EMBO/SourceData huggingface dataset:
Figure 6(A) IL-4 levels in paws from naive and zymosan-injected mice were determined by ELISA 8 and 24 hours after injection of zymosan in paws of control or eosinophil-depleted mice. Data are shown as mean ± SEM (n=6 mice). Student's t-test/Two-way ANOVA/Bonferroni, control vs anti-Siglec F antibody treatment: **p<0.01, ***p< 0.001.(B) FACS analysis of the eosinophil number in paws from control and eosinophil-depleted mice with or without IL-4c treatment 24h prior zymosan injection. Data are shown as the mean ± SEM (n=8 mice). Two-way ANOVA/Bonferroni ***p< 0.001.(C) Same as panel B except that resident (Ly6C-) and monocyte-derived macrophages (Ly6C+) were determined.Same as panel B except that M2-like (panel D) were determined. Data are shown as mean ± SEM (n=8 mice). Student's t-test/Two-way ANOVA/Bonferroni, control vs anti-Siglec F antibody treatment: *p<0.05, ***p<0.001.Same as panel B except that M1-like (panel E) were determined. Data are shown as mean ± SEM (n=8 mice). Student's t-test/Two-way ANOVA/Bonferroni, control vs anti-Siglec F antibody treatment: *p<0.05, ***p<0.001.Same as panel B except that M0 macrophages (panel F) were determined. Data are shown as mean ± SEM (n=8 mice). Student's t-test/Two-way ANOVA/Bonferroni, control vs anti-Siglec F antibody treatment: *p<0.05, ***p<0.001.
Figure 7(A) Concentrations of cytokines and chemokines were determined by multiplex cytokine assay 8 and 24 hours after injection of zymosan in paws from control or eosinophil-depleted mice. Data are mean ± SEM (n=6 mice). Student's One-way ANOVA, significance between control and anti-Siglec F antibody treatment *p<0.05; **p<0.01; ***p<0.001.FACS analysis of the neutrophil count in paws (panel B) from control or eosinophil-depleted mice with or without IL-4c treatment. Data are shown as the mean ± SEM (n=6-8). Two-way ANOVA/Bonferroni, **p<0.01, ***p<0.001.FACS analysis of the neutrophil count in peripheral blood (panel C) from control or eosinophil-depleted mice with or without IL-4c treatment. Data are shown as the mean ± SEM (n=6-8). Two-way ANOVA/Bonferroni, **p<0.01, ***p<0.001.(D) Same as panel B except that efferocytosis was determined. *p<0.05; ***p<0.001(E) FACS analysis of alive and dying neutrophils in paws from control or eosinophil-depleted mice. Data are shown as the mean ± SEM (n=5 mice).
Figure 8(C) Zymosan-induced thermal hypersensitivity in control or eosinophil-depleted mice with or without IL-4c treatment 24h hours prior zymosan injection. The right panel show the comparison of paw withdrawal latencies between the 4 treatment groups at the indicated time points. Data are shown as the mean ± SEM (n=6 mice). Two-way ANOVA/Bonferroni, *p< 0.05, **p< 0.01; ns, not significant.
Figure 1Northern blotting shows that Gasz depletion (Gasz KD) led to the accumulation of flam-piRNA precursors but reduced the level of mature idefix-piRNA arising from the precursors, as in the case of Zuc depletion (Zuc KD). U6 snRNA was detected as a loading control.Western blotting showing the protein levels of endogenous Armi, Piwi, SoYb, Yb, Vret, and Gasz in total OSC lysate (total), the mitochondrial fraction (mito), and the cytoplasmic fraction after mitochondrial isolation (cyto). β-Tubulin (β-Tub) and HSP60 were detected as markers for the "cyto" and "mito" fractions, respectively.Left: Western blotting comparing the abundances of Armi and Piwi in total OSC lysate (total) and the mitochondrial fraction (mito) before (control) and after (Zuc KD) Zuc knockdown in OSCs. Relative mito-localization intensity (*) shows the amount of proteins in "mito" normalized by the amount of proteins in "total" and HSP60. Right: Immunofluorescence shows that the Armi signal (green) is strongly overlapped with the mitochondrial signal (red) in Zuc-depleted OSCs (Zuc KD). Armi is localized to Yb bodies in control OSCs (control). The scale bar represents 5 μm. DAPI (blue) shows the nuclei.Left: Western blotting comparing the abundances of Armi and Piwi in total OSC lysate (total) and the mitochondrial fraction (mito) before (control) and after (Gasz KD) Gasz depletion in Zuc-depleted OSCs (Zuc KD). Relative mito-localization intensity (*) shows the amount of proteins in "mito" normalized by the amount of proteins in "total" and HSP60. Right: Immunofluorescence shows that the Armi mitochondrial signal (green) found in Zuc-depleted cells (Zuc KD/control) mostly disappeared after additional Gasz depletion in the cells (Zuc KD/Gasz KD), but was found at Yb bodies. Mitochondrial signal is shown in red. The scale bar represents 5 μm. DAPI (blue) shows the nuclei.
Figure 2Armi and Piwi co-immunoprecipitated with Flag-Gasz (F-Gasz) from the mitochondrial fraction (mito fraction) of OSCs (input). F-Gasz was ectopically expressed in the cells by transfection prior to immunoprecipitation. *: Heavy chains of antibodies used for immunoprecipitation. n.i.: Non-immune IgG used as a negative control.Co-immunoprecipitation from whole-cell lysate of OSCs before (control) and after (Zuc KD) Zuc depletion and subsequent western blotting show that the levels of Piwi and Armi in the Flag-Gasz (F-Gasz) complex increased upon Zuc depletion (Zuc KD).Northern blotting shows that the flam-piRNA precursors in the Flag-Gasz complex (Flag) accumulated upon Zuc depletion (Zuc KD).Upper: The flam-piRNA precursors associated with Piwi disappeared upon Armi depletion (Armi KD) in Zuc-lacking OSCs (Zuc KD). Lower: The abundance of immunopurified Piwi is shown by western blotting.
Figure 3Depletion of endogenous Piwi (Piwi KD) in Zuc-lacking OSCs (Zuc KD) caused Armi (green) to relocate to Yb bodies. Localization of Armi at Yb bodies is shown in B. Mitochondrial (upper panel) and Yb (lower panel) signals are shown in red. The scale bar represents 5 μm. DAPI (blue) shows the nuclei.The Armi complex was immunopurified from Zuc-lacking OSCs (Zuc KD) before (control) and after (Piwi KD) Piwi depletion and probed for detecting Armi and Gasz. Armi no longer stably associated with endogenous Gasz upon Piwi depletion in Zuc-lacking OSCs. Co-IP index (*) shows that the ratio of Gasz amount in the Armi complex isolated from Zuc-depleted cells (Zuc KD/control) and Zuc- and Piwi-depleted cells (Zuc KD/Piwi KD) is 1:0.05.The Flag-Gasz (F-Gasz) complex was immunopurified from Zuc-lacking OSCs (Zuc KD) before (control) and after (Piwi KD) Piwi depletion and probed for detecting Armi and F-Gasz. F-Gasz was ectopically expressed prior to immunoprecipitation. Co-IP index (*) shows that the ratio of Armi amount in the F-Gasz complex isolated from Zuc-depleted cells (Zuc KD/control) and Zuc- and Piwi-depleted cells (Zuc KD/Piwi KD) is 1:0.31.In vitro pull-down assays show that Armi-Flag (Armi-F) directly binds with GST-Gasz∆C113 but hardly with GST. Armi-F was immunopurified from Schneider 2 (S2) cells under harsh conditions. GST and GST-Gasz∆C113 were visualized by CBB staining, while Armi-F was detected by western blotting using anti-Flag antibodies.
Figure 4Upper: Northern blotting shows that Flag-Piwi WT (WT) and PAZ mutant (PAZmt) but not MID mutant (MIDmt) bound with flam-piRNA precursors in Zuc-depleted OSCs. Endogenous Piwi was depleted simultaneously with Zuc (Zuc KD + Piwi KD). Zuc was depleted by RNAi and so residual Zuc may have remained in cells. Therefore, a tiny amount of piRNAs was loaded onto Piwi. F-Piwi WT, PAZmt, and MIDmt were made to be RNAi-resistant by mutagenesis (see Materials and Methods). Lower: The abundance of immunopurified Flag-Piwi (F-Piwi) is shown by western blotting.Armi localization to Yb bodies observed in Zuc- and Piwi-depleted OSCs (Zuc KD + Piwi KD) (Figs 3A and B) was restored by ectopic expression of Flag-Piwi WT and PAZmt but not of MIDmt (see also Fig EV4B). Armi, mitochondria, and F-Piwi are shown in green, red, and yellow, respectively. The scale bar represents 5 μm. DAPI (blue) shows the nuclei.Western blotting shows that Flag-Piwi (F-Piwi) WT (WT) and PAZ mutant (PAZmt) but not MID mutant (MIDmt) bound with Armi in Zuc/Piwi-depleted OSCs (Zuc KD + Piwi KD).
Figure 5Armi-Flag (Armi-F) WT and the N756A mutant were expressed in OSCs where endogenous Armi and Zuc had been depleted by RNAi (Zuc KD + Armi KD). Immunoprecipitation and subsequent western blotting show that Armi-F WT bound with Piwi and Gasz. The Armi N756A mutant bound with Piwi strongly but only weakly with Gasz. Luciferase-Flag (Luc-F) was used as a negative control.Armi-Flag (Armi-F) WT and the N756A mutant were expressed in OSCs where endogenous Armi and Zuc had been depleted by RNAi (Zuc KD + Armi KD). Armi-F WT (green) localized onto mitochondria (red), whereas Armi-F N756A mutant (green) localized to Yb bodies (see also Fig EV5D). The scale bar represents 5 μm. DAPI (blue) shows the nuclei.In vitro pull-down assays show that both Armi-Flag (Armi-F) WT and N756A mutant directly bind with GST-Gasz∆C113 but not with GST. Armi-F WT and mutant were immunopurified from Schneider 2 (S2) cells under harsh conditions. GST and GST-Gasz∆C113 were visualized by CBB staining, while Armi-F WT and N756A mutant were detected by western blotting using anti-Flag antibodies.
Figure 6Armi-Flag (Armi-F) WT and the ∆N34 mutant were expressed in OSCs where endogenous Armi and Zuc had been depleted by RNAi (Zuc KD + Armi KD). Immunoprecipitation and subsequent western blotting show that Armi-F WT but not Armi ∆N34 mutant bound with Gasz in the cells. Both Armi WT and ∆N34 mutant interacted with Piwi to similar extents. Luciferase-Flag (Luc-F) was used as a negative control.Armi-Flag (Armi-F) WT and the ∆N34 mutant were expressed in OSCs where endogenous Armi and Zuc had been depleted by RNAi (Zuc KD + Armi KD). Armi-F WT (green) localized onto mitochondria (red), whereas Armi-F ∆N34 mutant (green) localized to Yb bodies (see also Fig EV6D). The scale bar represents 5 μm. DAPI (blue) shows the nuclei.In vitro pull-down assays show that both Armi-Flag (Armi-F) WT and ∆N34 mutant directly bind with GST-Gasz∆C113 but not with GST. Armi-F WT and mutant were immunopurified from Schneider 2 (S2) cells under harsh conditions. GST and GST-Gasz∆C113 were visualized by CBB staining, while Armi-F WT and mutant were detected by western blotting using anti-Flag antibodies.Immunoprecipitation and subsequent western blotting shows that Armi-Flag (Armi-F) but not Flag-Armi (F-Armi) strongly bound with Gasz in Zuc/Armi-depleted OSCs (Zuc KD + Armi KD). Piwi bound with both Armi (F-Armi and Armi-F). Luciferase-Flag (Luc-F) was used as a negative control.
Figure 1G IP of UbcH7 by PknG or PknGΔUbl in U937 cells. Cells were infected with WT, ΔpknG, or ΔpknG:pknGΔUbl Mtb strains as in B. H IP of UbcH7 by PknG or PknG E190A in U937 cells. Cells were infected with WT, ΔpknG, or ΔpknG:pknG E190A Mtb strains as in B.
Figure 2E In vitro ubiquitin conjugation assay of UbcH7 catalyzed by PknG or His-UBA1 at 37°C for 30 min. The reaction products were analyzed by immunoblotting with antibodies against Ub (top panel), the His-tag on UBA1 (middle panel), and PknG (bottom panel), or by staining with Coomassie Brilliant Blue (bottom). For reducing conditions, samples were treated with 500 mM β-mercaptoethanol (βME) prior to SDS-PAGE gel analysis.
Figure 3A MS/MS analysis of UbcH7 in the absence (top) or presence (bottom) of PknG to reveal ubiquitination sites. Tryptic peptides spanning residues ranging from 74 to 100 were identified as the high-confidence peptides possessing di-Gly modifications. The ubiquitinated residue (Lys82) in the peptide sequence is highlighted in red. The peak heights are the relative abundances of the corresponding fragmentation ions. Matched amino terminus-containing ions (b ions) are indicated in red, and matched carboxyl terminus-containing ions (y ions) are indicated in blue. Only the major peaks that were identified are labeled.
Figure 4A Luciferase assay of NF-κB activation in HEK293T cells transfected with WT or mutant Mtb PknG. The graph shows the mean ± SEM of three independent biological replicates, n = 3. *P < 0.05, **P < 0.01. Data were analyzed according to one-way analysis of variance (ANOVA) using GraphPad Prism 7.D Survival of Mtb strains in primary human monocyte-derived macrophages treated as in B and C. The graph shows the mean ± SEM of three independent biological replicates, n = 3. *P < 0.05, **P < 0.01. Data were analyzed according to one-way ANOVA using GraphPad Prism 7.
Figure 5B IP of TRAF2 and TAK1 by Mtb PknG in U937 cells. Cells were infected with the indicated Mtb strains at an MOI of 1. Non-infected cells were used as controls. After 4 hours, the cells were lysed and immunoprecipitated with the antibody against PknG. The immunoprecipitated proteins were immunoblotted with TRAF2, TAK1, TAB2 (negative control), or TAB3 (negative control) antibodies.E Immunoblotting of TRAF2, TAK1, and PknG from lysates of U937 cells infected with the indicated Mtb strains at an MOI of 1 for 8 hours. Cells were treated with CHX (50 μg/mL) with or without MG132 (5 μM). Cell lysates were analyzed by immunoblotting for TRAF2 (anti-TRAF2), TAK1 (anti-TAK1), and PknG (anti-PknG). GAPDH was used as the loading control. Densitometric quantification of immunoblots is indicated below the immunoblots.
Figure 6G, H In vivo ubiquitination assay of TRAF2 (G) and TAK1 (H) in the absence or presence of PknG in HEK293T cells transfected with the indicated plasmids for 24 hours. Cells were lysed and immunoprecipitated using the antibody against Flag. Immunoprecipitated proteins were immunoblotted with the antibody against HA.
Figure 7A, B qPCR analysis of Tnf mRNA (A) and Il6 mRNA (B) in splenic cells from C57BL/6 mice infected with WT or mutant Mtb stains for 0-20 days. Data are representative of one experiment with two independent biological replicates (mean ± SEM of n = 6 mice per group). *P < 0.05 and **P < 0.01. Data were analyzed according to one-way ANOVA using GraphPad Prism 7.D H&E staining of lungs and livers of C57BL/6 mice treated as in A. Arrows indicate puncta of cellular infiltration. Scale bars, 200 μm.
Figure 2A. Volcano plot showing fold change (log2 values) in transcript level of ESC+2i and ESC+serum with two consecutive treatments with siRNA-control and siRNA-Baz2a, each one lasting for 4 days. Gene expression values of three replicates were averaged and selected for 1.5 fold changes and P < 0.05. Statistical significance (P-values) was calculated using R package DEseq2.B. Validation by qRT-PCR of genes regulated by BAZ2A in ESC+2i but not in ESC+serum (upregulated genes in ESC+2i upon BAZ2A-KD are labeled in blue, downregulated genes are in red). Nanog, Rex1 and Actin B (ActB) are shown as genes not regulated by BAZ2A. mRNA levels were normalized to Rps12 mRNA and to ESCs transfected with siRNA-Control. Average values of three independent experiments. Error bars represent s.d. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (* < 0.05, ** < 0.01, *** < 0.001). Data without P values are statistically non significant.
Figure 3A. Western blot showing equal BAZ2A levels in wt-ESC and F/H-BAZ2A ESC line containing FLAG-HA sequences at the N-terminus of both Baz2a alleles. Whole cell lysates from equivalent amounts of cells were analysed. SNF2H, and Tubulin are shown as protein loading controls.B. Anti-FLAG ChIP of wt-ESCs and F/H-BAZ2A-ESCs cultured in 2i or serum. Data were measured by qPCR and normalized to input and a control region that is not bound by BAZ2A. Average values of three independent experiments. Error bars represent s.d. and statistical significance (P-values) was calculated using the paired two-tailed t-test (* < 0.05, ** < 0.01).C. Representative images showing the association of BAZ2A at regions enriched in H3K27ac and low in H3K27me3. The grey rectangles highlighted some of BAZ2A associated regions.D. Pearson correlation heat map for the indicated ChIPseqs in ESC+2i. BAZ2A ChIPseqs were performed with FLAG antibodies in F/H-BAZ2A-ESCs cultured in 2i or serum and with HA antibodies in F/H-BAZ2A-ESC+2i. Data of H3K4me3, H3K27ac and H3K27me3 are from this work. H3K36me3, H3K9me3 and H3K4me1 values in ESC+2i were taken from published data setsE. Boxplot showing the mean of normalized BAZ2A occupancies in ESC+2i and ESC+serum at BAZ2A-bound regions. Statistical significance (P-values) for the experiments was calculated using the unpaired two-tailed t-test (**** < 0.0001). Box plots depict the minimum and maximum values. The mean is represented by a horizontal line within the boxes.F. Differential BAZ2A-binding in 2i and serum correlates with H3K27ac occupancies. Boxplot showing the mean of normalized H3K27ac and H3K27me3 read counts in ESC+2i and ESC+serum at all BAZ2A-bound regions and BAZ2A-bound regions only in 2i were calculated and their log2-fold changes are plotted. H3K27ac in ESC+serum Statistical significance (P-values) was calculated using the paired two-tailed t-test (*** < 0.001). Box plots depict the minimum and maximum values. The mean is represented by a horizontal line within the boxes.G. Representative images showing 2i-specific BAZ2A-bound regions and H3K27ac and H3K27me3 in ESC+2i and ESC+serum. The grey box highlights strong TIP5-bound regions and corresponding histone modifications.H. Representative images showing that genes regulated by BAZ2A are enriched in H3K27me3, depleted of H3K27ac and not bound by BAZ2A.I. Heat map profiles of H3K27me3 and H3K27ac at ±5 kb from the TSS of genes differentially regulated by BAZ2A in ESC+2i and random genes. Differentially regulated genes are shown as upregulated or downregulated in ESC+2i upon BAZ2A knockdown. Data were ranked by H3K27me3 levels. Transcription start site (TSS).J. Boxplot showing the mean of normalized H3K27me3 and H3K27ac read counts over +/- 1kb of TSSs from upregulated, downregulated and random genes. Box plots depict the minimum and maximum values. The mean is represented by a horizontal line within the boxes.
Figure 4A. Number of BAZ2A-bound regions in A and B compartments of ESC+serum. B. Hi-C contact matrices for a zoomed in region on chromosome 3 at 100-kb resolution showing the presence of strong long-distance contacts corresponding to regions in A compartment bound by BAZ2A in ESC+serum. Low panels. HiC contacts of a zoomed region at 50-kb resolution in ESC+serum, NPCs and CNs are shown. Data are from (Bonev et al., 2017). HiC contacts were visualized using Juicebox C. BAZ2A-bound regions mark A subcompartments that are characterized by the strongest far-cis contacts in ESC+serum. Boxplots indicating observed/expected contact values between BAZ2A-bound regions and loci not bound by BAZ2A at near-cis and far-cis contacts in all genome or in A compartments. Statistical significance (P-values) was calculated using the unpaired two-tailed t-test (****< 0.0001). D. Long-range contacts marked by BAZ2A binding are specific for ESCs. Boxplots indicating observed/expected contact values between BAZ2A-bound regions and loci not bound by BAZ2A at far-cis contacts in all genome or in A compartments of ESC+serum, NPC and CN. Statistical significance (P-values) was calculated using the unpaired two-tailed t-test (**** < 0.0001). Box plots depict the minimum and maximum values. The mean is represented by a horizontal line within the boxes.E. Number of BAZ2A-bound regions in A and B compartments in ESC+2i. F. BAZ2A-bound regions mark A subcompartments that are characterized by the strongest far-cis contacts in ESC+2i. Boxplots indicating observed/expected contact values between BAZ2A-bound regions and loci not bound by BAZ2A at near-cis and far-cis contacts in all genome or in A compartments of ESC+2i. Statistical significance (P-values) was calculated using the unpaired two-tailed t-test (** < 0.001, ****< 0.0001). Box plots depict the minimum and maximum values. The mean is represented by a horizontal line within the boxes. G. BAZ2A binding correlates the best with eigenvector values. Heat map showing Pearson correlation values of ChIPseq data sets with the eigenvector values from HiC data in ESC+serum and ESC+2i, respectively.H. Boxplots showing H3K4me1 and H3K27ac content of BAZ2A-bound regions and loci not bound by BAZ2A at far-cis contacts in ESC+2i. Data from regions with lowest 10% and top 10% contact strength are shown. Box plots depict the minimum and maximum values. The mean is represented by a horizontal line within the boxes.
Figure 6A. Hi-C contact matrices for chromosome 1 of ESC+serum, ESC+2i/siRNA-control and ESC+2i/siRNA-Baz2a showing the presence of strong long-distance contacts corresponding to regions in A compartment bound by BAZ2A in ESC+serum. Cells were subjected to two consecutive treatments with siRNA-control and siRNA-Baz2a, each one lasting for 4 days. Eigenvector values are shown. Increase in far-cis contacts in ESC+2i upon BAZ2A-KD are highlighted with a rectangle. B. Relative contact frequencies over genomic distances in ESCs treated with siRNA-control and siRNA-BAZ2A. Statistical significance (P-values) was calculated using the Kolmogorov-Smirnov test. C. Boxplot indicating differences in eigenvector values at boundaries and centers of A and B compartments. Differences in eigenvector values were calculated from HiC data sets from ESC+2i treated with siRNA-Baz2a and siRNA-control. Statistical significance (P-values) for the experiments was calculated using the unpaired two-tailed t-test (*** < 0.001, ns: non significant). Box plots depict the minimum and maximum values. The mean is represented by a horizontal line within the boxes.
Figure 8B. BAZ2A-interacting proteins on chromatin. Mass spectrometry analysis of FLAG immunoprecipitates from wt and F/H-BAZ2A ESC+2i. Values of peptide number are shown as the difference between peptides obtained in FLAG-IP of F/H-BAZ2A and wt ESC chromatin extracts. Data are from three independent experiments. The proteins found enriched in FLAG-IP of F/H-BAZ2A ESCs in all three experiments are shown. Further BAZ2A-interacting proteins identified in only two or one of the immunoprecipitation (IP) experiments Error bars represent s.d.C. Anti-HA IP of wt- and F/H-BAZ2A-ESC+2i and ESC+serum. Western blot shows the interaction of BAZ2A with SNF2H, TOP2A and SMC1a.D. TOP2A and BAZ2A regulate gene expression in ESC+2i via a shared pathway. qRT-PCR of genes regulated by BAZ2A in ESC+2i (left panel) and in ESC+serum (right panel) upon TOP2A or BAZ2A knockdown by siRNA. ESCs were treated with the corresponding RNAs for 4 days. Upregulated genes in ESC+2i upon BAZ2A-KD are labeled in blue, downregulated genes are in red. Nanog, Rex1 and Actin B (ActB) are shown as genes not regulated by BAZ2A. mRNA levels were normalized to Rps12 mRNA and to ESCs transfected with siRNA-Control. Average values of three independent experiments. Error bars represent s.d. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (ns: non significant, * < 0.05, ** < 0.01, *** < 0.001, **** < 0.0001).E. Inhibition of type 2 topoisomerase activity phenocopies the alterations in gene expression of BAZ2A-regulated genes in ESC+2i observed upon TOP2A or BAZ2A knockdown. ESCs were treated with the TOP2 inhibitor ICF-193 for 24 hours. mRNA levels were normalized to Rps12 mRNA and to ESCs treated with DMSO. Upregulated genes in ESC+2i upon BAZ2A-KD are labeled in blue, downregulated genes are in red. Average values of three independent experiments. Error bars represent s.d. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (ns: non significant, ** < 0.01, *** < 0.001, **** < 0.0001).F. ChIP analysis showing that TOP2A regulates H3K27me3 occupancy in ESC+2i but not in ESC+serum. Data were measured by qPCR and normalized to input and to Tshz1 value in cells transfected with siRNA-control. #1-#4 indicated sequences with elevated H3K27me3 at CTCF boundary (H3K27me3hi/CTCF). Control represents an intergenic region that does not contain H3K27me3. Average values of three independent experiments. Error bars represent s.d. Statistical significance (P-values) for the experiments was calculated using the paired two-tailed t-test (ns: non significant, * < 0.05, ** < 0.01, *** < 0.001).