Journal: Epigenetics & Chromatin

Article Title: The cohesin-associated protein Wapal is required for proper Polycomb-mediated gene silencing

doi: 10.1186/s13072-016-0063-7

Figure Lengend Snippet: a ESCs were infected with lentiviruses encoding shRNAs to Sall4 and Wapal. Cells with a high multiplicity of infection (MOI) were selected by addition of puromycin for 6 days. Images were taken by bright field (BF, ×10) or after staining for the pluripotency marker alkaline phosphatase (AP, ×10). b Changes in pluripotency markers (Nanog, Oct4, Sall4, and Rex1) and Wapal were measured by RT-qPCR. Fold change was calculated relatively to cells infected with the empty vector and plotted linearly on the y -axis. Error bars represent SEM of at least two experiments. Asterisk indicates statistically significant reductions compared to empty vector ( p value <0.05). c Similar to b , but for differentiation markers Brachyury (mesoderm), Fgf5 (ectoderm), and Cdx2 (trophectoderm). a Log 10 scale for fold change is used because of strong induction of these markers. Asterisk indicates statistically significant increases compared to empty vector ( p value <0.05). d Protein levels after depletion of Wapal or Sall4 for core cohesin subunits Rad21 and Smc3. GAPDH is shown as a loading control. Intervening, unrelated lanes were removed, but all samples were run on the same gel. e Propidium iodide (PI) staining was used to measure DNA levels in cells by flow cytometry. The percent of cells within each phase of the cell cycle and which exhibited abnormal DNA content (<2N or >4N) is shown. The only statistically significant difference was in the fraction within G0/G1 after depletion of Sall4 or Wapal as compared to empty vector ( asterisk indicates p value <0.05)

Article Snippet: A homology donor repair (HDR) vector containing an IRES :EGFP cassette flanked by homology regions from Oct4 was obtained from Addgene (plasmid #48681) and co-transfected along with the guide RNA into 1–2 × 10 6 wild-type ESCs.

Techniques: Infection, Staining, Marker, Quantitative RT-PCR, Plasmid Preparation, Control, Flow Cytometry

Journal: Epigenetics & Chromatin

Article Title: The cohesin-associated protein Wapal is required for proper Polycomb-mediated gene silencing

doi: 10.1186/s13072-016-0063-7

Figure Lengend Snippet: a The 100 probes most upregulated ( red ) or downregulated ( green ) with shRNA #1 to Wapal were identified and the Log2 fold change (Log2 FC) calculated for Wapal shRNA #2, Smc3 shRNAs, and from published studies in which the pluripotency-associated TFs Nanog or Oct4 were depleted by siRNAs. Log2 FC is displayed, with the color scheme indicated. b GSEA was used (see “ ” section for details) to identify pathways globally dysregulated by Wapal depletion. Six gene sets from pluripotent cells which all exhibited substantial enrichment in Wapal-depleted samples are shown. Positive normalized enrichment score (NES) indicates that the gene set is enriched in Wapal-depleted samples as compared to samples infected with the empty vector. False discovery rate (FDR < 25 %) and p value <0.01 were used to determine statistically significant changes in gene set expression. c ChIP-seq datasets were used to generate a list of high-confidence targets of Polycomb-mediated gene silencing by the presence of both H3K27me3 and H2Aub1 in their promoters (1455 genes). For each gene, the expression changes after Wapal or Smc3 depletion were measured by microarray. For genes with multiple probes, maximal probe deviation was used. Genes that showed minimal change in their expression after Wapal depletion or the two shRNAs did not demonstrate similar changes were eliminated because of possible off-target effects. Log 2 fold change is displayed, with a color scheme shown below

Article Snippet: A homology donor repair (HDR) vector containing an IRES :EGFP cassette flanked by homology regions from Oct4 was obtained from Addgene (plasmid #48681) and co-transfected along with the guide RNA into 1–2 × 10 6 wild-type ESCs.

Techniques: shRNA, Infection, Plasmid Preparation, Expressing, ChIP-sequencing, Microarray

Journal: Nature cell biology

Article Title: A PAX5-OCT4-PRDM1 developmental switch specifies human primordial germ cells.

doi: 10.1038/s41556-018-0094-3

Figure Lengend Snippet: Fig. 1 | Global redistribution of OCT4 binding in PGCs compared with ESCs. a, Cross-section of a human fetal testis (22 weeks) immunostained for OCT4. The panel on the right is an enlargement of the region enclosed within the white broken lines of the left panel. Scale bars, 50 µm; inset, 100 μm. Immunostaining experiments were independently repeated a minimum of three times and similar results were obtained. b, Left panel: heatmap visualization of OCT4 ChIP–seq data, depicting all binding events centred on the peak region within a 5 kb window around the peak. Right panel: distribution and peak heights of OCT4 peaks around the transcription start site (TSS). Peak heights are reported in reads per million. c, Scatterplot comparing OCT4 binding in PGCs and ESCs. Selected genes known to be associated with pluripotency are highlighted in blue, and those highly expressed in the germline are highlighted in red. d, Genome browser representation of ChIP–seq tracks for OCT4 in ESCs (red) and PGCs (yellow) at the OCT4 and PIWIL1 loci. Regions that were bound exclusively by OCT4 in ESCs or PGCs are highlighted by pink shaded boxes. Y-axes represent ChIP-seq signals in units of SPMR (signals per million reads). ChIP–seq were independently repeated twice and similar results were obtained. e, Venn diagram of unique and shared genes bound by OCT4 in ESCs and PGCs. GO analysis results are shown to the right and bottom of the venn diagram. Y-axes represent each category of molecular functions and biological processes. The analysis was performed twice and similar results were obtained based on two independent ChIP–seq data.

Article Snippet: The recombinant protein OCT4 (Novus; H00005460-P01) was bound to glutathione–sepharose beads (Amersham) and incubated with recombinant PAX5 protein (Novus; H00005079-P01) overnight at 4 °C.

Techniques: Binding Assay, Immunostaining, ChIP-sequencing

Journal: Nature cell biology

Article Title: A PAX5-OCT4-PRDM1 developmental switch specifies human primordial germ cells.

doi: 10.1038/s41556-018-0094-3

Figure Lengend Snippet: Fig. 2 | Co-occurrence of OCT4 with PAX5 and PRDM1 in PGCs. a, The position weight matrix of an enriched motif found in OCT4 ChIP–seq data from PGCs. The motif resembles the binding motifs for PRDM1 and PAX5. b, Cross-section of a human fetal testis (22 weeks). Upper panel: immunostained for PAX5 (red) and OCT4 (green), and DAPI stained for nuclei (blue). Lower panel: immunostained for PRDM1 (red) and OCT4 (green), and DAPI stained for nuclei (blue). Enlarged panels on the right represent the region enclosed within the white broken lines of the far left panel. White arrows indicate co-localization of PAX5 and OCT4 or PRDM1 and OCT4. Scale bars (orignal images), 100 µm; (expanded images) 50 µm. Immunostaining experiments were independently repeated a minimum of three times and similar results were obtained. c, Venn diagram of unique and shared genes bound by OCT4, PRDM1 and PAX5 in PGCs. The number of genes bound exclusively by each transcription factor or co-bound by multiple transcription factors are labelled. d, Genome browser representation of ChIP–seq tracks for OCT4 (yellow), PAX5 (blue) and PRDM1 (green) at the TBX3 and PIWIL1 loci. Regions that are bound collectively by OCT4, PAX5 and PRDM1 in PGCs are highlighted by pink shaded boxes. Y-axes represent ChIP-seq signals in units of SPMR (signals per million reads). ChIP–seq were independently repeated twice and similar results were obtained. e, GO analysis of co-bound genes. The analysis was performed twice and similar results were obtained. f, GST pull-down assay performed using OCT4 and PAX5 recombinant proteins. Pull-down was repeated three times and similar results were obtained. Unprocessed scans of western blots are shown in Supplementary Fig. 8.

Article Snippet: The recombinant protein OCT4 (Novus; H00005460-P01) was bound to glutathione–sepharose beads (Amersham) and incubated with recombinant PAX5 protein (Novus; H00005079-P01) overnight at 4 °C.

Techniques: ChIP-sequencing, Binding Assay, Staining, Immunostaining, Pull Down Assay, Recombinant, Western Blot

Journal: Nature cell biology

Article Title: A PAX5-OCT4-PRDM1 developmental switch specifies human primordial germ cells.

doi: 10.1038/s41556-018-0094-3

Figure Lengend Snippet: Fig. 5 | PAX5 acts upstream of OCT4. a, Genome browser representation of ChIP–seq tracks at the OCT4 locus. Enhancer regions are highlighted by pink shaded boxes. Y-axes represent ChIP-seq signals in units of SPMR. ChIP–seq was independently repeated twice and similar results were obtained. b, OCT4 expression in hESCs, control cells and cells overexpressing PAX5 during in vitro differentiation. Data represent the mean ± s.d. of n = 3 independent replicates. P values were calculated by two-tailed Student’s t-test. c, Mouse testis xenografts immunostained for GFP and c-KIT. Scale bar, 50 µm. Immunostaining experiments were independently repeated a minimum of three times and similar results were obtained. DAPI stained for nuclei. d, Flow cytometry analysis for GFP and c-KIT of mouse testis xenografts. e, RT-qPCR analysis of OCT4 expression in hPGCs formed in mouse seminiferous tubules by cells overexpressing PAX5, PAX5 knockout cells or control hESCs. Data represent the mean ± s.d. of n = 3 independent replicates. P values were calculated by two-tailed Student’s t-test. f, Reporter construct used for measuring OCT4 enhancer activity is shown. The genomic fragment bound by PAX5 and OCT4 (red) was inserted upstream of a luciferase gene driven by a minimal promoter. The y axis represents the fold enrichment of luciferase activity. Data represent the mean ± s.d. of n = 3 independent replicates. Source data for b, e and f are provided in Supplementary Table 2.

Article Snippet: The recombinant protein OCT4 (Novus; H00005460-P01) was bound to glutathione–sepharose beads (Amersham) and incubated with recombinant PAX5 protein (Novus; H00005079-P01) overnight at 4 °C.

Techniques: ChIP-sequencing, Expressing, Control, In Vitro, Two Tailed Test, Immunostaining, Staining, Flow Cytometry, Quantitative RT-PCR, Knock-Out, Construct, Activity Assay, Luciferase

Journal: Nature cell biology

Article Title: A PAX5-OCT4-PRDM1 developmental switch specifies human primordial germ cells.

doi: 10.1038/s41556-018-0094-3

Figure Lengend Snippet: Fig. 6 | PAX5 and OCT4 act upstream of PRDM1. a, Genome browser representation of ChIP–seq tracks at the PRDM1 locus. Enhancer regions bound by OCT4 and PAX5 in PGCs are highlighted by pink shaded boxes. Y-axes represent ChIP-seq signals in units of SPMR. ChIP–seq was independently repeated twice and similar results were obtained. b, PRDM1 expression in control cells, cells overexpressing PAX5 and PAX5 knockout cells during in vitro differentiation. Data are represented as mean ± s.d. of n = 3 independent replicates. P values were calculated by two-tailed Student’s t-test. c, RT-qPCR analysis of PRDM1 expression in hPGCs formed in the mouse seminiferous tubules by PAX5 overexpressing, PAX5 knockout and control hESCs. Data are represented as mean ± s.d. of n = 3 independent replicates. P values were calculated by two-tailed Student’s t-test. d, Reporter construct used for measuring PRDM1 enhancer activity is shown. The genomic fragment bound by PAX5 and OCT4 (red) was inserted upstream of a luciferase gene driven by a minimal promoter. The y axis represents the fold enrichment of luciferase activity. Data represent the mean ± s.d. of n = 3 independent replicates. e, RT-qPCR analysis of the expression of genes associated with germline programming. Data represent the mean ± S.D. of n = 3 independent replicates. P values were calculated by two-tailed Student’s t-test. f, Model for gene regulation in pluripotency and germline programmes. In pluripotent stem cells, OCT4, together with other transcription factors (TFs) and cofactors, binds to its own enhancer to activate and maintain its high expression. While differentiating towards germline cells, PAX5 replaces OCT4 and binds to the enhancer of OCT4 to maintain a moderate expression of OCT4. Meanwhile, PAX5 and OCT4 bind to the enhancer of PRDM1 and activate its expression to initiate the germline programme. Source data for b, c, d and e are provided in Supplementary Table 2.

Article Snippet: The recombinant protein OCT4 (Novus; H00005460-P01) was bound to glutathione–sepharose beads (Amersham) and incubated with recombinant PAX5 protein (Novus; H00005079-P01) overnight at 4 °C.

Techniques: ChIP-sequencing, Expressing, Control, Knock-Out, In Vitro, Two Tailed Test, Quantitative RT-PCR, Construct, Activity Assay, Luciferase