Journal: Cell stem cell

Article Title: Deterministic Somatic Cell Reprogramming Involves Continuous Transcriptional Changes Governed by Myc and Epigenetic-Driven Modules

doi: 10.1016/j.stem.2018.11.014

Figure Lengend Snippet: A. Experimental scheme. B. Spearman correlation between expression profiles of Mbd3f/- system Calculated over all differential genes (n=8,042), showing an average correlation of R=0.93 between consecutive samples. C. As in B, but between Mbd3flox/- and Gatad2a-/- systems. D. Overlap between targets of OSKM in promoters and enhancers. Pixel shade indicates Jaccard Index. E. Correlation between consecutive samples in Mbd3f/- system (MEF-day1, day1-day2, day2..day8-iPS), measured over all ESPGs promoters (promoters with differential chromatin pattern, n=3,593, top), or all differential enhancers (n=40,174, bottom), for each chromatin mark. Negative controls were calculated between MEF and IPS, are marked with solid border. F. Overlap between binding targets of Oct4, Sox2, Klf4 or Myc, and previously published binding data of the same factors, calculated in ES and iPS samples. Percentage out of our measured binding targets is presented, along Fisher exact test p-values. G. Global transcriptional pattern of 8,042 differential genes (FC>4 & maximal FPKM value>1), sorted by their temporal pattern in Mbd3f/-system (the same gene order was applied for the other reprogramming systems). Heatmap represents unit-transformation of FPKM values. H. PCA analysis of all samples, alongside samples from previous publications (Polo et al., 2012). PCA was calculated on the same set of genes and normalization as in G. I. GO categories enriched among the genes that are active in each day. Gene is defined to be active in samples where RPKM is above 0.5 of the gene max value. P-values were calculated with Fisher exact test, and FDR corrected. Categories with corrected p-value<0.01 in at least two-time points are presented. Gray Shades represent FDR corrected p-values

Article Snippet: Secondary mouse embryonic fibroblast (MEF) from Mbd3 flox/- cell line (A12 clone: Mbd3 flox/- cell lines that carries the GOF18-Oct4-GFP transgenic reporter (complete Oct4 enhancer region with distal and proximal enhancer elements) (Addgene plasmid #60527)) and WT-1 cell line (WT-1 clone that carries the deltaPE-GOF18-Oct4-GFP reporter (Addgene plasmid#52382) were previously described ( Rais et al., 2013 ).

Techniques: Expressing, Binding Assay, Transformation Assay

Journal: Cell stem cell

Article Title: Deterministic Somatic Cell Reprogramming Involves Continuous Transcriptional Changes Governed by Myc and Epigenetic-Driven Modules

doi: 10.1016/j.stem.2018.11.014

Figure Lengend Snippet: A. ChIP-seq landscape of two examples. Promoters are marked in red, enhancers are marked in green. Signals are normalized to sample size (RPM). B. Overlap between binding of OSKM and active enhancers, in each day of reprogramming. Enhancer is defined as active in a specific day if its ATAC-seq z-score is above 1.5 STD in that day. Gray shades indicate Fisher exact test p-value for overlap between compared samples. Note that OSKM do not bind the enhancers that are active in MEF (D0, marked in red); these enhancers are not significantly bound by OSKM at any day during reprogramming. C. Number of enhancers bound by each of OSKM factors in each day of reprogramming. Upper row: out of enhancers that are bound by the factor in late stages (day8, iPS, ESC). Bottom row: out of enhancers that are bound by the factor in early stages (day1-day3). D. Probability to observe co-localized binding of transcription factors in promoters (gray) and enhancers (black). Calculated in days 1,8 and iPS (Error bars indicate S.E.M). Right – Myc binds 32% of promoters, and 8% of active enhancers. E. Significant motifs enriched in promoters and enhancers that are bound by each of Oct4, Sox2, Klf4 and c-Myc at different days of reprogramming, as detected by Homer/4.7 software. P-values, indicated by color shade, were reported by Homer, and are FDR corrected. Motifs which are significantly enriched (corrected p<10-30) in at least one time point are presented. F. a. Motifs enriched in differential enhancers that are active in each day of reprogramming (ATAC-seq z-score >1.5). P-values, indicated by color shade, were reported by Homer, and are FDR corrected. Motifs which are significantly enriched (corrected p<10-50) in at least one-time point are presented. G. Motifs found in “closed” vs. “open” binding targets of the indicated transcription factor. Accessibility of targets was calculated based on ATAC-seq. Motifs found in OSK binding targets calculated in Mbd3f/- day1. Motifs that are different between open and closed binding targets are marked in black line. Complementary motifs to canonical motif appear in reverse order. H. Spearman correlation between ATAC-seq profiles of the two efficient reprogramming systems: Mbd3f/- MEF and C/EBPaTg B cell systems calculated over 40,174 differential enhancers.

Article Snippet: Secondary mouse embryonic fibroblast (MEF) from Mbd3 flox/- cell line (A12 clone: Mbd3 flox/- cell lines that carries the GOF18-Oct4-GFP transgenic reporter (complete Oct4 enhancer region with distal and proximal enhancer elements) (Addgene plasmid #60527)) and WT-1 cell line (WT-1 clone that carries the deltaPE-GOF18-Oct4-GFP reporter (Addgene plasmid#52382) were previously described ( Rais et al., 2013 ).

Techniques: ChIP-sequencing, Binding Assay, Software

Journal: Cell stem cell

Article Title: Deterministic Somatic Cell Reprogramming Involves Continuous Transcriptional Changes Governed by Myc and Epigenetic-Driven Modules

doi: 10.1016/j.stem.2018.11.014

Figure Lengend Snippet: A. Distribution of low (<0.02), mid (0.02-0.98) and high (>0.98) methylated CpG sites, along reprogramming. Average and SEM are indicated in red plot. B. Methylation level measured in covered enhancers (n=18,072), in Mbd3f/-, Gatad2a-/- and WT-2 systems. Enhancers are clustered into eight clusters using k-means. Cluster 8 consists of enhancers that undergo fast demethylation, compared to clusters 3 and 7. C. Average methylation measured in promoters of genes that were highly methylated (>80%) in day0. Genes that change their expression level (red) are compared to genes that do not change their expression level (gray). Wilcoxon p-value indicates places where methylation of differential genes is significantly lower than methylation of non-differential genes. D. Left: Enrichment of enhancer clusters, as shown in panel B, for OSK binding, DNA accessibility, and super enhancers, showing that cluster 8 is highly enriched for OSK binding and overlaps with super enhancers. Color shades represent FDR corrected enrichment p-value. Right: Enrichment of the same enhancer clusters to transcription factor binding, taken from hmChip database. Cluster size is indicated on the right. E. Experimental scheme summary. Reprogramming efficiency was measured by Oct4-GFP+ cells percentage in Tet1/2/3 null(Δ) and Tet1/2/3fl/fl with and without Gatad2a expression, after 8 days. **p<0.01, ***p<0.001 (Student’s t-test), n=6, error bars indicate SD. F. Secondary MEF harboring Mir290-RGM and Nanog GFP-reporter were sorted after reprogramming to 3 different populations: RGM-SE-Mir290-tdTomato positive cells (sorted at day 5), Nanog-GFP and Mir290-RGM positive cells (sorted at d10-14), and "double negative" cells (sorted at d5). The cells were seeded as single cell-per-well, and were treated with medium either supplemented with Dox or lacking Dox. On day 14 colonies were inspected for GFP and mCherry (RGM) markers.

Article Snippet: Secondary mouse embryonic fibroblast (MEF) from Mbd3 flox/- cell line (A12 clone: Mbd3 flox/- cell lines that carries the GOF18-Oct4-GFP transgenic reporter (complete Oct4 enhancer region with distal and proximal enhancer elements) (Addgene plasmid #60527)) and WT-1 cell line (WT-1 clone that carries the deltaPE-GOF18-Oct4-GFP reporter (Addgene plasmid#52382) were previously described ( Rais et al., 2013 ).

Techniques: Methylation, Expressing, Binding Assay

Journal: Cell stem cell

Article Title: Deterministic Somatic Cell Reprogramming Involves Continuous Transcriptional Changes Governed by Myc and Epigenetic-Driven Modules

doi: 10.1016/j.stem.2018.11.014

Figure Lengend Snippet: A. Experimental flow describing three experimental perturbation settings: (i) Mbd3f/- MEFs were virally infected with cMyc over-expression (OE) cassette, OSK-OE cassette or both cassettes. Gene expression was measured on day4 following infection. (ii) Mbd3f/- MEFs carrying OSK Dox-dependent cassette were treated for knockdown of c-Myc, n-Myc and l-Myc. Gene expression was measured on days 3 and 7, and colony formation was measured on day 11. (iii) Mbd3f/- MEFs carrying OSKM Dox-dependent cassette were treated with inhibitor of cMyc (10058-F4) and with Dox. Gene expression and colony formation were measured on day 3. B. Distribution of Expression fold change (FC) compared to WT MEF of up/down regulated ESPGs (down regulated ESPGs are enriched for somatic genes), and CAPGs. Presented perturbations are over-expression of OSK cassette, over-expression of c-Myc cassette, or over-expression of the two cassettes together. (*p<10-5, **p<10-20, Wilcoxon test). C-D. Reprogrammed colony formation in Myc knockdown ort small molecule inhibition, measured 11-14 days after Dox. E. Distribution of expression fold change (FC, in log2 scale) compared to MEF of up/down regulated ESPGs and CAPGs. Presented perturbations are Myc knockdown, inhibition of Myc activity with small molecular inhibitor (10058-F4). (*p<10-5, **p<10-20, Wilcoxon test). F. Experimental scheme. G. IPSC Reprogramming efficiency in different cells expressing both endogenous and/or exogenous cMyc and nMyc. H. FACS analysis for surface expression of fibroblast surface marker Thy1 on the indicated Mbd3flox/- cell types. Dotted line indicates positive threshold for detection. I. Representative pictures of Mbd3fl/- cells harboring mCherry-NLS and ΔPE-GOF18 Oct4-GFP cassettes after 13 days of reprogramming in the presence of MYCi. Scale = 100μM. J. Left panel - iPSC reprogramming efficiency by applying highly efficient mouse B cell and WT CMP reprogramming protocols by OSKM in the presence or absence of MYC small molecule inhibitor (MYCi). Right panel – Human iPSC reprogramming efficiency by applying OKS lentiviral transduction in the presence of absence of MYCi. K. Expression fold-change distribution (log2 scale) of selected GO categories in Myc over-expression or Myc knockdown, showing that upon over-expression of Myc, processes such as ribosomal biogenesis and chromosome segregation are induced. L. Fraction of Myc targets in significantly induced and repressed GO categories, compared to what is expected by random (dashed line). M. Overlap between differential genes detected in Myc perturbation experiments, and differential genes detected in previous published perturbations (Scognamiglio et al., 2016). Fisher exact test p-values are presented. N. Expression fold change of selected chromatin modifiers.

Article Snippet: Secondary mouse embryonic fibroblast (MEF) from Mbd3 flox/- cell line (A12 clone: Mbd3 flox/- cell lines that carries the GOF18-Oct4-GFP transgenic reporter (complete Oct4 enhancer region with distal and proximal enhancer elements) (Addgene plasmid #60527)) and WT-1 cell line (WT-1 clone that carries the deltaPE-GOF18-Oct4-GFP reporter (Addgene plasmid#52382) were previously described ( Rais et al., 2013 ).

Techniques: Infection, Over Expression, Expressing, Inhibition, Activity Assay, Marker, Transduction

Journal: Cell stem cell

Article Title: Deterministic Somatic Cell Reprogramming Involves Continuous Transcriptional Changes Governed by Myc and Epigenetic-Driven Modules

doi: 10.1016/j.stem.2018.11.014

Figure Lengend Snippet: Key Resources Table

Article Snippet: Secondary mouse embryonic fibroblast (MEF) from Mbd3 flox/- cell line (A12 clone: Mbd3 flox/- cell lines that carries the GOF18-Oct4-GFP transgenic reporter (complete Oct4 enhancer region with distal and proximal enhancer elements) (Addgene plasmid #60527)) and WT-1 cell line (WT-1 clone that carries the deltaPE-GOF18-Oct4-GFP reporter (Addgene plasmid#52382) were previously described ( Rais et al., 2013 ).

Techniques: Recombinant, Protease Inhibitor, Sample Prep, Methylation, Transgenic Assay, Negative Control, Software

Journal: Circulation Research

Article Title: Jmjd3 Controls Mesodermal and Cardiovascular Differentiation of Embryonic Stem Cells

doi: 10.1161/circresaha.113.302035

Figure Lengend Snippet: Figure 1. Aberrant differentiation of Jmjd3-/- ESCs. (A) qPCR analysis of Jmjd3 in WT and Jmjd3-/- ESCs. (B) Western blot analysis of Jmjd3 and Histone marks in WT and Jmjd3-/- ESCs. Histone H3 is used as a loading control. Quantification is shown in the right panel (N=3-5). (C) Upper panel: Morphology of WT and Jmjd3-/- ESCs on feeder cells. Lower panel: Alkaline phosphatase staining of undifferentiated WT and Jmjd3-/- ESCs. (D) Western blot analysis of Jmjd3 and Oct4 in WT ESCs during differentiation. Į-Tubulin is used as a loading control. (E) Bright field image of EBs at day 5. Scale bar indicates 200μm. (F) Gene ontology analysis for more than 2-fold repressed genes in Jmjd3-/- ESCs compared to WT ESCs 4 days after differentiation. The most highly represented categories are presented with ontology terms on the y-axis and p-values for the significance of enrichment are shown on the x-axis. (G) Gene expression changes of pluripotency and lineage-specific markers in WT and Jmjd3-/- ESCs after spontaneous differentiation by LIF withdrawal. N=4.

Article Snippet: Immunofluorescence was performed as previously described 4 using antibody against Oct4 (2750 Cell Signaling), Nanog (ab80892 abcam), H3K27me3 (CS200603 Millipore), αMHC (sc-20641 Santa Cruz), Pecam-1 (550274 BD Biosciences) and Phalloidin (MFP-A1379 Mo Bi Tec).

Techniques: Western Blot, Control, Staining, Gene Expression

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