Lack of Top1 further increases the association of condensin with Pol III-transcribed genes when Swd2.2 and Sen1 are missing.

<p><b>A</b>. Serial dilutions of the indicated strains were plated on rich media at the indicated temperatures. <b>B</b>. Chromosome segregation in anaphase was monitored in the indicated strains after growing cells for one generation at 34°C. For each genotype, a minimum of 6 independent experiments was performed in which a minimum of 100 anaphase cells were scored (***<0.001; **<0.01 Wilcoxon - Mann Whitney). Anaphases were scored as defective when chromatin was detected lagging between the two main DNA masses <b>C</b>. ChIP qPCR of the indicated strains grown in cycling conditions at the indicated loci (mean ± standard deviation from 6 biological replicates. NS: not significant *P<0.05; **P<0.01; ***P<0.001 Wilcoxon - Mann Whitney). <b>D</b>. Model. Lack of Swd2.2 and Sen1 increases gene transcription at Pol III-transcribed genes. According to the twin supercoiled domain model, this results in more positive supercoils downstream of the polymerase and compensatory negative supercoils upstream of the polymerase. Negative supercoils favor the formation of R-Loops (reviewed in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004794#pgen.1004794-Drolet1" target="_blank">[41]</a>). Positive supercoils result in nucleosome eviction. This topological stress also facilitates the recruitment of condensin, either directly or indirectly.</p

R-Loops form in abundance at Pol III-transcribed genes but they do not significantly impact the association of condensin.

<p><b>A</b>. ChIP qPCR of the indicated strains grown in cycling conditions at the indicated loci (mean ± standard deviation from 3 biological replicates). <b>B</b>. As in <b>A</b>. Cells were grown in minimal medium for a minimum of 18 hours to promote the over-expression of RnhA driven by the nmt promoter. <b>C</b>. Genomic DNA was extracted from rnh1+rnh201+ and <i>rnh1Δrnh201Δ</i> cells in preparation for the DRIP procedure. Equal amount of genomic DNA were spotted on a nylon membrane and incubated with 2 µg/mL of purified S9.6 antibody. The amount of S9.6 bound to the DNA was revealed using chemiluminescence. <b>D</b>. DRIP-qPCR of the indicated strains grown in cycling conditions at the indicated loci (mean ± standard deviation from 3 biological replicates). <b>E</b>. Cells of the indicated genotypes were grown in minimum medium lacking thiamine for a minimum of 18 hours to drive the over-expression of RnhA. ChIP-qPCR was then performed (mean ± standard deviation from 3 biological replicates).</p

The double deletion of Swd2.2 and Sen1 facilitates the localization of condensin at Pol III-transcribed genes.

<p><b>A</b>. Serial dilutions of the indicated strains were plated on rich media at the indicated temperatures. <b>B</b>. Chromosome segregation in anaphase was monitored in the indicated strains after growing cells for one generation at 34°C. For each genotype, a minimum of 6 independent experiments was performed in which a minimum of 100 anaphase cells were scored (***<0.001; **<0.01 Wilcoxon - Mann Whitney). Anaphases were scored as defective when chromatin was detected lagging between the two main DNA masses <b>C</b>. ChIP-qPCR analysis of the amount of GFP-tagged Cut3 cross-linked to chromatin in cell populations of the indicated genotypes grown at 30°C (mean ± standard deviation from 6 biological replicates (NS: not significant, *P<0.05; **P<0.01; ***P<0.001 Wilcoxon - Mann Whitney). The primers used in this study are shown on <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004794#pgen.1004794.s013" target="_blank">Table S1</a>. <b>D</b>. Mitotic indexes of the cell populations used in <b>C</b>. Cells were fixed with cold methanol and processed for immuno-fluorescence using an anti-tubulin antibody. Cells with a spindle were counted as mitotic.</p

Lack of Swd2.2 and Sen1 results in local topological stress at Pol III-transcribed genes.

<p><b>A</b>. ChIP qPCR of the indicated strains grown in cycling conditions at the indicated loci (mean ± standard deviation from 6 biological replicates. NS: not significant *P<0.05; **P<0.01; ***P<0.001 Wilcoxon - Mann Whitney). <b>B</b>. Western blot analysis of the stability of Top1-3flag. Tubulin is used as a loading control. <b>C</b>. ChIP qPCR of the indicated strains grown in cycling conditions at the indicated loci (mean ± standard deviation from 6 biological replicates. NS: not significant *P<0.05; **P<0.01; ***P<0.001 Wilcoxon - Mann Whitney). <b>D</b>. Western blot analysis of the stability of Top2-GFP. Tubulin is used as a loading control. <b>E</b>. ChIP qPCR of histone H3 in the indicated strains grown in cycling conditions at the indicated loci (mean ± standard deviation from 6 biological replicates. NS: not significant *P<0.05; **P<0.01; Wilcoxon - Mann Whitney).</p

Transcription is enhanced at Pol III-transcribed genes when Swd2.2 and Sen1 are missing.

<p><b>A</b>. Sen1 is enriched at Pol III-transcribed genes. ChIP qPCR of the indicated strains grown in cycling conditions at the indicated loci (mean ± standard deviation from 3 biological replicates). NTS#2 is a site within the Replication Fork Barrier of the rDNA and is shown as a comparison. <b>B</b>. Flag-tagged Sen1 co-immunoprecipitates with Myc-tagged Rpc25. Whole cell extracts (WCE) and the immuno-precipitated material (Flag IP) of the indicated strains were analyzed by western blot. <b>C</b>. Rpc25 becomes more abundant at Pol III-transcribed genes when Swd2.2 and Sen1 are missing. ChIP qPCR of the indicated strains grown in cycling conditions at the indicated loci (mean ± standard deviation from 3 biological replicates). <b>D</b>. Western blot analysis of the stability of Rpc25-13myc. Tubulin is used as a loading control. <b>E</b>. Pol III transcripts are more abundant when Swd2.2 and Sen1 are missing. Total RNAs extracted from swd2.2+sen1+ or <i>swd2.2Δsen1Δ</i> cells grown in rich medium at 30°C were analyzed by RT-qPCR (3 biological replicates, 2 RT per replicate).</p

Media as A Source of Coping and Social, Psychological and Hedonic Well-being: A Longitudinal Qualitative Study during the COVID-19 Pandemic

The COVID-19 pandemic and lockdowns have provided an unprecedented opportunity to better understand the processes by which media are used to improve coping and alter well-being. Despite the important health issues at stake, the overall dynamic of media-based coping strategies (MBCS), their evolution over time according to their perceived efficacy, and their link with social well-being are poorly understood. The present longitudinal qualitative study, conducted in seven phases of interviews over a period of 36 weeks among a diverse population experiencing lockdown, lifting of lockdown, and then a second lockdown (N = 31; total duration 192 hours), shows how individuals implemented eight families of MBCS on two interdependent levels. On the first level, two families of MBCS developed “micro” and “macro” social processes, contributing to social well-being. Social media satisfied social needs usually satisfied offline. Two other families also improved psychological and hedonic well-being. Among these MBCS, the hedonic strategies in particular were perceived as being ineffective after about a month of confinement. Four families of second-level MBCS were then implemented and were perceived as effective in both the short and long term. Limitations and new perspectives opened by the results are discussed.</p

Endogenous <i>SOX9</i> is up-regulated in NT2/D1 cells in response to SRY.

<p>(<b>A</b>) <i>SOX9</i> is significantly upregulated in NT2/D1 cells transiently overexpressing SRY (1 µg), as measured by QRT-PCR and compared to empty vector (<i>n</i> = 3). (<b>B</b>) Immunostaining for endogenous SOX9 and exogenous SRY (Anti-Flag) reveals a positive correlation between SOX9 fluorescence and exogenous SRY fluorescence (R² = 0.835, <i>n</i> = 50). Scale bar represents 10 µm. Each point averages three fluorescence readings per NT2/D1 cell (Blue Diamond - SRY-transfected cells; Pink triangle - non-Flag cells). (<b>C</b>) (Upper panel) H3 Me₃K4 (red) is exclusively nuclear in NT2/D1 cells. Overlap of SRY (green) and H3 Me₃K4 in NT2/D1 cells reveals co-localization (yellow) within nuclear compartments. (Lower panel) SC-35 (red) is localized to alternate nuclear compartments, as overlap images of Flag-SRY and SC-35 staining do not show co-localization. DAPI (blue) stains nuclear DNA. Scale bar represents 10 µm. Error bars represent the standard error of mean values. Two-tail t-Test of paired sample means was performed. * <i>P</i>&lt;0.05.</p

Relating <i>SOX9</i> gonad-specific enhancer activity to disorders of sexual development caused by mutations to SRY, SF1 and SOX9.

<p>(<b>A</b>) Locations of four <i>de novo</i> and four familial SRY mutations causing 46,XY gonadal dysgenesis (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017751#pone-0017751-t001" target="_blank">Table 1</a>). CHO cells were co-transfected with <i>hTES</i> reporter and constructs expressing the SRY mutants (100 ng). Transcriptional activity was plotted as a percentage of SRY wild-type (WT) and statistical analysis performed. HMG, high mobility group domain. (<b>B</b>) Locations of two SF1 mutants causing 46,XY gonadal dysgenesis and adrenal failure (G35E and R92Q) and one mutant causing 46,XY gonadal dysgenesis (Δ8bp) (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017751#pone-0017751-t002" target="_blank">Table 2</a>). All SF1 mutants (20 ng) showed reduced transcriptional activity, plotted as a percentage of SF1 WT and statistically compared. DBD, DNA binding domain; FtzF1, FtzF1-box (A-box); LBD, ligand binding domain; AF2, activation function-2. (<b>C</b>) 46,XY gonadal dysgenesis mutant SOX9-A158T (26 ng) failed to activate <i>hTES</i> compared to SOX9 WT (26 ng), whereas 46,XY mutant SOX9-A76E (26 ng) (located in the dimerization domain (dim.)) had comparable activity, plotted as percentage of SOX9 WT. PQA, proline, glutamine and alanine-rich motif; PQS, proline, glutamine and serine-rich motif (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017751#pone-0017751-t003" target="_blank">Table 3</a>). All experiments were conducted three times in duplicate. Error bars represent the standard error of mean values. Two-tail t-Test of paired sample means was performed. *** <i>P</i>&lt;0.001, ** <i>P</i>&lt;0.01, * <i>P</i>&lt;0.05.</p

Summary of defects in SOX9 mutants.

<p>HMG – high mobility group domain. CGD – complete gonadal dysgenesis. CD – Campomelic Dysplasia. Biochemical defects obtained from respective references <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017751#pone.0017751-Preiss1" target="_blank">[40]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0017751#pone.0017751-Bernard1" target="_blank">[41]</a>.</p

Co-operative regulation of the <i>SOX9</i> enhancer by SRY, SF1 and SOX9.

<p>(<b>A</b>) Transfection of CHO cells with exogenous SF1 (20 ng) stimulates <i>hTES</i> ∼1.8-fold compared to vector. Together, SRY and SF1 co-operate to activate <i>hTES</i> by ∼16-fold. SOX9 (26 ng) activates <i>hTES</i> ∼2-fold compared to vector, increased to ∼3-fold by the presence of SF1. (<b>B</b>) Transfection of S203A SF1 phosphorylation mutant (100 ng) had similar transcriptional activity of the <i>hTES</i> reporter construct in comparison to SF1 WT (100 ng). Co-operative effect between SRY and SF1-S203A had abolished <i>hTES</i> reporter activity in comparison to SRY and SF1 WT co-transfected cells. (<b>C</b>) SOX9 truncation protein (amino acids 1–465) lacking the PQS transcriptional activation domain did not activate the <i>hTES</i> reporter construct to the same extent as SOX9 WT either alone or in the presence of SF1. Removal of the PQA transcriptional activation domain did not have any effect on activation of the <i>hTES</i> reporter construct compared to SOX9 WT. All experiments conducted three times in duplicate. Error bars represent the standard error of mean values. Two-tail t-Test of paired sample means was performed. * <i>P</i>&lt;0.05, *** <i>P</i>&lt;0.001.</p