Fig 2: Depletion of Cyrano Results in Altered Expression of Nanog and Lineage-Related Genes(A) Ranking of the top-10 decreased genes in RNA-seq based on FDR (black bars) shows significant decreases in Nanog levels with Cyrano KD. Fold change is also indicated (white bars).(B) Assessment of pluripotency regulators on Cyrano KD reveals that Nanog displays the most significant differential regulation.(C–J) UCSC genome browser plots shows RNA-seq reads mapped to mm9 and normalized to remove sequencing depth biases, and independent qRT-PCR examination upon Cyrano KD indicates increased expression of factors that antagonize self-renewal in mouse ESCs. Data are from three independent experiments with error bars representing 95% CI. ∗p < 0.05, ∗∗p < 0.01.See also Figure S4.

Fig 3: BRG1/BAF occupancy relies upon OCT4 but not SOX2 and NANOG binding.Genomic snapshots of OCT4 targets that retain SOX2 and NANOG binding in the absence of OCT4, but lose BRG1 and SS18 ChIP-seq signal.DOI: http://dx.doi.org/10.7554/eLife.22631.019

Fig 4: BRG1 supports pluripotency-associated transcription factor binding to functionally mature distal gene regulatory elements.(A) Genomic snapshots illustrating OCT4, SOX2 and NANOG ChIP-seq signal in Brg1fl/fl ESCs before (UNT) and after tamoxifen (TAM) treatment for 72 hr. Three examples of distal OCT4 targets are depicted, with affected sites highlighted in grey. (B) A heatmap analysis of OCT4, SOX2 and NANOG ChIP-seq signal at distal OCT4 targets (n = 11967) ranked by their relative change in OCT4 ChIP-seq after deletion of BRG1. (C) Piecharts depicting the significant changes in OCT4, SOX2 and NANOG ChIP-seq signal at distal OCT4 target sites, identified using the DiffBind package. Changes were deemed significant with FDR < 0.05 and a change greater than 1.5-fold. (D) Metaplot analysis of OCT4, SOX2 and NANOG binding at distal OCT4 target sites that are the most (20% most affected; BRG1-dependent) and the least (20% least affected; BRG1-independent) dependent on BRG1 for normal OCT4 binding. (E) A heatmap illustrating the changes in gene expression (log2 fold change in RNA-seq) for genes neighbouring distal OCT4 targets. Genes are sorted according to the change in OCT4 occupancy at neighbouring distal OCT4 target sites following BRG1 removal, as in (B). Gene expression changes for OCT4-depleted (OCT4cond) or BRG1-depleted (Brg1fl/fl) ESCs relative to wildtype ESCs are shown. (F) Quantitation of log2 fold change (log2FC) in Brg1fl/fl RNA-seq signal for genes neighbouring distal OCT4 target sites grouped into quintiles based on the change in OCT4 binding at neighbouring distal OCT4 target sites following removal of BRG1, as in (B). (G) Comparison of significant gene expression changes for genes neighbouring distal OCT4 target sites that show the largest (20% most affected; BRG1-dependent) and least (20% least affected; BRG1-independent) reductions in OCT4 ChIP-seq signal following BRG1 removal. Changes were deemed significant with FDR < 0.05 and a change greater than 1.5-fold, using DESeq2. (H) Quantitation of log2 fold change (log2FC) of OCT4, SOX2 and NANOG ChIP-seq signal at distal OCT4 targets in proximity to OCT4-dependent genes with decreased (↓; n = 468), unchanged (-; n = 639), or increased (↑; n = 816) RNA-seq signal after deletion of BRG1 (as identified in Figure 5—figure supplement 3B).DOI: http://dx.doi.org/10.7554/eLife.22631.012

Fig 5: PHC1 interacts with NANOG and organizes genome-wide chromatin interactions of the Nanog locus.a Co-immunoprecipitation (Co-IP) from total hESC extracts using antibodies against RING1B and PHC1 followed by immunoblotting of different proteins. b Co-IPs of nuclear extracts from HEK293T cells transfected with PHC1-FLAG and NANOG-HA followed by immunoblotting. c IP of nuclear extracts from mESCs using an antibody against Nanog and immunoblotting of Phc1, Ring1b, Rybp, and Nanog, respectively. d Top panel: Interaction profile showing the normalized interaction intensity (y axis) of regions with the Nanog promoter (anchor) in chr6: 122500000-122700000 of mm9 genome assembly (x axis) in Phc1+/+ (green line) and Phc1−/− (purple line) mESCs. Regions with significant decreases in Phc1−/− than Phc1+/+ mESCs were highlighted and statistical significance was indicated with asterisk. n = 4 independent experiments. Paired single-side t-tests were used (from left to right p values are 0.0090, 0.0234, 0.0379, and 0.0304, respectively). Middle panel: Analysis of the previously published 4C-seq datasets showing interaction profile of genomic regions near the Nanog promoter (empirical anchor) in chr6: 122500000-122700000 of mm9 genome assembly in control (green line) and Nanog KD (purple line) mESCs41; H3K27ac and Nanog ChIP-seq binding profiles in the corresponding regions8,37. Bottom panel: Hi-C interaction matrices of mouse cortical neurons (CNs) and mESCs in chr6: 122500000-122700000 of mm9 genome assembly, respectively58. Source data are provided as a Source Data file.