Myb-dependent regulation of thrombospondin 2 expression. Role of mRNA stability.
The nuclear transcription factor c-Myb, which is highly expressed in hematopoietic cells, has been shown to be functional in NIH 3T3 cells: cells that do not possess detectable levels of c-Myb. To identify endogenous target genes of c-Myb in fibroblasts, RNA isolated from NIH 3T3 cells stably transfected with a full-length or a dominant negative c-myb construct (GREMyb and GREMEn, respectively) was subjected to differential display analysis. 5'-Rapid amplification of cDNA ends of a selected band, sequencing, and a nucleotide homology search led to the identification of thrombospondin 2 (TSP 2) as the gene product repressed in GREMyb and induced in GREMEn cells. The pattern of TSP 2 expression during the cell cycle was consistent with c-myb-dependent regulation. The possibility that the identified transcript was TSP 1, a homologous product known to be repressed by v-Src, c-Jun, and v-Myc, was ruled out by using a TSP 2-specific DNA probe and by showing a distinct pattern of regulation of TSP 1 and TSP 2 expression. Nuclear run-on and TSP 2 promoter-reporter (chloramphenicol acetyltransferase) assays showed similar transcriptional levels in GREMyb and NIH 3T3 cells. However, mRNA stability studies showed a much shorter TSP 2 mRNA half-life in GREMyb compared with wild type NIH 3T3 cells, suggesting that c-myb affects TSP 2 expression via a post-transcriptional mechanism. The implications of a protooncogene-mediated suppression of TSP expression are discussed.
Angiogenesis Research Center, Cardiovascular Division, Department of Medicine Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts 02215, USA.
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Bein
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Ware
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Simons
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D005786:Gene Expression Regulation; D051379:Mice; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D020598:Proto-Oncogene Proteins c-myb; D012333:RNA, Messenger / Q000235:genetics; D019699:Thrombospondins / Q000235:genetics*; D015534:Trans-Activators / Q000378:metabolism*; D014158:Transcription, Genetic; D000099262:Thrombospondin 2
D017931:DNA Primers; D011518:Proto-Oncogene Proteins; D020598:Proto-Oncogene Proteins c-myb; D012333:RNA, Messenger; D019699:Thrombospondins; D015534:Trans-Activators; D000099262:Thrombospondin 2