Characterization of the promoter region of the human thrombospondin gene. DNA sequences within the first intron increase transcription.
Thrombospondin (TSP) is an extracellular matrix glycoprotein whose synthesis and secretion by mesenchymal cells is regulated at the level of gene transcription by platelet-derived growth factor. To examine the transcriptional regulation of the TSP gene at the molecular level, a genomic clone containing the human TSP promoter and flanking sequence was isolated and characterized. A 3.8-kilobase pair (kb) DNA fragment containing the first three exons, the first two introns, and 2.2 kb of 5'-flanking region was sequenced, and the site of transcription initiation was determined by both primer extension and S1 nuclease mapping. Consensus sequences for several potential regulatory elements were found in the 5'-flanking sequence, including a TATA box consensus sequence, TTTAAAA, located 24 base pairs upstream from the transcription start site. A chimeric gene was constructed containing the first intron, the first exon, and 2.0 kb of 5'-flanking sequence of the TSP gene fused to the promoterless gene for chloramphenicol acetyltransferase. When transfected into COS-1 or NIH3T3 cells this gene construct was transcribed, indicating the presence of a functional promoter in the TSP sequence. Transient transfection studies using deletion mutants of this TSP-chloramphenicol acetyltransferase construct were performed to locate cis-acting regulatory sequences. The deletion of flanking sequence 5' to position -234 had little or no effect on transcriptional activity, whereas deletion of 5'-flanking sequence extending further in the 3' direction resulted in the gradual loss of transcriptional activity. The removal of the first intron resulted in a 4-fold decrease in transcript levels, indicating the presence of a cis-acting positive element(s) in the first intron of the human TSP gene. This element(s) was further localized to the region between position +576 and position +727.
Laherty
C D
CD
Department of Pathology, University of Michigan Medical School, Ann Arbor, 48109.
Gierman
T M
TM
Dixit
V M
VM
D001483:Base Sequence; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D003001:Cloning, Molecular; D003360:Cosmids; D004247:DNA / Q000235:genetics*; D004262:DNA Restriction Enzymes; D004720:Endonucleases; D005091:Exons; D006023:Glycoproteins / Q000235:genetics*; D006801:Humans; D007438:Introns; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D011401:Promoter Regions, Genetic; D012045:Regulatory Sequences, Nucleic Acid; D012689:Sequence Homology, Nucleic Acid; D015719:Single-Strand Specific DNA and RNA Endonucleases; D019699:Thrombospondins; D014158:Transcription, Genetic; D014162:Transfection
D006023:Glycoproteins; D019699:Thrombospondins; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase; D004720:Endonucleases; D004262:DNA Restriction Enzymes; D015719:Single-Strand Specific DNA and RNA Endonucleases