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The proximal region of the rat PRL gene contains at least five transcription-stimulating elements that are located within a 170-basepair region up-stream of the TATA box. These cis-acting elements include four binding sites for the pituitary-specific transcription factor Pit-1 as well as another site for an unidentified factor. In this study interactions between different DNA elements have been examined through the construction of PRL-luciferase fusion genes containing mutations that disrupt various combinations of the individual DNA elements. In general, the disruption of multiple factor-binding sites had a much more than additive effect on expression of the luciferase constructs. Interestingly, comparison of the effects of disrupting pairs of binding sites demonstrated substantial differences in the effects of different combinations of mutations, suggesting that cooperative interactions may reflect specific interactions. Mutations that disrupted all five cis-elements of the PRL proximal region essentially abolished transcription from the proximal promoter. This finding suggests that there are no other DNA elements within the proximal 200 basepairs of the PRL gene that can independently stimulate transcription. Although there is strong functional cooperativity between different cis-elements in the PRL gene, DNase footprint studies failed to detect cooperative binding between different Pit-1 elements. Overall, the findings demonstrate that the normal transcription of the PRL gene involves strong cooperative interactions between individual DNA elements in the proximal region.

Analysis of functional cooperativity between individual transcription-stimulating elements in the proximal region of the rat prolactin gene.