Journal: eLife

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

doi: 10.7554/eLife.50087

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 Materials and methods). Each line represents one locus. Red line: mean. ( E ) Frequency of overlap (bar) and enrichment p-values (white digits) of the canonical OCT4::SOX2 motif in the four clusters as well as in unbound regions. The color shows the number of identified OCT4::SOX2 motifs per region. ( F ) Average RPKM-normalized OCT4 ChIP-seq signal in untreated ZHBTc4 cells 2 kb around loci in the four clusters. Statistics are available in . EG1: Early G1 phase; LG1: Late G1 phase; S: S phase; SG2: Late S and G2 phase.

Article Snippet: SNAP-MD-OCT4 and SNAP-MD*-OCT4 were further amplified and cloned by restriction cloning into pLV-rtTA3G-IRESBsd ( ) using AgeI and SalI. pLV-pCAGGS-Tir1-V5 was derived by amplification of pCAGGS-Tir1-V5 from pEN395 (Addgene #92141) ( ) and In-fusion cloning into pLV-PGK-SOX2-SNAP-IRES-Hygro ( ) digested using XhoI and XbaI restriction enzymes. pLEX-mCherry-OCT4-AID was derived by amplification of OCT4 from pLV-PGK-SNAP-MD-OCT4, AID 71–114 from pEN244 (Addgene #92140) , and mCherry from pLV-TRE3G-mCherry-PGK-Puro (Suter lab). mCherry and OCT4 were ligated using an XmaI restriction site and mCherry-OCT4 was cloned into the pLEX_305-C-dTAG backbone (Addgene #91798) ( ) using AgeI and NheI restriction sites.

Techniques: ChIP-sequencing

Journal: eLife

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

doi: 10.7554/eLife.50087

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. n = 45 cells from one replicate ( C ) Red fluorescence (mCherry) signal in mCherry-OCT4-AID treated with IAA for 2.5 hr and then washed out at t = 0 as measured by fluorescence microscopy. Gray lines: single cell traces; Black line: population average. n = 45 cells from one replicate ( 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. EG1: Early G1 phase; LG1: Late G1 phase; S: S phase; SG2: Late S and G2 phase. Statistics for ( D–E ) are available in . Figure 4—source data 1. Time-lapse microscopy source data of mCherry-OCT4-AID signal after IAA treatment and washout . Time is in hours; Signal is the background-subtracted mean intensity; Cell is the cell unique identifier of each tracked cell; Treatment indicates IAA or Washout just prior to imaging.

Article Snippet: SNAP-MD-OCT4 and SNAP-MD*-OCT4 were further amplified and cloned by restriction cloning into pLV-rtTA3G-IRESBsd ( ) using AgeI and SalI. pLV-pCAGGS-Tir1-V5 was derived by amplification of pCAGGS-Tir1-V5 from pEN395 (Addgene #92141) ( ) and In-fusion cloning into pLV-PGK-SOX2-SNAP-IRES-Hygro ( ) digested using XhoI and XbaI restriction enzymes. pLEX-mCherry-OCT4-AID was derived by amplification of OCT4 from pLV-PGK-SNAP-MD-OCT4, AID 71–114 from pEN244 (Addgene #92140) , and mCherry from pLV-TRE3G-mCherry-PGK-Puro (Suter lab). mCherry and OCT4 were ligated using an XmaI restriction site and mCherry-OCT4 was cloned into the pLEX_305-C-dTAG backbone (Addgene #91798) ( ) using AgeI and NheI restriction sites.

Techniques: Fluorescence, Microscopy, Derivative Assay, Time-lapse Microscopy, Imaging

Journal: eLife

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

doi: 10.7554/eLife.50087

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 ). Statistics for ( D–G ) are available in .

Article Snippet: SNAP-MD-OCT4 and SNAP-MD*-OCT4 were further amplified and cloned by restriction cloning into pLV-rtTA3G-IRESBsd ( ) using AgeI and SalI. pLV-pCAGGS-Tir1-V5 was derived by amplification of pCAGGS-Tir1-V5 from pEN395 (Addgene #92141) ( ) and In-fusion cloning into pLV-PGK-SOX2-SNAP-IRES-Hygro ( ) digested using XhoI and XbaI restriction enzymes. pLEX-mCherry-OCT4-AID was derived by amplification of OCT4 from pLV-PGK-SNAP-MD-OCT4, AID 71–114 from pEN244 (Addgene #92140) , and mCherry from pLV-TRE3G-mCherry-PGK-Puro (Suter lab). mCherry and OCT4 were ligated using an XmaI restriction site and mCherry-OCT4 was cloned into the pLEX_305-C-dTAG backbone (Addgene #91798) ( ) using AgeI and NheI restriction sites.

Techniques: Binding Assay, ChIP-sequencing

Journal: eLife

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

doi: 10.7554/eLife.50087

Figure Lengend Snippet: ( A ) Immunofluorescence of 2TS22C cells stained for DNA (DAPI), OCT4, and SOX2 without dox treatment (left), and after 26 hr (middle), and 40 hr (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 four biological replicates including two technical replicates; 26 hr: n = 42’298 cells from three biological replicates including two technical replicates; 40 hr: n = 32’342 cells from two technical replicates. Dots: mean; Vertical lines: standard deviation; p-values: Mann-Whitney U. ( C ) Immunofluorescence of ZHBTc4 cells stained for DNA (DAPI), OCT4, and SOX2 without dox treatment (left), and after 24 hr 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 three biological replicates. 24 hr: n = 23’157 cells from three biological replicates. Dots: mean; Vertical lines: standard deviation; p-values: Mann-Whitney U. ( 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 hr (x-axis) and 40 hr (y-axis) of dox treatment at SOX2 binding sites. Coefficient (R) and p-values are based on the Pearson correlation coefficient. Scale bars: 30 mm.

Article Snippet: SNAP-MD-OCT4 and SNAP-MD*-OCT4 were further amplified and cloned by restriction cloning into pLV-rtTA3G-IRESBsd ( ) using AgeI and SalI. pLV-pCAGGS-Tir1-V5 was derived by amplification of pCAGGS-Tir1-V5 from pEN395 (Addgene #92141) ( ) and In-fusion cloning into pLV-PGK-SOX2-SNAP-IRES-Hygro ( ) digested using XhoI and XbaI restriction enzymes. pLEX-mCherry-OCT4-AID was derived by amplification of OCT4 from pLV-PGK-SNAP-MD-OCT4, AID 71–114 from pEN244 (Addgene #92140) , and mCherry from pLV-TRE3G-mCherry-PGK-Puro (Suter lab). mCherry and OCT4 were ligated using an XmaI restriction site and mCherry-OCT4 was cloned into the pLEX_305-C-dTAG backbone (Addgene #91798) ( ) using AgeI and NheI restriction sites.

Techniques: Immunofluorescence, Staining, Standard Deviation, MANN-WHITNEY, Binding Assay

Journal: eLife

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

doi: 10.7554/eLife.50087

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: SNAP-MD-OCT4 and SNAP-MD*-OCT4 were further amplified and cloned by restriction cloning into pLV-rtTA3G-IRESBsd ( ) using AgeI and SalI. pLV-pCAGGS-Tir1-V5 was derived by amplification of pCAGGS-Tir1-V5 from pEN395 (Addgene #92141) ( ) and In-fusion cloning into pLV-PGK-SOX2-SNAP-IRES-Hygro ( ) digested using XhoI and XbaI restriction enzymes. pLEX-mCherry-OCT4-AID was derived by amplification of OCT4 from pLV-PGK-SNAP-MD-OCT4, AID 71–114 from pEN244 (Addgene #92140) , and mCherry from pLV-TRE3G-mCherry-PGK-Puro (Suter lab). mCherry and OCT4 were ligated using an XmaI restriction site and mCherry-OCT4 was cloned into the pLEX_305-C-dTAG backbone (Addgene #91798) ( ) using AgeI and NheI restriction sites.

Techniques: ChIP-sequencing, Binding Assay

Journal: eLife

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

doi: 10.7554/eLife.50087

Figure Lengend Snippet: ( A ) Classification of all OCT4 and SOX2 binding sites into OD, CD, and SD loci as well as loci that were discarded due to differences in untreated cells between conditions or cell lines (Discarded), due to incongruent effect on accessibility after depletion in different conditions (Incongruent), and those that were increased in accessibility or unaffected by depletion. ( B ) ChromHMM signal enrichment at OD, CD, and SD loci as well as bound loci that were unaffected by depletion and loci not bound by OCT4 or SOX2.

Article Snippet: SNAP-MD-OCT4 and SNAP-MD*-OCT4 were further amplified and cloned by restriction cloning into pLV-rtTA3G-IRESBsd ( ) using AgeI and SalI. pLV-pCAGGS-Tir1-V5 was derived by amplification of pCAGGS-Tir1-V5 from pEN395 (Addgene #92141) ( ) and In-fusion cloning into pLV-PGK-SOX2-SNAP-IRES-Hygro ( ) digested using XhoI and XbaI restriction enzymes. pLEX-mCherry-OCT4-AID was derived by amplification of OCT4 from pLV-PGK-SNAP-MD-OCT4, AID 71–114 from pEN244 (Addgene #92140) , and mCherry from pLV-TRE3G-mCherry-PGK-Puro (Suter lab). mCherry and OCT4 were ligated using an XmaI restriction site and mCherry-OCT4 was cloned into the pLEX_305-C-dTAG backbone (Addgene #91798) ( ) using AgeI and NheI restriction sites.

Techniques: Binding Assay

Journal: eLife

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

doi: 10.7554/eLife.50087

Figure Lengend Snippet: ( A ) Heatmap of RPKM-normalized OCT4 and SOX2 ChIP-seq binding profiles in untreated ZHBTc4 cells 5 kb around 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 (bar) and enrichment p-values (white digits) of 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 ) Relative enrichment values (bar) and p-values (white digits) for the closest genes 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 ). Statistics for ( B–C ), ( J–K ) are available in .

Article Snippet: SNAP-MD-OCT4 and SNAP-MD*-OCT4 were further amplified and cloned by restriction cloning into pLV-rtTA3G-IRESBsd ( ) using AgeI and SalI. pLV-pCAGGS-Tir1-V5 was derived by amplification of pCAGGS-Tir1-V5 from pEN395 (Addgene #92141) ( ) and In-fusion cloning into pLV-PGK-SOX2-SNAP-IRES-Hygro ( ) digested using XhoI and XbaI restriction enzymes. pLEX-mCherry-OCT4-AID was derived by amplification of OCT4 from pLV-PGK-SNAP-MD-OCT4, AID 71–114 from pEN244 (Addgene #92140) , and mCherry from pLV-TRE3G-mCherry-PGK-Puro (Suter lab). mCherry and OCT4 were ligated using an XmaI restriction site and mCherry-OCT4 was cloned into the pLEX_305-C-dTAG backbone (Addgene #91798) ( ) using AgeI and NheI restriction sites.

Techniques: ChIP-sequencing, Binding Assay

Journal: eLife

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

doi: 10.7554/eLife.50087

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 overlap (bar) and enrichment p-values (white digits) of the AP-2 motif 2 kb around OD, CD, and SD loci, and in background regions (BG). ( C ) Average ATAC-seq signal in TS cells 2 kb around OD, CD, and SD loci. ( D ) Average SOX2 ChIP-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 hr of OCT4 depletion. p-values: Fisher’s exact test. ( F ) Average ChIP-seq signal of ESRRB, NANOG, KLF4, and SALL4 in ES cells 2 kb around OD, CD, and SD loci. ( G ) Relative enrichment values (bar) and p-values (white digits) for the closest genes in the OD, CD, and SD groups, as well as loci upregulated upon SOX2 and OCT4 depletion, in the ‘Cell differentiation’ gene ontology set. ( H ) Average ChIP-seq signal of SOX2 2 kb around OD, CD, and SD loci in wt and PARP1 KO ES cells. Statistics for ( A ), ( C–D ), ( F ), ( H ) are available in .

Article Snippet: SNAP-MD-OCT4 and SNAP-MD*-OCT4 were further amplified and cloned by restriction cloning into pLV-rtTA3G-IRESBsd ( ) using AgeI and SalI. pLV-pCAGGS-Tir1-V5 was derived by amplification of pCAGGS-Tir1-V5 from pEN395 (Addgene #92141) ( ) and In-fusion cloning into pLV-PGK-SOX2-SNAP-IRES-Hygro ( ) digested using XhoI and XbaI restriction enzymes. pLEX-mCherry-OCT4-AID was derived by amplification of OCT4 from pLV-PGK-SNAP-MD-OCT4, AID 71–114 from pEN244 (Addgene #92140) , and mCherry from pLV-TRE3G-mCherry-PGK-Puro (Suter lab). mCherry and OCT4 were ligated using an XmaI restriction site and mCherry-OCT4 was cloned into the pLEX_305-C-dTAG backbone (Addgene #91798) ( ) using AgeI and NheI restriction sites.

Techniques: ChIP-sequencing, Cell Differentiation

Journal: eLife

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

doi: 10.7554/eLife.50087

Figure Lengend Snippet: ( A ) Correlation between log2 fold-change values of accessibility (x-axis) and OCT4 binding (y-axis) upon SOX2 depletion in 2TS22C cells with dox treatment for 26 hr. Coefficient (R) and p-value are based on the Pearson correlation coefficient. ( B–C ) Average RPKM-normalized ATAC-seq signal 2 kb around OD (n = 3’730), CD (n = 1’463), and SD (n = 273) loci that overlap with a canonical OCT4::SOX2 motif upon SOX2 ( B ) and OCT4 ( C ) depletion. ( D–E ) Average RPKM-normalized OCT4 ( D ) and SOX2 ( E ) ChIP-seq signal 2 kb around OD, CD, and SD loci that overlap with a canonical OCT4::SOX2 motif upon SOX2 ( D ) and OCT4 ( E ) depletion. ( F ) Average RPKM-normalized ATAC-seq signal upon SOX2 depletion 2 kb around loci that display a significant increase in accessibility and SOX2 binding upon OCT4 depletion (n = 3’270). ( G ) Average RPKM-normalized BRG1 ChIP-seq signal upon OCT4 depletion 2 kb around loci that display a significant increase in accessibility and SOX2 binding upon OCT4 depletion. Statistics for ( B–G ) are available in .

Article Snippet: SNAP-MD-OCT4 and SNAP-MD*-OCT4 were further amplified and cloned by restriction cloning into pLV-rtTA3G-IRESBsd ( ) using AgeI and SalI. pLV-pCAGGS-Tir1-V5 was derived by amplification of pCAGGS-Tir1-V5 from pEN395 (Addgene #92141) ( ) and In-fusion cloning into pLV-PGK-SOX2-SNAP-IRES-Hygro ( ) digested using XhoI and XbaI restriction enzymes. pLEX-mCherry-OCT4-AID was derived by amplification of OCT4 from pLV-PGK-SNAP-MD-OCT4, AID 71–114 from pEN244 (Addgene #92140) , and mCherry from pLV-TRE3G-mCherry-PGK-Puro (Suter lab). mCherry and OCT4 were ligated using an XmaI restriction site and mCherry-OCT4 was cloned into the pLEX_305-C-dTAG backbone (Addgene #91798) ( ) using AgeI and NheI restriction sites.

Techniques: Binding Assay, ChIP-sequencing

Journal: eLife

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

doi: 10.7554/eLife.50087

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; p-values: Mann-Whitney U. ( 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; p-values: paired t-test. ( 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 RT-qPCR 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; p-values: Mann-Whitney U. ( 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; p-values: paired t-test. ( 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; p-values: Mann-Whitney U. EG1: Early G1 phase; LG1: Late G1 phase; S: S phase; SG2: Late S and G2 phase.

Article Snippet: SNAP-MD-OCT4 and SNAP-MD*-OCT4 were further amplified and cloned by restriction cloning into pLV-rtTA3G-IRESBsd ( ) using AgeI and SalI. pLV-pCAGGS-Tir1-V5 was derived by amplification of pCAGGS-Tir1-V5 from pEN395 (Addgene #92141) ( ) and In-fusion cloning into pLV-PGK-SOX2-SNAP-IRES-Hygro ( ) digested using XhoI and XbaI restriction enzymes. pLEX-mCherry-OCT4-AID was derived by amplification of OCT4 from pLV-PGK-SNAP-MD-OCT4, AID 71–114 from pEN244 (Addgene #92140) , and mCherry from pLV-TRE3G-mCherry-PGK-Puro (Suter lab). mCherry and OCT4 were ligated using an XmaI restriction site and mCherry-OCT4 was cloned into the pLEX_305-C-dTAG backbone (Addgene #91798) ( ) using AgeI and NheI restriction sites.

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

Journal: eLife

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

doi: 10.7554/eLife.50087

Figure Lengend Snippet: ( A ) Violin plot of distance to closest TSS in the clusters from . Dots: mean; Vertical lines: standard deviation; p-values: Mann-Whitney U. ( B ) Heatmap of ChIP-seq signal of H3K4me3, H3K4me1, and H3K27ac in wt ES cells 5 kb around regions in the clusters from . Each row corresponds to one individual locus. ( C ) Average log2 fold-change values of H3K27ac ChIP-seq signal between MD-OCT4 and MD*-OCT4 cells in the clusters from (including 500 bp flanking regions at each side) at different cell cycle phases. Statistics are available in . ( D ) Genome browser tracks of RPKM-normalized H3K27ac profiles across the cell cycle for a cluster one locus (chr11:6894809–6895533) that decreases in accessibility and H3K27ac upon transient OCT4 depletion in M-G1. ( E ) Heatmap of RPKM-normalized H3K27ac ChIP-seq signal 2 kb around regions in the clusters from in MD*-OCT4 (MD*) and MD-OCT4 (MD) cells at different cell cycle phases. Each row corresponds to one individual locus. ( F ) log2 fold-change values of H3K27ac ChIP-seq signal between MD-OCT4 and MD*-OCT4 cells in the clusters from (including 500 bp flanking regions at each side) at different cell cycle phases. Each row corresponds to one individual locus. EG1: Early G1 phase; LG1: Late G1 phase; S: S phase; SG2: Late S and G2 phase.

Article Snippet: SNAP-MD-OCT4 and SNAP-MD*-OCT4 were further amplified and cloned by restriction cloning into pLV-rtTA3G-IRESBsd ( ) using AgeI and SalI. pLV-pCAGGS-Tir1-V5 was derived by amplification of pCAGGS-Tir1-V5 from pEN395 (Addgene #92141) ( ) and In-fusion cloning into pLV-PGK-SOX2-SNAP-IRES-Hygro ( ) digested using XhoI and XbaI restriction enzymes. pLEX-mCherry-OCT4-AID was derived by amplification of OCT4 from pLV-PGK-SNAP-MD-OCT4, AID 71–114 from pEN244 (Addgene #92140) , and mCherry from pLV-TRE3G-mCherry-PGK-Puro (Suter lab). mCherry and OCT4 were ligated using an XmaI restriction site and mCherry-OCT4 was cloned into the pLEX_305-C-dTAG backbone (Addgene #91798) ( ) using AgeI and NheI restriction sites.

Techniques: Standard Deviation, MANN-WHITNEY, ChIP-sequencing

Journal: eLife

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

doi: 10.7554/eLife.50087

Figure Lengend Snippet: ( A ) Percentage of the closest gene in the clusters from and at non-OCT4 bound accessible regions whose nascent RNA levels are downregulated or upregulated upon 24 hr of OCT4 depletion. p-values: Fisher’s exact test. ( B ) Relative enrichment values (Z-transformed –logP) for the closest genes in the clusters from and at non-OCT4 bound accessible regions, in all KEGG gene ontology sets with Z > 3, sorted by enrichment in cluster 1. ( C ) Percentage of loci in the clusters from and at non-OCT4 bound accessible regions overlapping typical enhancers (TE) and super-enhancers (SE) in mouse ES cells. ( D ) RPKM-normalized ATAC-seq signal in cells sorted for high and low endogenous OCT4 levels 2 kb around loci in the clusters from . Statistics are available in . ( G ) Percentage of loci in the clusters from overlapping OD, CD, and SD loci from .

Article Snippet: SNAP-MD-OCT4 and SNAP-MD*-OCT4 were further amplified and cloned by restriction cloning into pLV-rtTA3G-IRESBsd ( ) using AgeI and SalI. pLV-pCAGGS-Tir1-V5 was derived by amplification of pCAGGS-Tir1-V5 from pEN395 (Addgene #92141) ( ) and In-fusion cloning into pLV-PGK-SOX2-SNAP-IRES-Hygro ( ) digested using XhoI and XbaI restriction enzymes. pLEX-mCherry-OCT4-AID was derived by amplification of OCT4 from pLV-PGK-SNAP-MD-OCT4, AID 71–114 from pEN244 (Addgene #92140) , and mCherry from pLV-TRE3G-mCherry-PGK-Puro (Suter lab). mCherry and OCT4 were ligated using an XmaI restriction site and mCherry-OCT4 was cloned into the pLEX_305-C-dTAG backbone (Addgene #91798) ( ) using AgeI and NheI restriction sites.

Techniques: Transformation Assay

Journal: eLife

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

doi: 10.7554/eLife.50087

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. Coefficient (R) and p-values are based on the Pearson correlation coefficient. ( C ) Cluster predictions of regions in the test data based on a random forest model using mouse ES cell ChIP-seq data (see Materials and methods). The x‐axis shows the true cluster, and the y‐axis shows the fraction of regions predicted to belong to the clusters from (colors). ( D ) Average ChIP-seq signal of KLF4, KLF5, CHD4, SALL4, NANOG, ESRRB, MBD3, SOX2, DAX1, and TBX3 in ES cells 2 kb around regions in the clusters from . ( E ) Average ChIP-seq signal of CTCF and RAD21 in ES cells 2 kb around regions in the clusters from . Statistics for ( D–E ) are available in .

Article Snippet: SNAP-MD-OCT4 and SNAP-MD*-OCT4 were further amplified and cloned by restriction cloning into pLV-rtTA3G-IRESBsd ( ) using AgeI and SalI. pLV-pCAGGS-Tir1-V5 was derived by amplification of pCAGGS-Tir1-V5 from pEN395 (Addgene #92141) ( ) and In-fusion cloning into pLV-PGK-SOX2-SNAP-IRES-Hygro ( ) digested using XhoI and XbaI restriction enzymes. pLEX-mCherry-OCT4-AID was derived by amplification of OCT4 from pLV-PGK-SNAP-MD-OCT4, AID 71–114 from pEN244 (Addgene #92140) , and mCherry from pLV-TRE3G-mCherry-PGK-Puro (Suter lab). mCherry and OCT4 were ligated using an XmaI restriction site and mCherry-OCT4 was cloned into the pLEX_305-C-dTAG backbone (Addgene #91798) ( ) using AgeI and NheI restriction sites.

Techniques: ChIP-sequencing, Binding Assay

Journal: eLife

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

doi: 10.7554/eLife.50087

Figure Lengend Snippet: ( A ) Ratio of the number of colonies with and without dox treatment, for wt ZHBTc4 cells (Control) and ZHBTc4 cells expressing mCherry-OCT4-AID after one week in culture. n = 3 biological replicates; p‐values: paired t-test. ( B ) Representative alkaline phosphatase staining from cells in ( A ). ( C ) Histogram of mCherry signal in untreated mCherry OCT4-AID cells and treated with IAA for 2 hr 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. ( D–F ) 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 hr IAA treatment ( D ), 0.5 hr IAA treatment ( E ), and after 2.5 hr IAA treatment followed by 4.5 hr of washout ( F ). n = 3 biological replicates; p-values: Mann-Whitney U. EG1: Early G1 phase; LG1: Late G1 phase; S: S phase; SG2: Late S and G2 phase.

Article Snippet: SNAP-MD-OCT4 and SNAP-MD*-OCT4 were further amplified and cloned by restriction cloning into pLV-rtTA3G-IRESBsd ( ) using AgeI and SalI. pLV-pCAGGS-Tir1-V5 was derived by amplification of pCAGGS-Tir1-V5 from pEN395 (Addgene #92141) ( ) and In-fusion cloning into pLV-PGK-SOX2-SNAP-IRES-Hygro ( ) digested using XhoI and XbaI restriction enzymes. pLEX-mCherry-OCT4-AID was derived by amplification of OCT4 from pLV-PGK-SNAP-MD-OCT4, AID 71–114 from pEN244 (Addgene #92140) , and mCherry from pLV-TRE3G-mCherry-PGK-Puro (Suter lab). mCherry and OCT4 were ligated using an XmaI restriction site and mCherry-OCT4 was cloned into the pLEX_305-C-dTAG backbone (Addgene #91798) ( ) using AgeI and NheI restriction sites.

Techniques: Expressing, Staining, Flow Cytometry, Fluorescence, MANN-WHITNEY

Journal: eLife

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

doi: 10.7554/eLife.50087

Figure Lengend Snippet:

Article Snippet: SNAP-MD-OCT4 and SNAP-MD*-OCT4 were further amplified and cloned by restriction cloning into pLV-rtTA3G-IRESBsd ( ) using AgeI and SalI. pLV-pCAGGS-Tir1-V5 was derived by amplification of pCAGGS-Tir1-V5 from pEN395 (Addgene #92141) ( ) and In-fusion cloning into pLV-PGK-SOX2-SNAP-IRES-Hygro ( ) digested using XhoI and XbaI restriction enzymes. pLEX-mCherry-OCT4-AID was derived by amplification of OCT4 from pLV-PGK-SNAP-MD-OCT4, AID 71–114 from pEN244 (Addgene #92140) , and mCherry from pLV-TRE3G-mCherry-PGK-Puro (Suter lab). mCherry and OCT4 were ligated using an XmaI restriction site and mCherry-OCT4 was cloned into the pLEX_305-C-dTAG backbone (Addgene #91798) ( ) using AgeI and NheI restriction sites.

Techniques: Recombinant, Software