Fig. 1: The VP is an essential Chst8 enhancer.

a Schematic view of the model used in this study. NSCs were dissected from cerebral cortices of mouse fetal brains (E12.5) and cultured ex vivo (see methods). TGFβ addition leads to neuronal commitment. b NSCs were treated with TGFβ. Total RNA was prepared and the levels of the mRNA of the indicated genes were determined by qPCR. Values were normalized to the housekeeping gene Gapdh and the figure shows values relative to time 0 h. Results are the mean of three biologically independent experiments. Data are presented as mean values +/− SEM. **p < 0.01 (P values were calculated using two-tailed Student’s t test, p = 0.001131677 and p = 0.006143072). Source data are provided as a Source Data file. c UCSC captures showing the chromatin landscape and SMAD3 binding around the Chst8 gene promoter and the Chst8 putative enhancer (VP) in NSCs. Tracks display ChIP-seq in NSCs treated with TGFβ (SMAD3) or untreated NSCs (H3K4me1, H3K27ac, and H3K4me3). Promoter and VP enhancer are shaded in light orange and yellow respectively. d Schematic representation of the CRISPR/Cas9 experimental approach used to delete the Chst8 putative enhancer in NSCs. Two gRNAs flanking the Chst8 enhancer region were used to create the deletion (2.9 kb). Red arrows represent primers to test the deletion. PCRs using Chst8 deletion and G6pd2 pairs of primers are shown at the bottom of the figure in parental and ΔChst8 enh NSC lines. Results are representative of three independent experiments. Source data are provided as a Source Data file. e Parental and ΔChst8 enh cell lines were treated with TGFβ for 6 h. Total RNA was prepared and the levels of eRNA of the VP enhancer (left) or Chst8 mRNA (right) were determined by qPCR. mRNA and eRNA levels of Fapb4 were used as a control. Values were normalized to the Gapdh gene, and figure shows values relative to parental line. Data are presented as mean values +/− SEM. Results are representative of three biological independent experiments. ***p < 0.001 (P values were calculated using two-tailed Student’s t test, p = 2.2027E-05 and p = 1.71676E-08). Source data are provided as a Source Data file.