Bioinformatic and functional analysis reveals the common cis-acting code of two neuron-specific shadow enhancers

SOFIA NASIF; FLAVIO S. J. DE SOUZA; MARCELO RUBINSTEIN

Cataratas del Iguazú

Simposio; Gene Expression and RNA Processing Symposium; 2011

ICGEB-ANPCYT-CONICET

The proopiomelanocortin gene (POMC) is expressed in the arcuate nucleus of the hypothalamus to regulate food intake and energy balance. Hypothalamic POMC expression depends on two evolutionary unrelated transcriptional enhancers, named nPE1 and nPE2, which drive reporter gene expression to the same set of hypothalamic neurons. To reveal the cis-acting code shared by both shadow enhancers we combined bioinformatic with functional expression studies in transgenic mice. We identified a 140 bp region within nPE1 that is necessary and sufficient to drive reporter gene expression to POMC neurons. A comparative analysis of this critical nPE1 region with those identified in nPE2 evidenced a small number of short sequences that are present in both enhancers, suggesting that the transcriptional activity of nPE1 and nPE2 might be conferred by a similar set of transcription factors (TF). We tested the ability of several recombinant TF candidates to bind to the enhancers? regions in gel shift assays. To test the hypothesis that shared sequences are critical for enhancer function, we designed transgenic constructs carrying a set of mutations that simultaneously eliminated all the identified sites. These transgenes failed to drive reporter gene expression to hypothalamic neurons demonstrating that the mutated sites play a fundamental role in the neuronal-specific regulation of Pomc. Further ongoing studies are demonstrating the importance of each selected element present in nPE1 and nPE2 to the overall enhancer activity. Our results represent a step forward to understand the regulation of cell-specific gene expression by evolutionaty unrelated shadow enhancers.