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Isoform switching and exon skipping induced by the DNA methylation inhibitor 5-Aza-2'-deoxycytidine.

Ding XL, Yang X, Liang G, Wang K - Sci Rep (2016)

Bottom Line: By analyzing the time series RNA-seq data (days 5, 9, 13, 17) obtained from human bladder cells exposed to 5-Aza-CdR with 0.1 uM concentration, we showed that 5-Aza-CdR can affect isoform switching and differential exon usage (i.e., exon-skipping), in addition to its effects on gene expression.We identified more than 2,000 genes with significant expression changes after 5-Aza-CdR treatment.Particularly, exon-skipping event in Enhancer of Zeste Homologue 2 (EZH2) along with expression changes showed significant down regulation on Day 5 and Day 9 but returned to normal level on Day 13 and Day 17.

View Article: PubMed Central - PubMed

Affiliation: Co-Innovation Center for Sustainable Forestry in Southern China, College of Forestry, Nanjing Forestry University, Nanjing, Jiangsu, 210037, China.

ABSTRACT
DNA methylation in gene promoters leads to gene silencing and is the therapeutic target of methylation inhibitors such as 5-Aza-2'-deoxycytidine (5-Aza-CdR). By analyzing the time series RNA-seq data (days 5, 9, 13, 17) obtained from human bladder cells exposed to 5-Aza-CdR with 0.1 uM concentration, we showed that 5-Aza-CdR can affect isoform switching and differential exon usage (i.e., exon-skipping), in addition to its effects on gene expression. We identified more than 2,000 genes with significant expression changes after 5-Aza-CdR treatment. Interestingly, 29 exon-skipping events induced by treatment were identified and validated experimentally. Particularly, exon-skipping event in Enhancer of Zeste Homologue 2 (EZH2) along with expression changes showed significant down regulation on Day 5 and Day 9 but returned to normal level on Day 13 and Day 17. EZH2 is a component of the multi-subunit polycomb repressive complex PRC2, and the down-regulation of exon-skipping event may lead to the regain of functional EZH2 which was consistent with our previous finding that demethylation may cause regain of PRC2 in demethylated regions. In summary, our study identified pervasive transcriptome changes of bladder cancer cells after treatment with 5-Aza-CdR, and provided valuable insights into the therapeutic effects of 5-Aza-CdR in current clinical trials.

No MeSH data available.


Related in: MedlinePlus

Summary of differentially expressed exons after 5-Aza-CdR treatment.(a) Venn diagram showing overlapped differentially expressed exons found in each time point. (b) Venn diagram showing overlapped genes identified as both differentially expressed genes and genes containing differentially expressed exons. (c) Visualization of differentially expressed exons found in MAGEA3 (ENSG00000221867.4) after 5, 9, 13, and 17 days treatment (differentially expressed exons were highlighted in red).
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f2: Summary of differentially expressed exons after 5-Aza-CdR treatment.(a) Venn diagram showing overlapped differentially expressed exons found in each time point. (b) Venn diagram showing overlapped genes identified as both differentially expressed genes and genes containing differentially expressed exons. (c) Visualization of differentially expressed exons found in MAGEA3 (ENSG00000221867.4) after 5, 9, 13, and 17 days treatment (differentially expressed exons were highlighted in red).

Mentions: Identification of DE exons can inform us on how the demethylation treatment affects exons recognition, and it can shed lights on the potential regulatory role of DNA methylation on alternative splicing. In total, 5958 (up: 3362, down: 2596), 4766 (up: 2364, down: 2402), 3102 (up: 1940, down: 1162) and 4334 (up: 2557, down: 1777) DE exons were identified on Day 5, Day 9, Day 13 and Day 17, respectively (Padj < 0.05). Among them, 1103 exons were observed with significant changes across all time points (Fig. 2a). Unlike DE genes, the most abundant DE exons were identified on Day 5, followed with steady decrease on Day 9 and Day 13 and increased again on Day 17 (Fig. 2a). We also tried to measure the overlap between DE exons and DE genes. We found that 68, 73, 78 and 87 DE genes from Day 5, Day 9, Day 13 and Day 17 contained at least one DE exon (Fig. 2b). For instance, significant changes were found on exon 2, 3, 4 and 6 of tumor antigen MAGEA3 across all time points, while MAGEA3 itself was also known as a DE gene throughout 5-Aza-CdR treatment (Figs 2c and 1e). Interestingly, we also note that most DE genes do not contain any DE exon regardless of the exposure time (Fig. 2b). Thus, most of the genes may not be subject to isoform switching, despite that the overall expressions changed considerably. Functional analysis revealed that genes with DE exons were mainly associated with biological processes such as translational elongation, biopolymer methylation and DNA methylation. In summary, 5-Aza-CdR can not only induce changes in gene expression, but also exon-level changes for a small subset of DE genes.


Isoform switching and exon skipping induced by the DNA methylation inhibitor 5-Aza-2'-deoxycytidine.

Ding XL, Yang X, Liang G, Wang K - Sci Rep (2016)

Summary of differentially expressed exons after 5-Aza-CdR treatment.(a) Venn diagram showing overlapped differentially expressed exons found in each time point. (b) Venn diagram showing overlapped genes identified as both differentially expressed genes and genes containing differentially expressed exons. (c) Visualization of differentially expressed exons found in MAGEA3 (ENSG00000221867.4) after 5, 9, 13, and 17 days treatment (differentially expressed exons were highlighted in red).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4835787&req=5

f2: Summary of differentially expressed exons after 5-Aza-CdR treatment.(a) Venn diagram showing overlapped differentially expressed exons found in each time point. (b) Venn diagram showing overlapped genes identified as both differentially expressed genes and genes containing differentially expressed exons. (c) Visualization of differentially expressed exons found in MAGEA3 (ENSG00000221867.4) after 5, 9, 13, and 17 days treatment (differentially expressed exons were highlighted in red).
Mentions: Identification of DE exons can inform us on how the demethylation treatment affects exons recognition, and it can shed lights on the potential regulatory role of DNA methylation on alternative splicing. In total, 5958 (up: 3362, down: 2596), 4766 (up: 2364, down: 2402), 3102 (up: 1940, down: 1162) and 4334 (up: 2557, down: 1777) DE exons were identified on Day 5, Day 9, Day 13 and Day 17, respectively (Padj < 0.05). Among them, 1103 exons were observed with significant changes across all time points (Fig. 2a). Unlike DE genes, the most abundant DE exons were identified on Day 5, followed with steady decrease on Day 9 and Day 13 and increased again on Day 17 (Fig. 2a). We also tried to measure the overlap between DE exons and DE genes. We found that 68, 73, 78 and 87 DE genes from Day 5, Day 9, Day 13 and Day 17 contained at least one DE exon (Fig. 2b). For instance, significant changes were found on exon 2, 3, 4 and 6 of tumor antigen MAGEA3 across all time points, while MAGEA3 itself was also known as a DE gene throughout 5-Aza-CdR treatment (Figs 2c and 1e). Interestingly, we also note that most DE genes do not contain any DE exon regardless of the exposure time (Fig. 2b). Thus, most of the genes may not be subject to isoform switching, despite that the overall expressions changed considerably. Functional analysis revealed that genes with DE exons were mainly associated with biological processes such as translational elongation, biopolymer methylation and DNA methylation. In summary, 5-Aza-CdR can not only induce changes in gene expression, but also exon-level changes for a small subset of DE genes.

Bottom Line: By analyzing the time series RNA-seq data (days 5, 9, 13, 17) obtained from human bladder cells exposed to 5-Aza-CdR with 0.1 uM concentration, we showed that 5-Aza-CdR can affect isoform switching and differential exon usage (i.e., exon-skipping), in addition to its effects on gene expression.We identified more than 2,000 genes with significant expression changes after 5-Aza-CdR treatment.Particularly, exon-skipping event in Enhancer of Zeste Homologue 2 (EZH2) along with expression changes showed significant down regulation on Day 5 and Day 9 but returned to normal level on Day 13 and Day 17.

View Article: PubMed Central - PubMed

Affiliation: Co-Innovation Center for Sustainable Forestry in Southern China, College of Forestry, Nanjing Forestry University, Nanjing, Jiangsu, 210037, China.

ABSTRACT
DNA methylation in gene promoters leads to gene silencing and is the therapeutic target of methylation inhibitors such as 5-Aza-2'-deoxycytidine (5-Aza-CdR). By analyzing the time series RNA-seq data (days 5, 9, 13, 17) obtained from human bladder cells exposed to 5-Aza-CdR with 0.1 uM concentration, we showed that 5-Aza-CdR can affect isoform switching and differential exon usage (i.e., exon-skipping), in addition to its effects on gene expression. We identified more than 2,000 genes with significant expression changes after 5-Aza-CdR treatment. Interestingly, 29 exon-skipping events induced by treatment were identified and validated experimentally. Particularly, exon-skipping event in Enhancer of Zeste Homologue 2 (EZH2) along with expression changes showed significant down regulation on Day 5 and Day 9 but returned to normal level on Day 13 and Day 17. EZH2 is a component of the multi-subunit polycomb repressive complex PRC2, and the down-regulation of exon-skipping event may lead to the regain of functional EZH2 which was consistent with our previous finding that demethylation may cause regain of PRC2 in demethylated regions. In summary, our study identified pervasive transcriptome changes of bladder cancer cells after treatment with 5-Aza-CdR, and provided valuable insights into the therapeutic effects of 5-Aza-CdR in current clinical trials.

No MeSH data available.


Related in: MedlinePlus