ICER binds to a novel CRE sequence in the proximal NeuroD promoter.

<p>(<b>A</b>) Comparison of the proximal promoters of mouse (gi:3641530) and human (gi:7416051) NeuroD. The mouse TATA box (••••) at −30 bp and a potential CRE site (****) at −73 bp from the transcription initiation site (the arrow) are conserved. (<b>B</b>) A schematic model of reporter genes with −100 bp or −2.2 kb fragment of NeuroD promoter. Sequences for a putative wild type CRE and mutated (mCRE) are shown. (<b>C</b>) Reporter genes with the wild type CRE or mCRE (0.4 µg) were cotransfected with the CREB expression vector (0.15 µg) into HIT cells grown in 25 mM glucose. Forskolin (30 µM) was added for the indicated time before harvesting. The average luciferase activity from three independent experiments was shown as the fold ratio with respect to the basal activity of the reporter gene without CREB overexpression and forskolin. (*, <i>P</i><0.05; **, <i>P</i><0.01). (<b>D</b>) ICER Iγ directly repressed in a dose dependent manner the NeuroD reporter genes (0.4 µg) only with the wild type CRE but not with a mCRE in HIT cells grown in 5.5 mM glucose. (<b>E</b>) HIT cells grown in 5.5 mM glucose were cotransfected with NeuroD reporter genes (0.4 µg) and expression vectors for CREM τα (0.4 µg) and/or ICER Iγ (0.1 µg). The relative luciferase activity from three independent experiments is presented as the fold ratio with respect to the value of indicated conditions. (<b>F</b>) ChIP assays were performed to detect direct binding of endogenous ICER to the NeuroD CRE sequence at −73 bp using HIT cells grown in 5.5 mM or 25 mM glucose after stimulation with 30 µM forskolin for the indicated time. Long-term culture in 25 mM glucose enhanced the binding of ICER to CRE (right panels). The absence of immunoprecipitated CRE with a nonspecific IgG verified the specificity of the assay. Results from three independent ChIP assays were semi-quantitatively measured and presented as fold ratios with respect to the value obtained without forskolin in 5.5 mM glucose. Significant effects of forskolin (*, <i>P</i><0.05; **, <i>P</i><0.01) or chronic high glucose (<i>^#</i>, <i>P</i><0.05) are marked.