Reversible neuron-specific silencing of proopiomelanocortin (POMC) expression by targeting a loxP-flanked neo cassette to the distal upstream enhancer region of the Pomc gene.

WARREN BD; DE SOUZA FS; OTERO-CORCHON V; KRUSE-BEND R; RUBINSTEIN M; LOW MJ

San Diego, EEUU

Congreso; 37th annual meeting of the Society for Neuroscience; 2007

Society for Neuroscience

Abstract:Numerous experiments have established the importance of proopiomelanocortin (POMC) in the control of food intake, metabolism, and the HPA axis. POMC biological activity is highly regulated in characteristic cell-type specific patterns at both the posttranslational and transcriptional levels. Recently, we demonstrated that Pomc expression in the pituitary and the brain are independently controlled by cis-acting modular elements present in proximal or distal 5’ flanking regions, respectively. The neuronal module contains two enhancers, nPE1 and nPE2, and each of them has the full capacity to drive reporter gene expression to hypothalamic POMC neurons. In the present study, we developed a new knock-in mouse strain by targeting a lox-P flanked (“floxed”) PGK promoter-neomycin resistance cassette (Fneo) to the distal neuronal enhancer module of Pomc. This insertion retained sequences of the Pomc neuronal enhancer nPE1 but not nPE2. Mice that were homozygous for the Fneo-inserted allele (FneoÄ2/FneoÄ2) had no detectable POMC expression in the hypothalamus despite the presence of nPE1, but retained POMC expression in the pituitary gland. These mice developed a severe obesity phenotype identical to that previously observed in compound mutant mice with a pituitary-specific transgenic rescue of POMC on a global POMC-deficient background. In addition, male FneoÄ2/FneoÄ2 mice exhibited over-activity of their HPA axis characterized by enlarged adrenal glands and increased diurnal levels of plasma corticosterone. These data confirm that POMC expression in the brain is required to modulate HPA activity, independently of POMC-derived ACTH secretion from pituitary corticotrophs. Intercross of the FneoÄ2/FneoÄ2 mice to a CMV-Cre transgenic strain resulted in deletion of the Fneo cassette early in embryogenesis. The resulting Ä2/Ä2 mice had restored POMC expression in the hypothalamus, normal body weight and growth, and normal HPA function. While it is not known how the Fneo cassette is disrupting expression of the Pomc gene only in the brain, possibilities include disruption of the normal chromatin configuration of the enhancer region, blocked access of transcriptional co-activators, recruitment of co-repressors, or some combination of these mechanisms. However, it is clear that the distal upstream enhancer region of the POMC gene is not crucial for expression of POMC in the pituitary. The FneoÄ2/FneoÄ2 strain of mice provides a powerful new genetic tool to examine the functional significance of discrete subsets of POMC neurons after restoration of Pomc gene expression via spatially and temporally controlled delivery of Cre recombinase.