLC16A1, TLE1, WNT5B), and no reports were found for the other 10 genes (CNR1, FADS, FBXW3, FGD1, NPM2, P518, PDE4DIP, SNCB, TCEA3, VENTX2). To further validate our findings we are assessing the methylation status of these genes by bisulfite pyrosequencing. Analyses of the bone marrow samples from AML, ALL, CML and MDS patients are ongoing. Our preliminary data confirm methylation of H-cadherin precursor (CDH13), progesterone receptor (PGR) in AML and ALL and cannabinoid receptor 1 (CNR1) in ALL (Table). In conclusion, MCA-RDA identified methylation of 41 new and 10 previously reported promoter-associated CpG islands in leukemia. Functional studies of these may shed new light on the biology of leukemias, and these genes may be useful for methylation profiling of leukemias for prognosis and response to treatment. Promoter CpG Island Methylation Gene AML ALL Methylation levels over 10% for CDH13 and PGR and over 25% for CNR1 were scored as positive. CDH13 5/23 22% 12/19 63% PGR A Isoform 11/22 50% 12/18 67% PGR B Isoform 17/24 71% 5/13 38% CNR1 0/24 0% 3/18 17%

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