Figure 3.

Stress-induced GR binding change at Nr1d1 TSS E-box

Control (CT) and water avoidance stressed (WAS) mouse colon epithelium cells were isolated for CUT&Tag analysis; hypothesis-free analysis of GR cistrome.

(A) Gene body.

(B) Differential peak annotation.

(C) KEGG pathway enrichment of differential peaks.

(D) Each lane represents IECs pooled from four mice, stress reduced GR binding at the E-box (CACGTG) upstream of the Nr1d1 TSS.

(E) Pyrosequencing analysis of the genomic DNA CpGs within the gap in GR binding around the E-box.

(F and G) Nr1d1 promoter activity in differentiated mouse colon epithelium YAMC cells. 1 μM CORT treatment repressed Nr1d1 transcription, and the GR antagonist RU486 ameliorated this effect. Nr1d1 promoter TSS E-box mutation reduced Nr1d1 transcription.

(H) Western blot analysis of YAMC cells treated with CORT, RU486, and NR1D1 agonist SR9009. Data are expressed as means ± standard error. Statistical significance was determined using an unpaired t test with Welch’s correction (∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.01; ∗∗∗∗, p < 0.0001; N = 3).