A non-canonical-PPARγ/RXRα-binding sequence regulates leptin expression in response to changes in adipose tissue mass

Abstract

Leptin expression decreases after fat loss and is increased when obesity develops and its proper quantitative regulation is essential for the homeostatic control of fat mass. We previously reported that a distant leptin enhancer (LE1), 16kb upstream from the transcription start site (TSS), confers fat-specific expression in a BAC transgenic reporter mouse (BACTG). However this and the other elements that we identified do not account for the quantitative changes in leptin expression that accompany alterations of adipose mass. In this report, we used ATAC-seq to identify a 17bp non-canonical-PPARγ/RXRα-binding site leptin regulatory element 1 (LepRE1) within LE1, and show that it is necessary for the fat-regulated quantitative control of reporter (luciferase) expression. While BACTG reporter mice with mutations in this sequence still show fatspecific expression, luciferase is no longer decreased after food restriction and weight loss. Similarly the increased expression of leptin reporter associated with obesity in ob/ob mice is impaired. A functionally analogous LepRE1 site is also found in a second, redundant DNA regulatory element 13kb downstream of the TSS. These data uncouple the mechanisms conferring qualitative and quantitative expression of the leptin gene and further suggest that factor(s) that bind to LepRE1 quantitatively control leptin expression and might be components of a lipid sensing system in adipocytes.

Diet-dependent natriuretic peptide receptor C expression in adipose tissue is mediated by PPARγ via long-range distal enhancers

“…To further validate the functionality of the P2 element, the intact and mutated versions of P2 element were subcloned into the TK-Luc vector as triplet repeats (3xP2 and 3xP2M). Their activity was assayed with a previously established dualluciferase assay protocol in human embryonic kidney 293FT (HEK293FT) cells (28). As shown in Figure 5E, the triplet P2 element, but not its mutant P2M, displayed robust luciferase activity with rosiglitazone treatment.…”

“…Luciferase activity was assayed according to the manufacturer's instruction (Promega; E1501) and normalized to total protein concentration (bicinchoninic acid; Pierce). The HEK293FT dual-luciferase assay was performed according to a previous report (28). Briefly, HEK293FT cells were plated in 24-well plate at a density of 3 × 10 4 cells/well.…”