Figure 3.

Esrrg is expressed in FAE in PPs and is dependent on Rank–RankL signaling. (A) H3K27me3 occupancy at CpG islands spanning the promoter and first exon of the Esrrg gene in organoids treated with RankL (M cells) or inhibited with IWP2 (enterocytes) or treated with Wnt3a and Chir99021 (crypt/intestinal stem cells). Below, pre–messenger RNA (mRNA) expression of Esrrg in organoids treated as described earlier (y-axis: normalized tag count, ENR500 = R-spondin 500 ng/mL, R100 = Rankl 100 ng/mL). (B) Section of PP from wild-type mice stained with Esrrg antibody. Arrowheads indicating Esrrg expression in the nuclei of M cells in FAE. (C) RT-qPCR analysis of Esrrg and Gp2 in the FAE and villous epithelium (VE) from C57BL/6JRj mice (N = 3 from wild-type mice). (D) Organoids generated from wild-type mice were stimulated with 100 ng RankL for 4 days. Esrrg and Gp2 expression was examined by qPCR analysis. (E) Rank KO organoids and Scrambled organoids generated by lentiCRISPR v2 were incubated with RankL for 4 days, Esrrg and Gp2 expression was analyzed by quantitative RT-qPCR. (C–E) An unpaired 2-tailed Student t test was performed for 3 independent experiments. ∗∗∗∗P < .0005, ∗∗∗P < .005, and ∗∗P < .01. Values are presented as means ± SD. (F) RANK protein expression in RANK KO and Scrambled cells generated by lentiCRISPR v2 genome editing in C57BL/6JRj intestinal organoids. Organoid lysates were analyzed by Western blot. Gapdh, glyceraldehyde-3-phosphate dehydrogenase.