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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

PcG (EZH2) mediated gene expression alteration and validation of exon-skipping events.(a) Differentially expressed PcG (EZH2) targeted genes induced by exon-skipping changes in EZH2. Aberrant exon recognition changes in EZH2 found in Day 5 and Day 9 resulted in corresponding inhibition of targeted genes. (b) Experimental validations of seven differentially expressed exon-skipping events after 5-Aza-CdR treatment.
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f4: PcG (EZH2) mediated gene expression alteration and validation of exon-skipping events.(a) Differentially expressed PcG (EZH2) targeted genes induced by exon-skipping changes in EZH2. Aberrant exon recognition changes in EZH2 found in Day 5 and Day 9 resulted in corresponding inhibition of targeted genes. (b) Experimental validations of seven differentially expressed exon-skipping events after 5-Aza-CdR treatment.

Mentions: Finally, seven DE exon-skipping events were further validated by experimental verification. PCR results showed that bands with larger fragment sizes (fragment contained skipped exon) of EZH2, LAS1L, DPH7, TARBP2 and NUMA1 became lighter after 5-Aza-CdR treatment while bands with smaller fragment sizes (fragment without skipped exon) became darker, which indicated the down regulation of skipped exons. Among them, only DPH7 was not identified with protein sequence change. Opposite observations can be found in another two up-regulated skipped exons of PHKA1 and FAM13B, where expressions of fragment containing skipped exons were found to be increased after treatment (Fig. 4b, Table 1). Both of these two events resulted in protein sequence changes. In summary, 5-Aza-CdR treatments were also capable of inducing dynamic changes on exon-skipping events and most of the changes were consistent from Day 7 till Day 13. Some of these exon-skipping events may result in gene functional changes, such as truncated protein in EZH2.


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)

PcG (EZH2) mediated gene expression alteration and validation of exon-skipping events.(a) Differentially expressed PcG (EZH2) targeted genes induced by exon-skipping changes in EZH2. Aberrant exon recognition changes in EZH2 found in Day 5 and Day 9 resulted in corresponding inhibition of targeted genes. (b) Experimental validations of seven differentially expressed exon-skipping events after 5-Aza-CdR treatment.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: PcG (EZH2) mediated gene expression alteration and validation of exon-skipping events.(a) Differentially expressed PcG (EZH2) targeted genes induced by exon-skipping changes in EZH2. Aberrant exon recognition changes in EZH2 found in Day 5 and Day 9 resulted in corresponding inhibition of targeted genes. (b) Experimental validations of seven differentially expressed exon-skipping events after 5-Aza-CdR treatment.
Mentions: Finally, seven DE exon-skipping events were further validated by experimental verification. PCR results showed that bands with larger fragment sizes (fragment contained skipped exon) of EZH2, LAS1L, DPH7, TARBP2 and NUMA1 became lighter after 5-Aza-CdR treatment while bands with smaller fragment sizes (fragment without skipped exon) became darker, which indicated the down regulation of skipped exons. Among them, only DPH7 was not identified with protein sequence change. Opposite observations can be found in another two up-regulated skipped exons of PHKA1 and FAM13B, where expressions of fragment containing skipped exons were found to be increased after treatment (Fig. 4b, Table 1). Both of these two events resulted in protein sequence changes. In summary, 5-Aza-CdR treatments were also capable of inducing dynamic changes on exon-skipping events and most of the changes were consistent from Day 7 till Day 13. Some of these exon-skipping events may result in gene functional changes, such as truncated protein in EZH2.

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