FIGURE 6.

A putative FoxO1 binding site detected in a distal region of mouse Ins2 promoter. A, comparison of the putative FoxO1 binding elements identified in the mouse Ins2 promoter (a) to the consensus sequences, IRE and DBE (insulin response element, Daf-16 binding element) present in FoxO1-regulated genes. Relative position of the elements corresponds to the underlined nucleotide in the element, with respect to the transcription start site (b). B, chromatin immunoprecipitation (ChIP) of cross-linked DNA from lysed MIN6 cells using anti-FoxO1 antibody or immunoglobulin (IgG) and subsequent PCR amplification of short (∼200 bp) fragments around the putative FoxO1 binding regions. MIN6 cells (70% confluent) were cultured overnight in 3 mm glucose before incubating in 3 or 30 mm glucose for 6 h and subsequently formalin fixed for 15 min. ChIP experiments were done as described under “Experimental Procedures.” The agarose gel image is representative of ≥ 4 independent ChIP experiments. C, quantification of PCR products, as in B, normalized to the matched total, for regions 1 and 2. Bar represents mean ± S.E., n = 3.