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PMID: 7565693 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't

G10BP, an E1A-inducible negative regulator of Sp1, represses transcription of the rat fibronectin gene.

Molecular and cellular biology ·Vol. 15 ·No. 10 ·1995-10-00 ·Pages 5423-33

Suzuki M, Kuroda C, Oda E, Tsunoda S, Nakamura T, Nakajima T, Oda K

Abstract

Downregulation of the fibronectin (FN) gene in a rat 3Y1 derivative cell line, XhoC, transformed by the adenovirus E1A and E1B genes seems to be caused by the induction of a negative regulator, G10BP, which binds to three G-rich sequences in the promoter (T. Nakamura, T. Nakajima, S. Tsunoda, S. Nakada, K. Oda, H. Tsurui, and A. Wada, J. Virol. 66:6436-6450, 1992). These are the G10 stretch and two GC boxes consisting of the G10 stretch with one internal C residue insertion. The recognition sequences of G10BP and Sp1 (GGGCGG) overlap in these GC boxes. To analyze the mechanism of the downregulation, G10BP was purified by DNA affinity chromatography, and its molecular mass was estimated to be about 30 kDa. The promoter was modified by substituting the sequence GGGG with ATCC or CTTA in these G-rich sequences, leaving the Sp1 motif intact, and by replacing the Sp1 motif by the T stretch. Transcription of FN promoter-chloramphenicol acetyltransferase fusion genes carrying the base substitution in one or more of these G-rich sequences both in vivo and in vitro revealed that the base substitution in any G-rich sequence results in reduction of promoter activity, although the downstream GC box (GCd) plays a primary role. The addition of G10BP severely inhibited the activities of the FN promoters carrying the wild-type GCd in vitro, while the promoters carrying the mutant GCd were unaffected. The binding affinity of G10BP and Sp1 to each of the G-rich sequences, analyzed by gel shift assays, indicated that G10BP binds strongly to the GCd, moderately to the G10 stretch, and weakly to GCu, while Sp1 binds strongly to GCu, moderately to GCd, and weakly to the G10 stretch. Sp1 binding to GCd and the G10 stretch was inhibited by G10BP, while binding to GCu was unaffected. These results indicate that FN gene transcription is inhibited in XhoC cells primarily by exclusion of Sp1 binding to GCd by G10BP and that G10BP is a new class of Sp1 negative regulator.

MeSH Terms
Adenovirus E1A Proteins/physiology Animals Base Sequence Cell Line, Transformed DNA, Single-Stranded/metabolism Fibroblasts Fibronectins/genetics Gene Expression Regulation/physiology HeLa Cells Humans Molecular Sequence Data Molecular Weight Mutation Promoter Regions, Genetic/genetics Rats Rats, Inbred F344 Recombinant Fusion Proteins/biosynthesis Repressor Proteins/chemistry,isolation & purification,metabolism Retinoblastoma Protein/metabolism Sp1 Transcription Factor/metabolism Transcription, Genetic/physiology
Chemicals
Adenovirus E1A Proteins DNA, Single-Stranded Fibronectins Recombinant Fusion Proteins Repressor Proteins Retinoblastoma Protein Sp1 Transcription Factor
Authors & Affiliations
7 authors, click to expand affiliations / ORCID
Suzuki M
Department of Biological Science and Technology, Science University of Tokyo, Noda, Japan.
Kuroda C
Oda E
Tsunoda S
Nakamura T
Nakajima T
Oda K
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Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/MCB.15.10.5423
Published
1995-10-00
Pages
5423-33
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC230792
Subset
IM
External Links
PubMed source DOI: 10.1128/MCB.15.10.5423 PMC Full Text View journal details
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