Partially Redundant Enhancers Cooperatively Maintain Mammalian Pomc Expression Above a Critical Functional Threshold

Abstract

Cell-specific expression of many genes is conveyed by multiple enhancers, with each individual enhancer controlling a particular expression domain. In contrast, multiple enhancers drive similar expression patterns of some genes involved in embryonic development, suggesting regulatory redundancy. Work in Drosophila has indicated that functionally overlapping enhancers canalize development by buffering gene expression against environmental and genetic disturbances. However, little is known about regulatory redundancy in vertebrates and in genes mainly expressed during adulthood. Here we study nPE1 and nPE2, two phylogenetically conserved mammalian enhancers that drive expression of the proopiomelanocortin gene (Pomc) to the same set of hypothalamic neurons. The simultaneous deletion of both enhancers abolished Pomc expression at all ages and induced a profound metabolic dysfunction including early-onset extreme obesity. Targeted inactivation of either nPE1 or nPE2 led to very low levels of Pomc expression during early embryonic development indicating that both enhancers function synergistically. In adult mice, however, Pomc expression is controlled additively by both enhancers, with nPE1 being responsible for ∼80% and nPE2 for ∼20% of Pomc transcription. Consequently, nPE1 knockout mice exhibit mild obesity whereas nPE2-deficient mice maintain a normal body weight. These results suggest that nPE2-driven Pomc expression is compensated by nPE1 at later stages of development, essentially rescuing the earlier phenotype of nPE2 deficiency. Together, these results reveal that cooperative interactions between the enhancers confer robustness of Pomc expression against gene regulatory disturbances and preclude deleterious metabolic phenotypes caused by Pomc deficiency in adulthood. Thus, our study demonstrates that enhancer redundancy can be used by genes that control adult physiology in mammals and underlines the potential significance of regulatory sequence mutations in common diseases.

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

“…Similar treatment effects were observed in Pomc Δ2 mice, which maintain functional POMC activity (Lam et al., 2015) despite a ~40% reduction in constitutive ARC Pomc expression at the time of necropsy (Figure 2c). In Pomc wt and Pomc Δ2 , this change in body mass occurred in conjunction with an immediate decline in food intake (Figure 2d) that was followed by a slow rebound in energy intake by week 3 (Figure 2e).…”

“…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). A reduction in hypothalamic Pomc expression at or below ~30% of wild‐type controls in these mice results in a functional loss of Pomc ‐mediated regulation of body mass (Bumaschny et al., 2012; Lam et al., 2015; Zhan et al., 2013). Therefore, we hypothesized that if 17α‐E2 were to act selectively by increasing hypothalamic Pomc expression, the treatment effects on body mass and food intake would be disrupted in mutant mice lacking nPE1 ( Pomc Δ1 ) or those containing the Pomc transcription‐blocking neo selection cassette ( Pomc neo ).…”

Islet 1 specifies the identity of hypothalamic melanocortin neurons and is critical for normal food intake and adiposity in adulthood

“…Expression studies in transgenic mice showed that nPE1 and nPE2 drive completely overlapping spatiotemporal enhancer activities to the ∼3,000 POMC neurons present in the mouse ventromedial hypothalamus during embryogenesis as well as in adulthood (16) and only the concurrent removal of both enhancers from the mouse Pomc locus by targeted mutagenesis reduced Pomc expression to very low levels, leading to hyperphagia and early-onset obesity (17). Interestingly, nPE1 and nPE2 are unrelated sequences with distinct evolutionary origins; whereas nPE2 is derived from the exaptation (cooption) of a CORE-short interspersed nuclear element (SINE) retroposon in the lineage leading to mammals more than 166 million years ago (Mya) (18), nPE1 is a placental mammal novelty derived from the exaptation of a LTR-containing a mammalian apparent LTR retroposon that occurred between 150 and 90 Mya (16).…”

“…We have recently identified conserved C/ATTA motifs that might be recognized by homeodomain-containing transcription factors (HDTFs) within nPE2 and nPE1, which are essential for driving reporter gene expression to POMC neurons (17). Because nPE1 and nPE2 are functional analogs, it is conceivable that these sequences recruit a common, yet unidentified TF able to transactivate Pomc in the hypothalamus.…”

“…Because nPE1 and nPE2 are functional analogs, it is conceivable that these sequences recruit a common, yet unidentified TF able to transactivate Pomc in the hypothalamus. Pomc expression starts in the ventral lining of the developing mouse hypothalamus at embryonic day 10.5 (E10.5) (15)(16)(17), a precise spatiotemporal domain in which multiple HDTFs determine the early patterning of the mouse ventral hypothalamus (19,20). Therefore, we considered of interest to investigate whether the terminal differentiation of melanocortin-expressing neurons is regulated by a HDTF.…”