Cellular characteristics of MEF cells.

1<p>- Recovery of Cell Survival.</p>2<p>- As observed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003611#pgen.1003611-vanderHorst1" target="_blank">[38]</a>.</p>3<p>- Host Cell Reactivation Assay.</p>4<p>- As observed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003611#pgen.1003611-Harada1" target="_blank">[39]</a>.</p><p>The cellular characteristics of MEF cells studied in this report are summarized. In bold we highlighted the characteristics of MEF^DOT1L that differ from MEF^CSB.</p

DOT1L ensures binding of RNA Pol II to chromatin after UV irradiation.

<p>(<b>A</b>) Schematic diagram showing the FRAP assay. Cells were transfected with a GFP-RNA Pol II construct (on RPB1). A small region in the middle of the nucleus was bleached and the subsequent fluorescence recovery was followed in time. When indicated, cells were UV-irradiated, 1 hour before the photobleaching. (<b>B–C</b>) FRAP curves of RNA Pol II-GFP protein stably expressed in either MEF^WT (<b>B</b>) or MEF^DOT1L (<b>C</b>) cells untreated (green) or treated (red) with UV (16 J/m²), 1 hour before photobleaching. Cells were photobleached with a 488 nm laser at maximum power 4 sec after the beginning of the acquisition. One image per 20 msec was taken during 20 sec in the photobleached area. SEM is within the range of 1% and is shown is supplemental Data 1.</p

Host cell reactivation assay in MEF cells.

<p>MEF^WT, MEF^DOT1L and MEF^CSB were transfected with mock-treated (panels a–c, g–i, m–o) or irradiated (UV-C, 600 J/m²) (panels d–f, j–l, p–r) pEGFP reporter plasmid in combination with a non-irradiated pRFP reporter plasmid used as control. Twelve hours post transfection, GFP and RFP expression were determined. A ratio of 3/1 (pEGFP/pRFP) was used during transfection to ensure that any cell expressing the RFP expresses also the GFP. P-value was extrapolated using t-test (**<0.05, ***<0.005).</p

Inhibition of the initiation-to-elongation transition phase in the absence of DOT1L, after UV irradiation.

<p>(<b>A</b>) Schematic representation of measuring the initiation-to-elongation transition rate of RNA Pol II transcription <i>in vivo</i> on endogenous genes. We reversibly blocked gene transcription by incubating cells with DRB. Cells are depleted of their pre-mRNA pool within few hours of incubation with the drug <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003611#pgen.1003611-Singh1" target="_blank">[28]</a>. Following the chase of DRB, RNA Pol II is released from promoter-proximal regions and newly synthesized pre-mRNA appear; the level of pre-mRNA is measured using oligonucleotides targeting respectively the exon/intron junctions of the gene. (<b>B</b>) Schematic diagram showing the experimental approach used to measure the rate of initiation-to-elongation transition phase by RNA Pol II after UV irradiation. MEF cells were treated with DRB for 3 hours before chase and addition of fresh medium at t = 0 hour. When indicated, cells were UV irradiated at t = 0 hour, before the addition of fresh medium or treated with TSA for 12 hours before the addition of DRB. (<b>C</b>) Expression levels of the newly synthesized Exon1 of the <i>Utrophin</i> gene in MEF^WT and MEF^DOT1L cells treated with 100 µM of DRB for 3 hours before the addition of fresh medium. The cells were harvest at intervals of 10 minutes for RNA isolation and qRT-PCR was performed using oligonucleotides targeting respectively the Exon1 and Intron1 of the gene. Relative expression values compared to mock treated cells are plotted against time (±SD). AUC was determined and p-value was extrapolated using t-test. (<b>D</b>) Expression levels of the newly synthesized Exon1 of the <i>Utrophin</i> gene in either MEF^WT or MEF^DOT1L irradiated with UV-C (15 J/m²) after treatment as in (<b>C</b>). AUC was determined and p-value was extrapolated using t-test. (<b>E</b>) Expression levels of the newly synthesized Exon1 of the <i>Utrophin</i> gene in either MEF^WT or MEF^DOT1L treated with TSA (20 nM) for 12 hours before addition of DRB for 3 hours and UV-C irradiation (15 J/m²). TSA was maintained in the medium during DRB treatment and time course. AUC was determined and p-value was extrapolated using t-test. (<b>F</b>) MEF^WT, MEF^CSB and MEF^DOT1L were irradiated with increasing doses of UV-C light. Cell survival was determined 96 hours later, as detailed in the Experimental Procedures. Data were normalized to the mock treated controls (as value of 1). Means of three independent experiments are shown (± SD). When indicated, cells were treated with TSA (10 nM), 12 hours before UV irradiation, and TSA was maintained for the time of the experiment.</p

Mobility of TTDA-GFP and XPD-GFP after UV Irradiation

<div><p>(A) FRAP_abc of TTDA-GFP expressing cells untreated (blue line) and treated with 8J/m2 UV-C (red line). Error bars are included in the curves, and the <i>p</i>-value has been calculated for the two distinct datasets. </p> <p>(B) FRAP_abc of XPD-GFP-expressing cells untreated (blue line) and treated with 8J/m2 UV-C (red line). For each line at least 20 different cells were measured. Error bars are included in the curves, and the <i>p</i>-value has been calculated for the two distinct datasets. </p> <p>(C) Example of FRAP on local damage. A TTDA-GFP-expressing cell (left panel) containing a locally inflicted UV-damaged spot (shown by the white arrow). The locally damaged area is bleached by applying a high-pulse laser beam (middle panel), and the subsequent recovery of fluorescence is followed in time (right panel).</p> <p>(D) Curves of FRAP on local damage of TTDA-GFP- (red line), XPD-GFP- (blue line), and XPB-GFP-(green line) expressing cells. FLD, fluorescence measured on local damage; FNLD, fluorescence measured on untreated areas. Error bars are included in the curves.</p></div

DOT1L is involved in UV-survival in mammalian cells.

<p>(<b>A</b>) Fifty µg of whole cell extracts from MEF^WT or MEF^DOT1L cells were resolved by SDS-PAGE and Western-blotted for DOT1L. Asterisk indicates an unspecific cross-reacting band. Ten µg of fractions from histone acid-extraction performed on MEF^WT or MEF^DOT1L cells were resolved by SDS-PAGE and Western-blotted for H3, H3K79me1 or H3K79me3. (<b>B</b>) MEF^WT, MEF^DOT1L, MEF^XPG and MEF^CSB cells were tested in a UV-survival assay. Cells were treated with increasing dose of UV-C and cell survival was determined 96 hours later, as detailed in the Experimental Procedures. Data were normalized to the mock treatment controls (as value of 1). The values are the means of three independent experiments (± SD). P-value was extrapolated for MEF^DOT1L/MEF^WT or MEF^DOT1L/MEF^XPG and indicated on the graph (***<0.005).</p

Comparison of Accumulation of NER Proteins on Local UV Damage and on Localized ActD-Photosensitized Laser Damage

<div><p>(A–D) Left panel, local DNA damage infliction by UV-C irradiation through a micro-porous filter. Right panel, local laser-induced (488 nm) DNA lesions by photo-sensitization of ActD.</p> <p>(A) XPC-GFP-expressing cells showing accumulation at local UV-damaged area (left panel) and laser induced ActD-damaged area (right panel).</p> <p>(B) XPB-GFP expressing cells showing accumulation at local UV-damaged area (left panel) and laser induced ActD-damaged area (right panel).</p> <p>(C) TTDA-GFP expressing cells showing only accumulation on local UV-damaged area (left panel) but not on laser induced ActD-damaged area (right panel); drawn rectangle corresponds with the irradiated area.</p> <p>(D) GFP-XPA expressing cells showing only accumulation on local UV-damaged area (left panel) but not on laser induced ActD-damaged area (right panel); drawn rectangle corresponds with the irradiated area.</p></div

DNA repair takes place in the absence of DOT1L.

<p>(<b>A</b>) Unscheduled DNA Synthesis (UDS) assay. Left panel; UDS expressed as mean number of autoradiographic grains/nucleus. UDS was measured by incubating MEF^WT or MEF^DOT1L with radioactive [³H]thymidine before treatment with increasing doses of UV-C light and autoradiography <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003611#pgen.1003611-Stefanini1" target="_blank">[25]</a>. The values are the means of two independent experiments +/−SEM. AUC for each curve were determined and p-value was extrapolated using a t-test. Right Panel; Autoradiogram of the UDS experiment performed at 20 J/m² in either MEF^WT (a) or MEF^DOT1L (b) cells. Arrows indicate radioactive dots. (<b>B</b>) (6-4)PP removal was carried out in MEF^WT, MEF^DOT1L, MEF^XPG and MEF^CSB, harvested at different time points after UV irradiation at 20 J/m². Cells were labeled with an anti-(6-4)PP antibody and signals were measured using IN Cell 1000 analyzer (GE Healthcare). Graph represents the % of lesions removed from the genome at different time points. The values are the means of three independent experiments (± SD). For each time point, a mean of 4000 cells has been analysed. (<b>C</b>) MEF^WT, MEF^DOT1L and MEF^CSB cells were treated with increasing concentration of et743 and cell survival was determined 96 hours later, as detailed in the Experimental Procedures. Data were normalized to the mock treatment controls (as value of 1). The values are the means of three independent experiments (± SD). P-value is indicated (***<0.005) and was determined between MEF^DOT1L and either MEF^CSB.</p

FRAP_abc

<div><p>(A) TTDA-GFP-expressing fibroblast without treatment (left panel) and after (right panel) applying several high laser pulses in the cytoplasmic compartment (see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040156#s4" target="_blank">Materials and Methods</a> for details). </p> <p>(B) TTDA-GFP mobility in the cytoplasm (green line), in the nucleus without bleaching the cytoplasm (red line), in the nucleus after bleaching the cytoplasmic fraction (blue line), and XPB-GFP mobility in the nucleus (pink line). The <i>p</i>-value has been calculated for cytoplasmic and nuclear mobility curves of TTDA-GFP. </p> <p>(C) XPD-GFP mobility in the cytoplasm (green line), in the nucleus without bleaching the cytoplasm (red line), in the nucleus after bleaching the cytoplasmic fraction (blue line), and XPB-GFP mobility in the nucleus (pink line). The <i>p</i>-value has been calculated for cytoplasmic and nuclear mobility curves of XPD-GFP. </p> <p>(D) FRAP_abc on XPB-GFP (pink line), XPD-GFP (blue line), TTDA-GFP (red line), and free GFP (green line). Inset shows increased time resolution of the curves with error bars.</p></div

Model of TTDA Binding to TFIIH during Transcription, NER, and Abortive NER

<p>Schematic representation of a mammalian cell with the nucleus in gray and nuclear pores simplified by holes in the membrane. TTDA is represented as a green sphere, TFIIH is depicted as an orange ellipse, and XPC is illustrated as a yellow ellipse. Arrows indicate equilibrium (passage through) over the nuclear pores and equilibrium between different TTDA and/or TFIIH molecules. Colored arrows show the changes in the equilibrium after DNA-damage induction or ActD treatment. In “NER-induction” (right, upper), the UV lesion is depicted as a lightning-sign, in “Abortive NER” (right, bottom), ActD is depicted as a blue trapezoid, and the red cross represents the inhibition of transcription.</p