Cell-specific expression of the prolactin gene in transgenic mice is controlled by synergistic interactions between promoter and enhancer elements.

Abstract

Prolactin gene expression is restricted to the lactotrophic and somatomammotrophic cells of the anterior pituitary. In transgenic mice, a fusion gene consisting of 3 kb of prolactin 5'-flanking region fused to a firefly luciferase or human growth hormone {hGH) reporter gene is expressed at high levels with the strict tissue and cell-type specificity that is characteristic of the endogenous prolactin gene. High levels of expression require two c/s-acting regions: a distal enhancer (-1.8 to -1.5 kb) and a proximal region (-422 to + 33 bp). Each of these regions alone can direct low levels of fusion gene expression to prolactin-producing cell types in transgenic mice, but a synergistic interaction between these regions is necessary for high levels of expression. The ontogeny of the prolactin transgene expression closely follows the appearance of high levels of a POU homeo-domain transcription factor, Pit-l, that has been shown previously to bind structurally related sequences in both the distal enhancer and proximal regions and to activate the expression of the prolactin gene in vitro. Unexpectedly, transgenes containing the distal enhancer removed from its normal context are expressed in both the prolactin-producing lactotrophs and the thyroid-stimulating hormone (TSHJ-producing thyrotrophs, thereby suggesting that sequences flanking this enhancer are necessary to restrict expression to the correct cell type within the pituitary. These data indicate that distinct processes of gene activation and restriction are necessary for the fidelity of cell-type-specific expression within an organ.

Pituitary cell phenotypes involve cell-specific Pit-1 mRNA translation and synergistic interactions with other classes of transcription factors.

“…A detailed quantitation revealed that most (-80%), but not all, somatotrophs, lactotrophs, and thyrotrophs contained detectable Pit-1 protein (Table 2). These data were consistent with previous observations (Crenshaw et al 1989), suggesting that restrictive mechanisms, as well as Pit-l-dependent gene activation, were required for cell-specific pattems of growth hormone and prolactin gene expression.…”

“…Pit-1 gene transcripts are expressed in all five phenotypically distinct pituitary cell types, although Pit-1 protein is detected only in the nuclei of thyrotrophs, lactotrophs, and somatotrophs. The expression of Pit-1 protein in thyrotrophs, in concert with previous analyses of transgenic mice (Crenshaw et al 1989), suggests that specific restrictive mechanisms prevent the expression of GH and PRL genes in this cell type. Thus, transcriptional and posttranscriptional regulation of Pit-l, and the temporal lag in maximal expression of the estrogen receptor, could account for the ontogeny and physiological levels of PRL gene expression characteristic of the lactotroph cell type.…”

“…While GH gene expression was maximal by e19-e20, the PRL gene exhibited a gradual, marked (10-to 50-fold) increase in expression between the initial appearance at e17 and full expression -1 0 -1 5 days after birth. Because synergistic interactions between the proximal promoter and the distal enhancer regions of the PRL gene were necessary to achieve high levels of expression in vivo as established in transgenic expression in mice, and on the basis of analysis of specific interactions in the distal enhancer in GC pituitary cells (Nelson et al 1986(Nelson et al , 1988Crenshaw et al 1989), it became important to determine whether Pit-1 alone was capable of mediating these synergistic interactions between the proximal and distal regulatory regions of the PRL gene. We and others have established that purified or bacterially expressed Pit-1 protein binds effectively to elements in both the rat PRL and GH promoters and can activate transcription of both promoters in cell culture models (Ingraham et al 1988;Mangalam et al 1989;Fox et al 1990;Larkin et al 1990;D.…”

A mouse oocyte-specific protein that binds to a region of mZP3 promoter responsible for oocyte-specific mZP3 gene expression.

“…The presence of cis-acting elements in the vicinity of the transcription start site has been reported for several genes, including those encoding growth hormone (25), elastase (13), insulin (14), and prolactin (7). Our findings suggest that certain other potential cis-acting elements (20), located further upstream of the mZP3 gene transcription start site (e.g., a 54-nt consensus sequence, present as six tandem repeats between nt -826 and -508), are probably not involved in regulating oocytespecific, mZP3 gene expression.…”

“…The digest was fractionated on an agarose gel, and the fragment was recovered by electroelution, extracted with phenol-chloroform, precipitated with ethanol, resuspended at 1 Fg/ml in injection buffer (5 mM Tris-HCI [pH 7.4]-5 mM NaCl-1 mM EDTA), and stored at -20°C.…”