ERa Function by Image Analyses Figure 7. Temporal single-cell analyses of ER-S118A and ER-S118E transcriptional response at the PRL-array. PRL-HeLa cells transiently expressing the two phosphomutants, GFP-ERS118A (panel A and C) or GFP-ERS118E (panel B and D), were treated with either ethanol, E2 (panel A and B) or EGF (panel C and D) for different times. Subsequent to ligand treatment, the cells were fixed and subjected to RNA FISH using a biotinylated dsRED2 probe followed by fluorescent-tagged streptavidin. The FISH signal at the array was determined as described and the value graphed as fold induction over vehicle control cells (solid line). For each cell the area of the array was also determined and plotted as fold induction over vehicle control (dotted line). Data represent the mean 6SEM of three different experiments graphed as fold induction over time-matched vehicle-treated control cells. doi:10.1371/journal.pone.0002286.g007 several studies measuring the time-course of EGFR internaliza- tion following continuous EGF treatment correlate with our observations of the temporal response of PRL-array decondensa- tion to EGF. This suggests that the effect of EGF is modulated by down-regulation of EGFR at the plasma membrane [29,30]. However, EGF withdrawal results in a faster rate of condensa- tion of the PRL-array (Figure S2), suggesting an active yet time- limited role of the EGF-EGFR interaction in maintaining the promoter in an ‘‘open state’’ for transcription. Temporal cycling Our previous report focused upon the early events in PRL-array transcription activity (#2 hrs) and [19]. To obtain a more comprehensive temporal picture of ER function in response to ligand-dependent and –independent activation, we determined the relationship between large-scale chromatin alterations and mRNA accumulation over 24 hours. Our results suggest a time-depen- dent, stimulus-specific regulation of mRNA synthesis mediated by ER. Time course experiments show a cyclical pattern of reporter gene mRNA accumulation at the PRL-array in the continual presence of E2, which was markedly different to the non-cyclic pattern of transcription induced by sustained presence of EGF. An interesting question that we are addressing is: does ER phosphorylation cycle in phase with transcript accumulation? However, our data are in accord with data showing that P-Ser118 increased between 15 and 60 minutes following E2 treatment, as opposed to 15 and 30 minutes for EGF [31]. Cyclical ER function has been previously reported. Several groups have shown via chromatin immunoprecipitation (ChIP) analysis that levels of ER, coregulators, and RNA polymerase II crosslinked at endogenous promoters (c-Myc, pS2), can cycle at 20–40 minute intervals, dependent upon E2 treatment subsequent to transcription synchronization by promoter clearance [32–34]. To complement the ChIP studies, nuclear run-on assays performed on target genes [34] also revealed a significant cyclic pulse of E2-induced transcription at 45 minutes and 2 hours. Thus, both single cell studies and pooled biochemical studies demonstrate the cyclical nature of ER induction of transcription, although differing in frequency. An intriguing in vivo corollary is the finding that a single injection of estradiol in ovariectomized rats at noon on day zero induced a peak of prolactin mRNA content at 8:00 PM for three days after treatment [35]. This response to estrogen is vital for a daily surge of prolactin release by the pituitary, which is important for maintenance of the corpora lutea in pregnancy [36]. Accordingly, it will be important to determine if other E2 responsive promoters such as myc and pS2 demonstrate similar cyclic activity. The results obtained from biochemical approaches (ChIP) are obtained from millions of cells and, thus, are averaged responses PLoS ONE | www.plosone.org 9 May 2008 | Volume 3 | Issue 5 | e2286