ChIP-qPCR validation of Pol II and PIC subunit behavior on promoters of group A and B genes.

<p>ChIP was carried out 3 and 6-treated cells and analyzed by quantitative (q)-PCRs to monitor Pol II (dark blue bars) (in A), TBP (green bars) (in B), p62 (light blue bars) (in C) occupancy on the promoters of genes selected from group A (<i>ubc, rplp1</i>), group B (<i>p21, wdr24</i>) and on a negative control (<i>intergenic</i>) region, as indicated. Control ChIP (NoAb, red bars) was carried out with Sepharose G beads only. The occupancy values at the promoters are represented in input %. Error bars represent +/− standard deviations.</p

The protein level of Pol II and GTF subunits does not change after UVB treatment.

<p>(A) Western blot assays were carried out to measure the levels of total Pol II at the indicated time points after UVB treatment by using the N-20 antibody. (B) The different IIA and IIO forms of Pol II were densitometrically quantified in each time points from four independent experiments and are represented in a bar chart, where red bars indicate Pol IIA and the blue bars indicate Pol IIO forms. In each lane Pol IIA+IIO signals are taken as 100%. Error bars represent +/− standard deviation of the four independent experiments. (C) The phosphorylation levels of Pol II Rpb1-CTD Ser2, Ser5 and Ser7 were analyzed by western blot by using the indicated antibodies, respectively. (D) The levels of the indicted GTF subunits such as TFIIB, TBP and TFIIH/CDK7 were also tested in the above prepared extracts, as indicated. Tubulin-α was used as a loading control.</p

Different Pol II behavior patterns in time after UVB irradiation on 4500 expressed genes.

<p>From the ChIP-seq datasets heat maps were generated to follow Pol II behavior in time on the 4500 expressed genes at different genomic regions after UVB treatment in MCF7 cells. Tag numbers were calculated and visualized around the TSS (−/+300 bp), on the gene body (−100 bp from TSS until EAG) and downstream from EAG (from EAG to EAG+4 kb) of each gene. To be able to uniformly follow and represent the diverse Pol II occupancy changes on thousands of genes, the calculated Pol II reads at the different regions of different genes (as indicated) were converted into percentage values, where the control non-UVB treated values represent 100% reads (black color code) and the Pol II read alterations are either represented as % loss (green color) or as % gain (red color code). Each horizontal line represents one gene. The intensity of the color on the heatmap represents the magnitude of the Pol II tag number alteration (max −/+73%). Black color also refers to the lack of Pol II density change. In addition, genes were sorted into distinct groups and subgroups (Group Aa-Ag and Group B) based on the Pol II tag density values and patterns by using k-means clustering. Different time points following UVB irradiation are labeled at the top of the panels in hour (h). C = control, non-irradiated data set. For individual genes see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004483#pgen.1004483.s003" target="_blank">Figure S3</a>.</p

Global Pol II tag density changes dynamically in the gene body regions of all transcribed genes following UVB irradiation.

<p>Representation of Pol II read number changes obtained by ChIP-seq on MCF7 cell populations following UVB irradiation (as indicated) on the GB regions of the selected 4500 expressed genes when compared to that of the control sample. Pol II tag numbers were calculated on the GB (−100 bp from TSS until EAG) of the selected genes and represented in log 10 value on the y-axis. On the x axis, 4500 expressed genes are sorted based on their genomic lengths labeled in base pairs. Genes are classified from longest genes (from left) to shortest genes (at the right). Blue dots indicate tag numbers at genes in the control sample, and red dots indicate tag numbers at genes in samples harvested (A) 1 hour (h), (B) 2 h, (C) 3 h, (D) 4 h, (E) 5 h and (F) 6 h following UVB irradiation. See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004483#pgen.1004483.s002" target="_blank">Figure S2</a> for the total quantifications of the read numbers obtained in the different samples with the corresponding statistical tests.</p

Pol II behavior on annotated DNA damage repair and UV responsive genes after UVB irradiation.

<p>Pol II reads were calculated around the TSS (+/−300 bp), on the gene body (−100 bp from TSS until EAG) and downstream from EAG (from EAG to EAG +4 kb) for well characterized DNA repair and UV responsive genes (from the KEGG database) from each ChIP-seq dataset. From the collected values heat map was generated, and genes are organized into groups based on similar Pol II behavior by using k-means clustering. Pol II occupancy changes are represented as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004483#pgen-1004483-g004" target="_blank">Figure 4</a>. Red color shows increased; green color shows decreased Pol II tag numbers in percentages compared to data obtained from the control non-UV irradiated cells (black color). Black color also refers to no change in Pol II density. The figure is labeled similarly as <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004483#pgen-1004483-g004" target="_blank">Figure 4</a>.</p

Results of gene ontology analyses carried out using the D.A.V.I.D. software to identify differential gene-function categories for the detected gene groups with distinct Pol II behavior patterns from Figure 4.

<p>Results of gene ontology analyses carried out using the D.A.V.I.D. software to identify differential gene-function categories for the detected gene groups with distinct Pol II behavior patterns from <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004483#pgen-1004483-g004" target="_blank">Figure 4</a>.</p

In the absence of CSB UVB does not inhibit Pol II and TFIIH binding at promoters of group A genes.

<p>MCF7 cells were transfected with either scrambled siRNA or siRNAs targeting CSB (ERCC6) (see also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004483#pgen.1004483.s004" target="_blank">Figure S4</a>), 72 hours following siRNA transfection cells were treated with UVB, or not. ChIP was carried out 3 hours following UVB treatment of MCF7 cells as well as on non-treated cells. Q-PCRs were carried out to monitor Pol II (dark blue bars) (in A), and p62 (light blue bars) (in B) occupancy on the promoters of genes selected from group A (<i>ubc, rplp1</i>), group B (<i>p21, wdr24</i>) and on a negative control (<i>intergenic</i>) region in the presence or absence of CSB, as indicated. Control ChIPs (NoAb, red bars) was carried out with Sepharose G beads only. The occupancy values at the promoters are represented in input %. Error bars represent +/− standard deviations in two biological replicates.</p

Global ongoing transcription in MCF7 cells upon UVB irradiation.

<p>(A) Incorporation of 5-fluorouridine (5-FU) in nascent transcripts was assessed in untreated or UV irradiated cells by indirect immunofluorescence using an anti 5-FU antibody. Cell nuclei were counterstained using DAPI and the overlay between the DAPI and 5-FU signal is shown. (B) 5-FU signal intensity in the nucleus (excluding the nucleolar signal) was measured in n randomly chosen cell nuclei using the ImageJ software (NIH), where n is 59 in control, 54 in UVB+1 h, 84 in UVB+2 h, 69 in UVB+3 h, 58 in UVB+4 h, 57 in UVB+6 h. Results are presented as a boxplot where the min, max and median values are in red, purple and green respectively. P values (non-equal variance), calculated by comparing the non-irradiates sample (control) with the other time points following UVB irradiation, are the following: UVB+1 h 0,00126; UVB+2 h 0,03203; UVB+3 h 1,91441 E-13; UVB+4 h 5,72028 E-17; UVB+6 h 9,452619 E-20.</p

Table1_The use of progeroid DNA repair-deficient mice for assessing anti-aging compounds, illustrating the benefits of nicotinamide riboside.docx

Despite efficient repair, DNA damage inevitably accumulates with time affecting proper cell function and viability, thereby driving systemic aging. Interventions that either prevent DNA damage or enhance DNA repair are thus likely to extend health- and lifespan across species. However, effective genome-protecting compounds are largely lacking. Here, we use Ercc1Δ/− and Xpg−/− DNA repair-deficient mutants as two bona fide accelerated aging mouse models to test propitious anti-aging pharmaceutical interventions. Ercc1Δ/− and Xpg−/− mice show shortened lifespan with accelerated aging across numerous organs and tissues. Previously, we demonstrated that a well-established anti-aging intervention, dietary restriction, reduced DNA damage, and dramatically improved healthspan, strongly extended lifespan, and delayed all aging pathology investigated. Here, we further utilize the short lifespan and early onset of signs of neurological degeneration in Ercc1Δ/− and Xpg−/− mice to test compounds that influence nutrient sensing (metformin, acarbose, resveratrol), inflammation (aspirin, ibuprofen), mitochondrial processes (idebenone, sodium nitrate, dichloroacetate), glucose homeostasis (trehalose, GlcNAc) and nicotinamide adenine dinucleotide (NAD+) metabolism. While some of the compounds have shown anti-aging features in WT animals, most of them failed to significantly alter lifespan or features of neurodegeneration of our mice. The two NAD+ precursors; nicotinamide riboside (NR) and nicotinic acid (NA), did however induce benefits, consistent with the role of NAD+ in facilitating DNA damage repair. Together, our results illustrate the applicability of short-lived repair mutants for systematic screening of anti-aging interventions capable of reducing DNA damage accumulation.</p

Image2_The use of progeroid DNA repair-deficient mice for assessing anti-aging compounds, illustrating the benefits of nicotinamide riboside.JPEG

Despite efficient repair, DNA damage inevitably accumulates with time affecting proper cell function and viability, thereby driving systemic aging. Interventions that either prevent DNA damage or enhance DNA repair are thus likely to extend health- and lifespan across species. However, effective genome-protecting compounds are largely lacking. Here, we use Ercc1Δ/− and Xpg−/− DNA repair-deficient mutants as two bona fide accelerated aging mouse models to test propitious anti-aging pharmaceutical interventions. Ercc1Δ/− and Xpg−/− mice show shortened lifespan with accelerated aging across numerous organs and tissues. Previously, we demonstrated that a well-established anti-aging intervention, dietary restriction, reduced DNA damage, and dramatically improved healthspan, strongly extended lifespan, and delayed all aging pathology investigated. Here, we further utilize the short lifespan and early onset of signs of neurological degeneration in Ercc1Δ/− and Xpg−/− mice to test compounds that influence nutrient sensing (metformin, acarbose, resveratrol), inflammation (aspirin, ibuprofen), mitochondrial processes (idebenone, sodium nitrate, dichloroacetate), glucose homeostasis (trehalose, GlcNAc) and nicotinamide adenine dinucleotide (NAD+) metabolism. While some of the compounds have shown anti-aging features in WT animals, most of them failed to significantly alter lifespan or features of neurodegeneration of our mice. The two NAD+ precursors; nicotinamide riboside (NR) and nicotinic acid (NA), did however induce benefits, consistent with the role of NAD+ in facilitating DNA damage repair. Together, our results illustrate the applicability of short-lived repair mutants for systematic screening of anti-aging interventions capable of reducing DNA damage accumulation.</p