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Alternative transcript initiation and splicing as a response to DNA damage.

Sprung CN, Li J, Hovan D, McKay MJ, Forrester HB - PLoS ONE (2011)

Bottom Line: Dose-response and time course kinetics have also been characterized.Interestingly, most genes showing alternative transcript induction maintained these isoforms over the dose range and times tested.This study provides novel insights into alternative transcripts as a mechanism for response to DNA damage and cell stress responses in general.

View Article: PubMed Central - PubMed

Affiliation: Centre for Innate Immunity and Infectious Disease, Monash Institute for Medical Research, Monash University, Clayton, Victoria, Australia. carl.sprung@monash.edu

ABSTRACT
Humans are exposed to the DNA damaging agent, ionizing radiation (IR), from background radiation, medical treatments, occupational and accidental exposures. IR causes changes in transcription, but little is known about alternative transcription in response to IR on a genome-wide basis. These investigations examine the response to IR at the exon level in human cells, using exon arrays to comprehensively characterize radiation-induced transcriptional expression products. Previously uncharacterized alternative transcripts that preferentially occur following IR exposure have been discovered. A large number of genes showed alternative transcription initiation as a response to IR. Dose-response and time course kinetics have also been characterized. Interestingly, most genes showing alternative transcript induction maintained these isoforms over the dose range and times tested. Finally, clusters of co-ordinately up- and down-regulated radiation response genes were identified at specific chromosomal loci. These data provide the first genome-wide view of the transcriptional response to ionizing radiation at the exon level. This study provides novel insights into alternative transcripts as a mechanism for response to DNA damage and cell stress responses in general.

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PCR validation of ionizing radiation responsive genes in LCLs.(A) PCR was used to amplify the PLK2, SESN2 and XPC cDNA derived from the transcriptional products of cell lines that were irradiated with 10 Gy or sham irradiated. The amplified products were analysed by polyacrylamide gel electrophoresis. The relative amounts were calculated using densitometric analysis and expression levels were normalized to PGK expression. QRT-PCR was used to validate selected up- (B) and down- (C) regulated genes. PSRs that were used to assess intra-gene expression are indicated by the last three numerals of the gene-specific PSR. Error bars represent the SEM. Bar graphs represent CDKN1A-PSR189: n = 4 (p = 0.012); FBXO22-PSR527: n = 6 (p = 0.004); AEN-PSR256 (p = 0.003); XPC-PSR853 (p = 0.0001); H2AFX-PSR185: n = 6 (p = 0.001); CENPA-PSR000: n = 5 (p = 0.002); CENPE-PSR236: n = 4 (p = 0.002). (D) Example of individual cell lines that show increased or decreased expression at a specific PSR following radiation are shown. PLK2-PSR040 (induced) and CENPA-PSR000 (down-regulated) array data expression levels for each LCL tested at a representative PSR are shown at 0 (red) and 10 (blue) Gy. Lines link 0 Gy and 10 Gy for the individual cell lines. Boxes in box plots show 50% and whiskers to 80% of samples.
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pone-0025758-g003: PCR validation of ionizing radiation responsive genes in LCLs.(A) PCR was used to amplify the PLK2, SESN2 and XPC cDNA derived from the transcriptional products of cell lines that were irradiated with 10 Gy or sham irradiated. The amplified products were analysed by polyacrylamide gel electrophoresis. The relative amounts were calculated using densitometric analysis and expression levels were normalized to PGK expression. QRT-PCR was used to validate selected up- (B) and down- (C) regulated genes. PSRs that were used to assess intra-gene expression are indicated by the last three numerals of the gene-specific PSR. Error bars represent the SEM. Bar graphs represent CDKN1A-PSR189: n = 4 (p = 0.012); FBXO22-PSR527: n = 6 (p = 0.004); AEN-PSR256 (p = 0.003); XPC-PSR853 (p = 0.0001); H2AFX-PSR185: n = 6 (p = 0.001); CENPA-PSR000: n = 5 (p = 0.002); CENPE-PSR236: n = 4 (p = 0.002). (D) Example of individual cell lines that show increased or decreased expression at a specific PSR following radiation are shown. PLK2-PSR040 (induced) and CENPA-PSR000 (down-regulated) array data expression levels for each LCL tested at a representative PSR are shown at 0 (red) and 10 (blue) Gy. Lines link 0 Gy and 10 Gy for the individual cell lines. Boxes in box plots show 50% and whiskers to 80% of samples.

Mentions: Expression differences observed from microarrays at 4 hours after exposure to 10 Gy of radiation was validated by PCR using several different LCLs (3 to 12 for QRT-PCR) as indicated (Figures 3, 4, 5). Primers were designed within exon regions (Table S1). QRT-PCR primers to PGK and/or GAPDH transcripts were used for normalization controls. Amplicons from genes (e.g., PLK2, SESN2 and XPC) were run on polyacrylamide gels using cycle numbers determined to be in the linear amplification range (Figure 3A). Primers were prepared to selected PSRs and QRT-PCR was performed to compare transcript levels in sham-irradiated and 10 Gy at 4 hours post-IR. The results for a number of gene transcripts shown to be modulated from exon array data were confirmed to be induced (Figure 3B) or down-regulated (Figure 3C). Likewise, similar validation experiments were conducted to confirm microarray data obtained for fibroblast samples (Figure 5). Examples of relative expression levels for each of twelve cell lines for sham-irradiated and 4 hr post-IR at a specific PSR is shown for PLK2 PSR040 and CENPA PSR000 (Figure 3D).


Alternative transcript initiation and splicing as a response to DNA damage.

Sprung CN, Li J, Hovan D, McKay MJ, Forrester HB - PLoS ONE (2011)

PCR validation of ionizing radiation responsive genes in LCLs.(A) PCR was used to amplify the PLK2, SESN2 and XPC cDNA derived from the transcriptional products of cell lines that were irradiated with 10 Gy or sham irradiated. The amplified products were analysed by polyacrylamide gel electrophoresis. The relative amounts were calculated using densitometric analysis and expression levels were normalized to PGK expression. QRT-PCR was used to validate selected up- (B) and down- (C) regulated genes. PSRs that were used to assess intra-gene expression are indicated by the last three numerals of the gene-specific PSR. Error bars represent the SEM. Bar graphs represent CDKN1A-PSR189: n = 4 (p = 0.012); FBXO22-PSR527: n = 6 (p = 0.004); AEN-PSR256 (p = 0.003); XPC-PSR853 (p = 0.0001); H2AFX-PSR185: n = 6 (p = 0.001); CENPA-PSR000: n = 5 (p = 0.002); CENPE-PSR236: n = 4 (p = 0.002). (D) Example of individual cell lines that show increased or decreased expression at a specific PSR following radiation are shown. PLK2-PSR040 (induced) and CENPA-PSR000 (down-regulated) array data expression levels for each LCL tested at a representative PSR are shown at 0 (red) and 10 (blue) Gy. Lines link 0 Gy and 10 Gy for the individual cell lines. Boxes in box plots show 50% and whiskers to 80% of samples.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198437&req=5

pone-0025758-g003: PCR validation of ionizing radiation responsive genes in LCLs.(A) PCR was used to amplify the PLK2, SESN2 and XPC cDNA derived from the transcriptional products of cell lines that were irradiated with 10 Gy or sham irradiated. The amplified products were analysed by polyacrylamide gel electrophoresis. The relative amounts were calculated using densitometric analysis and expression levels were normalized to PGK expression. QRT-PCR was used to validate selected up- (B) and down- (C) regulated genes. PSRs that were used to assess intra-gene expression are indicated by the last three numerals of the gene-specific PSR. Error bars represent the SEM. Bar graphs represent CDKN1A-PSR189: n = 4 (p = 0.012); FBXO22-PSR527: n = 6 (p = 0.004); AEN-PSR256 (p = 0.003); XPC-PSR853 (p = 0.0001); H2AFX-PSR185: n = 6 (p = 0.001); CENPA-PSR000: n = 5 (p = 0.002); CENPE-PSR236: n = 4 (p = 0.002). (D) Example of individual cell lines that show increased or decreased expression at a specific PSR following radiation are shown. PLK2-PSR040 (induced) and CENPA-PSR000 (down-regulated) array data expression levels for each LCL tested at a representative PSR are shown at 0 (red) and 10 (blue) Gy. Lines link 0 Gy and 10 Gy for the individual cell lines. Boxes in box plots show 50% and whiskers to 80% of samples.
Mentions: Expression differences observed from microarrays at 4 hours after exposure to 10 Gy of radiation was validated by PCR using several different LCLs (3 to 12 for QRT-PCR) as indicated (Figures 3, 4, 5). Primers were designed within exon regions (Table S1). QRT-PCR primers to PGK and/or GAPDH transcripts were used for normalization controls. Amplicons from genes (e.g., PLK2, SESN2 and XPC) were run on polyacrylamide gels using cycle numbers determined to be in the linear amplification range (Figure 3A). Primers were prepared to selected PSRs and QRT-PCR was performed to compare transcript levels in sham-irradiated and 10 Gy at 4 hours post-IR. The results for a number of gene transcripts shown to be modulated from exon array data were confirmed to be induced (Figure 3B) or down-regulated (Figure 3C). Likewise, similar validation experiments were conducted to confirm microarray data obtained for fibroblast samples (Figure 5). Examples of relative expression levels for each of twelve cell lines for sham-irradiated and 4 hr post-IR at a specific PSR is shown for PLK2 PSR040 and CENPA PSR000 (Figure 3D).

Bottom Line: Dose-response and time course kinetics have also been characterized.Interestingly, most genes showing alternative transcript induction maintained these isoforms over the dose range and times tested.This study provides novel insights into alternative transcripts as a mechanism for response to DNA damage and cell stress responses in general.

View Article: PubMed Central - PubMed

Affiliation: Centre for Innate Immunity and Infectious Disease, Monash Institute for Medical Research, Monash University, Clayton, Victoria, Australia. carl.sprung@monash.edu

ABSTRACT
Humans are exposed to the DNA damaging agent, ionizing radiation (IR), from background radiation, medical treatments, occupational and accidental exposures. IR causes changes in transcription, but little is known about alternative transcription in response to IR on a genome-wide basis. These investigations examine the response to IR at the exon level in human cells, using exon arrays to comprehensively characterize radiation-induced transcriptional expression products. Previously uncharacterized alternative transcripts that preferentially occur following IR exposure have been discovered. A large number of genes showed alternative transcription initiation as a response to IR. Dose-response and time course kinetics have also been characterized. Interestingly, most genes showing alternative transcript induction maintained these isoforms over the dose range and times tested. Finally, clusters of co-ordinately up- and down-regulated radiation response genes were identified at specific chromosomal loci. These data provide the first genome-wide view of the transcriptional response to ionizing radiation at the exon level. This study provides novel insights into alternative transcripts as a mechanism for response to DNA damage and cell stress responses in general.

Show MeSH
Related in: MedlinePlus