Abstract
Previous studies indicate that a human chorionic somatomammotropin (hCS) gene enhancer (CSEn) associated with the growth hormone (hGH) gene locus is involved in directing cell-specific expression of the hCS genes in placenta. In the current studies, we report a detailed structural analysis of this enhancer. CSEn stimulated transcription of a variety of promoters, including the hCS, human growth hormone, thymidine kinase, and Rous sarcoma virus promoters, in human choriocarcinoma cell lines (BeWo and JEG-3) but not HeLa cells or rat somatolactotrophes (GC). Maximal enhancer activity was confined to a 242-base pair DNA segment. Of several CSEn subfragments, only the En 57/242 subfragment retained activity (33.5% wild-type). The CSEn DNA sequence contained direct and inverted repeat motifs and sequences related to the SV40 enhansons, GT-IIC, GT-I, and SphI/SphII. DNase I footprint analysis revealed that most of these sites were protected by nuclear proteins derived from BeWo, JEG-3, HeLa, and GC cells. Site-specific block mutation of the GT-IIC-related and inverted repeat motifs virtually abolished enhancer activity, and mutation of all but the GT-I-related motif resulted in significant loss (30-60%) of activity. These data demonstrate that the CS enhancer is comprised of multiple elements related to SV40 enhansons that interact cooperatively to generate enhancer function.
Highlights
To whom correspondence should be addressed4-407 Alfred, SMH, Mayo Clinic, Rochester, MN 55905. “el.: 507-255-6554; Fax: 507-255-
Previous studies indicate that a human chorionicso- to be a pseudogene (Hirt et al.,1987)
Activity was confined to a 242-basDeNpAasiergment.Of the hCS5’-flanking, promotor, and coding regions several CSEn subfragments,only the En-571242 subfrag- are nearly identical(93.5-96% sequence identity) to thecorrement retained activity(33.6%wild-type).The CSEn DNA sponding regions of thehGH gene(Miller andEberhardt, sequence contained direct and inverted repeat motifs 1983), its distinct cell-specific expression in placental syncyand sequences related to the SV40 enhansons, GT-IIC, tiotrophoblasts indicates that unique mechanisms account for GT-I, andSphYSphII.DNase I footprintanalysisre- its cell-specific expression
Summary
4-407 Alfred, SMH, Mayo Clinic, Rochester, MN 55905. “el.: 507-255-6554; Fax: 507-255-. CSEn consists of multipartite DNA sequences, all of which are with enhancer inserted in different orientations were designaEte&d required for maximal enhancer function These sequences bear p&LUC and EnB-pA,LUC, respectively. Mutation of the TEF-1binding motif, GT-IIC, virtually eliminated enhancer activity; a 49-nucleotide region containing this element lacked enhancer activity when linked to the hCS promoter, indicating that TEF-1 or related the promoterless vectors, p&LUC, E A p & L U C , a n d EnB_pA,LUC were indistinguishable from buffer background control assays (data not shown) due to thepoly(A) stops preceding the luciferase reporter gene (Maxwell et al, 1989). The hCS promoter was inserted into p&LUC, E&p&LUC, and EnB-p&LUC between the Sal and Hind111 sites, immediately upstream of the luciferase reporter gene using standard factors were necessary but not sufficient for enhancer activity.
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