Alternative Transcript Initiation and Splicing as a Response to DNA Damage

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

Photoinduced damage of AsLOV2 domain is accompanied by increased singlet oxygen production due to flavin dissociation

Flavin mononucleotide (FMN) belongs to the group of very efficient endogenous photosensitizers producing singlet oxygen, 1O2, but with limited ability to be targeted. On the other hand, in genetically-encoded photosensitizers, which can be targeted by means of various tags, the efficiency of FMN to produce 1O2 is significantly diminished due to its interactions with surrounding amino acid residues. Recently, an increase of 1O2 production yield by FMN buried in a protein matrix was achieved by a decrease of quenching of the cofactor excited states by weakening of the protein-FMN interactions while still forming a complex. Here, we suggest an alternative approach which relies on the blue light irradiation-induced dissociation of FMN to solvent. This dissociation unlocks the full capacity of FMN as 1O2 producer. Our suggestion is based on the study of an irradiation effect on two variants of the LOV2 domain from Avena sativa; wild type, AsLOV2 wt, and the variant with a replaced cysteine residue, AsLOV2 C450A. We detected irradiation-induced conformational changes as well as oxidation of several amino acids in both AsLOV2 variants. Detailed analysis of these observations indicates that irradiation-induced increase in 1O2 production is caused by a release of FMN from the protein. Moreover, an increased FMN dissociation from AsLOV2 wt in comparison with AsLOV2 C450A points to a role of C450 oxidation in repelling the cofactor from the protein

Expression levels of the top five DNA repair genes as determined by qRT-PCR in fibroblasts over different IR doses and times.

<p>Relative gene expression is shown for samples that were either sham irradiated or irradiated with 1 Gy, 2 Gy, 5 Gy, 10 Gy or 20 Gy of radiation at 4 hours post-IR, or sham irradiated or irradiated with 10 Gy at 2 h, 4 h, 8 h, 24 h or 48 h post-IR.</p

DNA repair genes transcriptionally modulated in primary fibroblasts at 4 hr post-IR (p&lt;0.05).

<p>DNA repair genes transcriptionally modulated in primary fibroblasts at 4 hr post-IR (p&lt;0.05).</p

Expression levels of the top five DNA repair genes as determined by qRT-PCR in LCLs over different IR doses and times.

<p>Relative gene expression is shown for samples that were either sham irradiated or irradiated with 1 Gy, 2 Gy, 5 Gy, 10 Gy or 20 Gy of radiation at 4 hours post-IR, or sham irradiated or irradiated with 10 Gy at 2 h, 4 h, 8 h, 24 h or 48 h post-IR.</p

Alternative transcripts in DNA repair genes are induced by IR.

<p>Alternative transcripts in the DNA repair genes, XPC (A) and RRM2B (B) in response to IR are shown. Graph axes are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053358#pone-0053358-g001" target="_blank">figure 1</a>; PCR products from 5′-RML-RACE were run on a 2% agarose gel. An arrow indicates the amplicon from the alternative initiated transcript that was sequenced (gel picture to the right of the panel; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053358#pone.0053358.s004" target="_blank">Figure S4</a>). Diagrams of the predominant transcripts (initiating by P1) and the alternatively initiated (P2) transcripts after IR are shown below. Primer locations for 5′ RLM-RACE are indicated below exon 2 (arrow pointing to the left).</p

Induction of DNA repair genes at the exon level four hours after treatment with 10 Gy IR in primary fibroblast cells.

<p>The DNA repair genes: <i>XPC</i> (A), <i>POLH</i> (B), <i>DDB2</i> (C), <i>PCNA</i> (D) and <i>RRM2B</i> (E) as identified using Partek Genomics Suite 6.6 statistical package. Relative fluorescence (y-axis; log₂) is plotted for each PSR (x-axis). Core PSRs are labelled numerically in a 5′ to 3′ direction (left to right). Samples were either sham irradiated (red) or irradiated (blue) with 10 Gy of radiation from a ¹³⁷Cs source. RNA was collected 4 hours following treatment. Arrow indicates the PSR region to which primers were designed for qRT-PCR used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053358#pone-0053358-g003" target="_blank">figure 3B</a>. Error bars = SEM (n = 12).</p

Dose response and time course of selected DNA repair genes in human cell lines.

<p>PSR hybridization signals are shown for a two DNA repair genes that are induced following radiation. These are <i>POLH</i> (A, B) and <i>DDB2</i> (C–F) in LCLs (A–D) or fibroblasts (E, F). Relative fluorescence is plotted on the y-axis and PSRs are plotted evenly across the x-axis in a 5′ to 3′ direction (left to right). Samples from different individuals were either sham irradiated (red) or irradiated with 1 Gy (blue), 2 Gy (green), 5 Gy (purple), 10 Gy (orange) or 20 Gy (aqua) of radiation (A, C and E; n = 4). RNA was collect 4 hours following treatment. Time course of DNA repair genes were either sham irradiated (red) or irradiated with 10 Gy and RNA isolated 2 hrs (blue), 4 hrs (green), 8 hrs (purple), 24 hours (orange) or 48 hours (aqua) after irradiation (B, D and F; n = 4). Error bars = SEM.</p

Induction of DNA repair genes at the exon level four hours after treatment with 10 Gy IR in LCLs.

<p>The top five DNA repair genes: <i>XPC</i> (A), <i>POLH</i> (B), <i>DDB2</i> (C), <i>PCNA</i> (D) and <i>RRM2B</i> (E) as identified using Partek Genomics Suite 6.6 statistical package. Relative fluorescence (y-axis; log₂) is plotted for each PSR (x-axis). Core PSRs are labelled numerically in a 5′ to 3′ direction (left to right). Samples were either sham irradiated (red) or irradiated (blue) with 10 Gy from a ¹³⁷Cs source. Arrow indicates the PSR region to which primers were designed for qRT-PCR used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053358#pone-0053358-g003" target="_blank">figure 3A</a>. Error bars = SEM (n = 12).</p

Validation of DNA repair gene expression modulation following 10 Gy IR using qRT-PCR.

<p>Ct values were normalized to <i>PGK</i>. Each bar represents data from 12 different cell lines for both LCL (A) and primary fibroblasts (B) with the following exceptions: 6 samples were used for <i>PCNA</i> and <i>RRM2B</i> in the LCL experiments; 10 samples were used for <i>XPC</i>, <i>RRM2B</i>, <i>REV3L</i> for the fibroblast experiments and 5 samples for, <i>EXO1</i>, <i>PALB2</i>, <i>LIG1</i> and <i>H2AFX</i> were used in the fibroblast experiments. Gene expression levels were averaged across multiple experiments. Four separate qRT-PCR runs were carried out for <i>POLH</i>, <i>DDB2</i>, <i>APTX</i>, <i>RAD51C</i>, and <i>PALB2</i> genes; three separate qRT-PCR runs were carried out for <i>PCNA</i>, <i>REV3L</i> and <i>EXO1</i> genes; and two separate qRT-PCR runs were carried out for <i>XPC</i>, <i>RRM2B</i>, <i>H2AFX</i>, and <i>RAD51</i> genes. Error bars = SEM (n = 12). Each value on an experiment was run in triplicate. All differences shown are statistically significant (p&lt;0.05) using a t-test. PSRs used for amplification are: <i>XPC</i>: PSR853; <i>POLH</i>: PSR124; <i>DDB2</i>: PSR663; <i>PCNA</i>: PSR213; <i>RRM2B</i>: PSR293; <i>REV3L</i>: PSR729; <i>LIG1</i>: PSR905; <i>APTX</i>: PSR338; <i>H2AFX</i>: PSR185; <i>RAD51C</i>: PSR786; <i>RAD51</i>: PSR100; <i>EXO1</i>: PSR239; <i>PALB2</i>: PSR346; <i>POLL</i>: PSR904 for which some are indicated by and arrow in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053358#pone-0053358-g001" target="_blank">figures 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053358#pone-0053358-g002" target="_blank">2</a> and all full PSR numbers can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053358#pone.0053358.s001" target="_blank">Figures S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053358#pone.0053358.s002" target="_blank">S2</a>.</p