Abstract
Binding sites for the tissue-specific transcription factor, Pit-1, are required for basal and hormonally induced prolactin gene transcription. Although Pit-1 is phosphorylated in response to several signaling pathways, the mechanism by which Pit-1 contributes to hormonal induction of gene transcription has not been defined. Recent reports suggest that phosphorylation of Pit-1 may not be required for hormonal regulation of the prolactin promoter. Analysis of the contribution of individual Pit-1 binding sites has been complicated due to the fact that some of the elements appear to be redundant. To better understand the role of Pit-1 sites in mediating hormonal regulation of the prolactin gene, we have performed enhancer tests using the three most proximal Pit-1 binding sites of the rat prolactin gene which are designated the 1P, 2P, and 3P sites. The results demonstrate that multimers of the 3P Pit-1 binding site are much more responsive to several hormonal and intracellular signaling pathways than multimers of the 1P or 2P sites. The 3P DNA element was found to contain a consensus binding site for the Ets family of proteins. Mutation of the Ets binding site greatly decreased the ability of epidermal growth factor, phorbol esters, Ras, or the Raf kinase to induce reporter gene activity. Mutation of the Ets site had little effect on basal enhancer activity. In contrast, mutation of the consensus Pit-1 binding site in the 3P element essentially abolished all basal enhancer activity. Overexpression of Ets-1 in GH3 pituitary cells enhanced both basal and Ras induced activity from the 3P enhancer. These data describe a composite element in the prolactin gene containing binding sites for two different factors and the studies suggest a mechanism by which Ets proteins and Pit-1 functionally cooperate to permit transcriptional regulation by different signaling pathways.
Highlights
The transcription of the prolactin gene is modulated by a number of hormones which bind to plasma membrane receptors
This approach is based on the observation that there appears to be functional redundancy in the DNA elements which permit hormonal regulation of the prolactin promoter [11, 35, 36] and the finding that multimers of individual Pit-1 binding sites can support transcriptional responses to some hormones or second messengers [14, 18]
Pit-1 binding to the 2P site has been demonstrated [10], there is evidence that this DNA element can interact with a ubiquitous factor [39] and that it plays a role in repressing the activity of the prolactin promoter in non-pituitary cell types [40]
Summary
The transcription of the prolactin gene is modulated by a number of hormones which bind to plasma membrane receptors. Oct-1 [24], Zn-15 [25], and TEF [26] can all interact with Pit-1 binding sites and activate transcription through these sites It is not clear if any of these other factors play a role in mediating hormonally regulated transcriptional activation. In the present study we have examined the ability of individual Pit-1 binding sites to permit responses to hormones and activators of specific signal transduction pathways. This functional redundancy has complicated analysis of the role of specific DNA elements in mediating hormonal responsiveness Because of this problem, we have used an enhancer test to compare the ability of the three most proximal Pit-1 binding sites of the prolactin gene to respond to hormones, intracellular second messengers and activated components of signal transduction pathways. The Ets site in the 3P DNA element is crucial for transcriptional responses to hormones and second messengers
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