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Figure 6. Pin1 and H1 phosphorylation are marks of transcriptionally competent chromatin. U2Os 263 cells harboring lac arrays followed by TRE, CMV promoter, and CFP-SKL gene were either transfected with mCherry LacR or mCherry ER-tTA. The former represented the transcriptionally inactive state while addition of tamoxifen (3 h) to the latter represented the transcriptionally active state of chromatin. Pin1 (A) and pS173H1.2 (B) levels were measured using immunofluorescence. Both Pin1 and pS173H1.2 levels were found to increase at sites of active transcription. α-Amanitin, was used to deplete RNA polymerase II levels in the cells. When transcription was activated in these competent, yet transcriptionally silent cells, Pin1 and H1 phosphorylation levels were elevated, suggesting that these were early events in the initiation of transcription. Bar, 5 µm (unless otherwise specified). (C) The volume occupied by the arrays was measured in both the transcriptionally inactive state (mCherry LacR alone) and in the transcriptionally active state (addition of tamoxifen to cells expressing mCherry ER-tTA for either 1 h or 3 h). Volume was measured through rapid acquisition of z-stacks in living cells. Whereas transcription caused an increase in the volume occupied by the arrays, treatment of cells with α-amanitin led to compact arrays. Both transcriptionally active and inactive arrays were found to occupy larger volumes when Pin1 was depleted by Pin1siRNA treatment. Significance between control vs. treated (amanitin or Pin1si-RNA) was analyzed using unpaired t test (95% confidence interval). Notation for significance: *** if P value is < 0.001; ** if P is between 0.001 and 0.01; * if P is between 0.01 and 0.05. Image published in: Raghuram N et al. (2013) © 2013 Raghuram et al. This image is reproduced with permission of the journal and the copyright holder. This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike license Larger Image Printer Friendly View

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