Analysis of topoisomerase I and II cleavage sites on the Drosophila actin and Hsp70 heat shock genes.
We have compared topoisomerase I and II cleavage sites on the actin 5C and 57A genes and the hsp70 genes in Drosophila Kc cells using the inhibitors camptothecin (topoisomerase I specific) and VM-26 (topoisomerase II specific) to assess the role of these enzymes in transcriptional regulation. Topoisomerase I cleavage sites were localized to the transcribed regions of the actin 5C and hsp70 genes and were present only when these genes were active. The actin 57A gene, shown previously to be inactive in Kc cells, had no detectable topoisomerase I cleavage sites. In contrast to topoisomerase I, topoisomerase II cleavage sites could be detected on transcriptionally active and inactive actin and hsp70 DNA sequences. Topoisomerase II cleavage sites on the inactive hsp70 gene were primarily localized to the very 5' end of the transcribed region of the gene. However, upon heat-induced activation of hsp70 transcription, topoisomerase II cleavage rapidly shifted from the 5' to the 3' end of the gene. Then, during the shutdown of hsp70 expression, there was a gradual reappearance of topoisomerase II cleavage at the 5' end of the gene that temporally correlated with the repression of hsp70 transcription. There was a similar preferential association of topoisomerase II with the 5' ends of transcriptionally repressed actin 5C and 57A genes. These results demonstrate that there are marked differences in how topoisomerases I and II interact with transcriptionally active and inactive regions of chromatin. In addition, we have identified an unusual type of topoisomerase II binding site that is preferentially associated with the 5' ends of inactive hsp70 and actin genes, suggesting that this enzyme may facilitate changes in chromatin structure that are associated with repression of gene transcription.
Kroeger
P E
PE
Department of Pharmacology and Therapeutics, University of Florida College of Medicine, Gainesville 32610-0267.
Rowe
T C
TC
D000199:Actins / Q000235:genetics*; D000818:Animals; D001665:Binding Sites; D002166:Camptothecin / Q000494:pharmacology; D004247:DNA / Q000378:metabolism*; D004264:DNA Topoisomerases, Type I / Q000378:metabolism*; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004330:Drosophila / Q000737:chemistry*; D005091:Exons; D015870:Gene Expression; D006360:Heat-Shock Proteins / Q000235:genetics*; D006358:Hot Temperature; D012333:RNA, Messenger / Q000096:biosynthesis; D015183:Restriction Mapping; D013713:Teniposide / Q000494:pharmacology; D059004:Topoisomerase I Inhibitors; D059005:Topoisomerase II Inhibitors; D014158:Transcription, Genetic
D000199:Actins; D006360:Heat-Shock Proteins; D012333:RNA, Messenger; D059004:Topoisomerase I Inhibitors; D059005:Topoisomerase II Inhibitors; D004247:DNA; D013713:Teniposide; D004264:DNA Topoisomerases, Type I; D004250:DNA Topoisomerases, Type II; D002166:Camptothecin