Identification of Neuronal Enhancers of the Proopiomelanocortin Gene by Transgenic Mouse Analysis and Phylogenetic Footprinting

Abstract

The proopiomelanocortin (POMC) gene is expressed in the pituitary and arcuate neurons of the hypothalamus. POMC arcuate neurons play a central role in the control of energy homeostasis, and rare loss-offunction mutations in POMC cause obesity. Moreover, POMC is the prime candidate gene within a highly significant quantitative trait locus on chromosome 2 associated with obesity traits in several human populations. Here, we identify two phylogenetically conserved neuronal POMC enhancers designated nPE1 (600 bp) and nPE2 (150 bp) located approximately 10 to 12 kb upstream of mammalian POMC transcriptional units. We show that mouse or human genomic regions containing these enhancers are able to direct reporter gene expression to POMC hypothalamic neurons, but not the pituitary of transgenic mice. Conversely, deletion of nPE1 and nPE2 in the context of the entire transcriptional unit of POMC abolishes transgene expression in the hypothalamus without affecting pituitary expression. Our results indicate that the nPEs are necessary and sufficient for hypothalamic POMC expression and that POMC expression in the brain and pituitary is controlled by independent sets of enhancers. Our study advances the understanding of the molecular nature of hypothalamic POMC neurons and will be useful to determine whether polymorphisms in POMC regulatory regions play a role in the predisposition to obesity.The proopiomelanocortin (POMC) gene encodes a prohormone expressed at significant levels in three discrete groups of cells of ectodermal neural plate origin that develop into either pituitary endocrine cells or brain neurons (54). Cell-specific posttranslational processing of the POMC prohormone generates a variety of biologically active peptides that mediate the mammalian stress response (46). In the pituitary gland, anterior lobe corticotrophs release adenocorticotropic hormone (ACTH) whereas intermediate lobe melanotrophs further process POMC to produce N-acetylated forms of ␣-melanocyte stimulating hormone (␣-MSH) and ␤-endorphin. In the brain, a specialized population of neurons concentrated in the hypothalamic arcuate nucleus also express Pomc, giving rise to ␣-, ␤-, and ␥-melanocortins and the potent analgesic opioid peptide ␤-endorphin (46, 50).During the last few years, POMC hypothalamic neurons have received a great deal of attention because they express receptors for the adipostatic hormone leptin and play a central role in the control of energy homeostasis and body weight regulation (10, 52, 56). Once leptin crosses the blood-brain barrier, it stimulates both the transcription of the POMC gene and the activity of POMC arcuate neurons, leading to the release of melanocortins which, in turn, stimulate central melanocortin receptors to decrease food intake and increase metabolic rate (4, 52). The importance of the central melanocortin pathway in feeding behavior is clearly observed in mice and humans with null Pomc homozygous mutations, which display hyperphagia and early-onset obesity (8,29,30,65). Even though total POMC defi...

Convergent evolution of two mammalian neuronal enhancers by sequential exaptation of unrelated retroposons

“…In previous work we defined nPE1 as a 630-bp human-mousedog conserved sequence able to direct reporter gene expression to hypothalamic POMC neurons of transgenic mice (15). Because the critical functional regions of cell-specific enhancers are usually under stronger selective constraint than neighboring nonfunctional regions, we performed an evolutionary divergence analysis to calculate the substitution rate along nPE1 sequences in every branch of a phylogenetic tree constructed from all available mammalian species ( Fig.…”

“…The physiological relevance of POMC-derived peptides in the brain can be readily appreciated in mice lacking central Pomc expression, which are hyperphagic and display earlyonset severe obesity (14). Transcriptional regulation of Pomc in neurons is conveyed by two upstream distal enhancers, named nPE1 and nPE2, which are highly conserved in mammals (15). Expression studies in transgenic mice showed that nPE1, as well as nPE2, is sufficient to independently drive reporter gene expression to POMC neurons when placed upstream of a minimal heterologous promoter (15).…”

“…Transcriptional regulation of Pomc in neurons is conveyed by two upstream distal enhancers, named nPE1 and nPE2, which are highly conserved in mammals (15). Expression studies in transgenic mice showed that nPE1, as well as nPE2, is sufficient to independently drive reporter gene expression to POMC neurons when placed upstream of a minimal heterologous promoter (15). Moreover, only the concurrent removal of nPE1 and nPE2 from transgenic constructs leads to the loss of reporter gene expression in POMC hypothalamic neurons (15).…”

“…Expression studies in transgenic mice showed that nPE1, as well as nPE2, is sufficient to independently drive reporter gene expression to POMC neurons when placed upstream of a minimal heterologous promoter (15). Moreover, only the concurrent removal of nPE1 and nPE2 from transgenic constructs leads to the loss of reporter gene expression in POMC hypothalamic neurons (15). However, whether both enhancers drive distinctive expression patterns within the arcuate nucleus still remains unknown.…”

17α‐estradiol acts through hypothalamic pro‐opiomelanocortin expressing neurons to reduce feeding behavior

“…Cooperative interactions between two POMC‐neuron‐specific enhancers, nPE1 and nPE2, promote expression of Pomc transcripts in the mouse ventromedial hypothalamus (Franchini et al., 2011; de Souza et al., 2005). Deletion of nPE2, nPE1, or insertion of a transcription‐blocking neo selection cassette into the vicinity of the two hypothalamic neuronal Pomc enhancers reduces hypothalamic Pomc expression to ~80%, ~30%, or ~2% of wild‐type controls, respectively (Lam et al, 2015).…”