SOX2 Co-Occupies Distal Enhancer Elements with Distinct POU Factors in ESCs and NPCs to Specify Cell State
Figure 4
Brn2 biases ES cells towards neural differentiation.
(A) Staining with DAPI (blue) and immunocytochemistry of NESTIN (green) in ESCs induced to differentiate in adherent cultures with or without ectopic Brn2. (B) qRT-PCR of the indicated genes in ESCs with (black lines) and without (gray lines) ectopic Brn2 expression through differentiation. y-axis represents relative expression normalized to Gapdh in 3 biological replicates, measured in triplicate. ESC time point is ESCs without doxycycline, and d1–d9 time points represent time in differentiation medium. * denotes p-value<0.05, ** denotes p-value<0.01 ANOVA with Bonferroni correction (C) Heatmap of OCT4 and SOX2 enrichment in ESCs and ectopic BRN2 and SOX2 in TetO-Brn2 cells of 701 genomic regions occupied by only ectopic BRN2 and SOX2. (D) GREAT GO biological processes enriched in 701 regions in (C). x-axis reflects negative log base 10 of raw binomial p-value for enrichment versus a whole genome background. (E) Gene plots depicting peaks of enrichment in indicated datasets at a locus distal to Lrrn1. y-axis corresponds to reads per million. Genomic positions reflect NCBI Mouse Genome Build 36 (mm8). (F) qRT-PCR of genes associated with SOX2-BRN2 binding in TetO-Brn2 cells and NPCs, in ESCs with (black lines) and without (gray lines) ectopic Brn2 expression through differentiation. y-axis represents relative expression normalized to Gapdh in 3 biological replicates, measured in triplicate. ESC time point is ESCs without doxycycline, and d1–d9 time points represent time in differentiation medium. * denotes p-value<0.05, ** denotes p-value<0.01 ANOVA with Bonferroni correction. (G) Pie chart reflecting overlap between SOX2-BRN2 regions and enhancer chromatin marks in TetO-Brn2 cells. Percentages in legend reflect fraction of 1,533 SOX2-BRN2 regions in each category. (H) Example region that was occupied by ectopic BRN2 in TetO-Brn2 cells, leading to recruitment of endogenous SOX2 and the deposition of H3K4me1 and H3K27Ac. y-axis corresponds to reads per million. Genomic positions reflect NCBI Mouse Genome Build 36 (mm8). Gray box indicates region of SOX2-BRN2 co-occupancy in TetO-Brn2 cells which was not occupied by SOX2 in control cells. (I) Example poised enhancer occupied by ectopic BRN2 in induced cells, leading to recruitment of endogenous SOX2, and deposition of H3K27Ac. y-axis corresponds to reads per million. Genomic positions reflect NCBI Mouse Genome Build 36 (mm8). Gray box indicates region of SOX2-BRN2 co-occupancy in TetO-Brn2 cells which was not occupied by SOX2 in control cells.