[Show abstract][Hide abstract] ABSTRACT: Cancer cells typically exhibit aberrant DNA methylation patterns that can drive malignant transformation. Whether cancer cells are dependent on these abnormal epigenetic modifications remains elusive. We used experimental and bioinformatic approaches to unveil genomic regions that require DNA methylation for survival of cancer cells. First, we surveyed the residual DNA methylation profiles in cancer cells with highly impaired DNA methyltransferases. Then, we clustered these profiles according to their DNA methylation status in primary normal and tumor tissues. Finally, we used gene expression meta-analysis to identify regions that are dependent on DNA methylation-mediated gene silencing. We further showed experimentally that these genes must be silenced by DNA methylation for cancer cell survival, suggesting these are key epigenetic events associated with tumorigenesis.
[Show abstract][Hide abstract] ABSTRACT: DNA methylation, histone modifications and nucleosome occupancy act in concert for regulation of gene expression patterns in mammalian cells. Recently, G9a, a H3K9 methyltransferase, has been shown to play a role in establishment of DNA methylation at embryonic gene targets in ES cells through recruitment of de novo DNMT3A/3B enzymes. However, whether G9a plays a similar role in maintenance of DNA methylation in somatic cells is still unclear.
Here we show that G9a is not essential for maintenance of DNA methylation in somatic cells. Knockdown of G9a has no measurable effect on DNA methylation levels at G9a-target loci. DNMT3A/3B remain stably anchored to nucleosomes containing methylated DNA even in the absence of G9a, ensuring faithful propagation of methylated states in cooperation with DNMT1 through somatic divisions. Moreover, G9a also associates with nucleosomes in a DNMT3A/3B and DNA methylation-independent manner. However, G9a knockdown synergizes with pharmacologic inhibition of DNMTs resulting in increased hypomethylation and inhibition of cell proliferation.
Taken together, these data suggest that G9a is not involved in maintenance of DNA methylation in somatic cells but might play a role in re-initiation of de novo methylation after treatment with hypomethylating drugs, thus serving as a potential target for combinatorial treatments strategies involving DNMTs inhibitors.
Full-text · Article · Jan 2012 · Epigenetics & Chromatin
[Show abstract][Hide abstract] ABSTRACT: Figure S2. Increased DNA hypomethylation of MAGE-A1 promoter in G9a knockdown cells upon treatment with 5-Aza-CdR. Methylation of MAGE-A1 promoter in G9a knockdown (shG9a5) and control (NS) HCT116 cells, treated with 5-Aza-CdR and PBS, was analyzed 72 h after drug treatment using bisulfite sequencing. CpG sites in the map of MAGE-A1 promoter are represented by the lower tick marks (top). Each straight line, with circles representing CpG sites, represents MAGE-A1 promoter sequence from a single cell (bottom). White circles indicate unmethylated CpG sites and black circles indicate methylated CpG sites. Cross indicates methylation status could not be determined. Residual DNA methylation levels were estimated by calculating the percentage of CpGs remaining methylated after drug treatment from the total number of CpGs assayed for the MAGE-A1 loci among all individual cells.
[Show abstract][Hide abstract] ABSTRACT: Increase in DNMT3A protein level in DKO cells, upon expression of different Myc-DNMTs, is independent of transcription. RT-PCR analysis was performed for analyzing endogenous DNMT3A mRNA levels in the (A) DKO8 and (B) DKO1 cells, 8 weeks after infection with different DNMTs. WT HCT116, 1KO and 3BKO cell lines were also included in the analysis. Results are normalized to PCNA mRNA levels. Data represents mean and standard deviation of triplicate PCR reactions from a single experiment, which is representative of two independent biological replicate experiments. E/V: Empty Vector.
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[Show abstract][Hide abstract] ABSTRACT: Author Summary
Proper inheritance of DNA methylation patterns is essential for preserving cellular identity and preventing malignant cellular transformation. In mammals, DNMT3A/3B, the de novo methyltransferases, establish the DNA methylation patterns during development and then maintain them in co-operation with the maintenance methyltransferase, DNMT1, through cell divisions. However, the mechanisms by which DNMT3A/3B assist DNMT1 in faithful inheritance of methylation patterns in somatic cells while guarding against aberrant de novo DNA methylation are still unclear. In this study, we present a novel principle of enzyme regulation where the levels of the catalyzing enzymes, DNMT3A/3B, are determined by the level of their own enzymatic product, i.e. 5-methylcytosine itself. Through biochemical analyses, we have shown that binding of DNMT3A/3B to nucleosomes with methylated DNA stabilizes these proteins, enabling faithful propagation of methylation patterns through cell divisions. However, reduction in DNA methylation results in diminished nucleosome binding of DNMT3A/3B and subsequent degradation of the free DNMT3A/3B proteins. This novel self-regulatory inheritance mechanism not only ensures faithful somatic propagation of methylated states but also prevents aberrant de novo methylation by causing degradation of free DNMT3A/3B enzymes.
[Show abstract][Hide abstract] ABSTRACT: Decreased stability of DNMT3A protein in DKO8 cells compared to WT HCT116 cells. Quantitation of protein bands shown in Figure 2B was done using Quantity One software (Bio-Rad). The data points represent DNMT3A levels, normalized to actin, at different time points presented as the fraction of protein remaining compared to levels present before CHX treatment. Straight lines represent linear regression adjustment of the individual time points. The half-life of the DNMT3A protein decreased from 16 hr in WT HCT116 to 7 hr in DKO8 cells. The data presented is from a single experiment, which is representative of two independent biological replicate experiments.
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[Show abstract][Hide abstract] ABSTRACT: Residual DNMT3A protein remains localized within nuclei in DKO cells. Co-cultured HCT116, DKO8 and DKO1 cells were immunostained for DNMT3A (green) using a rabbit polyclonal DNMT3A antibody and their nuclei (blue) were stained with 4,6-diamidino-2- phenylindole. Scale bar, 20 µm.
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[Show abstract][Hide abstract] ABSTRACT: Decrease in DNMT3A level in DKO cells is not due to protein mislocalization. Western blot analysis of whole cell extracts from various HCT116 derivative cell lines using DNMT3A antibody. Actin was used as the loading control.
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[Show abstract][Hide abstract] ABSTRACT: DNMT3B1 and ΔDNMT3B2 catalytically-inactive mutants interact with DNMT3A similar to wild-type DNMT3B1, ΔDNMT3B2, and DNMT3L. Western blot analysis was used to analyze proteins immunoprecipitated using Myc and DNMT3A antibodies in DKO8 cells expressing (A) Myc-DNMT3B1 or Myc- mut (mutant) DNMT3B1, (B) Myc-ΔDNMT3B2 or Myc- mut (mutant) ΔDNMT3B2 and (C) Myc- DNMT3L. IgG and CD-8 antibodies were used as a negative control. Antibodies used for immunoprecipitation (IP) are mentioned at the top and for immunoblotting on the left. Very faint bands of co-immunoprecipitated DNMT3A were visible in IPs with Myc antibody in (A) and (B), possibly due to very low levels of DNMT3A present in those cell lines. In (C), the upper strong band in the Myc and CD-8 IP lanes corresponds to the mouse IgG of the IP antibody while the lower band is for Myc-DNMT3L. Input denotes mononucleosomal digestes prepared for immunoprecipitation experiments. mut: mutant
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[Show abstract][Hide abstract] ABSTRACT: ΔDNMT3B2 catalytically-inactive mutant stimulates DNMT3A activity through their interaction upon nucleosomes. (A) Western blot analysis of nuclear extracts from DKO8 cells expressing wild-type and catalytically-inactive Myc-tagged ΔDNMT3B2 mutant (mut) using specific antibodies. (B) DNA methylation analysis of infected DKO8 cells using methylation-sensitive restriction enzymes. Genomic DNA was isolated from infected cells eight weeks after infection and methylation level was estimated as described in Figure 1. Data is presented as percentage of total genomic methylation present compared to WT HCT116 methylation levels. Data represents mean and SEM of three independent replicate experiments. (C) Mononucleosomes released from nuclei, extensively digested with MNase, were resolved by ultracentrifugation on a sucrose density gradient (5% to 25%) containing 300 mM NaCl. The gradients were fractionated and analyzed as described previously. E/V: Empty Vector; mut: mutant.
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[Show abstract][Hide abstract] ABSTRACT: Similar levels of mRNA expression of exogenous DNMTs in DKO8 and DKO1 cells. RT-PCR analysis was performed using primers for DNMT3B to check Myc-DNMT3B1 & Myc-ΔDNMT3B2 expression in DKO cells (see Table S1). The results are normalized to GAPDH mRNA levels. Data represents mean and standard deviation of triplicate PCR reactions from a single experiment, which is representative of two independent biological replicate experiments.
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[Show abstract][Hide abstract] ABSTRACT: Methylation of specific L1s across the bladder. (A) Tissue samples were taken from five patients of their tumors (red, T) and at increasing distances from the tumor (0.5 to 2 cm) in the surrounding normal-appearing tissue in multiple directions (light blue, a to d). Additionally, distant normal-appearing samples were taken at least 5 cm from the tumor (dark blue, C). (B) Methylation at L1-ACVR1C and (C) L1-RAB3IP was measured by pyrosequencing. The green line represents the mean methylation of 12 normal samples from cancer-free patients. While there are no error bars for the clinical sample analysis due to the extremely limited amount of sample DNA. the results show a consistent trend.
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[Show abstract][Hide abstract] ABSTRACT: ROC curves for specific L1s. (A) ROC curves using L1-MET methylation distinguish between normal bladder tissue (N) and corresponding normal bladder tissues (CN), N and bladder tumors (T), and CN and T. (B) ROC curves using L1-ACVR1C methylation, and (C) ROC curves using L1-RAB3IP methylation. *** represents p<0.001 and * represents p<0.05.
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[Show abstract][Hide abstract] ABSTRACT: Specific L1s with alternate transcripts located in intron of genes. Black boxes represent exons of the host gene while red boxes represent a specific L1. The black arrow represents the transcriptional start site of the host gene while the red arrow represents the alternate transcriptional start site within the potentially active L1 promoter. GenBank accession numbers for representative alternate transcripts are followed by the number in parentheses of similar transcripts transcribed from the individual L1. All L1s are antisense to their host genes, yielding alternate transcripts that are sense with their host genes.
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[Show abstract][Hide abstract] ABSTRACT: Bisulfite sequencing of L1-MET. Biphasic distribution of L1-MET methylation status in corresponding tissue from a patient with bladder cancer is revealed by plotting the number of DNA strands by the percent of CpG sites methylated.
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[Show abstract][Hide abstract] ABSTRACT: Chromatin remodeling occurs an active L1-MET promoter. Nucleosome positioning in an active fully unmethylated L1-MET promoter in T24 bladder carcinoma cells reveals a dinucleosomal structure, as determined by both M. SssI, a CpG methyltransferase and M. CviPI accessibility.
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[Show abstract][Hide abstract] ABSTRACT: The truncated MET protein encoded by L1-MET. (A) The functional domains of MET include the signal peptide (SP), sema domain at the N-terminus, the PSI domain, IPT repeats, the transmembrane domain (TM), and the kinase domain at the C-terminus. The structure of truncated MET proteins 1 and 2 are shown, encoded by transcripts derived from placenta (GenBank accession no. BX334980) and a bladder carcinoma cell line (BF208095), respectively. (B) The two L1-MET transcripts, truncated L1-MET-1 (T-MET-1) and truncated L1-MET-2 (T-MET-2), were cloned into a pMEV expression vector with 2 HA tags fused at the N-terminal. Hela cells were transfected with either the empty pMEV vector, pMEV T-MET-1, or pMEV T-MET-2 and protein was extracted after 48 hours. The expression of truncated MET-1 (90 kDa) and truncated MET-2 (60 kDa) was detected by western blot using an HA antibody. (C) Results of 5′RACE reveal the start site for L1-MET within the L1 element. The transcriptional start site of L1-MET was confirmed by 5′RACE in the T24 cell line which expressed L1-MET. The underlined sequence is located inside of the LINE-1. (D) RT–PCR analysis of reactivation of L1-MET by 1 or 3 µM of 5-Aza-CdR treatment for 24 hours (day 3 after treatment). β-actin expression level was used as a control.
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[Show abstract][Hide abstract] ABSTRACT: Methylation and expression of L1-RAB3IP correlates in cell lines. (A) Map of alternate transcripts from L1-RAB3IP. Exons are represented by black boxes while the specific L1s are represented by red boxes. The lower tick marks represent each CpG site. The left bent arrow indicates transcriptional start sites and ATGs indicate translational start sites. Green arrows indicate the primers used to amplify the pyrosequencing product and the black arrow in between indicates the location of the pyrosequencing primer for L1-RAB3IP. (B) L1-RAB3IP methylation (red bars) and L1 methylation (black bars) was analyzed by pyrosequencing in 6 normal tissues, one normal bladder fibroblast cell line (LD419), one non-tumorigenic urothelial cell lines (UROtsa), and 10 bladder carcinoma cell lines. Values are the average of one CpG site for L1 and an average of two CpG sites for L1-RAB3IP from two technical duplicates. (C) Expression of L1-RAB3IP was measured using real-time RT–PCR in one normal bladder fibroblast cell line, one normal urothelial cell line, and 10 bladder carcinoma cell lines. Values are also the average from two technical duplicates. Red bars indicate the methylation status of L1-RAB3IP, which is also represented in (B), and green bars represent the level of expression relative to GAPDH.
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