Studies on the role of the distal sequences involved in the expression of the human growth hormone/chorionic somatomammotropin gene family

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Surabhi, Rama Mohan
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The human growth hormone/chorionic somatomammotropin (GH/CS) gene family offers an interesting model to study mechanisms involved in tissue-specific transcriptional regulation of genes. Despite their high sequence homology, the members of GH/CS gene family are predominantly expressed in two different tissues: the GH-N gene is expressed in pituitary, whereas the CS-L, CS-A, GH-V and CS-B genes are expressed in placenta. A series of highly conserved regions within the GH/CS gene locus were identified by other investigators and were suggested to play a role in the differential and tissue-specific transcriptional regulation of the GH/CS genes. Presented here is the characterization of sequences/elements residing within the GH/CS locus, as well as those found in the flanking regions. Previous work in our laboratory identified PSF (PSF-A/-B) elements which are involved in the pituitary repressor mechanism for the CS/GH-V genes. Part of the present thesis work is aimed at the identification of the PSF-DNA binding proteins using conventional protein purification methods combi ed with PSF-specific DNA affinity chromatography. Characterization of partially purified proteins using southwestern blotting suggested that PSF proteins could be in the molecular range of 50-70 kDa. Further analysis of the PSF-DNA sequence revealed that these elements are recognized likely by nuclear factor-1 (NF-1) or NF-1-like proteins. NF-1 belongs to a family of proteins whose molecular sizes range from 50 to 70 kDa. Thus, these characteristics strongly suggest that PSFs could be NF-1 or NF-1-like. An AP-1 element is identified in the upstream sequences of the GH/CS genes using Nase I protection assays. Attempts were also made to characterize the role of this AP-I element through the use of transient transcription analysis. The enhancer/enhancer-like sequences located downstream of CS-L, CS-A and CS-genes were shown to contain multiple elements for transcriptional enhancer factor-1 (TEF-1). The presence of these sites implicated TEF-1, which is expressed ubiquitously, and TEF-5, which is expressed abundantly in placenta, in modulating the placenta-specific expression of the CS genes. A series of deletion mutants coding for truncated forms of TEF-1 and TEF-5 were generated and were tested in transient transfections. These studies reveal a HeLa (human cervical carcinoma) cell-specific potent transcriptional activator in the form of a truncated TEF-1. Further work on the characterization of remote sequences in the upstream as well as downstream flanking sequences of the GH/CS gene locus was done. Structural analysis of hypersensitive sites I and II, which are located ~15 kilobases upstream of the GH-N gene and form part of the GH/CS locus control region, led to the suggestion and identification ofthe involvement of the pituitary-specific transcription factor, growth hormone factor-1 (GHF-1)/Pit-1 in the pituitary-specific enhancer activity associated with these regions. Characterization of far-downstream sequences of the GH/CS gene loci revealed the location of two ubiquitously expressed genes: BAF60b (a gene coding for a subunit protein of SWI/SNF protein complex) and TRIP-1 (thyroid hormone receptor interacting protein-1). The pattern of this arrangement of genes, GH-BAF60b-TRIP-1, appears to be evolutionarily conserved among several species of vertebrate animals. A model is proposed to signify the presence of these ubiquitously expressing genes downstream of the GH/CS genes.