Transcriptional regulation of the proopiomelanocortin (POMC) gene in the pituitary has been evolutionary conserved across vertebrates

BUMASCHNY VF; DE SOUZA FSJ; SANTANGELO AM; BAETSCHER M; KATO-WEINSTEIN J; LOW MJ; RUBINSTEIN M

Boston, MA, EEUU

Congreso; The Endocrine Society’s 88th Annual Meeting (Endo 2006); 2006

The Endocrine Society

POMC is expressed in pituitary corticotrophs and melanotrophs and participates in stress responses and pigmentation in representative species of all vertebrate classes. To study whether the cis-acting transcriptional code has been conserved throughout vertebrate evolution we performed multiple local alignment comparisons of genomic sequences encompassing the POMC locus of several mammalian and fish species, the chicken, and the amphibian Xenopus tropicalis. We found that the only conserved sequences among all species were present in coding exon 3. This result indicates either that distant evolutionary species acquired novel molecular mechanisms to transcribe pituitary POMC or that existing bioinformatic tools are unable to detect functionally conserved transcriptional codes. To challenge this second hypothesis we generated transgenic mice expressing β-galactosidase driven by POMC 5’ flanking region of Tetraodon nigroviridis, a teleost fish that diverged from the lineage leading to mammals around 450 million years ago. T. nigroviridis genome has been sequenced and mapped and its size is 8 times smaller than those of mammals. A 900 bp Tetraodon POMC promoter was able to drive β-galactosidase expression to melanotrophs and corticotrophs of the pituitary of transgenic mice that greatly increased after adrenalectomy in parallel to expression of mouse Pomc. As functional conservation of pituitary specific elements and hormonal regulation was evident, we looked for homology in short sequences using the program Footprinter. Phylogenetic footprinting revealed a 20-bp sequence that was highly conserved between zebrafish, Tetraodon, Fugu, Xenopus, chicken, mouse and human. This element, that we named PPCE (Proximal POMC conserved element), has two consensus sites for homeodomain binding proteins. Deletion of PPCE reduced promoter activity by 70% in the mouse corticotrophic cell line AtT20. In AtT20 cells, Tetraodon POMC promoter was induced by the hormone CRH and the transcription factor Pitx1 in the presence or absence of PPCE. Our results indicate that the cis- and trans-acting regulatory elements controlling POMC pituitary expression have been remarkably conserved over 450 million years of vertebrate evolution. Also, our results show that functional analyses of promoters are more sensitive than existing bioinformatic programs to detect short conserved regulatory elements hidden within orthologous genes of evolutionary distant species.