Evolutionary and functional properties of the neuronal proopiomelanocortin (POMC) gene enhancers nPE1 and nPE2

LUCÍA F. FRANCHINI, FLÁVIO JS DESOUZA, RODRIGO LÓPEZ-LEAL, MALCOLM J. LOW, MARCELO RUBINSTEIN

San Diego, California, USA

Congreso; Society forNeuroscience; 2007

Society for Neuroscience

POMC is mainly expressed in pituitary cells and ventral hypothalamic neurons of all vertebrates where it gives rise to several bioactive peptides that include ACTH, a-, b-, and g-MSH (melanocyte stimulating hormone) and b-endorphin. These peptides participate in functions as diverse as the stress response, skin and hair pigmentation, analgesia and the regulation of food intake and energy balance. Recently, we demonstrated that a distal cis-acting module located upstream of POMC contains two neuronal enhancers, nPE1 and nPE2. Although nPE1 and nPE2 sequences are unrelated, their regulatory functions seem to overlap, since only the removal of both elements from transgenic constructs leads to the loss of reporter gene expression in hypothalamic POMC neurons. The aim of the present study was to determine the particular properties of each of these two enhancers in the control of central POMC expression. We first investigated the evolutionary history of nPE1 and nPE2 and second, we performed a comprehensive expression study of different reporter fluorescent proteins driven by nPE1 or nPE2 in transgenic mice. Taking advantage of the increasing availability of novel genome sequences we determined that nPE1 is present in the genomes of placental mammals but not in marsupials or monotremes. In turn, nPE2 is a highly conserved element in placental and non-placental mammals. None of these two enhancers is present in non-mammalian vertebrates. Thus, nPE1 is a placental novelty that appeared after the Methateria-Eutheria split around 150 million years ago (MYA) whereas nPE2 appeared in an ancestor to all extant mammals, before 200 MYA. By using an in silico paleogenomic strategy, we also demonstrated that the neuronal POMC enhancer nPE2 originated from the exaptation of a CORE-SINE retroposon, probably still active in marsupials, in the lineage leading to mammals where it remained under strong purifying selection. In parallel, we generated several independent pedigrees of transgenic mice expressing either the red fluorescent protein tdimer2 or the green fluorescent protein EGFP under the transcriptional control of nPE1 or nPE2, respectively. Several double transgenic mouse lines were generated carrying different integrations. An initial evaluation of coronal brain slices using confocal microscopy indicates that either nPE1 or nPE2 are able to drive reporter gene expression to all POMC hypothalamic neurons. In summary, two unrelated enhancer sequences with distinct evolutionary origin direct the expression of reporter genes to the same subset of hypothalamic neurons and probably bind the same specific combination of transcription factors.