The proximal promoter of the melanocortin 4 receptor harbors regulatory elements responsible for brain preferential expression.

Abstract

The melanocortin 4 receptor (MC4R) is a G-protein coupled receptor, which is widely, yet weakly, expressed throughout the CNS. Although the sites of MC4R expression are known, the field still does not understand how the gene is regulated temporally.

To determine key regulatory regions of the MC4R promoter, I have established in vitro and in vivo models for studying MC4R promoter activity. By transiently transfecting cell lines with mouse and human promoter-luciferase constructs, I identified regions within the proximal 900bp that correlate with positive expression in endogenous MC4R cell lines.

A 4.8kb promoter tauEGFP transgene transcript expression was demonstrated by Real Time RT-PCR to be restricted to the CNS, and similar to endogenous MC4R transcript expression in three of four independent lines. Using a Beta-Galactosidase reporter, 3.3kb 5’-flanking and 650bp 3’-flanking murine MC4R sequence also directed expression in a CNS specific manner. Interestingly, fetal tissues stained for Beta-Galactosidase activity showed non-CNS expression nearly identical to endogenous extra-neural MC4R expression in fetal rats. The luciferase transgenic mice, from the largest construct (identical in sequence to the LacZ construct) to the smallest (only containing 65bp of the 100% conserved region – CR-8) were found to express the transgene in the CNS.

These results suggest that the 5’-flanking proximal sequence of the MC4R gene is sufficient for brain preferential expression in vivo. Furthermore, the highly conserved CR-8 region is sufficient to target brain preferential expression in a heterologous promoter in vivo in a pattern similar to the larger constructs.