Leptin signaling regulates hypothalamic expression of nescient helix-loop-helix 2 (Nhlh2) through signal transducer and activator 3 (Stat3)

• A small 163 bp proximal promoter confers leptin-regulation of the Nhlh2 promoter.
• Three Stat3 sites mediate leptin-induced and basal Nhlh2 expression.
• Stat3 interacts with the Nhlh2 promoter in leptin-treated cell lines and mice.
• The results identify the mechanisms for leptin-responsiveness of the Nhlh2 promoter.

Mice with a deletion of the hypothalamic basic helix-loop-helix transcription factor Nhlh2 display adult onset obesity. We have previously shown that Nhlh2 expression is induced by leptin. In this study, we identify a small proximal leptin-responsive promoter region in the Nhlh2 gene. This 163 bp promoter contains five putative binding sites for the leptin-activated Stat3 transcription factor, and two putative binding sites for the NFκB transcription factor. Results of mutagenesis studies reveal that deletion of the NFκB sites have little effect, mutagenesis of the third Stat3 site eliminates both leptin-induced and basal expression of Nhlh2. Mutagenesis of the 4th and 5th sites eliminates leptin-induced expression, and increases basal expression above the WT promoter. Stat3 can be preferentially pulled down from leptin-treated mouse hypothalamic chromatin extracts. This study identifies leptin-induced Stat3 transcription factor as the major transcriptional regulator of Nhlh2. As Nhlh2 transcriptionally regulates genes within the melanocortin pathway, these findings have implications for human body weight control.

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