Journal: BMC Biology

Article Title: FACT regulates pluripotency through proximal and distal regulation of gene expression in murine embryonic stem cells

doi: 10.1186/s12915-023-01669-0

Figure Lengend Snippet: FACT and pluripotency factors colocalize at putative enhancers genome-wide. A – C SPT16-V5 CUT&RUN data visualized as one-dimensional heatmaps . Each row represents the average of technical replicates, while biological replicates are displayed separately ( n = 3 for untagged and n = 2 for each V5-tagged clone). Visualized at A H3K27ac ChIP-seq peaks ± 2 kb (GSE32218), B H3K4me1 ChIP-seq peaks ± 2 kb (GSE31039), C TSS-distal DNaseI hypersensitive sites ± 2 kb (GSM1014154) [ – ]. D SPT16-V5 CUT&RUN data visualized at SPT16-V5-bound putative enhancers, defined as DHSs (GSM1014154) overlapping H3K4me1 or H3K27ac ChIP-seq peaks (GSE32218 and GSE31039) ± 2 kb [ – ]. E SPT16-V5 CUT&RUN binding over TSS-distal OCT4 ChIP-seq peaks (ChIP-seq from GSE11724 ). Merged replicates are shown as heatmaps ± 2 kb from the center of the OCT4 ChIP-seq peak ( n = 3 for untagged, n = 2 for each V5-tagged clone). F OCT4, SOX2, or NANOG enrichment over SPT16-V5 CUT&RUN peaks. Averaged replicates shown ( n = 1 for OCT4, n = 2 for SOX2 and NANOG; ChIP-seq from GSE11724) . Significance of overlap between pluripotency factor binding and SPT16-V5 binding was assessed via two-tailed Fisher’s exact tests ( p = 0 for all remodelers, ratio = 61.597, 94.475, and 93.779 for OCT4, SOX2, and NANOG overlap with SPT16-V5, respectively). G Heatmaps displaying overlap between OCT4 ChIP-seq and SPT16-V5 CUT&RUN peaks. Clusters were assigned by direct overlap between peak datasets and are individually sorted by strength of SPT16-V5 binding (ChIP-seq from GSE11724) . Merged replicates are shown as heatmaps ± 2 kb from the center of the peak ( n = 3 for untagged, n = 2 for each V5-tagged clone, n = 1 for OCT4 ChIP-seq, n = 2 for other ChIP-seq experiments; ChIP-seq from GSE11724)

Article Snippet: Cells were incubated in anti-OCT4 primary antibody (ReproCELL 09–0023, lot J17070000000001) overnight at a 1:500 dilution in 1% BSA-DPBS.

Techniques: Genome Wide, ChIP-sequencing, Binding Assay, Two Tailed Test

Journal: BMC Biology

Article Title: FACT regulates pluripotency through proximal and distal regulation of gene expression in murine embryonic stem cells

doi: 10.1186/s12915-023-01669-0

Figure Lengend Snippet: Overlap between SPT16-V5 CUT&RUN peaks and OCT4, SOX2, and NANOG ChIP-seq peaks

Article Snippet: Cells were incubated in anti-OCT4 primary antibody (ReproCELL 09–0023, lot J17070000000001) overnight at a 1:500 dilution in 1% BSA-DPBS.

Techniques:

Journal: BMC Biology

Article Title: FACT regulates pluripotency through proximal and distal regulation of gene expression in murine embryonic stem cells

doi: 10.1186/s12915-023-01669-0

Figure Lengend Snippet: FACT depletion has distinct effects on chromatin accessibility and nucleosome occupancy at SPT16-V5 binding sites and gene regulatory regions. A Differential chromatin accessibility visualized over TSS-distal DHSs, ± 2 kb, after 24-h IAA treatment. Higher signal indicates more accessible chromatin in IAA-treated samples than in EtOH-treated samples at the indicated timepoint, with the exception of untagged samples (log IAA:IAA ratio) ( n = 1 per cell line). Significance of altered chromatin accessibility at gene-distal DHSs ( A ) was analyzed by Friedman tests and corrected for multiple comparisons via Dunn’s test ( p < 0.0001 overall, adj. p < 0.0001 for each individual comparison). B As in A but visualized over SPT16-V5 binding sites identified in Fig. . Significance of altered chromatin accessibility at SPT16-V5 binding sites was analyzed as in panel A ( p < 0.0001 overall, adj. p < 0.0001 for each individual comparison). C As in A but visualized over RefSeq mRNA TSSs, ± 2 kb, after 24-h IAA treatment. Significance of altered chromatin accessibility was analyzed as in panel A ( p < 0.0001 overall, adj. p < 0.0001 for both individual comparisons to Untagged. D Metaplot depicting change in chromatin accessibility at 24-h treatment over putative enhancer regions as defined in Fig. . Standard error is shaded in either direction. Significance of altered chromatin accessibility was analyzed as in panel A ( p < 0.0001 overall, adj. p < 0.05 for each individual comparison except Untagged [adj. p = 0.4046]). E – H Differential nucleosome occupancy following 24-h IAA treatment (MNase-seq, n = 3 for untagged samples, n = 2 for each tagged cell line). Visualized over E TSS-distal DNaseI hypersensitive sites (DHSs) ± 2 kb (DNase-seq from GSM1014154) [ – ]. Significance of altered chromatin accessibility was analyzed as in panel A ( p < 0.0001 overall, adj. p < 0.001 for each individual comparison). F Peaks called from SPT16-V5 CUT&RUN ± 2 kb. Significance of altered chromatin accessibility was analyzed as in panel A ( p < 0.0001 overall, adj. p < 0.0001 for each individual comparison). G SOX2 ChIP-seq binding sites, ± 2 kb (ChIP-seq from GSE11724) . Significance of altered chromatin accessibility was analyzed as in panel A ( p < 0.0001 overall, adj. p < 0.0001 for each individual comparison). H OCT4 ChIP-seq binding sites, ± 2 kb (ChIP-seq from GSE11724) . Significance of altered chromatin accessibility was analyzed as in panel A ( p < 0.0001 overall, adj. p < 0.0001 for each individual comparison)

Article Snippet: Cells were incubated in anti-OCT4 primary antibody (ReproCELL 09–0023, lot J17070000000001) overnight at a 1:500 dilution in 1% BSA-DPBS.

Techniques: Binding Assay, Comparison, ChIP-sequencing

Journal: BMC Biology

Article Title: FACT regulates pluripotency through proximal and distal regulation of gene expression in murine embryonic stem cells

doi: 10.1186/s12915-023-01669-0

Figure Lengend Snippet: Working model: FACT maintains ES cell pluripotency through regulation of pluripotency factor expression. FACT binds to gene-distal cis-regulatory elements (promoters and enhancers) and regulates both ncRNA transcription and nucleosome occupancy at these regulatory locations to permit appropriate expression of mRNAs. When FACT is depleted through IAA treatment, nucleosome occupancy at cis-regulatory elements is increased and mRNA expression is altered. These changes result in a loss of pluripotency and initiation of irregular differentiation across all 3 germ layers. OSN = OCT4, SOX2, and/or NANOG. Created with Biorender.com

Article Snippet: Cells were incubated in anti-OCT4 primary antibody (ReproCELL 09–0023, lot J17070000000001) overnight at a 1:500 dilution in 1% BSA-DPBS.

Techniques: Expressing