that are sometimes in contrast to single cell studies, which show nuclear receptors and other transcription factors rapidly exchange with a promoter array at a much faster rate (seconds [19,25,37– 40]). A reconciling model posits that transcription initiates by virtue of infrequent yet stable and productive association of factors at target genes (ChIP). These events are the result of many rapid and stochastic unproductive associations of transcription factors (FRAP). This model received substantial supportive evidence by a study of a naturally repeated gene in yeast [25]. Recent quantitative measurement in prokaryotic and eukaryotic cells also suggests that gene expression might involve intermittent pulses [41] that are stochastically regulated. Therefore, in order to achieve regulated transcription, activation-induced events must be synchronized in a spatiotemporal context. The result of synchrony would yield a higher mean transcript amount at the promoter level. Conversely, the loss of synchrony would result in periodic reduction (at 2 hrs and 12 hrs) of the mean transcription level of the population (cycling). In the context of the ‘‘visual-ChIP’’ system presented here, we demonstrate that synchronization is a natural process that is achieved by the addition of E2 or EGF. Furthermore, the mechanism of transcription synchronization in this model system is independent of large-scale chromatin modification. A disconnection between chromatin decondensation and transcription The discoveries that chromatin structure influences transcrip- tion have expanded upon the static model based on response elements as a straightforward platform for intermediate complexes and basal transcriptional machinery [42]. The understanding of how chromatin remodeling can mechanistically affect gene regulation requires additional insight into the kinetics of the process. This includes how quickly such changes can be generated, how long they are maintained, and if they correlate precisely with transcription. Transcriptional activity, visualized here as accumu- lation of transcripts at the PRL-array, correlates with the presence of active transcriptional machinery on the promoter that could sterically impede DNA rewrapping before the transcriptional activity ends. The condensed status of the array chromatin visualized in 4HT treated cells (see Figure 3A) link both the loss of RNA polymerase II recruitment and transcription with a repressed locus while E2 and EGF induce chromatin decondensation and increase transcription at the locus. By themselves, these results suggest a direct correlation between transcription and chromatin status. However, temporal analysis of chromatin condensation and transcription indicates a more complex picture. At basal chromatin condensation (unliganded ER, EGF-treated wt-ER after 12 hrs, or ER-S118A) reporter gene mRNA accumulation is indistinguishable. Despite the constant presence of a presumably open and transcriptionally permissive state of the chromatin (wt- ER upon E2 treatment, or ER-S118E transfected cells treated with E2 and EGF), a maximal sustained level of mRNA accumulation is not observed indicating that decondensed chromatin is necessary, but not sufficient for sustained high rates of transcription. In this scenario, the presence of an open array would allow continuous sampling of the stimuli exposure. The unexpected result of Ser118E transcriptional cycling in the presence of EGF leads us to attribute the higher response of this mutant to a lack of negative feedback on its activity rather than to a stronger activation of the mutant itself. Serine 118 of the ER plays a particularly important role both for E2 and EGF responses [16,30]. For ligand-independent ER activation, control of phosphorylation at this site may provide an important negative feedback function on transcriptional activity ERa Function by Image Analyses and consequently on the chromatin structure at the promoter level in ER ligand-independent activation. Note in Figure 6B, there is a sharp reduction in targeted arrays in cells expressing GFP-ER- S118A, which have been treated with EGF. This is consistent with results from cells expressing GFP-ER that have been treated with EGF and tyrphostin (Figure 5A), which is discussed further below. Indeed, the phosphorylation of ER Serine 118 appears necessary to permit EGF induced large-scale chromatin modifi- cation in PRL-HeLa (Figure 7) and it also plays a partial role in E2-dependent activation of transcription. Results using the mutant ER-S118E receptor provide strong evidence for the critical role of phosphorylation of this residue in contributing to PRL-array modification. This mutation mimics a phosphorylated state of serine and induces a sustained decondensed PRL-array. These results suggest that ER phosphorylation alters receptor function (promoter targeting, chromatin remodeling and transcriptional output) effecting changes that could lead to a more sensitive response to EGF stimuli, as reported for tamoxifen-resistant breast tumors [43,44]. Our results also clearly show that this residue is not only important for ER binding to the promoter, it also has a crucial role in ensuring a transcriptional response to EGF, and in regulating the temporal cycling of transcription accumulation in response to E2. The role of EGF signaling pathway on ER function It is widely accepted that EGF indirectly activates ER transcription in the absence of its cognate ligand through binding to the cell membrane EGF receptor (EGFR), dimerization of the receptor and signal transduction through kinase pathways (including Ras/Raf/MAPK pathway [45]). The direct inhibition of EGFR by Tyrphostin AG537, and its downstream signal transducer, MKK1 kinase, by UO126 or PD98589 reduces EGF activation of ER, as determined by the proportion of responsive cells in our assays. Interestingly, after EGF treatment, the reduction of PRL-array targeted cells in the presence of these inhibitors was less than in control cells and is consistent with reduced array occupancy by ER-S118A (Figure 6B) or the AF-1 deletion mutant (Figure 2). This antagonist-like effect of EGF (20% versus 60%, Table 1 and Figure 6) indicates the possibility that array occupancy is modulated when ER phosphorylation is indirectly, or directly, inhibited (Figure 5 and Figure 6 respectively). To dynamically control output signals, the EGFR signaling cascade may have evolved positive- and negative- feedback circuits [11]. The latter mechanism may participate in signal attenuation where cells can tune their response to various mixed stimuli. It is possible that when EGF and kinase inhibitor (or phosphomutant) are both present, the negative-feedback mode would dominate, perhaps by altering the phosphocode (and interactions) of ER and coregulators [46]. In this case, the end result would be a decrease in promoter targeting. In support of this argument, EGF stimulation of SRC-3 has been shown to alter its nuclear accumulation and occupancy of the PRL-array through changes in specific SRC-3 phosphorylation sites [47]. Perspectives The importance of transcription in cell growth and differenti- ation is underlined by the redundancy of feedback mechanisms that regulate those processes (different combination of events to achieve the same result in controlling gene expression). The results of this study further an emerging systems biology level view, which suggests an increased coherent understanding of gene transcription requires not only knowledge of which molecular players are involved but how they interact within the context of cell temporal/ structure/organization [44,48]. As the multiplex systems level PLoS ONE | www.plosone.org 10 May 2008 | Volume 3 | Issue 5 | e2286