Physical interaction between TRα1 and the β-catenin/Tcf4 complex.

<p>Nuclear extracts from Caco2 cells, maintained in T3-depleted (−) or T3-supplemented (+) serum, were immunoprecipitated with antibodies directed against endogenous Tcf4, β-catenin or TRα1 and analyzed by WB by using the antibodies as indicated. Rabbit IgG was used as negative control. Ponceau red was used as whole-protein (50 µg/lane) loading control. Histone H1 was used to check the enrichment and was the loading control for the nuclear proteins in the inputs. The pictures are representative of at least three independent experiments.</p

The Wnt3a ligand is not sufficient to impair TRα1 transcriptional activity ex vivo.

<p>The primary cultures of intestinal epithelial cells were treated with 10 ng/ml of Wnt3a and/or 10⁻⁷ M of T3 for 24 hours. (A) The number of proliferating cells in the different experimental conditions was analyzed by Ki67 immunolabeling; all of the nuclei were labeled by Hoechst. The percentage of Ki67-positive nuclei was determined by counting under a fluorescence microscope (Zeiss Axioplan). The histograms represent the summary (mean ± sd) of the scoring of specific immunolabeling in two independent experiments each conducted in triplicate (n = 50). (B, C) Analysis of β-catenin in intestinal epithelial primary cultures by immunolabelling (B) and WB (C). Cells were in control, T3, Wnt3a and T3+Wnt3a conditions as indicated. Pictures in B show the fluorescent staining of the nuclei (blue), β-catenin (red) and the merging of each simple labeling. Bar: 15 µm. For the WB (C), we used a specific antibody allowing the detection of activated non-phosphorylated β-catenin <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034162#pone.0034162-Staal1" target="_blank">[54]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034162#pone.0034162-vanNoort1" target="_blank">[55]</a>. Actin was used as loading control. The image is representative of two independent experiments. Each lane represents whole protein extracts (50 µg/lane). (D–F) RT-qPCR analysis to evaluate mRNA levels of <i>Ccnd1</i>, <i>Ctnnb1</i> and <i>Sfrp2</i>. Results are from three independent experiments, each conducted in duplicate. Values represent fold change ± sd after normalization to the control condition (Ctrl). *: P<0.05, **: P<0.01, ***: P<0.001 by two-tailed Student's t-test (n = 6).</p

β-catenin/Tcf4 complex interferes with TRα1 functionality in luciferase assay in vitro.

<p>(A) The DR4-luc luciferase reporter was transfected into Caco2 cells maintained in T3-depleted serum with or without supplementation with T3 as indicated, together with TRα1, Tcf4 or β-catenin expression vectors in different combinations. (B, C) The DR4-luc luciferase reporter (B) or TopFlash luciferase reporter (C) was transfected into Caco2 cells maintained in culture medium containing physiological concentrations of T3, together with the β-catenin/Tcf4 complex in the presence or absence of the TRα1 expression vector. Histograms represent mean ± sd from three independent experiments, each conducted in triplicate (n = 9). *: P<0.05, **: P<0.01 compared with the control condition (Ctrl); #: P<0.05, ##: P<0.01, compared with the TRα1 condition; $: P<0.05 compared with the TRα1+β-catenin condition; ££: P<0.01 compared with the β-catenin or β-catenin+Tcf4 condition, by two-tailed Student's t-test.</p

Analysis of TRα1 target genes in mice of different genotypes.

<p>RT-qPCR experiments were performed in the intestine of 6-month-old mice of the indicated genotype to analyze the mRNA levels of <i>Ctnnb1</i> (A), <i>Sfrp2</i> (B), <i>Ccnb1</i> (C) and <i>Cdc2a</i> (D). Values represent fold change ± sd after normalization to the wild-type [WT] animals. *: P<0.05, **: P<0.001, compared with the WT; #: P<0.05, ##: P<0.01 compared with the <i>vil</i>-TRα1 animals, by two-tailed Student's t-test (n = 4). N, normal mucosa; T, tumor.</p

Binding of NF-κB to remodeled and slid nucleosomes.

<p>(<b>A</b>) Nucleosomes, RSC-remodeled nucleosomes (remosomes), slid nucleosomes and naked 601_D₀ DNA were incubated with the indicated amount of NF-κB and separated on a 5% native PAGE. The positions of the different particles are shown on the left part of the gel. (<b>B</b>) UV laser footprinting of the indicated distinct NF-κB bound particles. The experiment was carried out as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003830#pgen-1003830-g002" target="_blank">Figure 2</a>. The NF-κB binding site is shown as vertical black line and the black arrow indicates the nucleosomal dyad; M, 10 bp DNA molecular marker (<b>C</b>) top, “zoom” of the NF-κB binding region from the footprints shown in (B); bottom, scan of the footprints; red, in presence NF-κB; green in absence of NF-κB.</p

NF-κB displaces H1 from the chromatosome and binds to its recognition sequence.

<p>(<b>A, upper panel</b>), schematics of the substrates used in each experiment. (<b>A, lower panel</b>), EMSA of the binding of NF-κB to depicted substrates. The bottom strand of the 255 bp 601_D₈ DNA (Supplementary <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003830#pgen.1003830.s001" target="_blank">figure S1</a>) was uniquely 5′-end labeled by ^32-P and used to reconstitute centrally positioned nucleosomes. Chromatosomes were assembled by using the NAP-1/H1 complex to properly deposit H1 on the nucleosome in H1/nuc ratio of ∼1.5. The first two panels show the NF-κB-DNA (lanes 1–4) and NF-κB-nucleosome (lanes 5–8) complexes formed upon incubation with increasing amount of NF-κB. The last panel illustrates both the interaction of chromatosomes with the indicated increasing amount of NF-κB (lanes 1′–5′) and the deposition of H1 on the already assembled (at increasing NF-κB concentration) NF-κB nucleosome complexes (lanes 6′–9′). (<b>B</b>) UV laser (upper panel) and •OH (lower panel) footprinting of the NF-κB binding region of NF-κB-DNA and distinct NF-κB-nucleosome complexes.</p

Dilution driven H2A–H2B dimer eviction allows binding of NF-κB to Nucleosome Core Particle.

<p>(<b>A</b>) 152 bp DNA fragment derived from <i>X. borealis</i> somatic 5 S RNA gene containing single NF-κB site near the dyad NF1 (53–56) was amplified by PCR and uniquely 3′ end labeled with α-³²P by Klenow. Nucleosomes were reconstituted on this labeled fragment as described previously. The DNA and nucleosomes at a concentration 40 nM were incubated with increasing amounts of NF-κB as indicated to allow the formation of stable complexes which were subsequently irradiated by a single high intensity UV laser pulse (<i>E</i>_pulse∼0.1 J/cm²). The formation of complexes was checked by EMSA (upper panel, DNA lane 1–3, nucleosomes lane 4–9), the positions of free DNA and nucleosomes are indicated, “cplx.” represents NF-κB – DNA/nuc complexes. The samples were split into two parts, DNA was purified and treated with Fpg glycosylase to cleave 8-oxoG (represented by ▸,lower panel, DNA lane 1–3 and nucleosome lane 4–9) and with T4 endonuclease V to cleave CPDs (represented by ◊, DNA lane 1′–3′ and nucleosome lane 4′–9′). The cleaved DNA fragments were visualized by 8% sequencing gel. (<b>B</b>) The same 152 bp 5S fragment was 5′ end labeled with γ-³²P by T4 polynucleotide kinase and used for nucleosome reconstitution. DNA and nucleosomes, at 10 nM final concentration, were incubated with increasing amounts of NF-κB as indicated to allow the formation of complexes. The assembly of the complexes was checked by EMSA (upper panel, DNA lane 1–5, nucleosomes lane 1′–5′). The samples were irradiated with a single high intensity UV laser pulse (<i>E</i>_pulse∼0.1 J/cm²), treated with a mix of Fpg glycosylase and T4 endonuclease V to cleave both the 8-oxoG (▸) and CPDs (◊). Finally, the cleaved products were visualized by 8% sequencing gel (DNA, lane 1–5; nucleosomes lane 1′–5′). The NF-κB binding sites (vertical bold lines) and the NF-κB recognition sequences are shown. The arrows designated the nucleosomal dyad.</p

Characterization of the reconstituted nucleosomes.

<p>(<b>A</b>) Schematics of the reconstituted nucleosomes. The canonical NF-κB site was inserted in the 255 bp 601 DNA fragment either at the dyad of the nucleosome (601_D₀ DNA) or at the nucleosomal end (601_D₇ DNA) or in the linker DNA (601_D₈ DNA); bold lines, free DNA arms; dashed line, core particle region. The vertical black line represents the dyad. The NF-κB binding sites (BS) are depicted by the red line. The length of each region is shown on top of the constructs. The very bottom schematics shows the location of the NF-κB binding site inserted in the 5S rDNA fragment of <i>Xenopus borealis</i> used for nucleosome reconstitution. (<b>B</b>) Electrophoretic analysis of the indicated purified recombinant histones and histone octamer. (<b>C</b>) Electrophoretic analysis of purified recombinant NF-κB (p50); M, molecular marker; p50, the p50 subunit of NF-κB. (<b>D</b>) Nucleosome reconstitution check by 5% native PAGE. (<b>E</b>) •OH radical and (<b>F</b>) DNase I footprinting of free 601 DNA and the indicated reconstituted nucleosomes.</p

FACT facilitates both RSC-induced remodeling and mobilization of nucleosomes.

<p>(<b>A)</b> DNase I footprinting. End-positioned nucleosomes, reconstituted on ³²P 5’-labeled 241 bp 601 DNA fragment, were incubated with 0.2 units of RSC in the absence (lane 3) or in the presence of 1.6 pmol of FACT (lane 4) for 50 min at 30°C; lane 5, same as lane 3, but with 1 unit of RSC; After arresting the remodeling reaction, the samples were digested with 0.1 units of DNase I for 2 min, the cleaved DNA was isolated and run on 8% PAGE under denaturing conditions; lanes 1 and 2, controls showing the DNase I cleavage pattern of nucleosomes (lane 1) alone or incubated with 1.6 pmol FACT under the conditions described above. (<b>B</b>) The presence of FACT increases the efficiency of RSC-induced nucleosome mobilization. Centrally positioned nucleosomes were incubated with 0.2 units of RSC in the presence of increasing amount of FACT, the reaction was arrested and the samples were run on native PAGE. The position of the non-mobilized and the slid end-positioned nucleosomes were indicated; lane 1 control nucleosomes; lane 2, nucleosomes incubated with RSC alone (in the absence of FACT). (<b>C</b>) Quantification of the data presented in (B).</p

FACT is implicated in the repair of oxidatively damaged DNA.

<p>(<b>A</b>) FACT is recruited to the sites of oxidative DNA lesions. HeLa cells expressing either OGG1-EGFP (left) or DsRed-SSRP1 (the smaller subunit of FACT, right) were locally irradiated with 405-nm laser in the absence (upper two panels) or presence (lower two panels) of photosensitizer Ro-19-8022 and the accumulation of the fusions at the bleached sites (indicated with white arrow) was observed 5 minutes post irradiation (lower panels). (<b>B</b>) Same as (A), but for Hela cells expressing either Ku80-EGFP or DsRed-SSRP1. (<b>C</b>) Treatment of cells with H₂O₂ results in release of FACT from transcribed chromatin and in binding to chromatin regions associated with both DNA repair proteins and chromatin remodeling factors. Stable Hela cell lines expressing a fusion of HA with SSRP1, treated or not with H₂O₂, were used to immunopurify the chromatin bound FACT complexes. Upper panel shows the silver stained SDS gel of the proteins associated with either control FACT chromatin bound complex (-) or with the FACT chromatin bound complex isolated from H₂O₂ treated cells (+). The lower panel shows the association of the indicated proteins identified by Western blotting in the respective complexes. (<b>D</b>) Mass spectrometry identification of the polypeptides associated with control FACT chromatin bound complex (-) or with the FACT chromatin bound complex, isolated from H₂O₂ treated cells (+). Proteins present in the e-SSRP1.com together with the number of identified peptides are indicated. Proteins involved in transcription are shown in red. DNA repair proteins and chromatin remodelers are shown in blue.</p