DNA sequences required for efficient transcription of the estrogen regulated Xenopus laevis vitellogenin promoter
Chang, Tsu-Chung
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Permalink
https://hdl.handle.net/2142/23610
Description
Title
DNA sequences required for efficient transcription of the estrogen regulated Xenopus laevis vitellogenin promoter
Author(s)
Chang, Tsu-Chung
Issue Date
1990
Department of Study
Biology, Molecular
Chemistry, Biochemistry
Discipline
Biology, Molecular
Chemistry, Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, Molecular
Chemistry, Biochemistry
Language
eng
Abstract
In liver cells of Xenopus laevis expression of the vitellogenin genes is tightly regulated by estrogen. Estrogen activates gene transcription by binding as estradiol-estrogen receptor complex to estrogen response elements (EREs) in the 5$\sp\prime$-flanking region of target genes. In order to identify the DNA sequences essential for estrogen-regulated and liver-specific vitellogenin gene expression, we employed site-specific mutagenesis to create a variety of deletions, mutations, and insertions in the 5$\sp\prime$-flanking region of the vitellogenin B1 promoter. The effects of these mutations on promoter activity were studied by using a homologous cotransfection system developed in our laboratory with cell lines derived from Xenopus hepatocytes and fibroblasts. The role of EREs and other cis-acting sequences in the B1 promoter has been characterized. Mutation of the CAAT box or deletion of a conserved nematode box or the entire region between -301 and -121 had no significant effect on vitellogenin promoter activity. An eight base DNA element with weak sequence homology to the NF1 element is essential for efficient transcription of the B1 promoter. Deletion or mutation of this vitellogenin activator (VA) element reduces promoter activity by at least an order of magnitude. Reinsertion of this sequence into the VA-deleted construct restores promoter activity in a position dependent manner. Gel mobility shift assays have been used to identify a protein in liver cell and oocyte extracts which binds to VA sequence with high affinity and specificity. Competition studies demonstrate that this factor is a member of an NF1-like protein family. The VA region linked to a TATA box can function as a more powerful independent transcription activator than the NF1 consensus sequence in a similar construct. These data indicate that the VA binding protein is related to, but probably not identical to NF1. (Abstract shortened with permission of author.)
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