EMBO/soda-nel huggingface dataset:
Homologous recombination is reduced in female embryonic stem cells by two active X chromosomes (2021)
Figure 2

Figure 2. HR efficiency is reduced in female ESCs

A-B Clonogenic assay for ESCs in response to etoposide (A) or camptothecin (B). Non-treated cells were set to 1.

C Nanog HR assay. The percentage of GFP-positive colonies after gene targeting to the Nanog locus is shown. The results for each cell line are shown at right.

D Nanog expression confirmed by RNA-seq.

E Detection of the number of X-linked Atrx gene foci by DNA-FISH in GFP-positive subclones of the Nanog-targeting assay (nine independent clones, N> 100 each), performed as described in Figure 1C.

Data information: The percentage survival (A, B), percentage of GFP-positive colonies (C), and FPKM by RNA-seq (D) are shown for biological replicates (n=3) with three different cell lines (Male: #A6, #A13, #A17; female: #A4, #A10, #A11). Data are presented as the mean ± SD. **p<0.01, *p<0.05, n.s.: not significant, Student's t-test. N represents number of cells in (E).
Homologous recombination is reduced in female embryonic stem cells by two active X chromosomes (2021)
Figure 3

Figure 3. Induced XCI restores lower HR efficiency

A Schematic representation showing the establishment of XJF1XTX ROSA26+/rtTA ESCs. The TX allele allows Xist expression from its endogenous locus by dox addition, eventually leading to XCI.

B Sexing of established ESCs (gDNA-qPCR, Zfy/Gapdh), performed as described in Figure 1B.

C Detection of the number of X-linked Atrx gene foci by DNA-FISH with or without dox addition for 96 h (N >100), performed as described in Figure 1C.

D Detection of the Xist cloud in #B3 by RNA-FISH, with or without dox addition for 96 h ( N >200 each). A representative image is shown [magenta: Xist (white arrowhead), cyan: DAPI. Scale bar: 10 μm].

E Allele-distinguishable RT-PCR for detecting Hprt, G6pdx, and Rnf12 X-linked genes. Bands with the anticipated sizes expressed from XJF1 or XTX alleles are shown.

F Nanog HR assay. The percentage of GFP-positive colonies after gene targeting to the Nanog locus is shown as described in Figure 2C. #B2 or #B3 were cultured with or without dox addition for 96 h. The combined number of GFP-positive cells and total colonies from all experiments are listed and summarized at right.

Data information: Data in (B) are derived from two technical replicates and are shown as the mean with single data points of both experiments. Data in (D) are derived from four biological replicates and are presented as the mean ± SD. The graph in (F) is derived from two (#B2) or four (#B3) biological replicates and is shown as the mean with single data points of each experiment. The error bars of #B3 data in (F) indicate SD. **p<0.01, Student's t-test. N represents number of cells in (C, D). Scale bar in (D) is 10 μm.
Homologous recombination is reduced in female embryonic stem cells by two active X chromosomes (2021)
Figure 4

Figure 4. Induced XCI restores HR but not NHEJ efficiency

A Diagrams of the reporter assay vectors DRGFP and EJ5GFP for measuring HDR and Distal-EJ, respectively.

B Detection of the Xist foci by RNA-FISH, with or without dox addition for 96 h (N >200 each). A representative image with percentage of Xist-positive cells is shown [magenta: Xist, cyan: DAPI.].

C Detection of the number of X-linked Atrx gene foci by DNA-FISH, with or without dox addition for 96 h (N >100 each), performed as described in Figure 1C.

D Estimated repair events. The flow cytometry plots are shown in Figure EV4. The percentage of GFP-positive cells in TX-DRGFP#11 and TX-EJ5GFP#23 was normalized to the transfection efficiency detected by the VENUS expression vector. Dox (-) was set to 1 and the ratio of relative GFP-positive cells is shown (n=3).

Data information: Data are presented as the mean ± SD in (D). *p<0.05, n.s.: not significant, Student's t-test. N and n represent the number of cells in (B, C) and the number of biological repeats (D), respectively. Scale bar in (B) is 10 μm.
Homologous recombination is reduced in female embryonic stem cells by two active X chromosomes (2021)
Figure 6

Figure 6. X-linked Brcc3 lowers HR efficiency in female ESCs

A-C Brcc3 expression confirmed by RNA-seq (A), RT-qPCR (B), and western blotting (C) [biological replicates (n=3) with three different cell lines (male: #A6, #A13, #A17; female: #A4, #A10, #A11)].

D-E Confirmation of Brcc3 expression from knockdown samples in (F) by RT-qPCR (technical duplicates) (D) or western blotting (E).

F Nanog HR assay with Brcc3 knockdown (three biological replicates). The percentage of GFP-positive colonies after gene targeting to the Nanog locus is shown, as shown in Figure 2C. The combined number of GFP-positive cells and total number of colonies are shown (please note that total cell number of #A13 for drug screening was 3.67×107 because of contamination of 1 out of 12 dishes, compared with the other colonies for which 4×107 cells were used).

Data information: Normalized to β-actin, average of male ESCs was set to 1 (B-E). RT: reverse transcription (B, D). The amount of BRCC3 normalized to β-actin is shown above the upper-panel (C, E). Data are represented as the mean ± SD (A-C, F) and data in (D) are shown as the mean with single data points of both experiments. **p< 0.01, Student's t-test.

Source data are available online for this figure.
Structural insights into the assembly and activation of the IL-27 signalling complex (2022)
Figure 3

Figure 3. Biophysical analysis of the assembly of the IL-27 receptor complex. (A-E) For SPR measurements, IL-27Rα or GP130 were immobilized on the chip surface by biotin-streptavidin interaction and IL-27 or p28 were flowed across the chip in solution. SPR data are representative of three biological replicates. Kinetic charts for IL-27Rα (A) and GP130 (B). Concentrations used are shown on the curves. Data traces were fitted using a 1:1 interaction model (black) to quantify the kinetics (kon, koff) and binding affinity (KD) of the interactions. (C, D) Equilibrium chart for p28 binding to IL-27Rα (C, left panel) and curve-fitting to data points generated at various concentrations of p28 (C, right panel). (D) Equilibrium chart for p28 binding GP130. (E) Table presenting kinetically derived (IL-27) and thermodynamically derived (p28) binding constants. Standard Error values (SE) are shown in parentheses. (F,G) Dose response curves for pSTAT1 (F) and pSTAT3 (G) in resting mouse CD8+ T cells. Cells were stimulated with mIL-27 or p28 for 15 minutes with the indicated doses. Data shown are the mean of four biological replicates with error bars depicting standard error of the mean.
DNA polymerase zeta contributes to heterochromatin replication to prevent genome instability (2021)
Figure 1

Figure 1. S phase progression is impaired in Rev3l-/- MEFs

A. Rev3l+/+ and Rev3l-/- MEFs were pulse-labelled with BrdU prior to harvesting and analyzed by flow cytometry at different time points. The analysis was focused on S phase divided in 3 parts: G1/early S, middle S and late S/G2 BrdU+ cells. Red arrow represents BrdU+ cells that re-entry in S phase and green arrow shows BrdU- cells in G2 phase. Histograms represent the percentage of cells in G1/early and late S/G2 phase after BrdU pulse. Error bars represent standard error for three independent experiments. ns: not significant, * p<0.05 by Student's t-test.

B. Rev3l+/+ and Rev3l-/- MEFs were pulse-labelled with EdU for 15min, permeabilized, fixed and stained for EdU incorporation (green). S phase sub-stages from I to IV were evaluated by visual inspection of the cycling population (>300 EdU+ cells, top panel). Scale bar= 5 μm. Dot plots and pie charts show the relative proportion (percentage of total S) of each sub-stage from I to IV (middle and bottom panel, respectively). Each dot represents the mean of two technical replicates.

C. Seventy-two hours after transfection with non-targeting (NT) or Rev3l siRNA, HeLa cells were pulse-labelled with BrdU for 15 min, permeabilized, fixed and stained for BrdU (red). As in (B), S phase sub-stages were evaluated by visual inspection of the cycling population (>300 BrdU+ cells, top panel). Scale bar= 5 μm. Dot plots and pie charts show the relative proportion (percentage of total S) of early, middle-late and late S phase (middle and bottom panel, respectively). Each dot represents the mean of two technical replicates.
DNA polymerase zeta contributes to heterochromatin replication to prevent genome instability (2021)
Figure 2

Figure 2. Loss of REV3L disrupts the replication timing in specific genomic loci.

A. Cells were pulse-labelled with BrdU for 1.5 h and sorted by flow cytometry in two fractions, S1 and S2, corresponding to early and late S phase fractions, respectively. Neo-synthesized DNA was immunoprecipitated with BrdU antibodies. Early and late neo-synthesized DNAs were labeled with Cy3 and Cy5, respectively and hybridized on microarrays. After processing analysis with the START-R software, replication-timing profiles were obtained from two replicates (see EV2.A). Shown are the zoomed microarray profiles of the timing of replication on chromosome 1 (136.6-143.5 Mb), chromosome 5 (127.3-134.7 Mb) and chromosome 9 (46.1-51.3 Mb) from Rev3l+/+ and Rev3l-/- MEFs overlaid. Blue lines represent replication timing from Rev3l+/+ MEFs and red lines represent replication timing from Rev3l-/- MEFs. Genomic regions displaying significant difference between Rev3l+/+ and Rev3l-/- MEFs by START-R are indicated by a pink line (p<0.01).

B. Analysis of GC and Line-1 content in early, mid, late and TTR compared to disturbed replicating regions found in Rev3l-/- MEFs. Bar in boxplot represents the median, and red points represents the mean. The limit of the boxes corresponds to the 0.25 to 0.75 quartiles with whiskers extending to the maximum value of 1.5 times the interquartile range. Graphs show data from two biological experiments.

C. Analysis of the active marks H3K27ac and H3K4me3 and facultative heterochromatin mark H3K27me3 content in early, mid, late and TTR compared to disturbed replicating regions found in Rev3l-/- MEFs. Bar in boxplot represents the median, and red points represents the mean. The limit of the boxes corresponds to the 0.25 to 0.75 quartiles with whiskers extending to the maximum value of 1.5 times the interquartile range. Graphs show data from two biological experiments.
DNA polymerase zeta contributes to heterochromatin replication to prevent genome instability (2021)
Figure 3

Figure 3. Inactivation of Rev3l impairs expression of numerous developmentally and imprinted genes

A. Heat map showing log2 fold change in differentially expressed genes in Rev3l+/+ and Rev3l-/- MEFs from 2 independent biological experiments (Rev3l+/+: samples 1 and 2, Rev3l-/-: samples 5 and 6) with 2 technical replicates (samples 3,4,7,8: technical repeats from samples 1,2,5,6 respectively). Several developmentally regulated genes and imprinting genes (paternally in blue and maternally in pink) downregulated in Rev3l-/- MEFs are indicated on the right. Yellow and blue indicate high and low mRNA expression levels, respectively.

B. Top five biological process gene ontology (GO) terms of genes found downregulated in Rev3l-/- MEFs transcriptome analysis.

C. Volcano plot shows in red genes involved in development and imprinting with high fold changes ≥ 3 (FDR<0.05).

D. Relative mRNA levels of four genes involved in development (Hoxb2, 8, 9 and WT1) were validated through qRT-PCR from Rev3l+/+ and Rev3l-/- MEFs samples. The data were normalized to the amount of GAPDH mRNA. Error bars indicate standard error of the mean for three independent experiments. ***, P<0.001 and **, P<0.005 by Student's t-test.

E. Relative mRNA levels of genes involved in imprinting (paternal expressed genes: Dlk1and Slc18a4 and maternal expressed genes: Dcn1 and H19) were validated through qRT-PCR from Rev3l+/+ and Rev3l-/- MEFs samples. The data were normalized to the amount of GAPDH mRNA. Error bars indicate standard error of the mean for three independent experiments. *** P<0.001 by Student's t-test.
DNA polymerase zeta contributes to heterochromatin replication to prevent genome instability (2021)
Figure 4

Figure 4. Loss of REV3L results in epigenetic changes

A. Total histones were acid extracted from Rev3l+/+ and Rev3l-/- MEFs. Samples were analyzed by western-blot using indicated antibodies specific to histone marks (right panel). An anti-pan H4 or H3 was used as loading control (left panel). Histograms show WT/KO Rev3l fold change relative to the immunoblot shown on the top panel (using 1X sample intensity).

B. Histone mark levels were examined at selected genes repressed in Rev3l-/- MEFs (Hoxb2, Hoxb8, WT1). Histograms represent enrichment of H3K9me3 and H3K27me3 at indicated loci assessed by ChIP-qPCR in Rev3l+/+ and Rev3l-/- MEFs. Error bars indicate standard error of the mean for three independent experiments.

C. H3K9me3 and H3K27me3 mark levels were evaluated at selected genes that localized in genomic loci displaying replication timing delay and downregulated in Rev3l-/- MEFs (FoxG1, GATA6). Histograms represent enrichment of H3K9me3 and H3K27me3 assessed by ChIP-qPCR at indicated loci in Rev3l+/+ and Rev3l-/- MEFs. Error bars indicate standard error of the mean for three independent experiments.

D. Southern blot analysis of genomic DNA extracted from Rev3l+/+ and Rev3l-/- MEFs and digested with the CpG methylation-sensitive enzyme MaeII (5' -ACGT- 3'). The membrane was hybridized with radiolabeled probes specific to major satellites. The presented data are representative of two repeats (see Source Data file for this figure).
DNA polymerase zeta contributes to heterochromatin replication to prevent genome instability (2021)
Figure 6

Figure 6: REV3L facilitates replication in pericentromeric heterochromatin, preventing DNA double-strand breaks.

A. Representative fibers of newly synthesized DNA labeled with IdU (red) for 30 minutes and CldU (green) for 30 minutes in HeLa cells transfected with non-targeting siRNA (siNT) or siRNA against Rev3l. SatIII probe is visualized in blue. Scale bar: 10 μm = 20kb. Relative Rev3l mRNA level normalized to GAPDH mRNA level is shown (top panel). Distribution of fork speeds (kb/min) and fork ratios (IdU/CldU track length) are shown in dot plots for SatIII and global DNA (bottom panel). The number of fibers analyzed is indicated in (n). Bars represent the median ± interquartile range (Mann-Whitney test. ns: not significant, * p<0.05; ** p<0.01, *** p< 0.001 and ****: p<0.0001). The presented data are representative of 3 biological repeats.

B. Asynchronous Rev3l+/+ and Rev3l-/- MEFs were fixed with PFA then subjected to in situ proximity ligation assay (PLA) using 53BP1 and HP1α antibodies then PLA foci were counted in both cell lines (more than 150 nuclei for each condition were counted). p-values were calculated by Mann-Whitney test ( ****: p<0.0001). Red lines indicate the mean values. Error bars: SEM. Controls with a single antibody are also shown. Experiments were repeated three times. Representative images are shown. Scale bar= 5 μm.

C. Asynchronous Rev3l+/+ and Rev3l-/- MEFs were subjected to PLA as by using γH2AX and HP1β antibodies and processed as in (B). p-values were calculated by Mann-Whitney test ( *: p<0.05). Red lines indicate the mean values. Error bars: SEM. Controls with a single antibody are also shown. Three independent experiments were performed.

D. RPE cells were transfected with non-targeting siRNA (siNT) or siRNA against Rev3l then 72h later cells were subjected to PLA by using 53BP1 and HP1α antibodies and processed as in (B). p-values were calculated by Mann-Whitney test ( ****: p<0.0001). Red lines indicate the mean values. Error bars: SEM. Three independent experiments were performed.

E. HeLa cells were transfected with non-targeting siRNA (siNT) or siRNA against Rev3l then 72 h later cells were subjected to PLA by using 53BP1 and HP1α antibodies and processed as in (B). p-values were calculated by Mann-Whitney test ( ***: p<0.001). Red lines indicate the mean values. Error bars: SEM. Three independent experiments were performed.

F and G. Rev3l+/+ and Rev3l-/- MEFs were incubated with or without 0.23 μM aphidicolin for 24h before metaphase spreading. FISH was performed using major satellite probe to quantify breaks in pericentromeric regions (F). Representative images of chromosomes showing abnormalities (see arrows) in pericentromeric regions from Rev3l-/- MEFs. Chromosomes were labelled with DAPI and breaks in non-pericentromic regions were quantified (G). Error bars indicate standard error of the mean for three independent experiments. Mann-Whitney test (ns: not significant, * p<0.05; *** p< 0.001).
DNA polymerase zeta contributes to heterochromatin replication to prevent genome instability (2021)
Figure 8

Figure 8: REV3L loss compromises heterochromatin-associated DSB repair.

A. Asynchronous Rev3l+/+ and Rev3l-/- MEFs were UV-irradiated at the indicated doses and harvested 6 h or 24 h later. Cell lysates were analyzed by immunoblotting with indicated antibodies.

B. Asynchronous Rev3l-/- MEFs complemented with empty vector (EV) or REV3L were UV-irradiated at the indicated doses and harvested 6 h or 24 h later. Cell lysates were analyzed by immunoblotting with indicated antibodies.

C-E. Asynchronous Rev3l+/+ and Rev3l-/- MEFs were pulse labelled for 15 min using 10 μM EdU then UV-irradiated at 4 J/m2. Cells pre-treated with CSK buffer were fixed and 53BP1, RPA and RAD51 were detected by immunofluorescence. Representative images in non-irradiated cells (NI) and quantification of 53BP1 foci in EdU positive cells at different time points are shown (C). Scale bar= 10 μm Quantification of the intensity of chromatin-bound RPA2 (D) and RAD51 foci (E) 24 hours after UV-irradiation was performed. The number of cells analyzed is indicated in (n) on each graph. ***p < 0.001 (Kruskal-Wallis test). The presented data are representative of 2 repeats). Bars represent the median ± interquartile range

F-H. Asynchronous Rev3l-/- MEFs complemented with empty vector (EV) or REV3L were processed as in (C). Quantification of 53BP1 foci in EdU positive cells at different time points is shown (F). Quantification of the intensity of chromatin-bound RPA2 (G) and RAD51 foci (H) 24 hours after UV-irradiation was performed. The number of cells analyzed is indicated in (n) on each graph. ***p < 0.001 (Kruskal-Wallis test). The presented data are representative of 2 repeats. Bars represent the median ± interquartile range.

I. Model that summarizes the results obtained in this study.

Expanded View Figure Legends
Retinoblastoma protein promotes uterine epithelial cell cycle arrest and necroptosis for embryo invasion (2020)
Figure 1

Fig 1. Mice with uterine deletion of Rb1 show subfertility.

A, Rb1 protein (RB) was expressed in uterine luminal epithelium and stroma during early pregnancy in mice. Scale bar = 100μm; arrowhead, embryo.

B, Phosphorylated RB (pRB) protein, the inactive form of RB, was expressed in the luminal epithelium on days 2 and 3 of pregnancy. Scale bar = 100μm.

C, RB was efficiently deleted in the uterus of mice with uterine deletion of Rb1 (Rb1d/d mice) on day 4 of pregnancy. Scale bar = 100μm.

D, RB expression was normal in the ovary of Rb1d/d mice on day 4 of pregnancy. Scale bar = 100μm.

E, The number of new-born pups was decreased in Rb1d/d dams (n=37 different dams) compared to their littermate controls (Rb1f/f mice, n=20 different dams) (mean ± SEM, Student's t test).

Data information: le, luminal epithelium; s, stroma
Retinoblastoma protein promotes uterine epithelial cell cycle arrest and necroptosis for embryo invasion (2020)
Figure 2

Fig 2. Rb1d/d mice show impaired pre-implantation epithelial cell cycle arrest and implantation failure.

A-C, Ovulation, fertilization and development of preimplantation embryos were normal in Rb1d/d mice (A&C, mean ± SEM, Student's t test; n=5 mice for each group; B, Fisher's exact test; n=5 mice for each group).

D-E, Number of Ki67-positive cells on day 4 in uterine epithelium of Rb1d/d mice was higher than that of Rb1f/f mice (mean ± SEM, Student's t test; n=5 mice for each group). Scale bar, 100μm; le, luminal epithelium; s, stroma.

F-G, Embryo attachment occurred normally in Rb1d/d mice at 0900h on day 5 (mean ± SEM, Student's t test; n=5 mice for each group). Arrow, implantation site; scale bar = 1 cm.

H-I, Rb1d/d mice showed higher rate of embryo resorption on day 8 (*P<0.05; Fisher's exact test; n=45 implantation sites for Rb1f/f mice and n=46 implantation sites for Rb1d/d mice). Arrow, implantation site; scale bar = 1 cm.

J, H&E staining showed embryo resorption in Rb1d/d mice on day 8 of pregnancy. Scale bar = 200 μm; arrowhead, embryo; arrow, broken embryo with blood cell infiltration.

K, Weight of implantation site was comparable between Rb1f/f and Rb1d/d mice on day 6, but reduced on day 8 in Rb1d/d mice compared to Rb1f/f mice (mean ± SEM, Student's t test; n=6 mice for each group).

L-M, Expression of Cox2 and Bmp2, markers of decidualization, was comparable in between Rb1d/d and Rb1f/f mice at 0900h on day 6 in implantation sites (mean ± SEM, Student's t test; n=4 mice for each group).
Retinoblastoma protein promotes uterine epithelial cell cycle arrest and necroptosis for embryo invasion (2020)
Figure 3

Fig 3. Rb1d/d mice show persistent alignment of luminal epithelium surrounding the embryo, impaired trophoblast phagocytosis and defected embryo invasion. A, Immunostaining of cytokeratin 8 (CK8), a marker of epithelial cell lineage, showed that epithelial alignment is collapsed and the trophoblast invades the stroma at implantation sites in Rb1f/f mice, while epithelial alignment is persistent and trophoblast invasion into the stroma is compromised in Rb1d/d mice. Scale bar = 200μm; arrowhead, an embryo.

B, Transmission electron microscopic analyses (TEM) showed that the uterine epithelium surrounding the embryo disappears and the trophoblast contacts directly with the stroma at 2000h on day 5 in Rb1f/f mice, while the uterine epithelium is persistent and the embryo does not attach to the stroma in Rb1d/d mice. Scale bar = 10µm; red dotted line, stroma; green dotted line, luminal epithelium; blue dotted line, trophoblast.

C, TEM showed that fragmented luminal epithelial cells with cytoplasmic lipid droplets are engulfed by the trophoblast in the control mice (Rb1f/f mice). Scale bar = 2µm. Arrowhead, cytoplasmic fragments engulfed by trophoblast; yellow dotted line circle, cytoplasmic lipid droplets in the luminal epithelium; red dotted line, stroma; green dotted line, luminal epithelium; blue dotted line, trophoblast.

D, TEM showed that the microvilli, markers of uterine receptivity, were observed at the surface of intact luminal epithelium on day 4 of pregnancy and epithelial cells with cytoplasmic lipid droplets were observed at 2000h on day 5 of pregnancy in the control mice (Rb1f/f mice). Scale bar = 2µm; red dotted line, stroma; green dotted line, luminal epithelium; blue dotted line, trophoblast.

E, Immunofluorescence of MFG-E8, a marker of programmed cell death, showed that MFG-E8 is not expressed at the implantation sites of Rb1f/f mice at 0900h on day 5 and Rb1d/d mice at 0900h and 2000h on day 5, but is expressed at the implantation sites of Rb1f/f mice at 2000h on day 5. Scale bar = 100μm; blue signal, nuclei stained by DAPI; yellow signal, MFG-E8; arrowhead, embryo.

Data information: le, luminal epithelium; s, stroma; tr, trophoblast; n, nucleus.