EMBO/soda-nel huggingface dataset:
Cold-induced chromatin compaction and nuclear retention of clock mRNAs resets the circadian rhythm (2020)
Figure 1

Fig. 1

Cold temperature induces changes to the higher-order chromatin architecture.

A) Upper panels: Representative single z-planes from 3D-SIM image stacks of the DAPI-stained nuclei of AC16 cells kept at 37°C or exposed to different cold temperatures for 24h. Lower panels: DAPI signal segmented into chromatin and interchromatin regions according to relative intensity. Scale bar: 5 μm. Arrows mark chromatin concentrated at the nuclear periphery (P) and around nucleoli (N).

B) Boxplots of the ratio of chromatin volume to nuclear volume for cells exposed to different temperature conditions. Median (central line), interquartile range (IQR, box edges), most extreme data point no more than 1.5x IQR from box edges (whiskers). Outliers outside the whiskers are shown as individual points. Asterisks mark mean ratios that are significantly (adjusted p-value < 0.05 (two-sided Mann-Whitney test)) different from the mean ratio of cells at 37°C (black asterisks) or cells exposed to 18°C for 24h (red asterisks). Number of nuclei: 37°C (31), 28°C 24h (26), 18°C 5h (19), 18°C 24h (22), 18°C 24h then 37°C 2h (25), 8°C 24h (23).

C) Left and central panels: representative single z-planes from 3D-SIM image stacks of the DAPI-stained nuclei of cells kept at 37°C or exposed to 18°C for 24h showing the spatial distribution of the immunofluorescence (IF) signal of hnRNPC (green) and nuclear pore complexes (NPCs, red). Right panel: DAPI signal overlaid with dots indicating IF signal centroid coordinates for centroids located in the z-plane shown. Scale bar whole nuclei: 5 μm. Scale bar enlarged section: 1 μm.

D) Bar graph of the mean NPC centroid to nearest hnRNPC centroid distance for each temperature condition. Error bars show the S.E.M. p-value (two-sided Welch's t-test) calculated for the comparison shown. Number of distances: 37°C (112511), 28°C 24h (94521), 18°C 5h (72340), 18°C 24h (74998), 18°C 24h then 37°C 2h (92896), 8°C 24h (80570).

E) Schematic illustrating how chromatin concentrated at the periphery after exposure to 18°C could widen the average NPC to nearest mRNP distance and thus hinder mRNA nuclear export. Data information: Actual p-values are presented in the source data file.
Cold-induced chromatin compaction and nuclear retention of clock mRNAs resets the circadian rhythm (2020)
Figure 3

Fig. 3

Rewarming following 18°C exposure releases nuclear-accumulated transcripts into the cytoplasm, including those of core circadian clock negative limb genes.

A) Boxplots of standardized nuclear and cytoplasmic RNA levels at time points during the transfer of AC16 cells from 37°C to 18°C for 24h and then back to 37°C for 24h for the group of 1285 genes showing significant upregulation in nuclear RNA levels at the 18°C for 24h time point. Range (whiskers), interquartile range (IQR, box edges), median +/-IQR/[square root(number of genes)] (notch) and median (central line).

B) Log2 fold change in cytoplasmic (black line, left axis) and nuclear (red line, right axis) RNA level of core circadian clock negative limb genes at time points during the transfer of AC16 cells from 37°C to 18°C for 24h and then back to 37°C for 24h relative to cells kept at 37°C. Error bars show the standard error.

C) Nuclear and cytoplasmic full-length RNA-Seq data from AC16 cells kept at 37°C or exposed to 18°C for 24h for REV-ERBα and CRY2. Data for each track have been normalized to show counts per million aligned counts (CPM) for each nucleotide. Panels were prepared using Integrative Genomics Viewer (IGV).

D) Western blot of Flag-tagged REV-ERBα levels (short and long exposure) in whole cell extracts from AC16 cells transferred from 37°C to 18°C for the time periods indicated and also returned after each of these time periods to 37°C for 2h. TP53 was used as a loading control as its transcript levels show minimal changes in response to 18°C exposure (Fig. 4C,G, EV3F). Ponceau stain is shown as an additional loading control. Quantification of the REV-ERBα/TP53 signal is shown below.

Data information: Number of 3' end RNA-seq sample replicates: 37°C (C: 6, N: 7), 18°C 5h (4), 10h (4), 24h (6), 18°C 24h then 37°C 2h (4), 5h (2), 10h (2), 24h (2). Number of cytoplasmic (C) and nuclear (N) samples are the same for each condition except 37°C (see also Appendix Table S4). Additional statistical details are presented in the source data file.
Cold-induced chromatin compaction and nuclear retention of clock mRNAs resets the circadian rhythm (2020)
Figure 4

Fig. 4

Nuclear accumulation upon 18°C exposure and release upon rewarming of core circadian clock negative limb gene transcripts.

A-D) Representative maximum intensity z-stack-projected images of DAPI-stained (blue) AC16 (A-C) or U2OS cells (D), exposed to 18°C and returned to 37°C for the time periods indicated, probed by RNA-FISH for REV-ERBα (A and D), CRY2 (B) or TP53 (C) transcripts (red dots). Scale bar: 10 μm.

E-H) Mean REV-ERBα (E and H), CRY2 (F) or TP53 (G) transcripts per AC16 (E-G) or U2OS (H) cell nuclear and cytoplasmic area across all images for each condition from quantification of the RNA-FISH data. p < 0.001 (Tukey's HSD) for all pairwise comparisons except those shown. All error bars (E-H) show S.E.M.

Data information: Number of images: AC16 cells (REV-ERBα): 37°C (65), 18°C 5h (30), 18°C 5h then 37°C 2h (21), 18°C 24h (63), 18°C 24h then 37°C 2h (64); AC16 cells (CRY2): 37°C (43), 18°C 24h (42), 18°C 24h then 37°C 2h (42); AC16 cells (TP53): 37°C (47), 18°C 24h (45), 18°C 24h then 37°C 2h (48); U2OS cells (REV-ERBα): 37°C (61), 18°C 5h (61), 18°C 5h then 37°C 2h (69), 18°C 24h (53), 18°C 24h then 37°C 2h (65) (see also Appendix Table S7). Counts per image and actual p-values are presented in the source data file.
Cold-induced chromatin compaction and nuclear retention of clock mRNAs resets the circadian rhythm (2020)
Figure 5

Fig. 5

Cold-induced REV-ERBα expression resets the phase and modulates the amplitude of the circadian rhythm.

A,B) Mean baseline-detrended bioluminescence profiles from plate wells containing either PER2::LUC U2OS cells with (WT, left panel) or without REV-ERBα (KO, right panel) recorded at 37°C before and after transfer to 18°C for 24h (A) or 5h (B) (grey region) from biological repeat 1. 6 differently colored profiles represent cells synchronized at 6 distinct phases of the circadian period prior to the start of 18°C exposure (time zero). Peaks marked "x" defined in (C).

C) Schematic showing the measurement of each profile from time zero to the peak marked "x" (this is the second peak after 18°C exposure or the corresponding peak in cells kept at 37°C (see (A),(B) and Fig. EV4D-F,H-J)). Plotting these times shows the effect of 18°C exposure on the spread of these profiles.

D) Boxplots and individual points for times measured as in (C) for each plate well profile for both WT and KO cells kept at 37°C or transferred to 18°C for 24h (top panel) or 5h (bottom panel) from biological repeats 1 and 2. Points are colored according to their distinct phase prior to 18°C exposure. Colored lines (bottom panel) show the change in the mean for the points from each phase. Pairwise comparisons test the significance of the difference in variance (Brown-Forsythe test (adjusted for multiple testing)) (ns = p > 0.05; * = 1x10-10 < p < 0.05; ** = 1x10-20 < p < 1x10-10 ; *** = p < 1x10-20). Boxplots display median (central line), interquartile range (IQR, box edges) and range (whiskers). Number of wells: top panel WT cells: 37°C (120), 18°C 24h (119); top panel KO cells: 37°C (120), 18°C 24h (120); bottom panel WT cells: 37°C (58), 18°C 5h (120); top panel KO cells: 37°C (60), 18°C 5h (120).

E) Bar graphs showing the mean amplitude change (left axis) following 18°C exposure for 5h for each group of plate well profiles (grouped according to their distinct phase (a-f) prior to 18°C exposure, 10 wells per group) for both WT and KO cells from biological repeats 1 and 2. Mean changes are plotted to approximately align with the position of the control profile at the point of rewarming within the circadian period that is represented by the PER2:LUC (dashed line) and predicted REV-ERBα expression level (right axis) (grey shaded region (light grey indicates REV-ERBα deletion)). Error bars show the standard deviation. Asterisks mark mean log2 amplitude fold changes significantly greater than +/-log2(1.4) (adjusted p < 0.05, one sample t-test). (see Appendix Fig. S2 (Fig. 5 extended data for more details)).

Data information: Actual p-values are presented in the source data file.

EV Figure legends:
Intrinsically disordered protein PID-2 modulates Z granules and is required for heritable piRNA-induced silencing in the C. elegans embryo (2020)
Figure 3

Figure 3. Loss of PID-2 causes a disbalance in 22G RNA populations.

A) MA-plot of log2 fold changes (Y-axis) versus the mean of normalized counts of 22G RNAs (X-axis) for pid-2(xf23) mutants, compared to wild type. Red dots: genes with adjusted p-value < 0.05 and fold-change > 2. Blue dots: genes with adjusted p-value < 0.05 and fold-change < -2.

B) Heatmap displaying overlap significance between different gene sets and genes that either up- or down-regulated in pid-2(xf23) mutants. Significance was tested with Fisher's exact test and p-values adjusted with the Benjamini-Hochberg Procedure. Colour scheme represents the odds ratio of overlaps representing the strength of association. N.S.: not significant.

C-D) Cumulative 22G coverage along the gene body of Mutator and CSR-1 targets. Values represent 22G coverage normalized to the total coverage of each gene, for wild type (N2) and pid-2(xf23) mutants. Gene sets are previously defined 22G RNA target sub-types (see Appendix). The lines represent the average of biological replicates, whereas the shading represents the standard deviation of biological replicates. a.u.: arbitrary units.
Intrinsically disordered protein PID-2 modulates Z granules and is required for heritable piRNA-induced silencing in the C. elegans embryo (2020)
Figure 4

Figure 4. IP-MS on PID-2 identifies two novel interacting proteins, PID-4 and PID-5, and reveals the existence of two distinct complexes.

A) Volcano plot representing the enrichment of proteins interacting with PID-2, isolated by immunoprecipitation of eGFP::PID-2 protein, followed by quantitative label-free mass spectrometry. As control, eGFP IPs were performed on protein extracts from wild-type, non-transgenic animals. For each IP-MS experiment, quadruplicates were measured and analysed. The dashed line reflects a significance threshold of p-value < 0.05 at two-fold enrichment.

B) Volcano plot, as described in A. In this experiment, the endogenous PID-2 protein was immunoprecipitated, and pid-2(xf23) mutant protein extracts were used as control.

C) Schematic representation of the predicted domain structure of PID-4 and PID-5. The eTudor domains were identified using HHpred (Zimmermann et al., 2018). The X-prolyl aminopeptidase domain was identified using BLAST (https://blast.ncbi.nlm.nih.gov/Blast.cgi).

D) Volcano plot, as described in A, representing the enrichment of proteins interacting with PID-4, by immunoprecipitating endogenously tagged PID-4 protein. IPs from protein extracts from wild-type, non-tagged animals were used as control.

E) Volcano plot, as described in A, representing the enrichment of proteins interacting with PID-5, by immunoprecipitating endogenously tagged PID-5 protein. IPs from protein extracts from wild-type, non-tagged animals were used as control.
Intrinsically disordered protein PID-2 modulates Z granules and is required for heritable piRNA-induced silencing in the C. elegans embryo (2020)
Figure 5

Figure 5. PID-2 and its interactors, PID-4 and PID-5, are required for maintenance of an immortal germline.

A) Expression of the 21U sensor in the indicated genetic backgrounds. Gonads are outlined by a dashed line. The mCherry signal is represented in pseudo-colours [LUT fire (ImageJ)] to reflect differences in the intensity of the signal. The panels on the right are DIC images of the respective animals. Scale bar: 25 μm.

B) 22G RNA coverage of the 21U sensor(RNAe) in the indicated genetic backgrounds. Quantification of the secondary and tertiary 22G RNAs is provided in Fig EV4A.

C) MA-plot of log2 fold changes (on the Y-axis) versus the mean of normalized counts of 22G RNAs (on the X-axis) for pid-4 mutants, compared to wild type. Red dots: genes with adjusted p-value < 0.05 and fold-change > 2. Blue dots: genes with adjusted p-value < 0.05 and fold-change < -2.

D) Heatmap displaying overlap significance between different gene sets and genes that either up- or down-regulated in pid-4 mutants. Significance was tested with Fisher's exact test and p-values corrected with the Benjamini-Hochberg Procedure. Colour scheme represents the odds ratio of overlaps representing the strength of association.

E) As panel C, but for pid-5 mutant animals.

F) As panel D, but for pid-5 mutant animals.

G) As panel C, but for pid-4;pid-5 double mutant animals.

H) As panel D, but for pid-4;pid-5 double mutant animals.

I, J) Line plots representing fertility over generations for the indicated genetic backgrounds at 25oC. Six L2-L3 larvae for each of the indicated backgrounds were hand-picked to a fresh plate every four or six days, counting as two or three generations, respectively, until no larvae were present on the plate to be picked. *: two plates of N2 were contaminated and were excluded from the assay.
Intrinsically disordered protein PID-2 modulates Z granules and is required for heritable piRNA-induced silencing in the C. elegans embryo (2020)
Figure 6

Figure 6. PID-2 is expressed in Z granules, whereas PID-4 and PID-5 localize to P granules, and their expression partially overlaps.

A-C) Expression of PID-2::eGFP(xfSi145) (A), PID-4::mTagRFP-T (B) and PID-5::mTagRFP-T (C) in perinuclear granules in the germline. PGL-1::mTagRFP-T (A) and DEPS-1::GFP (B, C) mark P granules. The indicated dashed boxes reflect zoom-ins on specific nuclei to better visualize the granules, and their overlaps. One L4 gonad is shown for each animal. Note that most of the L4 gonad is in pachytene stage. Arrow heads indicate individual condensates. Scale bar: 25 µm.

D, E) Expression of PID-4::mTagRFP-T (D) and PID-5::mTagRFP-T (E) together with 3xFLAG::GFP::ZNFX-1, a Z granule marker. The indicated dashed boxes reflect zoom-ins on specific nuclei to better visualize the granules, and their overlaps. One L4 gonad is shown for each animal. Note that most of the L4 gonad is in pachytene stage. Arrow heads indicate individual condensates. Scale bar: 25 µm.

F) Box-plots representing the distance (µm) between the centres of two fluorescent signals from the indicated fusion proteins, as represented in panels A-E, G, H. The distance between each pair of fluorescent proteins is represented by a dot. Between 4-10 different gonads were analyzed for each condition. The median is represented by a line. The interquartile range (IQR), 25th to 75th percentile, is represented by the upper and lower lines, respectively, and whiskers represent the first quartile (down to -1.5*IQR) or the third quartile (up to +1.5*IQR). P-values were calculated using an unpaired t-test (two-tailed).

G, H) Expression of PID-2::eGFP (xfSi145) together with either PID-4::mTagRFP-T (G) or PID-5::mTagRFP-T (H). The indicated dashed boxes reflect zoom-ins on specific nuclei to better visualize the granules, and their overlaps. One L4 gonad is shown for each animal. Note that most of the L4 gonad is in pachytene stage. Arrow heads indicate individual condensates. Scale bar = 25 µm.
Intrinsically disordered protein PID-2 modulates Z granules and is required for heritable piRNA-induced silencing in the C. elegans embryo (2020)
Figure 7

Figure 7. Z granules are affected by PID-2, PID-4 and PID-5.

A-E) Expression of 3xFLAG::GFP::ZNFX-1 and PGL-1::mTagRFP-T in a wild type (A), pid-2 (B), pid-4;pid-5 (C), pid-4 (D), and pid-5 (E) mutant backgrounds. The indicated dashed boxes reflect zoom-ins on specific nuclei to better visualize the granules, and their overlaps. One L4 gonad is shown. Arrow heads indicate individual condensates. Scale bar: 25 µm.

F) Quantification of the ratio Z / P granules in wild type, pid-2, pid-4/-5, pid-4 and pid-5 mutant backgrounds. The number of granules is indicated in brackets, next to the genotype. P-values were calculated using a two-proportion z-test and adjusted for multiple comparisons with the Holm method.

G, H) Expression of PID-4::mTagRFP-T (G) and PID-5::mTagRFP-T (H) together with 3xFLAG::GFP::ZNFX-1, a Z granule marker, in a pid-2 mutant background. The indicated dashed boxes reflect zoom-ins on specific nuclei to better visualize the granules, and their overlaps. One L4 gonad is shown for each animal. Scale bar: 25 µm.

I) Box-plots representing the distance (µm) between the centres of two fluorescent signals from the indicated fusion proteins, as represented in panels G, H and in Fig 6D, E. Please note that the wild-type distance measurements are the same represented in Fig 6F. The distance between each pair of fluorescent proteins is represented by a dot. Between 4-10 different gonads were analyzed for each condition. The median is represented by a line. The interquartile range (IQR), 25th to 75th percentile, is represented by the upper and lower lines, respectively, and whiskers represent the first quartile (down to -1.5*IQR) or the third quartile (up to +1.5*IQR). P-values were calculated using a t-test (two-tailed).
Break-induced replication plays a prominent role in long-range repeat-mediated deletion (2019)
Figure 1

Figure 1. BIR is involved in RMD when a DSB is close to one repeat.

A, Schematic drawing of the EGFP-based SSA reporter, EGFP-SSA. The EGFP coding sequence is split into two fragments, which share a 315 bp overlapping homology (repeat) and are separated by a 2,035 bp DNA sequence. An I-SceI cleavage sequence is inserted in the intervening sequence, 9 bp away from the 3' repeat. The EGFP open reading frame is restored after DSB-induced RMD of the two EGFP fragments.

B, RMD assays performed after DSBs have been generated by sgRNAs/Cas9. The distance between the sgRNA/Cas9 cleavage sites and the repeat sequences is indicated. U2OS cells carrying a single copy of the EGFP-SSA reporter (3427-17) were transfected with sgRNA/Cas9 plasmids, and the percentage of GFP-positive cells was determined (left). U2OS cells transfected with the indicated reporters EGFP-SSA-17/6 bp, EGFP-SSA-0.3 kb, EGFP-SSA-0.6 kb and EGFP-SSA-1.0 kb were pooled as a population after selection of the drug marker on the reporters, followed by sgRNA/Cas9 cleavage (right).

C, RMD is strongly induced when a DSB is generated close to one repeat. The U2OS (EGFP-SSA-3427-17) cell line carrying a single copy of the reporter was transfected with sgRNA/Cas9 plasmids as indicated, and the percentage of GFP-positive cells was determined.

D, Models of RMD mediated by SSA (RMD/SSA) or BIR (RMD/BIR). When a DSB is created in the middle of two repeats, the DNA ends of both sides are resected, generating 3' ssDNA overhangs, until the repeat sequences on opposite sides are both exposed. Then ssDNA of the repeat sequences anneal, resulting in a product with internal sequences deleted. When a DSB is generated close to one repeat, ssDNA formed in one repeat first can invade the other repeat still in duplex using BIR, which can also give rise to a deletion product identical to the SSA product after cleavage of D-loop. Pink arrow: putative structure-specific endonucleases to cleave non-homologous tails; blue arrow: putative structure-specific endonucleases to cleave the D-loop.

E, F and G, Determination of RMD frequency in U2OS (EGFP-SSA-3427-17) cells when POLD3 (E), RAD52 (F) or RAD51 (G) is depleted by lentiviral expression of shRNA. Cells infected with a lentiviral empty vector were used as control. The knockdown level of the indicated proteins was shown by Western blot, using KU70 as the loading control.

Data information: Error bars represent standard deviation (SD) of at least three independent experiments. *P < 0.05, **P < 0.01, ns, not signiﬁcant, two-tailed non-paired t-test.
Break-induced replication plays a prominent role in long-range repeat-mediated deletion (2019)
Figure 2

Figure 2. Effect of the distance between the DSBs and the repeats on RMD frequency.

A, A diagram indicating the position of DSBs generated by sgRNAs/Cas9 on the EGFP-SSA reporter.

B, RMD efficiency is decreased when the distance from a DSB to one repeat is increased. RMD assays were performed in U2OS (EGFP-SSA-3427-17) cells using the indicated sgRNAs/Cas9. The distance between the sgRNA/Cas9 cleavage sites and the repeat sequences is shown.

C and D, Knockdown of POLD3 impairs RMD when a DSB is close to one repeat. RMD frequency was determined in U2OS (EGFP-SSA-3427-17) cells using the indicated sgRNAs/Cas9 with or without depletion of POLD3 by lentivirus-expressed shRNA. Cells infected with a lentiviral empty vector were used as control. The distance between the sgRNA/Cas9 cleavage sites and the repeat sequences is indicated. The knockdown level of POLD3 was shown by Western blot, and KU70 was used as the loading control.

Data information: Error bars represent standard deviation (SD) of at least three independent experiments. *P < 0.05, **P < 0.01, ns, not signiﬁcant, two-tailed non-paired t-test.
Break-induced replication plays a prominent role in long-range repeat-mediated deletion (2019)
Figure 3

Figure 3. BIR/RMD is stimulated by oncogene overexpression and replication fork collapse.

A, Cyclin E overexpression results in increased RMD when the DSB is close to one repeat. The U2OS (EGFP-SSA-3427-3) cell line carrying Tet-on-cyclin E plasmid was used, and RMD frequency was determined with or without induction of cyclin E expression by doxycycline (DOX, 0.5 µg/mL), which was added 24 hours before transfection of the indicated sgRNAs/Cas9. Cyclin E expression levels were validated by Western blot, using KU70 as the loading control.

B, ATR is required for BIR/RMD upon cyclin E overexpression. RMD frequency was determined in U2OS (EGFP-SSA-3427-3) cells infected with lentivirus-expressed ATR shRNA or the control vector (Ctrl) with and without induction of cyclin E expression by DOX (0.5 µg/ml) after R29 sgRNA/Cas9 cleavage. ATR knockdown was shown by Western blot with KU70 as the loading control.

C, A diagram showing the BIR/RMD model when a single-ended DSB is generated after replication fork collapse.

D, RMD is stimulated when Cas9-D10A is used to create a nick near the repeat. The repair frequencies of the U2OS (MMEJ) reporter cell line (left) and U2OS (EGFP-SSA-3427-17) cell line (right) were determined after expression of the indicated sgRNAs/Cas9 and sgRNAs/Cas9-D10A. The distance between the sgRNA cleavage sites and the left and right repeat sequences is indicated (right bottom).

E, RMD induced by Cas9-D10A is dependent on POLD3. U2OS (EGFP-SSA-3427-17) cells were infected with lentiviruses expressing shRNA for POLD3 or vector control and RMD was determined after cleavage by the indicated sgRNAs/Cas9-D10A. The distance between sgRNA/Cas9-D10A cleavage sites and the left and right repeat sequences is indicated at the bottom. The knockdown level of POLD3 was shown by Western blot, and KU70 was used as the loading control.

F, p27 suppresses BIR/RMD and SSA/RMD. U2OS (EGFP-SSA-3427-3) cells carrying the Tet-on-cyclin E allele were infected with and without p27 lentiviral viruses. RMD was determined before and after induction of cyclin E overexpression by DOX (0.5 µg/mL) added 24 hours before transfection with the indicated sgRNAs/Cas9. Cyclin E expression levels and cell cycle profiles are shown in Appendix Fig S6.

Data information: Error bars represent standard deviation (SD) of at least three independent experiments. *P < 0.05, **P < 0.01, ns, not signiﬁcant, two-tailed non-paired t-test.