The phenomenon of global changes in transcription is correlated with increased phosphorylation of the histone selleck catalog H3 Serine 10 at the heat induced loci and with a sharp decrease of the global level of H3S10 phosphorylation at other loci. We hypothesized that the global changes of transcrip tion may involve changes in chromatin insulators at a global level. We thus monitored the distribution of mRFP CP190 and GFP CP190BTB D proteins in cells of the salivary gland after heat shock. We found that after 30 minutes of heat shock at 37 C, significant amounts of mRFP CP190 localized to the extra chromosomal space, although association of the protein with chromosomes was still obvious. After 50 minutes of heat shock, the mRFP CP190 signals were mostly diffused and the protein was clearly present at extra chromosomal spaces.
The result indicates that the heat treatment induced dissocia tion of the Cp190 protein from the originally bound insulator sites on chromosomes. On the other hand, we did not detect significant changes of the distribution of the GFP CP190BTB D protein which remained bound to polytene chromosomes as sharp bands and was not detectable in the extra chromosomal spaces. To determine if Cp190 tightly associates with chromo some without heat shock treatment, we analyzed the exchange rates of GFP CP190BTB D and mRFP CP190mRFP using the Fluorescence Recovery After Photobleaching technique. We did not detect significant recovery of both GFP CP190BTB D and mRFP CP190 signals in the bleached area two minutes after photobleaching, indicating that no significant exchanges of the two Cp190 proteins within two min utes on chromosomes.
In the cells heat shocked for 30 minutes, we detected signals of extra chromosomal mRFP CP190. The signals were significantly weaker in the bleached area right after photobleaching, indicating that the extra chromosomal signals were not background and were real signals representing the mRFP CP190 molecules which were not associated with chromosomes. The result is consistent with the conclusion above that Cp190 may dissociate from chro mosomes in response to a heat shock treatment. In contrast with the non heat shocked cells, we detected significant recovery of mRFP CP190 signals in the bleached area within 2 minutes, indicating that a fraction of the mRFP CP190 rapidly moved into the bleached area.
The result indi cates that the heat shocked cells contained a fraction of fast moving mRFP CP190 which was not present in cells before the heat treatment. The redistributed mRFP CP190 molecules in the bleached area were either in extra chromosomal space where Cp190 may move more freely or were associated with chromosomes during the recovering period. It is noticeable that the distribution pattern of the recovered signals in the bleached area was different from the pat tern before photobleaching. In most of the bleached area, the signals that reappeared lacked GSK-3 distinct bands.