<i>CSA</i> and <i>HR23A</i> expression is up-regulated in XB2 mouse keratinocytes after repetitive UV irradiation.

<p>(A) Quantification of <i>CSA</i> and <i>CSB</i> expression following UV-irradiation (8×10 J/m²) was determined by RT-qPCR (ΔΔCT method). Results are expressed relative to control (no UV treatment) and normalized to an <i>HPRT</i> transcript standard (comparable results were obtained with other reference genes: <i>GAPDH</i>) n = 3. (B) Western blotting analysis and quantification of CSA protein level in XB2 cells irradiated as previously described and in UV-irradiated cells following a pretreatment or not with α-amanitin. Tubulin (α-Tub) is included as a loading control. Signals are detected using LAS-3000 Imaging System (Fujifilm) and quantified with ImageJ. CSA quantified data are reported in the subpanel, where (▪) corresponds to UV-irradiated samples and () to UV-irradiated samples pre-treated with α-amanitin. The bar graphs compare the intensity of CSA protein normalized to the loading control. (C) Quantification of <i>HR23A</i> and <i>HR23B</i> mRNA expression following UV-irradiation (8×10 J/m²) determined as previously by RT-qPCR (ΔΔCT method) n = 4. (D) Western blotting analysis of HR23A and HR23B protein levels as described previously in irradiated XB2 cells, pre-treated (▪) or not with α-amanitin (). HSC70 is included as a loading control. For all results errors bars indicate s.e.m.; one asterisk, <i>P</i><0,05 <i>n</i> = 3; two asterisks, <i>P</i><0.01; three asterisks, <i>P</i><0.001.</p

USF family members interact with <i>CSA</i> and <i>HR23A</i> proximal promoters.

<p>(A) Graphic representation of human, mouse, dog and zebrafish <i>CSA</i> proximal promoter. Conserved E-boxes are represented in dark grey. (B) <i>In vivo</i> chromatin immunoprecipitation assays (ChIP) with HaCaT cells using USF-1, USF-2 antibodies or non-specific IgG. Recovered DNA under basal or UV-irradiation conditions was subjected to PCR or quantitative PCR using specific primers of both proximal and distal region (negative control) of the <i>CSA</i> promoter. (C) <i>In vitro</i> Electrophoretic Mobility Shift Assay (EMSA) experiments were performed using HaCaT nuclear extract and radiolabeled probes centered on the E-box motif present in the <i>CSA</i> proximal promoter (−246) (shifted complex (→)). Competition assays were performed in the presence or not of cold competitors (WT or mutated cold probe). Supershift assays were obtained in the presence of anti-USF-1, anti-USF-2, and anti-TBX2 antibodies or IgG as non-specific controls ( = >). (D) Graphic representation of human, mouse and dog <i>HR23A</i> and human <i>HR23B</i> proximal promoters. Conserved E-box motifs are represented in dark grey and GC-rich regions in light grey. (E) ChIP assays were performed as in (B) targeting proximal <i>HR23A</i> or <i>HR23B</i> promoters and the distal region of <i>HR23A</i> promoter (−3 kb). (F) EMSA experiments were performed as described in (C) using radiolabeled probes centered on each E-box motif (−154 and −36) present in the <i>HR23A</i> proximal promoter. (G) ChIP assay using SP3 antibody or non-specific IgG were performed as previously described for <i>HR23A</i> and <i>HR23B</i> promoter occupancy. (H) EMSA experiments were performed as previously with HaCaT nuclear extract and radiolabelled probes centered on the GC box present in the <i>HR23A</i> proximal promoter (−131) (shifted complex (→)).</p

<i>In vitro</i> transcriptional regulation of human <i>CSA</i> and <i>HR23A</i> by USF.

<p>(A) Schematic representation of the <i>CSA</i> promoter-luciferase constructs. The construct contains the sequence from −847 to +1 of the <i>CSA</i> promoter linked to the luciferase reporter. E-box is represented in dark gray and its position is indicated on top. Cross shows mutated E-box. (B) <i>CSA</i> promoter-luciferase activity measured after co-transfection of XB2 keratinocytes with WT or mutated CSA promoter-luciferase constructs with pCMV-USF-1, pCMV-USF-2 expression vectors or pCMV empty vector (control). (C) <i>CSA</i> promoter-luciferase activity measured 30 min or 5 h after UV induction (6×10 J/m²) of XB2 cells transfected with WT or mutated CSA promoter-luciferase constructs. (D) Schematic representation of the <i>HR23A</i> promoter-luciferase constructs. The construct contains the sequences from −186 to +73 of the <i>HR23A</i> promoter linked to the luciferase reporter. E-boxes are represented in dark gray, GC-box in light gray, and positions are indicated on top. Crosses show mutated boxes. (E) <i>HR23A</i> promoter-luciferase activity measured after co-transfection of XB2 keratinocytes with pCMV-USF-1 or pCMV-USF-2 expression vectors or empty vector. (F) WT and mutated <i>HR23A</i> promoter-luciferase activities in XB2 cells following UV-irradiation (6×10 J/m²). (G) WT and mutated <i>HR23A</i> promoter-luciferase activities transfected in XB2 cells with pCMV-USF-1 expression vector and UV irradiated (6×10 J/m²). Error bars indicate s.e.m.; <i>n</i> = 3; one asterisk, <i>P</i><0.05, two asterisks, <i>P</i><0.01, three asterisks, <i>P</i><0.001.</p

UV-induced <i>CSA</i> and <i>HR23A</i> expression is impaired in <i>USF-1</i> knock-out (KO) mice.

<p>(A) Expression analysis of <i>CSA</i> and <i>CSB</i> were performed by RT-qPCR after UV-irradiation (4×50 J/m²) of cultured punch biopsy samples from WT (dark color) or <i>USF</i>-1 KO mice (light color). Results for UV-treated samples are expressed relative to controls (no irradiation) with the <i>HPRT</i> transcript used as a standard. (B) Expression analysis of <i>HR23A</i> and <i>HR23B</i> were performed by RT-qPCR as previously described. (C) Expression analysis of the UV response positive control gene, <i>Gadd45α</i>, was performed by RT-qPCR as previously described. (D) 36 hours kinetics of CPD DNA-damage removal (ELISA quantification) in cultured skin punch biopsies from WT (black) or <i>USF-1</i> KO mice (grey). The yellow band corresponds to the irradiation protocol (4×50 J/m²). Skin punch biopsies were analyzed immediately after 3 UV-pulses (3×50 J/m²) (time 1 h), and after 4 UV-pulses (4×50 J/m²) (at the following times: 3–7–24–36 h). Error bars indicate s.e.m.; <i>n</i> = 3.</p

<i>NIPBL-AS1</i> does not influence <i>NIPBL</i> transcription.

<p>A) Overview of the genomic position of <i>NIPBL</i> and <i>NIPBL-AS1</i> genes. Strand-specific read coverage of RNA-sequencing data (positive in green; negative in red) from HEK293T cells shows the transcription of <i>NIPBL-AS1</i> antisense to <i>NIPBL</i> [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007137#pgen.1007137.ref001" target="_blank">1</a>]. CTCF binding sites in HEK293 cells (ENCODE hg18) are shown. Primers used in the transcript analysis are indicated as green bars. (B-C) Transcript levels of (B) <i>NIPBL-AS1</i> and (C) <i>NIPBL</i> after antisense oligonucleotide knockdown (ASO2, ASO3) of <i>NIPBL-AS1</i> in HEK293T cells. ASO C was used as control. Transcript levels were normalized against the control sample (ASO C) and the housekeeping <i>SNAPIN</i> using the ΔΔCt method (mean n = 3, error bars +/- s.d., p-values determined with t-Test).</p

Interactions of <i>NIPBL</i> and <i>NIPBL-AS1</i> with a potential distal enhancer.

<p>A) Long-range chromosomal interactions of the <i>NIPBL</i> and <i>NIPBL-AS1</i> promoter detected by chromosome conformation capture (3C-seq) in HEK293T cells using an ApoI digest. The positions of the different viewpoints used are marked in yellow. Three different viewpoints at the promoter (VP4, blue track) and the candidate enhancers regions R1 (VP5, green track) and R2 (R2—VP6, red track) were used. B) CTCF ChIP sequencing track from HEK293 cells (ENCODE). The orientations of the CTCF motifs as determined with JASPAR are shown below the track (red triangle–forward orientation, green triangle–reverse orientation). The CTCF sites involved in the promoter-enhancer interaction are indicated with yellow triangles above the track. C) DNAse clusters as well as histone modification profiles—H2A.z, H3K4me1, H3K4me2 and H3K4me3—of six different cell lines (G312878, K562, HeLa-S3, HEMEC, HSMM and HUVEC, available from ENCODE) are displayed as density graph. Black represents areas with the highest enrichment of the signals.</p

Implications for CdLS.

<p>A) Transcript levels of the genes <i>BBX</i>, <i>GLCCI1</i> and <i>ZNF695</i> that were described as dysregulated genes in CdLS [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007137#pgen.1007137.ref020" target="_blank">20</a>] and previously confirmed as NIPBL-dependent genes with NIPBL binding sites at the promoter [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007137#pgen.1007137.ref008" target="_blank">8</a>] were analysed in the different enhancer deletion clones D1 and D2 (mean n = 5 for D1 and n = 4 for D2, error bars +/- s.d., p-values determined with t-Test, the transcript levels of the individual clones are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007137#pgen.1007137.s007" target="_blank">S7 Fig</a>). B) Average transcript levels of <i>NIPBL</i> and <i>NIPBL-AS1</i> in lymphoblastoid cell lines (LCLs) derived from CdLS patients and controls. The details of the four LCL controls and three CdLS LCLs as well as the individual transcript levels are shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007137#pgen.1007137.s008" target="_blank">S8 Fig</a> and in [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007137#pgen.1007137.ref008" target="_blank">8</a>,<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007137#pgen.1007137.ref020" target="_blank">20</a>]. Two primer pairs for <i>NIPBL</i> and one for <i>NIPBL-AS1</i> were used. Transcript levels were normalized against the housekeeping gene <i>NADH</i> (mean n = 4 for control LCLs and n = 3 for CdLS LCLs, error bars +/- s.d., p-values determined with t-Test).</p