The bgalactosidase units produced per minute were estimated using the following protocol. Briefly, about 300 ml of the culture drawn at each time point were centrifuged at 14000 rpm at 4uC for 20 min

ses GR-mediated chromatin remodeling and transcription initiation and that methylation and acetylation at histone H3R17 and H3K18 respectively, decreased within minutes of iAs addition. Both of these histone PTMs are associated with transcriptional activation at steroid hormone-regulated promoters. Additionally, it was determined that CARM1 was absent from the promoter after treatment with iAs. Unexpectedly, while CARM1 may be a target for iAs, GRIP1 is also a probable target even though unlike CARM1 it was still associated with the promoter when cells were treated with iAs. Finally, the data suggest iAs-inhibited transcription is mediated through an indirect effect on one or both of these coactivator proteins that may be via deregulation of a cell signaling pathway. . This suggests that the MMTV promoter shuts down progressively with time, in agreement with the nuclear run-on experiments where transcript is still associated with the promoter at 60 minutes but is not by 120 minutes when iAs is present. We do not view the seeming discrepancy in promoter accessibility and the presence of initiated transcripts at 60 minutes a problem because transcripts detected at 60 minutes would have initiated before the chromatin template was shut down and thus there should be a lag in when promoter access is inhibited and when transcript can be detected. Thus iAs inhibits transcription initiation and associated chromatin remodeling at the GRregulated MMTV promoter. Accumulated CAT mRNA was measured by qRT-PCR and by 2 hours there was significantly more CAT mRNA with Dex alone than with Dex plus 8 mM iAs, in agreement with the pattern of transcript initiation observed. Transcription at the endogenous GR-regulated serum glucocorticoid kinase promoter was also inhibited by iAs which indicates that the inhibitory effect of iAs on the stably integrated MMTV promoter recapitulates events on an endogenous promoter. Treatment with 8 mM iAs alone showed no change in the amount of CAT or SGK transcripts from background levels. Together, these data raised the possibilities that iAs may inhibit GR binding or stability at the glucocorticoid response element, or alternatively, the binding of another promoterassociated protein essential for initiation and activation. GR binds to promoter DNA in the presence of iAs GRs are predominantly cytoplasmic prior 13679187 to ligand binding and translocate to the nucleus and to targeted GREs when ligand is bound to the receptor. It was previously shown that low levels of iAs do not significantly alter GR translocation into the nucleus, but whether iAs Pomalidomide web affects GR binding to the GRE was not tested. To determine if iAs affects GR/GRE binding, 1470.2 cells were treated with 5 nM Dex68 mM iAs for 15, 30, 60, 120, or 180 min. Chromatin immunoprecipitation analysis was done to determine GR association with the MMTV promoter on nucleosome B that has 4 GREs. GR was associated with NucB by 1530 min of treatment with no detectable difference in cells treated with Dex6iAs. These data confirm that GR translocates to the nucleus, and binds to the MMTV GRE in the presence of iAs. To determine whether iAs affects the DNA-binding kinetics 24678947 of ligand-bound GR, electromobility shift assay competitions were done. Nuclear extracts made from cells treated for 30 min with 50 nM Dex alone or 50 nM Dex plus 8 mM iAs were incubated with a radiolabeled consensus GRE with 0, 5x, 15x, or 30x molar excess of unlabeled competitor GRE. 50 nM versus 5 nM Dex was used i

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