EMBO/soda-nel huggingface dataset:
JNK-dependent gene regulatory circuitry governs mesenchymal fate. (2016)
Figure 1

Figure 1. JNK signaling is activated during a distinct phase of EMT and becomes a critical determinant of mesenchymal identity. (A) Heatmap showing standardized expression (Z-scores) of genes differentially expressed upon TGF-β-induced EMT in NMuMG cells. (B) Venn diagram showing overlap of upregulated genes at individual time-points. (C) Same as in B for downregulated genes. (D) Representative immunoblots (n=3) showing phosphorylation state of Smad2 and JNK during TGF-β-induced EMT in NMuMG cells. Lamin B serves as loading control. (E) Quantification of one set of representative immunoblots (in figure D) and its graphical representation showing dynamic of Smad2 and JNK activation during TGF-β-induced EMT in NMuMG. (F) Representative immunohistochemistry image for pSmad2 and pJNK in non-invasive (n=34) and invasive (n=14) breast cancer samples. Scale bars for 10X and 40X images are 200 µm and 50 µm respectively. (G) Quantification of Immunohistochemical staining for p-JNK and p-Smad2 as in (F) in invasive tumor samples (n=34). The pie chart shows the percentage of tumors with high p-Smad2 and low p-JNK or high p-JNK and low p-Smad2 or equal p-Smad2 and p-JNK. (H) Immunofluorescence microscopy for changes in the localization and expression levels of EMT markers in epithelial NMuMG cells (d0) and cells treated with TGF-β for seven days in the presence of JNK inhibitor (d7 JNKi) or control (d7 DMSO). Staining was performed with antibodies against Fibronectin 1, with Phalloidin to visualize the actin cytoskeleton and against Paxillin to detect focal adhesion plaques. It is important to note that the untreated epithelial cells are highly proliferative and appear small in size, round in shape and adhere to surrounding cells whereas day 7 TGF- β-treated (mesenchymal) cells are much bigger in size, elongated in shape and detached from the nearby cells. Scale bar, 100 μm, 63X magnification. (I) mRNA levels for classical EMT markers in cells treated as in (F) were measured by qRT-PCRs relative to Ctcf and fold change as compare to untreated and plotted on the y-axis. Mean and SEM is plotted from three independent biological replicates. (J) Transwell migration assay for NMuMG cells treated as in G. Plotted Mean ± SEM of three biological replicates. * p<0.05, ** p<0.01, *** p< 0.001, Student’s t-test.
JNK-dependent gene regulatory circuitry governs mesenchymal fate. (2016)
Figure 2

Figure 2. JNK signaling defines epigenome and transcriptome to specify mesenchymal fate. (A) Volcano plots showing significantly differentially expressed genes upon treatment with the JNK inhibitor SP600125 (JNKi) as compared to DMSO at day 1, 2, 3, 4 and 7 during TGF-β-induced EMT in NMuMG cells. X-axis indicates fold change in log2 scale while y-axis denotes -log 10 of the P-value. Differentially expressed genes are depicted with red circles. Blue horizontal line represents FDR adjusted Pvalue of 0.1. (B) Venn diagram showing the overlap of upregulated genes in JNKi versus DMSO treated cells. (C) Same as in (B) for genes downregulated upon JNKi. (D) A principal component analysis (PCA) using RNA-Seq data was performed comparing two replicates (r1, r2) each of DMSO and JNKi treated samples at different time points during TGF-β-induced EMT (d1, d2, d3, d4, d7). The first two principal components are presented to display the variation between samples. The distances on the x- and y-axis explain 47% and 19% of variation in the data respectively. Biological duplicates (r1 and r2) are depicted in the same color. (E) ChIP assay using H3K27me3-specific antibody in epithelial cells (day 0)) or NMuMG cells treated for seven days with TGF-β (mesenchymal) and DMSO or JNKi. qRT-PCRs were performed for indicated gene promoters and enrichments are plotted on the right y-axis as ratio of precipitated DNA (bound) to total input DNA. On the left y-axis RNA levels of analyzed genes derived from RNA-seq data (fold change to untreated normalized tag counts ) are plotted. Mean and SEM is plotted from two independent biological replicates. (F) FAIRE assay was performed in cells treated as in (D) and qRT-PCRs were performed for the same gene promoters and plotted as in (E).
JNK-dependent gene regulatory circuitry governs mesenchymal fate. (2016)
Figure 3

Figure 3. JNK-induced transcription factors are critically required for EMT. (A) Venn diagram showing overlap of genes upregulated upon JNKi treatment during EMT and downregulated during normal EMT at day 7 that contains 5 transcription factors. Right panel shows RNA levels (normalized tag counts) for these factors. (B) Same overlap as in (A) for genes downregulated upon JNKi treatment during EMT and upregulated during normal EMT. Right panel displays RNA levels as in (A) for 10 transcription factors identified in this overlap. (C) Representative bright field and immunofluorescence images for localization and expression levels of EMT marker proteins after four days (d4) of siRNA-mediated depletion of factors compared to non-targeting control (NTC). Staining was performed with antibodies against epithelial markers E-cadherin and ZO1, mesenchymal markers Fibronectin 1, with Phalloidin to visualize the actin cytoskeleton and against Paxillin to detect focal adhesion plaques. Scale bar, 100 μm, 40X magnification. Plotted Mean ± SEM of three biological replicates. * p<0.05, ** p<0.01, *** p< 0.001, Student’s t-test.
JNK-dependent gene regulatory circuitry governs mesenchymal fate. (2016)
Figure 4

Figure 4. Depletion of Prkrir, Pcbp4 and Tsc22d1 rescues TGF-β-induced reduction in the rate of cell division. (A) Lineage trees tracked by live imaging under control, Non treated control (NTC) and siRNA-mediated depletion of factors (X= cell death/cells could not be tracked till the end). (B) Average number of rounds of division per clone in NTC compared with control and siRNA-mediated depletion of factors (ANOVA, Tukeys post-test, *=p<0.05,**=p<0.01).
JNK-dependent gene regulatory circuitry governs mesenchymal fate. (2016)
Figure 5

Figure 5. PRKRIR, PCBP4 and TSC22D1 are crucial for maintenance of the mesenchymal fate. (A) Scratch assay in MDA-MB-231 cells treated for four days with DMSO or JNKi. Scale bar, 200 μm; 20 X magnification. Mean and SEM is plotted from three independent biological replicates. (B) mRNA levels for key EMT marker genes in MDA-MB-231 treated with DMSO or JNKi for 4 days were measured by qRT-PCR relative to Ctcf and plotted on the y-axis. Mean and SEM is plotted from three independent biological replicates (C) mRNA levels for transcription factors in isogenic epithelial (MDA-MB-361) and mesenchymal (MDA-MB-231) breast cancer cells were measured by qRT-PCR relative to Ctcf and plotted on the y-axis. Mean and SEM is plotted from three independent biological replicates. (D) Scratch assay in MDA-MB-231 cells transfected with NTC or siRNA against PRKRIR, NFIL3, PCBP4 and TSC22D1 for 4 days as in A. Mean and SEM is plotted from three independent biological replicates as bar plot on right . (E) Invasion assay in MDA-MB-231 cells treated as in (D). Mean and SEM is plotted from three independent biological replicates. (F) mRNA levels of key EMT marker genes in MDA-MB-231 and MDA-MB-231 transfected with either empty vector or vector overexpressing PCBP4 or TSC22D1 for 48 hours and then treated with the JNK inhibitor (SP600125) for 24 hours were measured by qRT-PCRs relative to Ctcf and plotted on the y-axis. Mean and SEM is plotted from three independent biological replicates. Plotted Mean ± SEM of three biological replicates. * p<0.05, ** p<0.01, *** p< 0.001, Student’s t-test.
JNK-dependent gene regulatory circuitry governs mesenchymal fate. (2016)
Figure 6

Figure 6. PRKRIR, PCBP4 and TSC22D1 regulate expression of distinct set of genes in mesenchymal breast cancer cells. (A) Volcano plots showing significantly changing genes upon siRNA-mediated depletion of PRKRIR, PCBP4 and TSC22D1 as compared to NTC. The x-axis and y-axis show fold expression changes (log2 scale) and associated P-value (-log10 scale) respectively. The blue horizontal line marks the cut-off for genes considered significantly changing upon factor depletion. Red dots indicate genes previously implicated in a similar context (Supplementary Table 1). (B) Circle plots of genes highlighted in (A) (red) and all significantly changing genes (grey). (C) mRNA levels for representative genes highlighted in (A) for cells treated like in (Fig 5D) were measured by qRT-PCR relative to Ctcf and plotted on the y-axis. Mean and SEM is plotted from three independent biological replicates. (D-E) Venn diagrams showing the overlap of the genes differentially upregulated and down regulated following knockdown of the three factors mentioned in (A). (F) Representative immunofluorescence images showing localization of Tsc22d1 in NMuMG cells following transient expression of Flag-HA tagged Tsc22d1 and detection using an anti-Flag antibody, Bar plot shows quantification of Tsc22d1 localization with respect to DAPI density within the nucleus. Y-axis shows the percentage of total fluorescence intensity for Tsc22d1 in DAPI dense and light regions. SEM is derived from independent biological replicates. (G) ChIP assay using anti-Flag antibody following expression of Flag-HA tagged TSC22D1 in MDA-MB-231 cells. Quantitative PCRs were performed for indicated gene promoters and enrichments are plotted on the y-axis as ratio of precipitated DNA (bound) to total input DNA and then further divided by the same obtained in the empty vector transfected cells. SEM is derived from independent biological replicates. (H) mRNA levels for PRKRIR, PCBP4, TSC22D1 in non-invasive (DCIS) (n=20) and invasive tumor (n=20) samples were measured by qRT PCRs relative to TBP and plotted on the y-axis. ZEB1 was used as an established positive control for invasive tumors. Plotted Mean ± SEM of three biological replicates. * p<0.05, ** p<0.01, *** p< 0.001, Student’s t-test.
JNK-dependent gene regulatory circuitry governs mesenchymal fate. (2016)
Figure 7

Figure 7. Newly identified transcription factors are similarly upregulated during neurogenesis and function in neuronal migration. (A) RT-qPCR analysis for key EMT markers in embryonic stem cells (ES), neuronal progenitors (NP) and terminally differentiated neurons (TN). mRNA levels were measured by RT-qPCR relative to Ctcf and plotted on the y-axis. Error bars represent SEM from independent biological replicates. (B) Western blot analyzing total JNK and phospho-JNK (p-JNK) during neuronal differentiation of stem cells. β-actin serve as a loading control. (C) RNA levels of Pcbp4, Prkrir and Tsc22d1 during neuronal differentiation as shown in A, measured by RT-qPCR relative to Ctcf and plotted on the y-axis. Error bars represent SEM from independent biological replicates. (D) RNA levels of Pcbp4, Prkrir, Tsc22d1, represented by average counts derived from previously published microarray data of independent biological replicates from neurons treated with DMSO or JNK inhibitor SP600125 (JNKi) for 6 hours. (E) RNA levels of Tsc22d1 and three Tsc22d1 target genes during various stages of neuronal differentiation shown in (A), measured by RT-qPCR relative to Ctcf and plotted on the y-axis. Error bars represent SEM from independent biological replicates. (F) RNA levels of Tsc22d1 and the same three target genes is shown as average normalized tag counts derived from RNA-seq data from Ventricular Zone (VZ), Sub-Ventricular Zone (SVZ) and Cortical plate (CP) of E14.5 mouse Cortex. (G) RNA levels of genes shown in Figure (E), represented by average normalized tag counts derived from RNA-seq data of TGF-β-induced EMT in NMuMG cells and plotted on the y-axis. Error bars represent SEM from three biological replicates. (H) Graphical representation of In Utero Electroporation (IUE) performed at the embryonic stage 12.5 (E12.5) during murine development. E12.5 mouse embryos were electroporated with GFP-tagged constructs either with an established non-target control (NTC) shRNA or a validated shRNA against Tsc22d1. Four days later (E16.5), embryos were sacrificed and brains were fixed for immunofluorescence analysis with antibodies against GFP and Tbr1 or Satb2 (early neuronal markers). (I) A representative image from immunofluorescence analysis performed with anti-GFP and anti-Tbr1 antibody that shows retention of GFP positive cells below the Tbr1 layer in Tsc22d1 knockdown brain as compared to the control brain. The bar plot on the right side shows quantification of migrated GFP positive cells in control and Tsc22d1 depleted mouse brain with respect to the Tbr1 staining. y axis represent percentage of cells above or below the Tbr1 stain region. Error bars represent SEM from 3 independent biological replicates. * p<0.05, ** p<0.01, *** p< 0.001, Student’s t-test.
SUMOylated PRC1 controls histone H3.3 deposition and genome integrity of embryonic heterochromatin (2020)
Figure 1

Figure 1. Parent-of-origin specific targeting pathways for DAXX and ATRX to PCH in mouse zygotes.

(A) Still images of time-lapse imaging of H3.3-mCherry and H3.2-EGFP proteins in wild-type zygotes (n=17).

(B) IF of DAXX and ATRX in zygotes at different pronuclear (PN) stages (PN0, n=8; PN2, n=6; PN5, n= 33).

(C) Boxplot displaying signal intensity of DAXX at pat- and mat-PCH in mouse zygotes at PN5 stage, relative to mean signal at pat-PCH. In boxplots, the enter lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Datapoints are indicated by circles. Experiments were replicated 3 times. ***P<0.001, n= 28, paired t-test.

(D) IF of DAXX and ATRX in PN5 zygotes upon siRNA-mediated knockdown of Daxx (n=14) or with control siRNAs (n=8).

(E) IF of DAXX and ATRX in control (n=15) and Atrxm-z+ (n=14) PN5 zygotes.

Data information: All panels: images of paternal and maternal pronuclei at given stage/condition are from same zygote. (D, E) Yellow dashed circles represent the contours of the pronuclei. All scale bars, 10µm.
SUMOylated PRC1 controls histone H3.3 deposition and genome integrity of embryonic heterochromatin (2020)
Figure 2

Figure 2. Targeting of DAXX to pat-PCH is dependent on PRC1.

(A) IF of DAXX and EZH2 in paternal pronuclei of Ezh1m-z+ (n=5), Ezh2m-z+ (n=3) and Ezh1m-z+; Ezh2m-z+ (n=4) late zygotes. Yellow dashed circles represent the contours of the pronuclei.

(B) IF of DAXX and RNF2 in PN0 zygotes (n=10).

(C) IF of DAXX in wild type (n=30), Ring1m-z+ (n=12), Rnf2m-z+ (n=11) and Ring1m-z+; Rnf2m-z+ (n=12) paternal pronuclei of PN5 zygotes.

(D) Anti-Flag co-IP of DAXXMycHis, RNF2 and H3K27me3 with CBX23×Flag from extracts of HEK293 cells in presence or absence of benzonase, followed by immuno-blot analyses for indicated antigens.

Data information: All scale bars, 10µm.
SUMOylated PRC1 controls histone H3.3 deposition and genome integrity of embryonic heterochromatin (2020)
Figure 3

Figure 3. CBX2-cPRC1 dependent targeting of DAXX to Polycomb regulated genes in human cells.

(A) Cartoon displaying recruitment of DAXXMycHis by GAL4DBDCBX2 (CBX2.1 or CBX2.2) to a 5×UAS site integrated in the first intron of the NCOA5 gene in HEK2935xUAS cells.

(B) Schematic diagram of CBX2.1 and CBX2.2 human isoform genes and proteins. * highlights the alternative chimeric exon 4.

(C) Occupancy (% of IP-ed DNA / input DNA) of GAL4DBDCBX2, RNF2, DAXXMycHis, H3.3, and H3 proteins at 5×UAS site as measured by ChIP-qPCR of chromatin from HEK2935xUAS cells. Data are means ± SEM (n = 3).

(D) Immunoblots for the indicated proteins in HEK2935xUAS cells from Fig. 3C.

(E) ChIP-seq analysis of HEK2935xUAS cells expressing full length GAL4DBDCBX2.1 and DaxxMycHis showing CBX2.1 dependent recruitment of DAXX to promoters of CCND2 and RUNX1 genes.

(F) ChIP-seq analysis of GAL4DBDCBX2.1 occupancy in HEK2935xUAS cells expressing DAXXMycHis (X-axis) or GAL4DBDCBX2.1 and DAXXMycHis (Y-axis), showing (log2) enrichment for GAL4DBDCBX2.1 at many TSSs (± 1 kb) in cells expressing the CBX2.1 protein (Y-axis).

(G) ChIP-seq analysis of GAL4DBDCBX2.1 versus DAXXMycHis occupancy in HEK2935xUAS cells expressing GAL4DBDCBX2.1 and DAXXMycHis, revealing very comparable enrichment levels (log2) of both proteins at TSSs. TSSs highlighted in red show enrichment with anti-Myc antibody independently of GAL4DBDCBX2.1 occupancy.

(H) ChIP-seq analysis of DAXXMycHis occupancy in HEK2935xUAS cells expressing DAXXMycHis (X-axis) versus GAL4DBDCBX2.1 and DAXXMycHis (Y-axis) showing strong enrichment (log2) for DAXXMycHis at many TSSs of genes, but only when both proteins are expressed (Y-axis), indicating that CBX2.1 targets DAXX to chromatin. TSSs highlighted in red show enrichment with anti-Myc antibody independently of GAL4DBDCBX2.1 occupancy.
SUMOylated PRC1 controls histone H3.3 deposition and genome integrity of embryonic heterochromatin (2020)
Figure 4

Figure 4. Sumoylation of CBX2 mediates DAXX targeting to pat-PCH.

(A) IF of CBX2 and SUMO2/3 in PN0 (n=10) and PN5 (n=12) zygotes.

(B) ChIP-qPCR analyses of HEK2935xUAS cells co-expressing GAL4DBDCBX2.1 either with DAXXMycHis or DAXXΔSIM1/2MycHis. Data are means ± SEM (n = 3).

(C) IF of EGFP and DAXX in Daxxm-z+ PN5 zygotes micro-injected with Daxx-EGFP or DaxxΔSIM1/2-EGFP mRNA (100ng/µl).

(D) Left: Boxplot displaying EGFP signal intensities at PCH relative to euchromatin in Daxxm-z+ zygotes micro-injected with Daxx-EGFP or DaxxΔSIM1/2-EGFP mRNA. Right: Boxplot displaying signal intensity of EGFP at euchromatin in same samples. In boxplots, the enter lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Datapoints are indicated by circles. Experiments were replicated 3 times. ***P<0.001, N.S.: no statistical difference, P>0.05, t-test.

(E) Sequence alignment of mouse and human CBX2 proteins with SUMO sites indicated.

(F) Ni-pulldown of 6xHisSUMO1/2/3-conjugated proteins from HEK293 extracts expressing 6xHis-tagged SUMO1, SUMO2 or SUMO3 in combination with CBX2-EGFP. CBX2 and SUMO-conjugated CBX2 were detected with an anti-GFP antibody.

(G) Ni-pulldown of 6xHis-SUMO2 conjugated proteins from HEK293 extracts transiently co-expressing 6xHis-SUMO2 with CBX2-EGFP or CBX24KR-EGFP. Detection of CBX2 as in (F).

(H) IF of DAXX and EGFP in wild-type PN5 zygotes, micro-injected with Cbx2 or Cbx24KR-EGFP mRNA (100ng/µl).

(I) Left: Boxplot displaying signal intensity of DAXX at PCH relative to mean intensity at pat-PCH in wild-type zygotes micro-injected with Cbx2 or Cbx24KR-EGFP mRNA. Right: Absolute intensities of EGFP in same samples. In boxplots, the enter lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Datapoints are indicated by circles. Experiments were replicated 2 times. ***P<0.001, t-test.

Data information: Scale bars, 10µm.
SUMOylated PRC1 controls histone H3.3 deposition and genome integrity of embryonic heterochromatin (2020)
Figure 5

Figure 5. CBX2 and the Suv39h/Hp1 pathway control DAXX targeting to pat-PCH in mouse zygotes.

(A) IF of DAXX and ATRX in PN5 stage pronuclei of wild-type (n=19), Hp1βm-z+ (n=10) and Suv39h2m-z+ (n=9) zygotes.

(B) Boxplots displaying signal intensities of DAXX or ATRX at pat- and mat-PCH in wild-type (n=5) and Hp1βm-z+ (n=10) mouse zygotes at PN5. In boxplots, the enter lines show the medians; box limits indicate the 25th and 75th percentiles as determined by R software; whiskers extend 1.5 times the interquartile range from the 25th and 75th percentiles. Datapoints are indicated by circles. Experiments were replicated 2 times. *P<0.05, **P<0.01, t-test.

(C) IF of DAXX in PN5 stage pronuclei of zygotes expressing CBX2-EGFP (n=9) or CBX2F12A-EGFP (n=17). mRNAs (100ng/µl) were injected into mouse PN2 stage zygotes. Yellow dashed circles represent the contours of the pronuclei..

Data information: Panels A and C: Paternal and maternal pronuclei of a given condition originate from same zygotes. All scale bars 10µm.
SUMOylated PRC1 controls histone H3.3 deposition and genome integrity of embryonic heterochromatin (2020)
Figure 7

Figure 7. DAXX and PRC1 control nucleosome density and chromatin stability at PCH in early mouse embryos.

(A) RNA FISH of forward and reverse strand major satellite sequences in Daxxm+z+ and Daxxm-z+ PN5 zygotes. Scale bars, 10µm.

(B) Box plots indicating the foci number (left) and total intensity (right) of forward (top) and reverse (bottom) transcription as measured by RNA FISH on 3D paternal pronuclei in Daxxm+z+ and Daxxm-z+ PN5 zygotes. Lower hinge, central line and upper hinge in boxplots represent 25th, 50th (median) and 75th percentiles respectively. Upper/lower whiskers extend to the largest/smallest values no further than 1.5 * IQR from the upper/lower hinge, where IQR is inter-quartile range or distance between 25th and 75th percentiles. Datapoints are indicated by circles. Experiments were replicated 3 times. ***P<0.001; t-test.

(C, D) MA-plots showing Fold Change (FC) (in log2) in gene expression between indicated samples as a function of averaged expression across all samples. Dotted lines indicated log2(FC) of 1.

(E) Histogram showing classification of genes into 3 groups according to distribution of ChIP enrichment (log2) of H3K27me3 around TSS in MII oocytes (Liu et al., 2016).

(F) MA-plot showing FC (in log2) in gene expression between indicated samples as a function of averaged expression across all samples. Dotted lines indicated log2(FC) of 1.

(G - I) Boxplots showing FC (log2) in gene expression between indicated samples for 3 groups of genes classified according to H3K27me3 enrichments (as shown in E). Lower hinge, central line and upper hinge represent 25th, 50th (median) and 75th percentiles respectively. Upper/lower whiskers extend to the largest/smallest values no further than 1.5 * IQR from the upper/lower hinge, where IQR is inter-quartile range or distance between 25th and 75th percentiles. Outliers are not displayed. The notches extend $1.5*IQR/\sqrt{n}$ where n is a number of genes in each H3K27me3 class. Number of genes (n) in "Low", "Medium" and "High" H3K27me3 class used for boxplots are 9824, 2311, 2647 respectively. Expression changes were estimated using 2 or 3 biological replicates for each condition. Statistical significance was estimated using two-sided t-tests.(J) Boxplot showing FC (log2) in gene expression between Daxx deficient and control MII oocytes for five groups of genes classified according to FC (log2) in gene expression between Ring1 Rnf2 deficient and control GV oocytes. Boxplots were created similarly to (G - I) and number of genes used for each boxplot is displayed below. Expression changes were estimated using 3 biological replicates for each condition. Statistical significance was estimated using two-sided t-tests.

(K) Schematic diagram showing molecular pathways controlling repression and chromatin integrity at paternal and maternal major satellite sequences in mouse early pre-implantation embryos.

Expanded View figure legends