EMBO/soda-nel huggingface dataset:
PML-NB-dependent type I interferon memory results in a restricted form of HSV latency (2021)
Figure 1

Fig. 1. Type I IFN induces the formation of PML-NBs in primary peripheral neurons.

(A) Representative images of primary neurons isolated from superior cervical ganglia (SCG) and sensory trigeminal ganglia (TG) of postnatal (P6) and adult (P28) mice stained for PML and the neuronal marker BIII-tubulin.

(B) SCG and TG neurons isolated from P6 and P28 mice were treated with interferon (IFN)α (600 IU/ml) for 18h and stained for PML and BIII-tubulin.

(C-F) Quantification of detectable PML puncta in P6 and P28 neurons following 18h treatment with IFNα (150 IU/ml, 600 IU/ml), IFNγ (150 IU/ml, 500 IU/ml) and IFNλ2 (100 ng/ml, 500 ng/ml). Data information: Data represent the mean ± SEM. n=60 cells from 3 biological replicates. Statistical comparisons were made using a one-way ANOVA with a Tukey's multiple comparison (ns not significant, **** P<0.0001). Scale bar, 20μm.
PML-NB-dependent type I interferon memory results in a restricted form of HSV latency (2021)
Figure 2

Fig. 2. Type I IFN treatment solely at time of infection inhibits LY294002-mediated reactivation of HSV-1 in primary sympathetic SCG neurons.

(A) Schematic of the primary postnatal sympathetic neuron-derived model of HSV-1 latency.

(B) Reactivation from latency is quantified by Us11-GFP expressing neurons following addition of the PI3K inhibitor LY294002 (20μM) in the presence of WAY-150168, which prevents cell-to-cell spread. The arrow indicates the time of LY294002 treatment at 5 days post-establishment of latency. n=9 biological replicates.

(C) Number of Us11-GFP expressing neurons at 3 days post-LY294002-induced reactivation in P6 SCG neuronal cultures infected with HSV-1 in the presence or absence of IFNα (600 IU/ml), then treated with an α-ΙFNAR1 neutralizing antibody. n=9 biological replicates.

(D) RT-qPCR for viral mRNA transcripts at 3 days post- LY294002-induced reactivation of SCGs infected with HSV-1 in the presence or absence of IFNα. n=9 biological replicates.

(E) RT-qPCR for viral mRNA transcripts at 20 hours post-LY294002-induced reactivation in SCGs infected with HSV-1 in the presence of absence of IFNα. n=9 biological replicates.

(F) Relative amount of viral DNA at time of reactivation (8dpi) in SCG neurons infected with HSV-1 in the presence or absence of IFNα (600 IU/ml). n=9 biological replicates.

(G) Quantification of vDNA foci detected by click chemistry at time of reactivation (8dpi) in SCG neurons infected with HSV-1 in the presence or absence of IFNα (600 IU/ml). n=60 genomes from 3 biological replicates.

(H) LAT mRNA expression at time of reactivation (8dpi) in neurons infected with HSV-1 in the presence or absence of IFNα (600 IU/ml). n=9 biological replicates.

Data information: Data represent the mean ± SEM. Statistical comparisons were made using a Mann-Whitney test (ns not significant, ** P<0.01, **** P<0.0001).
PML-NB-dependent type I interferon memory results in a restricted form of HSV latency (2021)
Figure 3

Fig. 3. Type I IFN-induced PML-NBs persist in primary sympathetic neurons despite resolution of IFN signaling.

(A-C) Kinetics of ISG15, IRF7 and PML mRNA expression at 0.75, 1.75, 3.75 and 8.75 days post-ΙFNα (600 IU/ml) treatment. Arrow indicates the time of HSV-1 infection at 18hr post-interferon treatment. n=9 biological replicates.

(D) Quantification of PML puncta at 1-, 3-, 6-, 8-, 10- and 15-days post-infection with HSV-1 in untreated and IFNα (600 IU/ml)-treated SCG neurons. n≥60 cells from 3 biological replicates.

(E) Representative images of P6 SCG neurons treated with IFNα (600 IU/ml) and stained for STAT1 at 18 hours and 8 days post-treatment. Scale bar, 100μm.

(F) Representative images of P6 SCG neurons treated with IFNα (600 IU/ml) and stained for Mx1 at 18 hours and 6 days post-treatment. Scale bar, 20μm.

Data information: Data represent the mean ± SEM. Statistical comparisons were made using one-way ANOVA with a Tukey's multiple comparison (A-C) or mixed-effects analysis with a Tukey's multiple comparison (D). (ns not significant, ** P<0.01, **** P<0.0001).
PML-NB-dependent type I interferon memory results in a restricted form of HSV latency (2021)
Figure 4

Fig. 4. Type I IFN induced PML-NBs stably entrap vDNA throughout a latent HSV-1 infection of primary sympathetic neurons.

(A) Representative images of vDNA foci detected by click chemistry to PML at 1, 3, 6 and 8 dpi in P6 SCG neurons infected with HSV-1EdC in the presence or absence of IFNα (600 IU/ml). Z-stack images of individual vDNA foci were acquired. Scale bar, 20μm. Zoom scale bar, 1 μm.

(B) Percent colocalization of vDNA foci detected by click chemistry to PML at 1, 3, 6 and 8 dpi in SCG neurons infected with HSV-1EdC in the presence or absence of IFNα (600 IU/ml). Each point represents the percentage of 20 vDNA foci that colocalized to PML from 3 biological replicates.

(C, D) (C) 3D reconstruction of a high-resolution Z-series confocal image showing PML entrapment of a HSV-1EdC vDNA foci. Scale bar, 2μm. (D) Enlargement of PML entrapped vDNA outlined by white dashed box. Scale bar, 0.5μm.

(E) Quantification of PML-NB volume in SCG neurons infected with HSV-1EdC in the presence or absence of IFNα (600 IU/ml). n=64 biological replicates.

Data information: Data represent the mean ± SEM. Statistical comparisons were made using a 2way ANOVA (B) or a one way ANOVA with a Tukey's multiple comparison (E) (**** P<0.0001).
PML-NB-dependent type I interferon memory results in a restricted form of HSV latency (2021)
Figure 5

Fig. 5. HSV-1 genomes only associate with PML-NBs when type I IFN is present during initial infection.

(A) Number of Us11-GFP expressing P6 SCG neurons infected with HSV-1 following IFNα treatment for 18 hours prior to infection or for 18 hours at 3 days prior to infection. n=9 biological replicates.

(B) Percent colocalization of vDNA foci detected by click chemistry to PML at 8 dpi in SCG neurons infected with HSV-1EdC following IFNα treatment for 18 hours prior to infection or for 18 hours at 3 days prior to infection. Each point represents the percentage of 20 vDNA foci that colocalized to PML from 3 biological replicates.

(C) Quantification of PML puncta at time of infection in P6 SCG neurons treated with IFNα (600 IU/ml) for 18 hours prior to infection or for 18hours at 3 days prior to infection. n=60 cells from 3 biological replicates.

(D) Percent colocalization of vDNA foci detected by click chemistry to PML at 3 dpi in SCG neurons with HSV-1EdC infected with ICP0-null mutant HSV-1, n212, or a rescued HSV-1 virus, n212R, in P6 SCG neurons treated with IFNα for 18 hours prior to infection or for 18hours at 3 days prior to infection. Each point represents the percentage of 20 vDNA foci that colocalized to PML from 3 biological replicates.

Data information: Data represent the mean ± SEM. Statistical comparisons were made using a one way ANOVA with a Tukey's multiple comparison (A-C) or a 2way ANOVA (D) (ns not significant, ** P<0.01, **** P<0.0001).
PML-NB-dependent type I interferon memory results in a restricted form of HSV latency (2021)
Figure 6

Fig. 6. Depletion of PML with shRNA-mediated knockdown prior to infection restores HSV-1 reactivation in type I interferon-treated primary sympathetic neurons.

(A) Quantification of PML puncta in P6 SCG neurons transduced with either control non-targeting shRNA or shRNA targeting PML for 3 days, then treated with IFNα (600 IU/ml) for 18 hours. n=60 cells from 3 biological replicates.

(B) Quantification of colocalization of vDNA foci detected by click chemistry to PML in primary SCG neurons transduced with shRNA targeting PML for 3 days prior to being infected with HSV-1EdC in the presence or absence of IFNα (150 IU/ml). Each point represents the percentage of 20 vDNA foci that colocalized to PML from 3 biological replicates.

(C-F) Number of Us11-GFP expressing neurons at 3 days post-LY294002-induced reactivation in P6 SCG neuronal cultures transduced with shRNA targeting PML for 3 days prior to infection with HSV-1 in the presence or absence of IFNα (150 IU/ml). n=9 biological replicates.

Data information: Data represent the mean ± SEM. Statistical comparisons were made using a one way ANOVA with a Tukey's multiple comparison (ns not significant, ** P<0.01, **** P<.0001).
PML-NB-dependent type I interferon memory results in a restricted form of HSV latency (2021)
Figure 7

Fig. 7. Depletion of PML with shRNA mediated knockdown post-infection partially restores LY294002-mediated HSV-1 reactivation in type I interferon-treated primary sympathetic neurons.

(A-D) Number of Us11-GFP expressing neurons at 3 days post-LY294002-induced reactivation in P6 SCG neuronal cultures transduced with shRNA targeting PML at 1 day post-infection with HSV-1 in the presence or absence of IFNα (150 IU/ml). n=9 biological replicates. Statistical comparisons were made using a Mann-Whitney test (ns not significant, * P<0.05).

(E) Quantification of colocalization of vDNA foci detected by click chemistry to PML in primary SCG neurons transduced with shRNA targeting PML at 1 day post-infection with HSV-1EdC in the presence or absence of IFNα (150 IU/ml). Each point represents the percentage of 20 vDNA foci that colocalized to PML from 3 biological replicates.

(F) Number of Us11-GFP expressing neurons at 3 days post- reactivation in P6 SCG neuronal cultures transduced with shRNA targeting PML at 1 day post-infection with HSV-1 in the presence of IFNα (150 IU/ml). Reactivation was induced by LY294002 in the presence of the DLK inhibitor GNE-3511 (4μM). n=9 biological replicates.

Data information: Data represent the mean ± SEM. Statistical comparisons were made using a Mann-Whitney test (A-E) or a one way ANOVA with a Tukey's multiple comparison (F) (ns not significant, * P<0.05, **** P<.0001).

Expanded View Figure Legends
Transient exposure to miR-203 enhances the differentiation capacity 
of established pluripotent stem cells (2020)
Figure 2

Figure 2. Transient exposure to miR-203 in vitro improves the in vivo developmental potential of iPSCs and ESCs.

A, Teratoma volume (mm3) 20-25 days after subcutaneous injection of wild-type iPSCs or miiPSCs expressing GFP. Data are represented as mean ± s.e.m. (n=8 tumors per genotype; 292,8 ± 66,67 versus 1216 ±140,1; difference between means: 923,5 ± 155,2; 95% confidence interval: 1256 to -590,7); ***P<0.001 (Student´s t-test). Representative images are shown in Fig. EV2.

B, Incidence and representative images of specific highly differentiated tissues in teratomas. The number of tumors included in the analysis is indicated in the panel. ***P<0.001 (Student´s t-test). Scale bars, 50 μm.

C, Table showing the frequency of nude mice with embryo-like structures (E-Ls) in their abdominal cavity 20-30 days after intraperitoneal (i.p.) injection of 400.000-500.000 in vivo (iv)-generated wild-type iPSCs, un-induced iPSCs, or miiPSCs. The number of independent clones tested per condition is indicated in the panel (each animal was inoculated with a different clone).

D, Representative example of E-Ls generated after i.p. injection of GFP-expressing miiPSCs. H/E, hematoxylin and eosin. The following antigens were detected by immunohistochemistry: Sox2 (ectoderm), Cd34 (mesoderm), Gata4 (endoderm), AFP and CK8 (visceral endoderm of the yolk sac) and Ter119 (nucleated erythroid cells). Scale bars, 500 μm and 100 μm for higher magnifications.

E, Embryo tetraploid complementation assays with un-induced iPSCs, miiPSCs or wild-type ESCs (n=3 clones per condition). Pictures on the right show a representative example of a viable "all-miiPSC" mouse (black) generated from miiPSCs and litters obtained from "all-miiPSC" adult mice, which efficiently contributed to germline transmission.
Transient exposure to miR-203 enhances the differentiation capacity 
of established pluripotent stem cells (2020)
Figure 3

Figure 3. miR-203-exposed human iPSCs efficiently contribute to human-mouse interspecies chimeras.

A, Summary of chimera assays in which one single Td-Tomato fluorescent-labeled human iPSC, either control or transiently transfected with miR-203-expressing vectors (miiPSCs) was injected into 8C-stage mouse embryos and their contribution analyzed 48-60 h later. P values are indicated in the Figure (Student´s t-test).

B, Representative images showing the contribution of human control iPSCs or miiPSCs cells to mouse blastocysts. These structures were co-immunostained with (human) anti-OCT4 and anti-HuNu antibodies. Td-Tomato, red; Scale bars, 50 μm.

C, Interspecies chimera assays in which the 8C-stage mouse embryos indicated in (A, B), injected with one single human iPSCs and cultured in vitro for 48 h to reach the blastocyst stage, were then transfected to 2.5 days post coitum pseudo-pregnant females. The post-implantation mouse conceptuses were dissected at the E9.5 developmental stage and analyzed by immunofluorescence. Representative images showing the integration of control human iPSCs (top) or miiPSCs (bottom) into mouse E9.5 embryos. Anti-human HuNu antibody was co-stained with Td-Tomato direct fluorescence to detect human cells in E9.5 mouse embryos. Scale bars, 20 μm.

D, E, Quantification of the Td-Tomato-(D) or HuNu-(E) positive area in 40-50 different cryosections of these embryos randomly and blindly selected for IF analysis. Data are mean ± s.e.m. **P<0.01; ***P< 0.001 (Student´s t-test).
Transient exposure to miR-203 enhances the differentiation capacity 
of established pluripotent stem cells (2020)
Figure 4

Figure 4. DNA methyltransferases 3a and 3b are miR-203 targets involved in the regulation of PSCs potential.

A, Venn Diagrams representing common genes down-regulated in miiPSCs, predicted as miR-203 targets and also involved in the epigenetic regulation of gene transcription (GO:0040029). The list of the common 18 transcripts (including Dnmt3a and Dnmt3b) is presented as Appendix Table S2.

B, C, Relative Luciferase Units (RLU; normalized to Renilla luciferase and relative to DNA amount) in 293T cells transfected with DNA constructs carrying the wild-type 3´UTRs from the indicated transcripts (B) or the mutated versions of Dnmt3a and Dnmt3b 3´UTRs, downstream of the luciferase reporter (C). Cells were co-transfected with Renilla luciferase as a control of transfection, and a plasmid expressing GFP or miR-203-GFP. Data are represented as mean ± s.d. (n=3 independent experiments).

D, Principal Component Analysis from RNAseq data including profiles from wild-type iPSCs, miiPSCs, and wild-type iPSCs transfected with either control siRNAs (siC), or siRNAs specific against Dnmt3a (siDnmt3a), Dnmt3b (siDnmt3b) or both (siDnmt3a/b).

E, Representative images of embryoid bodies (EBs) derived from wild-type iPSCs in which the expression of Dnmt3a and Dnmt3b was transiently repressed by siRNAs. Scale bars, 500 μm. Plots show the quantification of the size of EBs and the percentage of EBs with large cavities or beating at different time points during the differentiation process. Data are represented as mean ± s.e.m. (n=3 independent experiments).

F, Expression levels of Dnmt3a or Dnmt3b transcripts after transfection of wild-type iPSCs with specific siRNAs either against Dnmt3a, Dnmt3b or a combination of both (Dnmt3a/b) (as indicated in E). RNA expression was measured 24 hours after the transfection protocols and was normalized by GAPDH mRNA levels. Data are represented as mean ± s.e.m. (n=3 independent experiments).

G, Representative images of EBs derived from miiPSCs that were transiently and simultaneously transduced with Dnmt3a and Dnmt3b cDNAs or empty vectors, and simultaneously treated with Dox to induce miR-203 expression. Scale bars, 500 μm. Plots show the quantification of EB size, percentage of EBs with large cavities, and beating EBs at different time points during differentiation. Data are represented as mean ± s.e.m. (n=3 independent experiments).

H, Expression levels of miR-203, Dnmt3a or Dnmt3b transcripts in iPSCs after induction of miR-203 (miiPSCs) and transduced with Dnmt3a and Dnmt3b cDNAs or empty vectors (as indicated in G). RNA expression was measured 24 hours after the transfection protocols and was normalized by a control miRNA (miR-142) or GAPDH mRNA, respectively. Data are represented as mean ± s.e.m. (n=3 independent experiments).

Data information: In (B, C, E-H) *P<0.05; **P<0.01; ***P<0.001 (Student´s t-test).
Transient exposure to miR-203 enhances the differentiation capacity 
of established pluripotent stem cells (2020)
Figure 5

Figure 5. Transient expression of miR-203 induces genome-wide hypomethylation in iPSCs.

A, Experimental design for the genome-wide DNA methylation analysis of wild-type and miiPSC as well as embryoid bodies (EBs) derived from them. Cells (two independent miiPSC clones and two iPSC technical replicates) were transiently treated with Dox for 5 days and then subjected to Dox withdrawal for 20 additional days before starting the EB formation protocol. Samples for DNA and RNA analysis were collected at the indicated time points before Dox (t=0), 5 days after Dox withdrawal (t=10), 20 days after Dox withdrawal (t=25) or 7 days after starting the EB generation protocol (t=32 days).

B, Genome-wide DNA methylation data showing the number and total size of DNA methylation valleys (DMVs) and partially methylated domains (PMDs).

C, DNA methylation distribution of the indicated samples, smoothed over 100 kb-blocks.

D, Principal Component Analysis showing the distribution of methylation profiles in the indicated samples (color code as in panel B).

E, Number of differentially methylated single CpG sites (DMPs) and differentially methylated regions (DMRs) in the indicated comparisons (color code as in panel B).

F, Heat-scatterplots representing the mean methylation over all transcripts (transcription start sites, TSSs) in mm10, pairwise for "t=10 vs. t=0" (left panel) and "t=25 vs. t=0" (right panel). Note that especially for "t=25 vs. t=0" most of the points are below the diagonal, indicating broad hypomethylation across most of the genome.

G, Heat-scatterplots representing the mean methylation over the TSS of the 282 genes included in the 2-cell signature (as in Fig. 1D). Analysis as in (F).

H, The specific differentially methylated regions (DMR) at the Sirt6 locus were analyzed by PCR amplification and sequencing of bisulphite-modified DNA. Ten independent clones were sequenced per condition. The quantification of methylated (filled circles) vs. unmethylated (empty circles) CpGs is shown in the histogram in the indicated conditions.

I, Heatmaps representing the methylation levels at the top Differentially Methylated Regions (DMRs; n=100; upper panel) or the Imprinting Control Regions (ICRs; n=103 different imprinting loci; lower panel) in the indicated samples (referred to panels A,B). Color code and scale of DNA methylation, from 0.0 (red) to 1.0 (green) is applicable to both heatmaps.
Transient exposure to miR-203 enhances the differentiation capacity 
of established pluripotent stem cells (2020)
Figure 6

Figure 6. Transient exposure to miR-203 enhances differentiation into mature cardiomyocytes and improves cardiac regeneration.

A, Experimental protocol followed for the differentiation of cardiomyocytes from iPSCs in the absence or presence of miR-203 mimics and Dnmt3a/b cDNAs.

B, Representative immunofluorescences showing cardiac Troponin T (cTnT, green) and nuclei (Hoechst, blue) staining of in vitro-generated cardiomyocytes derived from wild-type iPSCs transiently transfected with either control mimics, miR-203 mimics or miR-203 mimics + Dnmt3a/b cDNAs. Pictures were taken at day 15 of differentiation. Scale bars, 68 μm. The cTnT-positive area in these cardiomyocytes is shown in the right histogram. Data are represented as mean ± s.d. (n=2 independent experiments with 6 replicates each).

C, Beating frequency (measured as number of beats per 5 seconds) of these cardiomyocytes at day 15 of differentiation. Data are represented as mean ± s.d. (n=8 independent replicates).

D, miRNA or mRNA levels as determined by quantitative PCR of the indicated transcripts at different time points during cardiomyocyte differentiation in the indicated samples. Data are represented as mean ± s.d. (n=2 independent experiments with 6 replicates each).

Data information: In (B, C, D), *P<0.05; **P<0.01; ***P<0.001 (Student´s t-test).

List of Expanded Material
ATP-Dependent DNA Binding, Unwinding, and Resection by the Mre11/Rad50 Complex (2015)
Figure 6

Figure 6. Effects of ATP-dependent binding of DNA to the MR complex.

(A) Endonuclease activities of the TmMR WT and mutant proteins towards a duplex DNA in the absence (left panel) or presence of ATP (right panel). Major products are marked with dots. The 5’-32P-label is indicated by a star. See Appendix Fig S4A and S4B for the quantified values. (B) Quantification of a bubble DNA binding by WT or mutant TmMR in the presence or absence of AMP-PNP. See Appendix Fig S3E and S3F for the original data. (C) Endonuclease activities of the WT and mutant TmMR proteins towards a bubble DNA molecule in the absence (left panel) or presence of ATP (right panel). See Appendix Fig S4C and S4D for the quantified values. (D) The effects of single or multiple point mutations in the DNA-contacting or neighboring residues of S. cerevisiae rad50 on the sensitivity to chemical drugs. Sensitivities of rad50 expressing the mutants towards indicated concentrations of CPT, PHL, and MMS were examined. (E) The effects of Rad50 mutation on recombination stimulated by inverted Alu repeats. See Appendix Table S1 for the quantified values.
Unloading of homologous recombination factors is required for restoring double-stranded DNA at damage repair loci (2016)
Figure 1

Figure 1. Srs2 is not required for the recovery from the DNA damage-induced arrest.

A. On the left, schematic of chr. VIIL variants, either with 2 or with 20 kb between a DSB and a telomere, used to study the effect of DNA damage checkpoint activation on cell viability in panels. Triangles represent HO sites, dashed lines – telomeric sequences, TG81 – 81 bp of (TG1-3)n and grey boxes – genes with the grey arrows above showing promoters. The diagram on the right outlines the DNA damage response (DDR) activation as the reaction of the two different stains on DSB induction by the addition of galactose (GAL).

B. Analysis of cell cycle arrest (in G2) in response to DSB induction assayed by flow cytometry.

C. Rad53 phosphorylation (Rad53-P) in response to DSB assayed by western blotting.

D. Cell survival upon DSB induction. Average ± SD (n=4) is shown for each genotype.

Strains used: NK4230, NK4231; NK4264, NK4265; NK1949; NK4268, NK4269.