Bhlhe40 is a critical repressor downstream of REV-ERB that regulates RORγt ⁺ expression in Treg cells. (A) Venn diagram of overlapping REV-ERBα–bound genes from CUT&RUN and genes with upregulated expression in REV-ERB–deficient Treg cells from RNA-seq. (B) RT-qPCR analysis of Bhlhe40 and Nfil3 expression in WT and REV-ERB–deficient Treg cells. ***P = 0.0002. Data are representative of two independent experiments. Statistical significance was determined using Student’s t test analysis. (C) CUT&RUN tracks showing H3K27Ac- and REV-ERBα–bound peaks at the Bhlhe40 locus, with underlines detected as peaks by HOMER. (D) Flow cytometric analysis of RORγt expression in Foxp3⁺iTreg cells with ectopic expression of Bhlhe40 or control vector (n = 3 biologically independent replicates per group). ****P < 0.0001, *P = 0.0131. (E) Flow cytometric analysis of RORγt expression in Foxp3⁺iTreg cells transduced with sgNT and sgBhlhe40. **P = 0.0094, ****P < 0.0001. Data in D and E are representative of at least two independent experiments. Statistical significance was determined using two-way ANOVA.