Regulation of transcription of the TATA-less human complement component C4 gene

AK Vaishnaw, TJ Mitchell, SJ Rose, MJ Walport, Bernard J Morley

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

The 5'-sequences flanking the human complement component C4 genes (C4A and C4B) have been analyzed for their ability to direct expression of a reporter gene in cell lines that constitutively express or do not express C4. No difference in the level of reporter gene expression was detected in cells transfected with C4A- or C4B-specific constructs. A series of reporter constructs containing progressively truncated C4 promoter fragments transfected into the hepatocyte Hep G2 cell line, identified the sequence contained within the region -178 to -39 as that associated with maximal reporter gene expression. This region contains consensus binding motifs for nuclear factor 1 (-110 to -97), Sp1 (-57 to -49), and three basic helix-loop-helix (-137 to -132, -98 to -93, and -78 to -73)-like transcription factors. Electromobility shift assays and DNase I footprinting analysis showed specific DNA-protein interactions of the C4 promoter at the nuclear factor 1, two E box (-98 to -93 and -78 to -73), and Sp1 binding domains. Site-directed mutagenesis of the Sp1 binding site resulted in total abrogation of reporter gene expression and mutation of the E box (-78 to -73) resulted in a 8-fold reduction in expression. We conclude that the Sp1 binding site at position -57 to -49 is critical for accurately initiated, basal transcription of C4.
Original languageEnglish
Pages (from-to)4353-4360
Number of pages8
JournalThe Journal of Immunology
Volume160
Publication statusPublished - May 1998

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Complement C4
Reporter Genes
NFI Transcription Factors
Gene Expression
Genes
Binding Sites
5' Flanking Region
Deoxyribonuclease I
Hep G2 Cells
Site-Directed Mutagenesis
Hepatocytes
Transcription Factors
Cell Line
Mutation
DNA
Proteins

Cite this

Vaishnaw, AK., Mitchell, TJ., Rose, SJ., Walport, MJ., & Morley, B. J. (1998). Regulation of transcription of the TATA-less human complement component C4 gene. The Journal of Immunology, 160, 4353-4360.

Regulation of transcription of the TATA-less human complement component C4 gene. / Vaishnaw, AK; Mitchell, TJ; Rose, SJ; Walport, MJ; Morley, Bernard J.

In: The Journal of Immunology, Vol. 160, 05.1998, p. 4353-4360.

Research output: Contribution to journalArticle

Vaishnaw, AK, Mitchell, TJ, Rose, SJ, Walport, MJ & Morley, BJ 1998, 'Regulation of transcription of the TATA-less human complement component C4 gene', The Journal of Immunology, vol. 160, pp. 4353-4360.
Vaishnaw, AK ; Mitchell, TJ ; Rose, SJ ; Walport, MJ ; Morley, Bernard J. / Regulation of transcription of the TATA-less human complement component C4 gene. In: The Journal of Immunology. 1998 ; Vol. 160. pp. 4353-4360.
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AB - The 5'-sequences flanking the human complement component C4 genes (C4A and C4B) have been analyzed for their ability to direct expression of a reporter gene in cell lines that constitutively express or do not express C4. No difference in the level of reporter gene expression was detected in cells transfected with C4A- or C4B-specific constructs. A series of reporter constructs containing progressively truncated C4 promoter fragments transfected into the hepatocyte Hep G2 cell line, identified the sequence contained within the region -178 to -39 as that associated with maximal reporter gene expression. This region contains consensus binding motifs for nuclear factor 1 (-110 to -97), Sp1 (-57 to -49), and three basic helix-loop-helix (-137 to -132, -98 to -93, and -78 to -73)-like transcription factors. Electromobility shift assays and DNase I footprinting analysis showed specific DNA-protein interactions of the C4 promoter at the nuclear factor 1, two E box (-98 to -93 and -78 to -73), and Sp1 binding domains. Site-directed mutagenesis of the Sp1 binding site resulted in total abrogation of reporter gene expression and mutation of the E box (-78 to -73) resulted in a 8-fold reduction in expression. We conclude that the Sp1 binding site at position -57 to -49 is critical for accurately initiated, basal transcription of C4.

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