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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

Exon recognition changes induced by 5-Aza-CdR treatment.(a) Venn diagram showing overlapped differentially expressed skipped exon events found in each time point. (b) Sashimi plot showing the expression changes of EZH2 exon skipping (skipped exon was highlighted with orange rectangle) events at each time point. (ΔΨ > 0.15) (c) Visualization of differentially expressed exons found in EZH2 after 5, 9, 13, and 17 days treatment (differentially expressed exons were highlighted in red).
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f3: Exon recognition changes induced by 5-Aza-CdR treatment.(a) Venn diagram showing overlapped differentially expressed skipped exon events found in each time point. (b) Sashimi plot showing the expression changes of EZH2 exon skipping (skipped exon was highlighted with orange rectangle) events at each time point. (ΔΨ > 0.15) (c) Visualization of differentially expressed exons found in EZH2 after 5, 9, 13, and 17 days treatment (differentially expressed exons were highlighted in red).

Mentions: We next tried to investigate whether demethylation treatment would induce alternative splicing in bladder cancer cells, since it has been reported in other cell lines14. Alternative splicing events can be classified into different types: skipped exon, mutually exclusive exon, alternative 5′ splice site, alternative 3′ splice site23. Here, we focused on exon-skipping events, since they are likely to be affected by methylation. DE exon-skipping were identified by comparing RNA-seq data from each time point with control using MISO24. Initially, we found 43, 26, 14 and 25 exon-skipping events, which showed considerable changes on Day 5, Day 9, Day 13 and Day 17 (Fig. 3a). To further validate our findings, we compared our DE exon-skipping results with previous DE exons. Intuitively, the DE exon-skipping events should involve some DE exons. We identified 28 (up:8 down:11), 10 (up:4 down:6), 5 (up:3 down:2) and 8 (up:5 down:3) overlapped DE exon-skipping events while 19 of them altered its coding sequence (Table 1). All five DE exon-skipping events found on Day 13 were included on Day 9 while four of them were also found on Day 5 except RRBP1 (Table 1). In addition, nine exon-skipping events on Day 9 were also found on Day 5, again except RRBP1. However, eight exon-skipping events found on Day 17 together with ten events on Day 5 could not be observed at any other time points. Consequently, it seemed that exon-skipping events on Day 5 continued to show similar exon-skipping behavior on Day 9 and Day 13. After that, 17 days treatment resulted in eight other new DE exon-skipping events. One particular down regulated DE exon-skipping event was found in EZH2 gene on Day 5 and Day 9 which was located in one of the EZH2 transcripts annotated as being subject to nonsense-mediated decay by Ensemble (ENST00000483012.1) (Fig. 3b,c). EZH2 belongs to PRC2/EED-EZH2 complex which catalyzes ‘Lys-27′ methylation of histone H3 and leads to transcriptional repression of the target genes. Therefore, this exon-skipping event is expected to cause the regain of functional EZH2 gene.


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)

Exon recognition changes induced by 5-Aza-CdR treatment.(a) Venn diagram showing overlapped differentially expressed skipped exon events found in each time point. (b) Sashimi plot showing the expression changes of EZH2 exon skipping (skipped exon was highlighted with orange rectangle) events at each time point. (ΔΨ > 0.15) (c) Visualization of differentially expressed exons found in EZH2 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

f3: Exon recognition changes induced by 5-Aza-CdR treatment.(a) Venn diagram showing overlapped differentially expressed skipped exon events found in each time point. (b) Sashimi plot showing the expression changes of EZH2 exon skipping (skipped exon was highlighted with orange rectangle) events at each time point. (ΔΨ > 0.15) (c) Visualization of differentially expressed exons found in EZH2 after 5, 9, 13, and 17 days treatment (differentially expressed exons were highlighted in red).
Mentions: We next tried to investigate whether demethylation treatment would induce alternative splicing in bladder cancer cells, since it has been reported in other cell lines14. Alternative splicing events can be classified into different types: skipped exon, mutually exclusive exon, alternative 5′ splice site, alternative 3′ splice site23. Here, we focused on exon-skipping events, since they are likely to be affected by methylation. DE exon-skipping were identified by comparing RNA-seq data from each time point with control using MISO24. Initially, we found 43, 26, 14 and 25 exon-skipping events, which showed considerable changes on Day 5, Day 9, Day 13 and Day 17 (Fig. 3a). To further validate our findings, we compared our DE exon-skipping results with previous DE exons. Intuitively, the DE exon-skipping events should involve some DE exons. We identified 28 (up:8 down:11), 10 (up:4 down:6), 5 (up:3 down:2) and 8 (up:5 down:3) overlapped DE exon-skipping events while 19 of them altered its coding sequence (Table 1). All five DE exon-skipping events found on Day 13 were included on Day 9 while four of them were also found on Day 5 except RRBP1 (Table 1). In addition, nine exon-skipping events on Day 9 were also found on Day 5, again except RRBP1. However, eight exon-skipping events found on Day 17 together with ten events on Day 5 could not be observed at any other time points. Consequently, it seemed that exon-skipping events on Day 5 continued to show similar exon-skipping behavior on Day 9 and Day 13. After that, 17 days treatment resulted in eight other new DE exon-skipping events. One particular down regulated DE exon-skipping event was found in EZH2 gene on Day 5 and Day 9 which was located in one of the EZH2 transcripts annotated as being subject to nonsense-mediated decay by Ensemble (ENST00000483012.1) (Fig. 3b,c). EZH2 belongs to PRC2/EED-EZH2 complex which catalyzes ‘Lys-27′ methylation of histone H3 and leads to transcriptional repression of the target genes. Therefore, this exon-skipping event is expected to cause the regain of functional EZH2 gene.

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