Prolactin is a hormone with diverse biological functions. This diversity of function seems to necessitate the wide range of regulators that have been reported to influence prolactin production. We are proposing to study the effects of estrogen on prolactin biosynthesis in normal and hyperplastic pituitary cells. We will use pituitary cells of rats because they are like the human, in that regulation of the biosynthetic machinery is in part due to estrogens. Also, many pituitary hyperplasias in the human are characterized by high prolactin production and thus are similar to the Fischer 344 rat model. Recent data show that, within minutes after estrogen administration into the rat, the transcription rate of the prolactin gene is stimulated. We proposed to determine what structural changes in prolactin gene chromatin occur after estrogen is administered. Chromatin structure will be examined by nuclease sensitivity with particular emphasis on hypersensitive sites previously detected in the upstream or 5' flanking region of the gene. Density gradient centrifugation and two-phase partitioning will be used to assess structural changes and to enrich prolactin chromatin fractions that are being actively transcribed. Finally, gene transfer experiments will be used to characterize the chromatin domains essential for estrogen regulation of prolactin gene transcription. Cells that do or do not contain estrogen receptors will be tranfected by plasmid constructs containing regulatory sequences from within or flanking the prolactin gene. Transient expression systems will permit the use of primary cell cultures in some experiments. These studies will be extended to the development of extrachromosomal plasmid constructs that might permit the isolation and characterization of prolactin chromatin fragments. (D)
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