Control Elements in the Neighboring ATPase Gene Influence Spatiotemporal Expression of the Human Agouti-Related Protein

The agouti-related protein (AgRP) is an orexigenic peptide that plays a significant role in the regulation of energy balance. It is expressed in the hypothalamus, the adrenal glands, and the testis, but sequences determining its spatial and temporal expression have not been identified. Using an elaborate in vitro screening approach, we show here that two adjacent enhancers inside the first intron of the neighboring (1.4 kb downstream) ATPase gene (ATP6V0D1) modulate the human AgRP promoter with profound spatiotemporal variation despite their diminutive sizes (221 and 231 nt). In transgenic mice, the proximal enhancer displayed specificity for the testis, tail, and ears, and the distal one for the testis, front feet, bone, heart, muscle, brain, spinal cord, and tongue, while dietary fat and overnight fasting had differential effects on enhancer activities. AgRP in the testis was localized to pachytene spermatocytes and in the tongue to epithelial cells. Comparative sequence analysis showed that the AgRP-ATP6V0D1 intergenic region is two times longer in humans than in mice and that the two enhancers are conserved in the rhesus monkey genome but not in the mouse genome. These data show that spatiotemporal expression of the human AgRP gene is influenced by diversified primate-specific intronic sequences in its neighboring ATP6V0D1 gene.