The Abolition of Collagen Gene Expression in SV40-transformed Fibroblasts is Associated with Trans-acting Factor Switching

Overview

Journal Nucleic Acids Res

Publisher Oxford University Press

Specialty Biochemistry

Date 1992 Nov 11

PMID 1333588

Citations 4

Authors

M I Parker

A A Smith

K Mundell

M Collins

S Boast

F Ramirez

Affiliations

Soon will be listed here.

Abstract

The goal of this study was to determine whether alpha 2(1) procollagen gene expression is modulated by positive or negative trans-acting DNA-binding proteins. Previous studies have shown that a clone of SV40-transformed WI-38 fibroblasts (SVWI-38) does not produce any alpha 2(1) procollagen mRNA (Parker et al (1989), J. Biol Chem. 264, 7147-7152). In order to elucidate the mechanism(s) responsible for such inactivation, we have examined the activity of a transfected wild type COL1A2 promoter in SVWI-38 cells. A set of 5' promoter deletions was linked to the chloramphenicol acetyltransferase (CAT) gene and transfected into SVWI-38 and other cell lines expression type I collagen. The resulting CAT assays confirmed the importance of several upstream regions for promoter activity and documented the decreased transcriptional activity from an exogenous COL1A2 promoter in the SVWI-38 cell line. Competition experiments with an excess of COL1A2 promoter DNA fragment and a constant amount of COL1A2/CAT construct displayed a linear relationship between excess COL1A2 fragment and CAT activity in SVWI-38 cells, suggesting the involvement of a titratable negative effector. Electrophoretic mobility shift assays revealed the presence of a specific DNA-protein complex which was present in SVWI-38 cells and almost absent in control fibroblasts. Methylation interference analysis mapped the region of binding of this factor between nucleotides -80 and -72, relative to the transcription start site. Thus the data presented provide strong evidence for the existence of a negative trans-acting factor that may play a role in the repression of COL1A2 expression in SVWI-38 fibroblasts.

Citing Articles

A novel role of annexin A2 in human type I collagen gene expression.

Schafer G, Hitchcock J, Shaw T, Katz A, Parker M J Cell Biochem. 2014; 116(3):408-17.

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Regulation of the human alpha 2(1) procollagen gene by sequences adjacent to the CCAAT box.

Collins M, Leaner V, Madikizela M, Parker M Biochem J. 1997; 322 ( Pt 1):199-206.

PMID: 9078262 PMC: 1218177. DOI: 10.1042/bj3220199.

Evidence for three major transcription activation elements in the proximal mouse proalpha2(I) collagen promoter.

Hasegawa T, Zhou X, Garrett L, Ruteshouser E, Maity S, de Crombrugghe B Nucleic Acids Res. 1996; 24(16):3253-60.

PMID: 8774909 PMC: 146084. DOI: 10.1093/nar/24.16.3253.

Human COL2A1-directed SV40 T antigen expression in transgenic and chimeric mice results in abnormal skeletal development.

Cheah K, Levy A, Trainor P, Wai A, Kuffner T, So C J Cell Biol. 1995; 128(1-2):223-37.

PMID: 7822417 PMC: 2120328. DOI: 10.1083/jcb.128.1.223.

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