Abstract
To study when and where active genes replicated in early S phase are transcribed, a series of pulse-chase experiments are performed to label replicating chromatin domains (RS) in early S phase and subsequently transcription sites (TS) after chase periods of 0 to 24 h. Surprisingly, transcription activity throughout these chase periods did not show significant colocalization with early RS chromatin domains. Application of novel image segmentation and proximity algorithms, however, revealed close proximity of TS with the labeled chromatin domains independent of chase time. In addition, RNA polymerase II was highly proximal and showed significant colocalization with both TS and the chromatin domains. Based on these findings, we propose that chromatin activated for transcription dynamically unfolds or “loops out” of early RS chromatin domains where it can interact with RNA polymerase II and other components of the transcriptional machinery. Our results further suggest that the early RS chromatin domains are transcribing genes throughout the cell cycle and that multiple chromatin domains are organized around the same transcription factory.
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Acknowledgements
The authors are grateful to Dr. Daniela S. Dimitrova who first demonstrated in our laboratory the lack of colocalization between replication and transcription sites using standard epifluorescence microscopy. The authors thank Dr. George Mayers and Dr. Richard B. Bankert (University at Buffalo, NY) for providing anti-BrdU mouse monoclonal antibodies. These studies were supported by the National Institutes of Health (NIH) grant GM 072131 awarded to R.B.
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S1
Proximity analysis of 0.5 μm Fluoresbrite beads. Fluoresbrite beads of 0.5 μm diameter, which could fluoresce in both red and green colors, were imaged and analyzed with our segmentation and proximity programs. Including the sub-pixel error associated with CCD imaging, ∼85% of the beads were at a distance of 1 pixel to their nearest neighbor. Below is the proximity plot which describes their spatial association. (60.5 KB)
S2
Line profile analysis of B3 and ARNA3 antibodies. Comparison of the staining patterns produced by B3 (green, specific for hyperphosphorylated form of the RNA polymerase II large subunit) and ARNA3 (red, specific for total RNA polymerase II large subunit) antibodies. Line profile analysis of the merged image and two enlarged portions A and B, indicates the overlap between the stained patterns in the red and green channels. (153 KB)
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Malyavantham, K.S., Bhattacharya, S., Alonso, W.D. et al. Spatio-temporal dynamics of replication and transcription sites in the mammalian cell nucleus. Chromosoma 117, 553–567 (2008). https://doi.org/10.1007/s00412-008-0172-6
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DOI: https://doi.org/10.1007/s00412-008-0172-6