Fig. 1

Motif identification in Gsc promoter using ChIP-Seq and CRISPR/Cas9. a Schematic representation of the proximal promoter and distal enhancer sites of mouse and human Gsc showing the GC-rich region, CAGAC, and the FoxH1 binding sites. b Heatmap of ChIP-Seq tag densities for Smad2/3 (GSM1782914⁶²) and Smad4 (GSM2746361, this work) located at −600 bp from the TSS of Gsc, showing how the signal is centered at the GC-rich region. Coordinates referred to the mm9 genome assembly. Data are displayed for chromosome 12 between 105,711,900 and 105,711,700 bases. c Scheme of the CRISPR/Cas9-mediated mutagenesis of the Gsc proximal promoter region. CRISPR-mediated deletions of a 10 bp region of the Gsc GC1 site (Clone C1) and of the GC1-FoxH1 region (155 bp, Clone C2) are indicated with dashed lines. The DNA sequence of targeted regions is represented as blue horizontal bars. Deletions were confirmed by deep sequencing and TA cloning (Supplementary Fig. 1e, f). d The effects of the deletions are shown as the relative mRNA levels of Gsc and Smad7 used as a control of the TGF-beta signaling pathway. qRT-PCR analysis of Gsc mRNA expression in wild type (WT) or Gsc mutant clones and of Smad7 expression in activin A- (green) or SB431542- (gray) treated d3 cells. Gene expression level is normalized to WT samples, n = 3. Error bars represent s.e.m., P < 0.05, Mann–Whitney test using Prism 6 software (GraphPad Software). e Smad4 ChIP-qPCR data (n = 2) in ES cells showing that the 10 bp deletion in the GC-rich region abolished Smad4 interaction with this region without affecting Smad4 interaction with the Gsc +6 kb distal enhancer element. Error bar represents s.e.m