Abstract
The proopiomelanocortin (POMC) gene is highly expressed in adult mouse pituitary anterior lobe corticotrophs and intermediate lobe melanotrophs. To identify the DNA elements important for this tissue-specific expression, we analyzed a series of POMC reporter genes in transgenic mice. A DNA fragment containing rat POMC 5'-flanking sequences from -323 to -34 recapitulated both basal pituitary cell-specific and hormonally stimulated expression in adult mice when fused to a heterologous thymidine kinase promoter. Developmental onset of the reporter gene expression lagged by 1 day but otherwise closely paralleled the normal ontogeny of murine POMC gene expression, including corticotroph activation at embryonic day 14.5 (E14.5) followed by melanotroph activation at E15.5 to E16.5. AtT20 corticotroph nuclear protein extracts interacted with three specific regions of the functional POMC promoter in DNase I protection assays. The positions of these protected sites were -107 to -160 (site 1), -182 to -218 (site 2), and -249 to -281 (site 3). Individual deletions of these footprinted sites did not alter transgene expression; however, the simultaneous deletion of sites 2 and 3 prevented transgene expression in both corticotrophs and melanotrophs. Electrophoretic mobility shift and Southwestern (DNA-protein) assays demonstrated that multiple AtT20 nuclear proteins bound to these footprinted sites. We conclude that the sequences between -323 and -34 of the rat POMC gene promoter are both necessary and sufficient for correct spatial, temporal, and hormonally regulated expression in the pituitary gland. Our data suggest that the three footprinted sites within the promoter are functionally interchangeable and act in combination with promoter elements between -114 and -34. The inability of any reporter gene construction to dissociate basal and hormonally stimulated expression suggests that these DNA elements are involved in both of these two characteristics of POMC gene expression in vivo.
Highlights
The proopiomelanocortin (POMC) gene is highly expressed in adult mouse pituitary anterior lobe corticotrophs and intermediate lobe melanotrophs
From a deletion analysis of the rat POMC (rPOMC) gene, we found that DNA sequences between -323 and -34 of the 5'-flanking region had the ability to confer all of the information required for cell-specific and hormoneregulated transgene expression from the heterologous thymidine kinase (TK) promoter in the pituitary glands of transgenic mice
Our results suggested that the sites may be functionally interchangeable and that at least two of the sites are needed for the activation of transgenes in pituitary gland POMC-producing cells
Summary
The proopiomelanocortin (POMC) gene is highly expressed in adult mouse pituitary anterior lobe corticotrophs and intermediate lobe melanotrophs. AtT20 corticotroph nuclear protein extracts interacted with three specific regions of the functional POMC promoter in DNase I protection assays. The positions of these protected sites were -107 to -160 (site 1), -182 to -218 (site 2), and -249 to -281 (site 3). The accumulated data suggest that the DNA promoter and/or enhancer elements mediating cell-specific and hormone-regulated POMC gene expression in the pituitary gland are located in the 5'-flanking region of the gene [19, 40]. Binding activity, consisting of one or more distinct proteins, was confirmed by gel retardation and Southwestern (DNA-protein) assays with synthetic double-stranded oligonucleotides corresponding to the footprinted sites The functions of these sites were investigated in transgenic mice by selective block deletions of the protected sequences from the 290 bp of DNA. We propose that a cooperative interaction of the three sites and their associated protein factors is essential for POMC gene transcription in the pituitary gland
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