Transcriptional Regulation of Pituitary POMC Is Conserved at the Vertebrate Extremes Despite Great Promoter Sequence Divergence

Viviana F Bumaschny,FláVio S J De Souza,Andrea M Santangelo,Diego H Levi,Marcelo Rubinstein,Malcolm J Low,Manfred Baetscher,Rodrigo A LóPez Leal

doi:10.1210/me.2006-0557

Abstract

The stress response involves complex physiological mechanisms that maximize behavioral efficacy during attack or defense and is highly conserved in all vertebrates. Key mediators of the stress response are pituitary hormones encoded by the proopiomelanocortin gene (POMC). Despite conservation of physiological function and expression pattern of POMC in all vertebrates, phylogenetic footprinting analyses at the POMC locus across vertebrates failed to detect conserved noncoding sequences with potential regulatory function. To investigate whether ortholog POMC promoters from extremely distant vertebrates are functionally conserved, we used 5'-flanking sequences of the teleost fish Tetraodon nigroviridis POMCalpha gene to produce transgenic mice. Tetraodon POMCalpha promoter targeted reporter gene expression exclusively to mouse pituitary cells that normally express Pomc. Importantly, transgenic expression in mouse corticotrophs was increased after adrenalectomy. To understand how conservation of precise gene expression mechanisms coexists with great sequence divergence, we investigated whether very short elements are still conserved in all vertebrate POMC promoters. Multiple local sequence alignments that consider phylogenetic relationships of ortholog regions identified a unique 10-bp motif GTGCTAA(T/G)CC that is usually present in two copies in POMC 5'-flanking sequences of all vertebrates. Underlined nucleotides represent totally conserved sequences. Deletion of these paired motifs from Tetraodon POMCalpha promoter markedly reduced its transcriptional activity in a mouse corticotropic cell line and in pituitary POMC cells of transgenic mice. In mammals, the conserved motifs correspond to reported binding sites for pituitary-specific nuclear proteins that participate in POMC transcriptional regulation. Together, these results demonstrate that mechanisms that participate in pituitary-specific and hormonally regulated expression of POMC have been preserved since mammals and teleosts diverged from a common ancestor 450 million years ago despite great promoter sequence divergence.

Highlights

THE PROOPIOMELANOCORTIN GENE (POMC) encodes a prohormone that gives rise to several bioactive peptides including ACTH, the melanocortins ␣, ␤, and ␥-MSH, and the opioid peptide ␤-endor
Extensive expression studies performed in the mouse corticotropic-derived cell line AtT20 led to the characterization of several cis-acting sequences that participate in the transcriptional control of pituitary POMC, including binding sites for the following transcription factors: paired-like homeodomain 1 (Pitx1), pituitary-restricted transcription factor (Tpit), Ikaros, neurogenic differentiation 1 (NeuroD1)/␤2, Nurr1/NR4A2, Nur77/NR4A1, signal transducer and activator of transcription 3 (STAT3), and glucocorticoid receptor [10,11,12,13,14,15]
To investigate whether the POMC promoter of a teleost fish is functionally conserved in a mammalian expression system, we sought to study the expression of Tetraodon POMC gene constructs in transgenic mice

Summary

Introduction

THE PROOPIOMELANOCORTIN GENE (POMC) encodes a prohormone that gives rise to several bioactive peptides including ACTH, the melanocortins ␣-, ␤-, and ␥-MSH, and the opioid peptide ␤-endor-. Stress-induced release of the hypothalamic peptide CRH stimulates the release of ACTH from pituitary corticotrophs, which in turn promotes glucocorticoid release from the adrenal gland cortex. Transgenic mouse studies revealed that 400 bp of the rat POMC proximal promoter direct pituitary cell-specific and hormonally regulated expression to corticotrophs and melanotrophs [9]. Extensive expression studies performed in the mouse corticotropic-derived cell line AtT20 led to the characterization of several cis-acting sequences that participate in the transcriptional control of pituitary POMC, including binding sites for the following transcription factors: paired-like homeodomain 1 (Pitx1), pituitary-restricted transcription factor (Tpit), Ikaros, neurogenic differentiation 1 (NeuroD1)/␤2, Nurr1/NR4A2 (nuclear receptor subfamily 4, group A, member 2), Nur77/NR4A1 (nuclear receptor subfamily 4, group A, member 1), signal transducer and activator of transcription 3 (STAT3), and glucocorticoid receptor [10,11,12,13,14,15]. 415 bp of the zebrafish proximal POMC promoter have been shown to direct specific expression of a reporter gene to the pituitary of transgenic zebrafish [16]

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Conclusion