The transcriptional tissue specificity of the human proα1(I) collagen gene is determined by a negative cis-regulatory element in the promoter

C. P. Simkevich, J. P. Thompson, H. Poppleton, Rajendra Raghow

Research output: Contribution to journalArticle

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Abstract

The transcriptional activity of plasmid pCOL-KT, in which human proα1(I) collagen gene upstream sequences up to -804 and most of the first intron ( +474 to +1440) drive expression of the chloramphenicol acetyltransferase (CAT) gene [Thompson, Simkevich, Holness, Kang and Raghow (1991) J. Biol. Chem. 266, 2549 2556], was tested in a number of mesenchymal and non-mesenchymal cells. We observed that pCOL-KT was readily expressed in fibroblasts of human (IMR-90 and HFL-I), murine (NIH 3T3) and avian (SL-29) origin and in a human rhabdomyosarcoma cell line (A204), but railed to be expressed in human erythroleukaemia (K562) and rat pheochromocytoma (PC12) cells, indicating that the regulatory elements required for appropriate tissue-specific expression of the human proα1(I) collagen gene were present in pCOL-KT. To delineate the nature of cis-acting sequences which determine the tissue specificity of proα1(I) collagen gene expression, functional consequences of deletions in the promoter and first intron of pCOL-KT were tested in various cell types by transient expression assays. Cis elements in the promoter-proximal and intronic sequences displayed either a positive or a negative influence depending on the cell type. Thus deletion of fragments using EcoRV (nt-625 to -442 deleted), Xbal (-804 to -331) or SstII (+670 to +1440) resulted in 2-10-fold decreased expression in A204 and HFL-I cells. The negative influences of deletions in the promoter-proximal sequences was apparently considerably relieved by deleting sequences in the first intron, and the constructs containing the EcoRV/SstII or Xbal/SstII double deletions were expressed to a much greater extent than either of the single deletion constructs. In contrast, the Xbal(*) deletion (nt -804 to -609), either alone or in combination with the intronic deletion, resulted in very high expression in all cells regardless of their collagen phenotype; the Xbal(*)/(SstII) construct, which contained the intronic SstII fragment (+670 to +1440) in the reverse orientation, was not expressed in either mesenchymal or non-mesenchymal cells. Based on these results, we conclude that orientation-dependent interactions between negatively acting 5'-upstream sequences and the first intron determine the mesenchymal cell specificity of human proα1(I) collagen gene transcription.

Original languageEnglish (US)
Pages (from-to)179-185
Number of pages7
JournalBiochemical Journal
Volume286
Issue number1
DOIs
StatePublished - Jan 1 1992

Fingerprint

Organ Specificity
Collagen
Genes
Introns
Tissue
A 204
Chloramphenicol O-Acetyltransferase
Transcription
Fibroblasts
Gene expression
Leukemia, Erythroblastic, Acute
Rats
Assays
Rhabdomyosarcoma
PC12 Cells
Plasmids
Pheochromocytoma
Cells
Phenotype
Gene Expression

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

The transcriptional tissue specificity of the human proα1(I) collagen gene is determined by a negative cis-regulatory element in the promoter. / Simkevich, C. P.; Thompson, J. P.; Poppleton, H.; Raghow, Rajendra.

In: Biochemical Journal, Vol. 286, No. 1, 01.01.1992, p. 179-185.

Research output: Contribution to journalArticle

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