Journal: bioRxiv

Article Title: Dynamic regulation of chromatin accessibility by pluripotency transcription factors across the cell cycle

doi: 10.1101/698571

Figure Lengend Snippet: (A) Experimental strategy to compare the effect of OCT4 and SOX2 depletion on chromatin accessibility. (B) Number of regions significantly changed in accessibility upon OCT4 (left) and SOX2 (right) depletion in distal (>1 kb from TSS) and promoter-proximal (<1 kb from TSS) elements. (C) log2 fold-change values of accessibility between dox-treated and untreated cells upon OCT4/SOX2 depletion at OCT4/SOX2 binding sites with decreased accessibility. Loci are grouped into those significantly affected upon OCT4 depletion (OD), SOX2 depletion (SD), or depletion of either factor (CD). Each row corresponds to one individual locus, and each column to a different experimental condition. (D-E) Average RPKM-normalized ATAC-seq signal 2 kb around OD, CD, and SD loci upon SOX2 depletion (D) and OCT4 depletion (E). (F-G) Average RPKM-normalized H3K27ac ChIP-seq signal 2 kb around OD, CD, and SD loci upon SOX2 depletion (F) and OCT4 depletion (G).

Article Snippet: pLV-PGK-YPet-MD was derived from pLVTRE3G-Sox2-YPet-MD ( ) by amplification of YPet-MD and restriction cloning into pLV-rtTA3G-IRESBsd using AgeI and SalI. pLV-PGK-SNAP-MD-OCT4 and pLV-PGK-SNAP-MD*-OCT4 were derived by amplification of MD or MD* from SNAP-MD-SOX2 (Addgene #115687) and SNAP-MD*-SOX2 (Addgene #115688) ( ) and restriction cloning into pLVTRE3G-Oct4-YPet ( ) using SalI and XbaI.

Techniques: Binding Assay, ChIP-sequencing

Journal: bioRxiv

Article Title: Dynamic regulation of chromatin accessibility by pluripotency transcription factors across the cell cycle

doi: 10.1101/698571

Figure Lengend Snippet: (A) Immunofluorescence of 2TS22C cells stained for DNA (DAPI), OCT4, and SOX2 without dox treatment (left), and after 26 hours (middle), and 40 hours (right) of dox treatment. (B) Violin plot of background-subtracted log values of immunofluorescence signal in OCT4 (left) and SOX2 (right) channels upon SOX2 depletion. Control: n=45’601 cells from 4 biological replicates including 2 technical replicates; 26 hours: n=42’298 cells from 3 biological replicates including 2 technical replicates; 40 hours: n= 32’342 cells from 2 technical replicates. Dots: mean; Vertical lines: standard deviation. (C) Immunofluorescence of ZHBTc4 cells stained for DNA (DAPI), OCT4, and SOX2 without dox treatment (left), and after 24 hours of dox treatment (right). (D) Violin plot of background-subtracted log values of immunofluorescence signal in OCT4 (left) and SOX2 (right) channels upon OCT4 depletion. Control: n=26’119 cells from 3 biological replicates. 24 hours: n=23’157 cells from 3 biological replicates. Dots: mean; Vertical lines: standard deviation. (E) Correlation between the log2 fold-change values of accessibility upon OCT4 depletion in S2iL (x-axis) and SL (y-axis) at OCT4-bound sites. (F) Correlation between the log2 fold-change values of accessibility upon SOX2 depletion after 26 hours (x-axis) and 40 hours (y-axis) of dox treatment at SOX2 binding sites. R is Pearson correlation coefficient. Scale bars: 30 μm.

Article Snippet: pLV-PGK-YPet-MD was derived from pLVTRE3G-Sox2-YPet-MD ( ) by amplification of YPet-MD and restriction cloning into pLV-rtTA3G-IRESBsd using AgeI and SalI. pLV-PGK-SNAP-MD-OCT4 and pLV-PGK-SNAP-MD*-OCT4 were derived by amplification of MD or MD* from SNAP-MD-SOX2 (Addgene #115687) and SNAP-MD*-SOX2 (Addgene #115688) ( ) and restriction cloning into pLVTRE3G-Oct4-YPet ( ) using SalI and XbaI.

Techniques: Immunofluorescence, Staining, Standard Deviation, Binding Assay

Journal: bioRxiv

Article Title: Dynamic regulation of chromatin accessibility by pluripotency transcription factors across the cell cycle

doi: 10.1101/698571

Figure Lengend Snippet: Heatmaps of RPKM-normalized ATAC-seq and ChIP-seq binding profiles upon OCT4 (A) and SOX2 (B) depletion 5 kb around OCT4-regulated (A) and SOX2-regulated (B) loci. Each row represents one individual locus and each column represents one experimental condition.

Article Snippet: pLV-PGK-YPet-MD was derived from pLVTRE3G-Sox2-YPet-MD ( ) by amplification of YPet-MD and restriction cloning into pLV-rtTA3G-IRESBsd using AgeI and SalI. pLV-PGK-SNAP-MD-OCT4 and pLV-PGK-SNAP-MD*-OCT4 were derived by amplification of MD or MD* from SNAP-MD-SOX2 (Addgene #115687) and SNAP-MD*-SOX2 (Addgene #115688) ( ) and restriction cloning into pLVTRE3G-Oct4-YPet ( ) using SalI and XbaI.

Techniques: ChIP-sequencing, Binding Assay

Journal: bioRxiv

Article Title: Dynamic regulation of chromatin accessibility by pluripotency transcription factors across the cell cycle

doi: 10.1101/698571

Figure Lengend Snippet: (A) Experimental strategy used to assess the impact of OCT4 depletion at the M-G1 transition. (B-C) Genome browser tracks of RPKM-normalized accessibility profiles across the cell cycle for one locus that decreases (B) at chr11:6894809-6895533 and one that increases (C) at chr9:41247953-4124841 in accessibility upon transient OCT4 depletion in M-G1. (D) log2 fold-change values of accessibility between MD-OCT4 and MD*-OCT4 (control) cells in different cell cycle phases at all accessible OCT4-bound sites, grouped into four clusters by k-means clustering (see Methods). Each line represents one locus. Red line: mean of each cluster. (E) Frequency of overlap with the canonical OCT4::SOX2 motif in the four clusters as well as in background regions (BG). (F) Average RPKM-normalized OCT4 ChIP-seq signal in untreated ZHBTc4 cells 2 kb around loci in the four clusters.

Article Snippet: pLV-PGK-YPet-MD was derived from pLVTRE3G-Sox2-YPet-MD ( ) by amplification of YPet-MD and restriction cloning into pLV-rtTA3G-IRESBsd using AgeI and SalI. pLV-PGK-SNAP-MD-OCT4 and pLV-PGK-SNAP-MD*-OCT4 were derived by amplification of MD or MD* from SNAP-MD-SOX2 (Addgene #115687) and SNAP-MD*-SOX2 (Addgene #115688) ( ) and restriction cloning into pLVTRE3G-Oct4-YPet ( ) using SalI and XbaI.

Techniques: ChIP-sequencing

Journal: bioRxiv

Article Title: Dynamic regulation of chromatin accessibility by pluripotency transcription factors across the cell cycle

doi: 10.1101/698571

Figure Lengend Snippet: (A) Heatmap of RPKM-normalized OCT4 and SOX2 ChIP-seq binding profiles in untreated ZHBTc4 cells at OD, CD, and SD loci. Each row represents one individual locus. (B-C) Average RPKM-normalized BRG1 ChIP-seq signal 2 kb around OD, CD, and SD loci upon SOX2 depletion (B) and OCT4 depletion (C). (D-F) Frequency of overlap with motifs at OD, CD, and SD loci as well as in background regions (BG) for the canonical OCT4::SOX2 motif (D), the OCT motif (E), and the SOX motif (F). (G-I) Enrichment (-log(p)) values for the closest gene in the OD, CD, and SD groups in the gene ontology sets PluriNetWork (G), ESC Pluripotency Pathways (H), and the KEGG gene set “Signaling pathways regulating pluripotency” (I). (J-K) Average RPKM-normalized OCT4 (J) and SOX2 (K) ChIP-seq signal 2 kb around OD, CD, and SD loci upon SOX2 depletion (J) and OCT4 depletion (K)

Article Snippet: pLV-PGK-YPet-MD was derived from pLVTRE3G-Sox2-YPet-MD ( ) by amplification of YPet-MD and restriction cloning into pLV-rtTA3G-IRESBsd using AgeI and SalI. pLV-PGK-SNAP-MD-OCT4 and pLV-PGK-SNAP-MD*-OCT4 were derived by amplification of MD or MD* from SNAP-MD-SOX2 (Addgene #115687) and SNAP-MD*-SOX2 (Addgene #115688) ( ) and restriction cloning into pLVTRE3G-Oct4-YPet ( ) using SalI and XbaI.

Techniques: ChIP-sequencing, Binding Assay

Journal: bioRxiv

Article Title: Dynamic regulation of chromatin accessibility by pluripotency transcription factors across the cell cycle

doi: 10.1101/698571

Figure Lengend Snippet: (A) Average ATAC-seq signal 2 kb around OD, CD, and SD loci in BRG1fl cells that were treated with tamoxifen (TAM) or left untreated. (B) Frequency of the AP-2 motif 2 kb around OD, CD, and SD loci, and in background regions (BG). (C) Average SOX2 ChIP-seq signal in TS cells 2 kb around OD, CD, and SD loci. (D) Average ATAC-seq signal in TS cells 2 kb around OD, CD, and SD loci. (E) Percentage of the closest gene in the OD, CD, and SD groups as well as all other accessible regions (Other) whose nascent RNA levels are downregulated or upregulated upon 24 hours of OCT4 depletion (F) Average ChIP-seq signal of ESRRB, NANOG, KLF4, and SALL4 in ES cells 2 kb around OD, CD, and SD loci. (G) Enrichment (-log(p)) values for the closest gene in the OD, CD, and SD groups in the “Cell differentiation” gene ontology set. (H) SOX2 binding profiles 2 kb around OD, CD, and SD loci in wt and PARP1 KO ES cells.

Article Snippet: pLV-PGK-YPet-MD was derived from pLVTRE3G-Sox2-YPet-MD ( ) by amplification of YPet-MD and restriction cloning into pLV-rtTA3G-IRESBsd using AgeI and SalI. pLV-PGK-SNAP-MD-OCT4 and pLV-PGK-SNAP-MD*-OCT4 were derived by amplification of MD or MD* from SNAP-MD-SOX2 (Addgene #115687) and SNAP-MD*-SOX2 (Addgene #115688) ( ) and restriction cloning into pLVTRE3G-Oct4-YPet ( ) using SalI and XbaI.

Techniques: ChIP-sequencing, Cell Differentiation, Binding Assay

Journal: bioRxiv

Article Title: Dynamic regulation of chromatin accessibility by pluripotency transcription factors across the cell cycle

doi: 10.1101/698571

Figure Lengend Snippet: (A) Gate used to sort SNAP-MD-OCT4 (left) and SNAP-MD*-OCT4 (right) cells for the same average SNAP-Cell 647-SiR signal. Y-axis: Signal amplitude at 405 nm excitation and 526/52 nm emission (negative control). X-axis: Signal amplitude at 640 nm excitation and 671/30 nm emission (SNAP signal). (B) Example of a sorting experiment for different phases of the cell cycle in cells expressing YPet-MD and stained for Hoechst33258. Y-axis: Integrated signal at 488 nm excitation and 525/50 nm emission (YPet). X-axis: Signal amplitude at 355 nm excitation and 450/50 nm emission (Hoechst) (C) Correlation between YPet-MD and SNAP-MD-OCT4 expression in MD-OCT4 cells as measured by flow cytometry. Y-axis: Integrated signal at 640 nm excitation and 670/14 nm emission (SNAP). X-axis: Integrated signal at 488 nm excitation and 525/50 nm emission (YPet). (D) Violin plot of log2 fold-change values of accessibility between MD-OCT4 and MD*-OCT4 cells in significantly downregulated and upregulated loci (see ) in unsorted cells in the absence of dox. Dots: mean; Vertical lines: standard deviation. (E) Percentage of dome-shaped colonies as assessed by microscopy in the ZHBTc4 cell line upon dox treatment and with overexpression of SNAP-MD*-OCT4 or SNAP-MD-OCT4. n=3 biological replicates. (F) Representative alkaline phosphatase staining from cells in (E). (G) Fold-change of expression levels of differentiation markers (Dlx3, Eomes and Esx1) and Nanog, measured by qRT-PCR in dox-treated versus untreated cells, in MD-OCT4 and MD*-OCT4 cells. Each sample is normalized to the expression of Rps9. n=4 biological replicates. (H) Percentage of cells in EG1/LG1/S/SG2 phases as determined by flow cytometry in MD-OCT4 and MD*-OCT4 cells. n=4 biological replicates. (I-J) Violin plot of log2 fold-change values of accessibility between MD-OCT4 and MD*-OCT4 cells in different cell cycle phases at significantly downregulated (I) and upregulated (J) loci (see ). Dots: mean; Vertical lines: standard deviation.

Article Snippet: pLV-PGK-YPet-MD was derived from pLVTRE3G-Sox2-YPet-MD ( ) by amplification of YPet-MD and restriction cloning into pLV-rtTA3G-IRESBsd using AgeI and SalI. pLV-PGK-SNAP-MD-OCT4 and pLV-PGK-SNAP-MD*-OCT4 were derived by amplification of MD or MD* from SNAP-MD-SOX2 (Addgene #115687) and SNAP-MD*-SOX2 (Addgene #115688) ( ) and restriction cloning into pLVTRE3G-Oct4-YPet ( ) using SalI and XbaI.

Techniques: Negative Control, Expressing, Staining, Flow Cytometry, Standard Deviation, Microscopy, Over Expression, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Dynamic regulation of chromatin accessibility by pluripotency transcription factors across the cell cycle

doi: 10.1101/698571

Figure Lengend Snippet: (A) Violin plot of distance to closest TSS in clusters from . Dots: mean; Vertical lines: standard deviation. (B) Heatmap of ChIP-seq signal of H3K4me3, H3K4me1, and H3K27ac in wt ES cells in the different clusters. (C) Average log2 fold-change values of H3K27ac ChIP-seq signal between MD-OCT4 and MD*-OCT4 cells in the different clusters (including 500bp flanking regions at each side) at different cell cycle stages. (D) Genome browser tracks of RPKM-normalized H3K27ac profiles across the cell cycle for a cluster 1 locus (chr11:6894809-6895533) that decreases in accessibility and H3K27ac upon transient OCT4 depletion in M-G1. (E) Percentage of loci in the different clusters and at non-OCT4 bound regions overlapping typical enhancers (TE) and super-enhancers (SE) in mouse ES cells. (F) ATAC-seq signal in cells sorted for high and low endogenous OCT4 levels in the different clusters. (G) Percentage of loci in the different clusters overlapping OD, CD, and SD loci (see ).

Article Snippet: pLV-PGK-YPet-MD was derived from pLVTRE3G-Sox2-YPet-MD ( ) by amplification of YPet-MD and restriction cloning into pLV-rtTA3G-IRESBsd using AgeI and SalI. pLV-PGK-SNAP-MD-OCT4 and pLV-PGK-SNAP-MD*-OCT4 were derived by amplification of MD or MD* from SNAP-MD-SOX2 (Addgene #115687) and SNAP-MD*-SOX2 (Addgene #115688) ( ) and restriction cloning into pLVTRE3G-Oct4-YPet ( ) using SalI and XbaI.

Techniques: Standard Deviation, ChIP-sequencing

Journal: bioRxiv

Article Title: Dynamic regulation of chromatin accessibility by pluripotency transcription factors across the cell cycle

doi: 10.1101/698571

Figure Lengend Snippet: (A) Correlation between the log of normalized OCT4 ChIP-seq reads per bp (x-axis) and the log2 fold-change values of accessibility loss upon OCT4 depletion (y-axis) at all OCT4 binding sites in ZHBTc4 cells. (B) Correlation between the log of normalized OCT4 ChIP-seq reads per bp (x-axis) and the log of normalized ATAC-seq reads per bp (y-axis) at all OCT4 binding sites in ZHBTc4 cells. R is Pearson correlation coefficient.

Article Snippet: pLV-PGK-YPet-MD was derived from pLVTRE3G-Sox2-YPet-MD ( ) by amplification of YPet-MD and restriction cloning into pLV-rtTA3G-IRESBsd using AgeI and SalI. pLV-PGK-SNAP-MD-OCT4 and pLV-PGK-SNAP-MD*-OCT4 were derived by amplification of MD or MD* from SNAP-MD-SOX2 (Addgene #115687) and SNAP-MD*-SOX2 (Addgene #115688) ( ) and restriction cloning into pLVTRE3G-Oct4-YPet ( ) using SalI and XbaI.

Techniques: ChIP-sequencing, Binding Assay

Journal: bioRxiv

Article Title: Dynamic regulation of chromatin accessibility by pluripotency transcription factors across the cell cycle

doi: 10.1101/698571

Figure Lengend Snippet: (A) Experimental strategy used to assess the impact of OCT4 depletion and recovery at different cell cycle phases. (B) Red fluorescence (mCherry) signal in mCherry-OCT4-AID cells treated with IAA at t=0 as measured by fluorescence microscopy. Gray lines: single cell traces; Black line: population average; Red line: exponential fit. Red text: half-life value derived from the exponential fit. (C) Red fluorescence (mCherry) signal in mCherry-OCT4-AID treated with IAA for 2.5h and then washed out at t=0 as measured by fluorescence microscopy. Gray lines: single cell traces; Black line: population average. (D) Average log2 fold-change values of accessibility between IAA-treated and untreated OCT4-AID cells in the four clusters from at each cell cycle phase. (E) Average log2 fold-change values of accessibility between cells first treated with IAA and then washed out, compared to untreated OCT4-AID cells for the four clusters from at each cell cycle phase.

Article Snippet: pLV-PGK-YPet-MD was derived from pLVTRE3G-Sox2-YPet-MD ( ) by amplification of YPet-MD and restriction cloning into pLV-rtTA3G-IRESBsd using AgeI and SalI. pLV-PGK-SNAP-MD-OCT4 and pLV-PGK-SNAP-MD*-OCT4 were derived by amplification of MD or MD* from SNAP-MD-SOX2 (Addgene #115687) and SNAP-MD*-SOX2 (Addgene #115688) ( ) and restriction cloning into pLVTRE3G-Oct4-YPet ( ) using SalI and XbaI.

Techniques: Fluorescence, Microscopy, Derivative Assay

Journal: bioRxiv

Article Title: Dynamic regulation of chromatin accessibility by pluripotency transcription factors across the cell cycle

doi: 10.1101/698571

Figure Lengend Snippet: (A) Histogram of mCherry signal in untreated mCherry OCT4-AID cells and treated with IAA for 2 hours as well as mCherry negative E14 ES cells as measured by flow cytometry. X-axis: Integrated signal at 561 nm excitation and 610/20 nm emission. Y-axis: Counts. (B-D) Fold-change of red fluorescence (mCherry) signal between treated and untreated mCherry-OCT4-AID cells as determined by flow cytometry in different cell cycle phases upon 2 h IAA treatment (B), 0.5 h IAA treatment (C), and after 2.5 h IAA treatment followed by 4.5 h of washout (D). n=3 biological replicates. (E-F) Genome browser tracks of accessibility profiles of a cluster 1 locus at chr10:95455826-95456819 after IAA treatment (E) and after IAA treatment followed by washout (F).

Article Snippet: pLV-PGK-YPet-MD was derived from pLVTRE3G-Sox2-YPet-MD ( ) by amplification of YPet-MD and restriction cloning into pLV-rtTA3G-IRESBsd using AgeI and SalI. pLV-PGK-SNAP-MD-OCT4 and pLV-PGK-SNAP-MD*-OCT4 were derived by amplification of MD or MD* from SNAP-MD-SOX2 (Addgene #115687) and SNAP-MD*-SOX2 (Addgene #115688) ( ) and restriction cloning into pLVTRE3G-Oct4-YPet ( ) using SalI and XbaI.

Techniques: Flow Cytometry, Fluorescence

Journal: bioRxiv

Article Title: Dynamic regulation of chromatin accessibility by pluripotency transcription factors across the cell cycle

doi: 10.1101/698571

Figure Lengend Snippet: (A) Experimental strategy to compare the effect of OCT4 and SOX2 depletion on chromatin accessibility. (B) Number of regions significantly changed in accessibility upon OCT4 (left) and SOX2 (right) depletion in distal (>1 kb from TSS) and promoter-proximal (<1 kb from TSS) elements. (C) log2 fold-change values of accessibility between dox-treated and untreated cells upon OCT4/SOX2 depletion at OCT4/SOX2 binding sites with decreased accessibility. Loci are grouped into those significantly affected upon OCT4 depletion (OD), SOX2 depletion (SD), or depletion of either factor (CD). Each row corresponds to one individual locus, and each column to a different experimental condition. (D-E) Average RPKM-normalized ATAC-seq signal 2 kb around OD, CD, and SD loci upon SOX2 depletion (D) and OCT4 depletion (E). (F-G) Average RPKM-normalized H3K27ac ChIP-seq signal 2 kb around OD, CD, and SD loci upon SOX2 depletion (F) and OCT4 depletion (G).

Article Snippet: pLV-PGK-YPet-MD was derived from pLVTRE3G-Sox2-YPet-MD ( ) by amplification of YPet-MD and restriction cloning into pLV-rtTA3G-IRESBsd using AgeI and SalI. pLV-PGK-SNAP-MD-OCT4 and pLV-PGK-SNAP-MD*-OCT4 were derived by amplification of MD or MD* from SNAP-MD-SOX2 (Addgene #115687) and SNAP-MD*-SOX2 (Addgene #115688) ( ) and restriction cloning into pLVTRE3G-Oct4-YPet ( ) using SalI and XbaI.

Techniques: Binding Assay, ChIP-sequencing

Journal: bioRxiv

Article Title: Dynamic regulation of chromatin accessibility by pluripotency transcription factors across the cell cycle

doi: 10.1101/698571

Figure Lengend Snippet: (A) Immunofluorescence of 2TS22C cells stained for DNA (DAPI), OCT4, and SOX2 without dox treatment (left), and after 26 hours (middle), and 40 hours (right) of dox treatment. (B) Violin plot of background-subtracted log values of immunofluorescence signal in OCT4 (left) and SOX2 (right) channels upon SOX2 depletion. Control: n=45’601 cells from 4 biological replicates including 2 technical replicates; 26 hours: n=42’298 cells from 3 biological replicates including 2 technical replicates; 40 hours: n= 32’342 cells from 2 technical replicates. Dots: mean; Vertical lines: standard deviation. (C) Immunofluorescence of ZHBTc4 cells stained for DNA (DAPI), OCT4, and SOX2 without dox treatment (left), and after 24 hours of dox treatment (right). (D) Violin plot of background-subtracted log values of immunofluorescence signal in OCT4 (left) and SOX2 (right) channels upon OCT4 depletion. Control: n=26’119 cells from 3 biological replicates. 24 hours: n=23’157 cells from 3 biological replicates. Dots: mean; Vertical lines: standard deviation. (E) Correlation between the log2 fold-change values of accessibility upon OCT4 depletion in S2iL (x-axis) and SL (y-axis) at OCT4-bound sites. (F) Correlation between the log2 fold-change values of accessibility upon SOX2 depletion after 26 hours (x-axis) and 40 hours (y-axis) of dox treatment at SOX2 binding sites. R is Pearson correlation coefficient. Scale bars: 30 μm.

Article Snippet: pLV-PGK-YPet-MD was derived from pLVTRE3G-Sox2-YPet-MD ( ) by amplification of YPet-MD and restriction cloning into pLV-rtTA3G-IRESBsd using AgeI and SalI. pLV-PGK-SNAP-MD-OCT4 and pLV-PGK-SNAP-MD*-OCT4 were derived by amplification of MD or MD* from SNAP-MD-SOX2 (Addgene #115687) and SNAP-MD*-SOX2 (Addgene #115688) ( ) and restriction cloning into pLVTRE3G-Oct4-YPet ( ) using SalI and XbaI.

Techniques: Immunofluorescence, Staining, Standard Deviation, Binding Assay

Journal: bioRxiv

Article Title: Dynamic regulation of chromatin accessibility by pluripotency transcription factors across the cell cycle

doi: 10.1101/698571

Figure Lengend Snippet: Heatmaps of RPKM-normalized ATAC-seq and ChIP-seq binding profiles upon OCT4 (A) and SOX2 (B) depletion 5 kb around OCT4-regulated (A) and SOX2-regulated (B) loci. Each row represents one individual locus and each column represents one experimental condition.

Article Snippet: pLV-PGK-YPet-MD was derived from pLVTRE3G-Sox2-YPet-MD ( ) by amplification of YPet-MD and restriction cloning into pLV-rtTA3G-IRESBsd using AgeI and SalI. pLV-PGK-SNAP-MD-OCT4 and pLV-PGK-SNAP-MD*-OCT4 were derived by amplification of MD or MD* from SNAP-MD-SOX2 (Addgene #115687) and SNAP-MD*-SOX2 (Addgene #115688) ( ) and restriction cloning into pLVTRE3G-Oct4-YPet ( ) using SalI and XbaI.

Techniques: ChIP-sequencing, Binding Assay

Journal: bioRxiv

Article Title: Dynamic regulation of chromatin accessibility by pluripotency transcription factors across the cell cycle

doi: 10.1101/698571

Figure Lengend Snippet: (A) Experimental strategy used to assess the impact of OCT4 depletion at the M-G1 transition. (B-C) Genome browser tracks of RPKM-normalized accessibility profiles across the cell cycle for one locus that decreases (B) at chr11:6894809-6895533 and one that increases (C) at chr9:41247953-4124841 in accessibility upon transient OCT4 depletion in M-G1. (D) log2 fold-change values of accessibility between MD-OCT4 and MD*-OCT4 (control) cells in different cell cycle phases at all accessible OCT4-bound sites, grouped into four clusters by k-means clustering (see Methods). Each line represents one locus. Red line: mean of each cluster. (E) Frequency of overlap with the canonical OCT4::SOX2 motif in the four clusters as well as in background regions (BG). (F) Average RPKM-normalized OCT4 ChIP-seq signal in untreated ZHBTc4 cells 2 kb around loci in the four clusters.

Article Snippet: pLV-PGK-YPet-MD was derived from pLVTRE3G-Sox2-YPet-MD ( ) by amplification of YPet-MD and restriction cloning into pLV-rtTA3G-IRESBsd using AgeI and SalI. pLV-PGK-SNAP-MD-OCT4 and pLV-PGK-SNAP-MD*-OCT4 were derived by amplification of MD or MD* from SNAP-MD-SOX2 (Addgene #115687) and SNAP-MD*-SOX2 (Addgene #115688) ( ) and restriction cloning into pLVTRE3G-Oct4-YPet ( ) using SalI and XbaI.

Techniques: ChIP-sequencing

Journal: bioRxiv

Article Title: Dynamic regulation of chromatin accessibility by pluripotency transcription factors across the cell cycle

doi: 10.1101/698571

Figure Lengend Snippet: (A) Heatmap of RPKM-normalized OCT4 and SOX2 ChIP-seq binding profiles in untreated ZHBTc4 cells at OD, CD, and SD loci. Each row represents one individual locus. (B-C) Average RPKM-normalized BRG1 ChIP-seq signal 2 kb around OD, CD, and SD loci upon SOX2 depletion (B) and OCT4 depletion (C). (D-F) Frequency of overlap with motifs at OD, CD, and SD loci as well as in background regions (BG) for the canonical OCT4::SOX2 motif (D), the OCT motif (E), and the SOX motif (F). (G-I) Enrichment (-log(p)) values for the closest gene in the OD, CD, and SD groups in the gene ontology sets PluriNetWork (G), ESC Pluripotency Pathways (H), and the KEGG gene set “Signaling pathways regulating pluripotency” (I). (J-K) Average RPKM-normalized OCT4 (J) and SOX2 (K) ChIP-seq signal 2 kb around OD, CD, and SD loci upon SOX2 depletion (J) and OCT4 depletion (K)

Article Snippet: pLV-PGK-YPet-MD was derived from pLVTRE3G-Sox2-YPet-MD ( ) by amplification of YPet-MD and restriction cloning into pLV-rtTA3G-IRESBsd using AgeI and SalI. pLV-PGK-SNAP-MD-OCT4 and pLV-PGK-SNAP-MD*-OCT4 were derived by amplification of MD or MD* from SNAP-MD-SOX2 (Addgene #115687) and SNAP-MD*-SOX2 (Addgene #115688) ( ) and restriction cloning into pLVTRE3G-Oct4-YPet ( ) using SalI and XbaI.

Techniques: ChIP-sequencing, Binding Assay

Journal: bioRxiv

Article Title: Dynamic regulation of chromatin accessibility by pluripotency transcription factors across the cell cycle

doi: 10.1101/698571

Figure Lengend Snippet: (A) Average ATAC-seq signal 2 kb around OD, CD, and SD loci in BRG1fl cells that were treated with tamoxifen (TAM) or left untreated. (B) Frequency of the AP-2 motif 2 kb around OD, CD, and SD loci, and in background regions (BG). (C) Average SOX2 ChIP-seq signal in TS cells 2 kb around OD, CD, and SD loci. (D) Average ATAC-seq signal in TS cells 2 kb around OD, CD, and SD loci. (E) Percentage of the closest gene in the OD, CD, and SD groups as well as all other accessible regions (Other) whose nascent RNA levels are downregulated or upregulated upon 24 hours of OCT4 depletion (F) Average ChIP-seq signal of ESRRB, NANOG, KLF4, and SALL4 in ES cells 2 kb around OD, CD, and SD loci. (G) Enrichment (-log(p)) values for the closest gene in the OD, CD, and SD groups in the “Cell differentiation” gene ontology set. (H) SOX2 binding profiles 2 kb around OD, CD, and SD loci in wt and PARP1 KO ES cells.

Article Snippet: pLV-PGK-YPet-MD was derived from pLVTRE3G-Sox2-YPet-MD ( ) by amplification of YPet-MD and restriction cloning into pLV-rtTA3G-IRESBsd using AgeI and SalI. pLV-PGK-SNAP-MD-OCT4 and pLV-PGK-SNAP-MD*-OCT4 were derived by amplification of MD or MD* from SNAP-MD-SOX2 (Addgene #115687) and SNAP-MD*-SOX2 (Addgene #115688) ( ) and restriction cloning into pLVTRE3G-Oct4-YPet ( ) using SalI and XbaI.

Techniques: ChIP-sequencing, Cell Differentiation, Binding Assay