In vivo interactions of RNA polymerase II with genes of Drosophila melanogaster.
We describe a method for examining the in vivo distribution of a protein on specific eucaryotic DNA sequences. In this method, proteins are cross-linked to DNA in intact cells, and the protein-DNA adducts are isolated by immunoprecipitation with antiserum against the protein. Characterization of the DNA cross-linked to the precipitated protein identifies the sequences with which the protein is associated in vivo. Here, we applied these methods to detect RNA polymerase II-DNA interactions in heat-shocked and untreated Drosophila melanogaster Schneider line 2 cells. The level of RNA polymerase II associated with several heat shock genes increased dramatically in response to heat shock, whereas the level associated with the copia genes decreased, indicating that both induction of heat shock gene expression and repression of the copia gene expression by heat shock occur at the transcriptional level. Low levels of RNA polymerase II were present on DNA outside of the transcription units, and for at least two genes, hsp83 and hsp26, RNA polymerase II initiated binding near the transcription start site. Moreover, for hsp70, the density of RNA polymerase II on sequences downstream of the polyadenylate addition site was much lower than that observed on the gene internal sequences. Examination of the amount of specific restriction fragments cross-linked to RNA polymerase II provides a means of detecting RNA polymerase II on individual members of multigene families. This analysis shows that RNA polymerase II is associated with only one of the two cytoplasmic actin genes.
D000818:Animals; D002467:Cell Nucleus / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D004262:DNA Restriction Enzymes; D004331:Drosophila melanogaster / Q000235:genetics*; D005796:Genes; D006360:Heat-Shock Proteins / Q000235:genetics; D006657:Histones / Q000235:genetics; D009693:Nucleic Acid Hybridization; D010957:Plasmids; D011485:Protein Binding; D012319:RNA Polymerase II / Q000378:metabolism*; D012091:Repetitive Sequences, Nucleic Acid; D014158:Transcription, Genetic
D006360:Heat-Shock Proteins; D006657:Histones; D004247:DNA; D012319:RNA Polymerase II; D004262:DNA Restriction Enzymes