Chromatin dynamics at nano-scale resolution and long-range correlation within the nucleus during transcription

ENS de Lyon - CBP
18/12/2017

Haitham A. Shaban, LBME/CBI-CNRS, University of Toulouse

Intrinsic high dynamics of chromatin is an essential underpinning of gene regulation. Control of gene expression largely occurs at the transcription stage and involves modifications of chromatin structure and transcription factors binding along with RNA polymerase II dynamics. However, it remains unclear how global and local motion of chromatin interleaves with the choreography of other transcription players. And also, how chromatin clusters respond dynamically through coordinate movements to regulate genomic processes.

For this purpose, we developed a method to estimate motion at sub-pixel resolution with high accuracy over the entire nucleus in live human cells. Although the fluorescence intensity of chromatin images is diffraction-limited, our method reveals sub-diffraction vectorial information based on reconstructed dense global flow fields of fluorescent images. This approach allows us parallel quantification of key biophysical properties (such as diffusion coefficient and the underlying modes of motion) of distinct labeled entities in one single cell: H2B-GFP and SiR- Hoechst as chromatin and DNA markers, in human cultured cells. We were thereby able to study nucleus-wide chromatin motion in healthy culture cells, or upon treatment with drugs that inhibits transcription at distinct stages (initiation or elongation), or upon serum deprivation which is also known to globally affect transcription.

Our method points first to a major heterogeneity in chromatin motion over the entire nucleus, whatever transcription state. This heterogeneity strongly suggests that nucleosome clustering (density & dynamics within clusters) along chromatin fibers is itself highly heterogeneous. We observed a striking correlation in transcriptional state between chromatin areas that display coherent motions over the entire nucleus. We further observed that coordinated movements within domains differentially correlate with RNA pol II activity. Similar trends with regard to transcriptional state were observed for global spatial correlation of chromatin dynamics upon pausing transcription at initiation, whereas correlation was significantly altered when elongation was halted. Finally, quantification of spatial smoothness in nuclear space over large time intervals points to clear-cut boundaries between distinct motion modes, while smooth transitioning in a small neighborhood instead dominate for short time intervals.