LH and CG are synthesized in pituitary and placenta, respectively, and are essential to mammalian reproduction. They are heterodimeric glycoprotein hormones composed of a common alpha-subunit noncovalently linked to a unique beta-subunit. In contrast to primates, the equine LH and CG beta- subunits have identical amino acid sequences and are encoded by a single- copy gene. Therefore, the eLH/CGbeta gene must possess elements that confer both pituitary- and placenta-specific expression. Little is known about the mechanisms that regulate expression of gonadotropin beta- subunits. Pituitary expression of the eLH/CGbeta gene has been shown to be regulated by multiple regions within the proximal promoter. Furthermore, 448 and 3000 bp of eLH/CGbeta 5 '-flanking sequence direct expression of a reporter gene to the pituitary of transgenic mice. Therefore, these constructs must contain the regulatory elements required for pituitary- specific expression. The overall goal of the present proposal is to elucidate these regulatory elements and their cognate trans-acting factors in order to develop a better understanding of the physiological events that regulate gene expression in pituitary. This will be addressed in the following aims: 1) Determine the identity of the elements and factors that direct pituitary expression of the equine LH/CGbeta subunit gene. An 85 bp region of the eLH/CGbeta promoter is required for basal transcription in a pituitary cell line. Elements contained within this region will be identified and their cognate trans-acting factors cloned and characterized. Experimental approaches include DNase footprinting, methylation interference, UV crosslinking, southwestern analysis, transient transfections, and recognition site cloning. 2) Determine the physiological relevance of the elements identified from in vitro studies. Transgenic mice will be used to determine the temporal and spatial patterns of expression for e-beta constructs containing 3000 or 443 bp of 5'-flanking sequence. These studies will begin to reveal the regions required for correct spatiotemporal expression. Transgenic mice will also be used to evaluate the physiological significance of elements identified in Aim l. The role these elements play in directing the correct spatiotemporal expression pattern of the eLH/CGbeta gene will also be examined. These experiments will involve the use of transgenic mice which will be evaluated for transgene expression by performing chemiluminescent assays, X-gal staining, immunohistochemistry and in situ hybridization.
