Figure 2: Nrf2 binds and activates enhancer elements of mouse and human miR-29a/b1 genes.

(a) Diagram showing the locations of Nrf2-bound AREs in the 5′ regions of the mouse Mir29ab1 (top) and human MIR29AB1 (bottom) genes. Note that the human MIR29AB1 cluster is located within the annotated lncRNA MIR-29A-001, whereas the location of the TSS of mouse Mir29ab1 is based on conservation analysis²⁷. The TSS of the human gene and the predicted TSS of the murine gene are indicated with bent arrows. (b,c) Nrf2 binds to the AREs of the Mir29ab1 and MIR29AB1 genes in the epidermis of K5Cre mice (b) and in human keratinocytes treated with compound 1a (c). Primers used for ChIP experiments hybridized to the AREs marked with the asterisks in a. Binding of Nrf2 to the ARE of Nqo1 served as a positive control. Primers spanning a ns region located 2 kb away from the ARE of each gene were used as negative control. The average of at least three independent ChIP experiments is shown as percentage of input bound. (d) Levels of primary transcripts of the Mir29ab1 and the Mir29b2c genes (Pri-miR29ab1 and Pri-miR29bc2) were measured in the skin of control and K5Cre-CMVcaNrf2 mice at P0.5, P2.5 and P32 by qPCR (N=5 per genotype and time point). (e) Expression of NQO1, Pri-MIRAB1 and Pri-MIRB2C was analysed by qRT-PCR in HaCaT cells treated with compound 1a. N=3. (f–h) ChIPs using antibodies against total histone H3 and the modified histones H3K27Ac (f) or H3K4me1/2 (g,h) using epidermal lysates from K5Cre (f) K5Cre-CMVcaNrf2 (f–h) and K14-dnNrf2 mice (h). For H3K27Ac analysis, the promoter of the Hnrnpa2 gene served as a positive control. Non-specific control regions (ns) were used as negative controls. The average of the values from at least three independent ChIP experiments is calculated as percentage of input bound and as a fold of H3K27Ac, H3K4me1 or H3K4me2 over total histone H3 occupancy. Error bars represent s.d., t-test P value *<0.05; **<0.01. RT–PCR, reverse transcriptase-PCR.