EMBO/SourceData huggingface dataset:
Figure 1A) Representative nuclear spreads of wild-type pachytene and Rec8−/− pachytene-like spermatocytes. Nuclear spreads were immunostained for SYCP3 and ACA. Magnified views are indicated by dashed areas. Schematic representation on magnified chromosomes represents tightly associated sister-AEs (separated for easier representation) in paired wild-type homologs, and the appearance of two distinguishable/separated sister-AEs once REC8-mediated cohesion is lost in Rec8−/− univalents. Bars, 10 μm in spreads and 1 μm on insets.B) Representative nuclear spread of zygotene-like Stag3 mutant spermatocytes. Nuclear spreads were immunostained for SYCP3 and ACA. Magnified views are indicated by dashed areas. Schematic representation on magnified univalents represents: separation of sister-AEs (centre) and close association of sister-AEs (right). Bellow: graph showing the percentages of axes with: separation of AEs (dark grey area) and closely associated AEs (light grey area). 414 axes analysed, from 11 nuclei. Bars, 10 μm in spreads and 1 μm on insets.C) Magnified views of zygotene-like Stag3 mutant univalents with LSAEs. Nuclear spreads were immunostained for SYCP3 and ACA. Schematic representation indicates sites of LSAEs. Bellow: graph showing the percentages of axes with: LSAEs (red area), extensive separation of AEs (dark grey area) and closely associated AEs (light grey area). 414 axes analysed, from 11 nuclei. Bars, 1 μm.D) Representative nuclear spread of zygotene-like Smc1β−/− spermatocytes. Nuclear spreads were immunostained for SYCP3 and ACA. Magnified views are indicated by dashed areas. Schematic representation on magnified univalents represents sites of LSAEs. Bellow: graph showing the percentages of axes with: LSAEs (red area), extensive separation of AEs (dark grey area) and closely associated AEs (light grey area). 404 axes analysed, from 10 nuclei. Bars, 10 μm in spreads and 1 μm on insets.E) STED images of representative zygotene-like Stag3 mutant and Smc1β−/− univalents displaying LSAEs. Nuclear spreads were immunostained for SYCP3. Bars, 1μm.F) Graph indicating the inter-axis distances measured in wild-type pachytene bivalents; pachytene-like Rec8−/− and zygotene-like Stag3 mutant and Smc1β−/− univalents. Each measurement in wild-type and Rec8−/− corresponds to the median of 3 distances measured along one homolog (n=60). Each measurement in Stag3 mutant and Smc1β−/− mutant axes correspond to 1 distance measured at sites of local separation of AEs (n=40 and n=20, respectively). Horizontal lines indicate median.G) Graph indicating the inter-axis distances measured at terminally and centrally located sites of LSAEs in zygotene-like Stag3 mutant univalents. Each measurement corresponds to 1 distance measured at terminally (n=20) and centrally (n=20) located sites of local separation of axial elements. Horizontal lines indicate median.
Figure 2A) Representative wild-type pachytene bivalents; pachytene-like Rec8−/−, zygotene-like Stag3 mutant and Smc1β−/− univalents. Nuclear spreads were immunostained for SYCP1, SYCP3 and ACA. Filled arrowheads indicate sites of LSAEs with detectable SYCP1. Bars, 1 μm. Bellow: graphs showing the percentages of sites of LSAEs with detectable SC assembly along sister-AEs of Stag3 mutant and Smc1β−/− univalents (n=120 and n=68, respectively).B) Representative zygotene-like Stag3 mutant univalents. Nuclear spreads were immunostained for SYCP3, ACA and: SYCE1, SYCE2 and TEX12. Filled arrowheads indicate SC assembly between sites of LSAEs. Bars, 1 μmC) Quantification of signal distribution within sites of LSAEs, in Stag3 mutant spermatocytes. Signal distribution measured for SYCP3 and: SYCP1, SYCE1, SYCE2 and TEX12. Empty arrowheads indicate signal intensity peaks that correspond to each of the two sister-chromatid axes, filled arrowheads indicate SYCP1 peaks.
Figure 3A) Representative zygotene-like Stag3 mutant and Smc1β−/− univalents. Nuclear spreads were immunostained for SYCP3, SYCE1 and REC8. Bellow: graph showing the percentages of Stag3 mutant axes with sites of LSAEs with and without flanking REC8 foci. 78 axes with LSAEs were analysed. Bars, 1 μm.B) Representative pachytene-like Rec8−/−, zygotene-like Stag3 mutant and Smc1β−/− univalents. Nuclear spreads were immunostained for SYCP3, SYCE1 and RAD21L. Bars, 1 μm.C) Representative zygotene-like Stag3 mutant and Smc1β−/− univalents. Nuclear spreads were immunostained for SYCP3, SYCE1 and RAD21. Bars, 1 μm.
Figure 4A) Representative wild-type pachytene sex chromosomes. Nuclear spreads of pachytene spermatocytes were immunostained for SYCP3. Filled arrowheads indicate the PAR. Dashed area indicated in centre panel corresponds to the magnified view on the right panel. Bars, 10 μm in spreads and 1 μm on insets.B) Graph indicating the inter-axis distances measured at sites of local separation of axial elements in zygotene-like Stag3 mutant univalents and along pachytene wild-type X-chromosomes. Each measurement in the X-chromosomes corresponds to the median of 3 distances (according to the scheme in figure) (n=54). Each measurement in Stag3 mutant corresponds to 1 distance measured at sites with local separation of axial elements (n=40). Horizontal lines indicate median. p < 0.0001, obtained with two-tailed Mann-Whitney test. Bellow: schematic representation of the XY pair: sister-AEs are coloured in black and grey for easier visualization. Filled arrowhead indicates PAR. Purple bars in scheme indicate regions where inter-axis distances are measured.C) Representative pachytene wild-type sex chromosomes. Nuclear spreads of pachytene spermatocytes were immunostained for SYCP3 and SYCE2. Dashed area indicated in centre panel corresponds to the magnified view on the right panel. Empty arrowhead indicates two discernable sister-AEs without SYCE2. Bars, 10 μm in spreads and 1 μm on insets.
Figure 5A) Analysis of REC8 distribution in representative pachytene wild-type sex chromosomes. Nuclear spreads of pachytene spermatocytes were immunostained for SYCP3 and REC8. Bars, 10 μm in spreads and 1 μm on insets. Magnified XY pair is indicated by dashed area. Scatterplot showing the number of REC8 foci per X-chromosome. 225 foci identified in 14 X-chromosomes. Horizontal line indicates the mean, error bars indicate s.d. 3.02.B) Top: representative computationally straightened pachytene wild-type X-chromosomes. Nuclear spreads were immunostained for SYCP3 and REC8. Chromosomes were aligned at the PAR, and subdivided into 25 regular intervals. Bar, 1 μm. Bellow: histogram showing the distribution of the percentages of REC8 foci (n=225) among 25 regular intervals along 14 X-chromosomes. Dotted line indicates mean.C) Histogram showing the relative frequency of inter-REC8 distances along X-chromosomes.211 inter-REC8 distances from 14 X-chromosomes. Grey area represents 15% of the chromosome axis length. The respective cumulative distribution function is shown.D) Histogram showing the best fit of the observed inter-REC8 distances to the gamma distribution. Green curve indicates the best fit of the observed inter-REC8 distances.  indicates the value for which the best fit was obtained, with estimated standard error 0.27.E) Graph comparing the median distance of REC8 foci with computationally predicted even and random distributions. Median distances between foci subdivided in three categories (11-14, 15-18 and 19-22 REC8 foci per chromosome). Purple asterisk indicates the median distance of all REC8 foci.F) Scatterplot comparing inter-REC8 distances along on X-chromosomes with the distances between flanking REC8 foci at sites of LSAEs in the Stag3 mutant. 211 inter-REC8 distances measured along X-chromosomes and 10 distances measured between flanking REC8 foci in Stag3 mutant. Grey area represents 15% of the chromosome axis length. Horizontal lines indicate the median. p < 0.0001, obtained with two-tailed Mann-Whitney test.
Figure 6A) Representative yet-unsynapsed autosome from wild-type zygotene spermatocytes. Nuclear spreads were immunostained for SYCP3 and SYCE2. Magnified autosome is indicated by dashed area. Filled arrowheads indicate yet-unsynapsed axes regions. Bars, 10 μm in spreads and 1 μm on insetsB) Representative unsynapsed autosome from wild-type zygotene spermatocytes. Nuclear spreads were immunostained for SYCP3, REC8 and ACA. Magnified autosome is indicated by dashed area. Bars, 10 μm in spreads and 1 μm on insets.C) Scatterplot comparing the average number of REC8 foci along zygotene autosomes and X-chromosomes. Number of REC8 foci were quantified along 15 zygotene autosomes and 14 X-chromosomes. Each dot represents the number of REC8 foci on each chromosome. Total number of foci identified: zygotene autosomes (343), X-chromosomes (225). Horizontal lines indicate the mean, error bars indicate s.d.: 7.42 and 3.02 for zygotene autosomes and X-chromosomes, respectively. p = 0.0060, obtained with two-tailed Mann-Whitney test.D) Histogram showing the relative frequency of inter-REC8 distances along yet-unsynapsed zygotene autosomes. 294 inter-REC8 distances analyzed in 14 chromosomes. Grey area represents 15% of the chromosome axis length. The respective cumulative distribution function is shown.E) Scatterplot comparing the measured inter-REC8 distances on yet-unsynapsed zygotene autosomes, X-chromosomes and sites of LSAEs in Stag3 mutant. Number of inter-REC8 distances for yet-unsynapsed zygotene autosomes, X-chromosomes and sites of local separation of axial elements in Stag3 mutant, n=294, n=211 and n=10, respectively. Horizontal lines indicate the median. p < 0.0001, obtained with two-tailed Mann-Whitney test.
Figure 1Immunoblot of whole cell lysates from multiple human lineage-committed cell-lines using a C-terminal anti-JARID2 antibody. The size of canonical form is 140kDa but an additional strong band was detected at around ~80kDa. The blot is divided according to cell-types, which are indicated in the figure.qPCR measurements of JARID2 mRNA levels in different cell lines. The RNA levels were measured relative to 18S rRNA. Means and SEs are calculated over at least 2 independent experiments and 3 technical replicates. The data is represented as mean ± SE.Immunoblot showing that the ~80kDa band disappears upon treatment of HEK293T and HaCaT cells with siRNAs against exon 3 and exon 15 of JARID2 RNA. Non-silencing control (NSC) was used as a transfection control.
Figure 2qPCR measurements of RNA levels (n=3) for the three isoforms of JARID2 in HaCaT cells. The RNA levels are plotted relative to 18S rRNA. Level of JARID2 isoform-1 is significantly higher than that of isoform-2 (***P<0.001) and isoform-3 (****P<0.0001).Agarose gel showing RT-PCR products amplified using primers in exon3 and exon15 (2.9kb) as well as RT-PCR product corresponding to JARID2 isoform-1 (3.7kb) amplified using the first (exon 1) and the last exon (exon18). Only one product at the right size is observed indicating that it is unlikely that JARID2 might be a product of an alternatively spliced isoform of JARID2.CRISPR-Cas9 knockout of JARID2 using a sgRNA guide designed to target main translation start site as seen in (E). Immunoblot revealed that ~80kDa form was removed from JARID2 KO lines.Immunoblot after a full length JARID2 isoform-1 was expressed from an exogenous vector in HaCaT cells showed an increase (2-3 fold) in ~80kDa level.Immunoprecipitation using anti-JARID2 antibody and followed by mass spectrometry identification of the ~80kDa band detected JARID2 peptides spanning only C-terminus of JARID2. The identified peptides are shown as red arrows on schematic of JARID2 sequence.
Figure 3Immunoblot showing increase in expression levels of differentiation markers Involucrin (IVL) and Transglutaminase-1 (TGase-1) as differentiation progressed indicating that keratinocyte differentiation protocol was successful. Protein levels on day 0 (D0), day 1 (D1), day 3 (D3) and day 6 (D6) of differentiation are shown. Immunoblot for JARID2 during D0, D1, D3 and D6 of differentiation (as in B) are shown.Effect of JARID2 removal on levels of differentiation markers Involucrin (IVL), Keratin-1 (KRT1) and Keratin-10 (KRT10) mRNAs as measured in qPCR experiment relative to 18S rRNA and the rescue using exogenous expression of ΔN-JARID2. The effect of exogenous expression of full-length JARID2 (FL-JARID2) in JARID2 knockout is also shown. Data from wildtype HaCaTs, two independent JARID2 knockout (KO1 and KO2) HaCaT lines as well as KO cells exogenously expressing empty vector control, ΔN-JARID2 and FL-JARID2 are shown. Levels of Involucrin (IVL), Keratin-1 (KRT1) and Keratin-10 (KRT10) mRNAs as above but measured on day 3 of differentiation.
Figure 4A metagene plot showing enrichment of full-length JARID2 in human induced pluripotent cells at down-regulated genes (blue) as compared to up-regulated genes (grey) in JARID2 KO cells. The plot is centered on Transcription Start Sites (TSS) of genes and distance from TSS is indicated on the x-axis.Metagene plots of average enrichment of EZH2 in ES cells and keratinocytes at down-regulated genes (blue) as compared to up-regulated genes (grey) in JARID2 KO cells. The plots are centered on Transcription Start Sites (TSS) of genes and distance from TSS is indicated on the x-axes.Levels of H3K27me3 in WT, JARID2 KO lines and JARID2 KO lines expressing ΔN-JARID2. Histone H3 and Ponceau staining is used as a loading control.A bar-plot showing percentage of down- and up-regulated genes among genes which lose H3K27me3 vs which do not lose H3K27me3 modification during differentiation.
Figure 1Geometric mean-centered, hierarchical cluster heat map from microarray data. 1077 annotated lncRNAs (p<0.05) were represented in liver CSC (CD13+CD133+) compared with non-CSC (CD13-CD133-) cells sorted from three HCC primary cells of three patients. Top ten upregulated lncRNAs in CSCs are shown.In situ hybridization of HAND2-AS1 in HCC tumor tissues. HAND2-AS1 staining is shown in tumor tissues (left panel). Quantitation of HAND2-AS1+ cells in liver tissues was examined in 10 high-power fields (HPF) of sections from ten different HCC patients (right panel). Results are shown as means ± SEM. Scale bar, 100 μm.Macroscopic appearance of livers in 12-month-old DEN-treated lncHand2+/+ and lncHand2 knockout (lncHand2-/-) mice (left panel). Black arrows indicate liver tumors. Quantitation of tumor foci numbers and areas in DEN-treated livers (right panel). Results are shown as means ± SD (n=12).Representative H&E and immunohistological staining with Ki67 antibody of liver sections of 12-month-old DEN-treated lncHand2+/+ and lncHand2-/- mice. Scale bar, 100 μm.Quantitation of Ki67-positive cells in non-treated and DEN-treated mice. Bars represent average percentages and SD of cells positively staining for Ki67 cells examined in 10 HPF of sections from five different mice. Data are shown as means ± SD.Representative immunofluorescence staining of livers in 5.5-month-old DEN-treated and untreated lncHand2RFP reporter mice for indicated molecules (lower panel). Scale bar, 100 μm. Upper panel: scheme of targeting strategy for IRES-RFP knockin allele.
Figure 2HAND2-AS1 knockout causes a declined oncosphere-forming capacity in HCC cells. The right panel represents statistical results as means ± SD (n=3 per group). Overexpression of HAND2-AS1 (oelnc) rescued the sphere formation reduced by HAND2-AS1 deletion. Scale bar, 100 μm.CD13+CD133+ (CSC) subpopulations were detected in HAND2-AS1 knockout cells by FACS analysis. Results are shown as means ± SD (n=4 per group).Limiting diluted HAND2-AS1 depletion or Ctrl HCC cells were subcutaneously implanted into BALB/c nude mice. n=12 for each group.Estimated frequency of TICs in HAND2-AS1 deficiency and control cells after the first transplantation using the extreme limiting dilution analysis. Data are shown as the mean and 95% confidence interval. (n=12 grafted tumors per dilution).Estimated frequency of TICs in HAND2-AS1 deficiency and control HCC cells during serial transplantations. Grey area indicates the 95% CI.Estimated frequency of TICs in HAND2-AS1 deficiency and control HCC cells during serial transplantations. Grey area indicates the 95% CI.Estimated frequency of TICs in different populations after the first transplantation (n=15 grafted tumors per dilution). Data represent the mean and 95% confidence interval.Representative whole-body imaging of Huh7-Luc cells transduced with control or HAND2-AS1 KO #1 or KO #2 vectors. Quantification of tumor numbers of 6 weeks after tumor cells orthotopically implanted to B-NSG mice. Data are shown as means ± SD. One-way ANOVA with Dunnett's correction for multiple comparisons.HAND2-AS1 expression levels (normalized to 18S rRNA) in livers from BALB/c nude mice subcutaneously transplanted xenograft primary HCC cells then intraperitoneally administered 25 mg kg−1 ASOs on days 15, 20, 25, 30 and 35 (n = 5 per group). Three ASOs (1, 2, 3) against HAND2-AS1 were used to treat mice and showed similar depletion effects. Results are shown as means ± SD. n=6 mice per group.Mice were killed at the 40th day after HCC cell injection, and the tumors were excised and weighed. Representative tumors are shown. Scale bar, 1 cm. Statistical data are shown as means ± SD (n = 6 mice per group) (right panel).HAND2-AS1 overexpression enhances the capacity of oncosphere formation. Scale bar, 100 μm. oe: overexpression. Right panel: Data are shown as means ± SD (n = 3 per group).Estimated frequency of TICs in HAND2-AS1 overexpression and control HCC cells during serial transplantations.Representative whole-body imaging of Huh7-Luc cells transduced with control or HAND2-AS1 overexpression vectors. Quantification for tumor numbers one month after tumor cells orthotopically implanted to B-NSG mice. (n = 5 mice per group).
Figure 3Biotin-RNA pulldowns were performed with nuclear extracts of oncosphere cells using full-length HAND2-AS1 transcript (sense), antisense, and one HAND2-AS1 intron control, followed by mass spectrometry. Band 1: INO80, band 2: RUVBL2.Two core components of the INO80 complex were confirmed by immunoblotting.Immunoprecipitation assays of biotin-labeled CHIRP probes incubated with liver CSC lysates.HAND2-AS1 was visualized by RNA FISH, followed by immunofluorescence staining of INO80 in HCC primary oncosphere cells. Scale bar, 100 μm.Mapping analysis of INO80-binding domains of HAND2-AS1. Schematic diagram of HAND2-AS1 full-length and truncated fragments (top panel); Western blot of INO80 and RUVBL2 in RNA pulldown samples by different HAND2-AS1 fragments (middle panel); Different HAND2-AS1 fragments (bottom panel).EMSA of biotin-labeled HAND2-AS1 (nt 83-242) probes incubated with INO80 protein.Binding affinity of HAND2-AS1 with INO80 was determined by five independent EMSA assays. Non-linear regression curves were generated by GraphPad Prism. Results are shown as means ± SD.Interaction regions of INO80 with HAND2-AS1 mutations at nt 83-242 of exon 2 were detected using RNA pulldown assays. LR, loop region.Representative whole-body imaging of Huh7-Luc cells transduced with control or INO80 shRNA #1 or shRNA #2 vectors. Quantification of tumor numbers of 4 weeks after tumor cells orthotopically implanted to B-NSG mice. Data are shown as means ± SD (n = 5 mice per group). One-way ANOVA with Dunnett's correction for multiple comparisons.Macroscopic appearance of livers in 10.5-month-old DEN-treated Ino80flox/flox (Ino80+/+) and Ino80 knockout (Ino80-/-) (left panel). Black arrows indicate tumors. Quantitation of tumor foci numbers and areas in DEN-treated livers. Results are shown as means ± SD (n=10).
Figure 4Heatmap results for HAND2-AS1 or INO80 deficiency in HCC cells.Based on GSEA results, significantly changed genes due to depletion of HAND2-AS1 and INO80 were attributed to the BMP signaling in HCC primary cells. NES, normalized enrichment score; FWER, familywise error rate.ChIRP-seq analysis of HAND2-AS1 genomic binding at target sites in liver CSCs, using LacZ probes as negative control. A 10-kb interval centered on the called HAND2-AS1 peak is shown.Representative ChIRP-seq of HAND2-AS1 binding of BMPR1A.Gel analysis of HAND2-AS1 RNA binding BMPR1A promoter in liver CSCs. LacZ and PVT1 served as negative controls.HAND2-AS1 deletion in HCC cells decreases BMPR1A expression and inactivates BMP signaling.ChIRP-immunoblotting analysis of BMPR1A interaction with the INO80 complex in control and HAND2-AS1-knockout liver CSCs.EMSA analysis of the interaction of HAND2-AS1, INO80 and RUVBL2 with the BMPR1A promoter region.BMPR1A promoter binding region to HAND2-AS1/INO80 was deleted (BMPR1A-p-KO) in liver CSCs using lenti-Cas9, followed by HAND2-AS1 and INO80 overexpression (oeHAND2-AS1, oeINO80) for 36 h. BMPR1A mRNA levels were examined by realtime PCR. Data are shown as means ± SD. n=5. ** P < 0.01 by two-tailed Student's t test.BMP signaling was examined in INO80-silenced liver CSCs by Western blot.
Figure 5BMPR1A is highly expressed in HCC tumor tissues provided by Wang's cohort (GSE14520).Kaplan-Meier analyses of liver cancer outcomes in the cancer genome atlas (TCGA). P value for Kaplan-Meier curves was determined using a Mantel-Cox log-rank test.Representative immunohistochemistry staining of BMPR1A was detected in HCC samples. Scale bar, 100 μm.BMPR1A overexpression rescues sphere formation ability reduced by HAND2-AS1 depletion in HCC samples. Representative sphere formation is shown on the left panel. Scale bar, 100 μm. Percentages of sphere-forming cells were calculated as means ± SD (right panel). n=4.BMPR1A depletion reduces tumor-initiating capacity. n=12 mice per group.Representative whole-body imaging of Huh7-Luc cells transduced with scramble (Ctrl) or BMPR1A shRNAs. Quantification for tumor numbers of 7 weeks after tumor cells orthotopically implanted to B-NSG mice. Calculated data are shown as means ± SD (n = 5 mice per group).Macroscopic appearance of livers from 10-month-old mice treated by DEN or vehicle. Scale bar, 1 cm. Black arrows indicates tumors.Representative H&E and immunohistological staining with Ki67 antibody in liver sections of 10-month-old DEN-treated WT and Bmpr1a-/- mice. Scale bar, 100 μm. Right: Quantitation of Ki67-positive cells in DEN-treated mice. Data are shown as means ± SD. n=5.BMP signaling inhibitor noggin decreased tumor sphere formation. Scale bar, 100 μm. Right: Data are shown as means ± SD (n=4).
Figure 6Representative whole-body imaging of Huh7-Luc cells in different groups. Mice treated with scramble ASOs and/or siRNAs obtained similar results to vehicle treatment. n≥5 mice in each group.Quantification of tumor numbers treated with ASOs and/or siRNAs after Huh7-Luc tumor cells orthotopically implanted to B-NSG mice. Calculated data are shown as means ± SD (n = 5).Tumors were excised from livers of PDX liver cancer models. Scale bar, 0.5 cm.Representative H&E staining in liver sections from tumor-bearing mice treated with ASO1 and siBMPR1A in orthotopic transplants of tumors from HCC#50 and HCC#51. Scale bar, 100 μm.Representative immunohistological staining with Ki67 antibody of tumor-bearing mice treated with ASO1 and siBMPR1A. Scale bar, 100 μm. Lower panel: Quantitation of Ki67-positive cells in different groups. Data are shown as means ± SD (n=5 per group).Northern blotting of HAND2-AS1 and immunobloting of BMPR1A in tumors from PDX HCC#50 after treatment.Kaplan-Meier survival analysis of B-NSG mice orthotopically transplanted with PDXs HCC#50 (I) and HCC#51 (J). Treatment with ASO1 combined siBMPR1A causes synergistic therapeutic effect than each treatment alone (n = 10 per group). P value for Kaplan-Meier curves was determined using a Mantel-Cox log-rank test.