ANalysis of transcription-induced chromosome DYnamics – ANDY

in situ DNA tagging
fluroescence microscopy
quantitive image analysis programs
microfluidic devices
bbiophysical modeling

We have completed a study which describes how to computationally define the conformational changes in a cell type specific manner in yeast using three independent labels (Lassadi, Kamgoué et al. PloS Comp bio 2015).
- We have successfully labeled single ectopic and endogenous genes in living human breast cancer cells using the ANCHOR technology to label DNA and the MS2 system to visualize nascent RNA. We were able to determine that estrogen induced transcription initiation locally reduced DNA mobility which relates to a reorganization of the chromatin domain including the activated gene (manuscript in prep).
JMV/partner 3:
We have applied our unique Langevin dynamics algorithm (P. Carrivain et al. PLoS Comp. Biol. 2015) to DNA condensation (Cortini, R.et al. J. Chem. Phys. 2015). In parallel with this simulation we worked out the finite-size effects of the coil-globule transition of chromatin in higher eukaryotes (Caré, B et al. Communications in Theoretical Physics 2014). Taken that chromosomes are block copolymers made of epigenetic domains, we argued that there should exist a dynamic equilibrium between euchromatin (coils) and heterochromatin (globules) at body temperature.

Analysis of effects of inhibitors and various conditions on chromatin dynamics;
optimisation of our microfluidic set-up, especially for human cells;
high resolution imaging;
apply biophysical models to collected data, adapt data collection to obtain finer models

1. Bystricky K.# “Chromosome dynamics and folding in eukaryotes: insights from live cell microscopy” FEBS Letters Special Issue 3D Genome Structure, in press pii: S0014-5793(15)00611-0. doi: 10.1016/j.febslet.2015.07.012. 2015 review
2. Lassadi I.*, Kamgoué A*., Goiffon I., Tanguy-le-Gac N. and Bystricky K.# “Differential chromosome conformations as hallmarks of cellular identity revealed by mathematical polymer modeling. ” PLoS Computational Biology, DOI 10.1371/journal.pcbi.1004306, 2015
3. Caré, B.; Carrivain, P.; Forné, T.; Victor, JM; Lesne, A. (2014), Finite-size conformational transitions: a unifying concept underlying chromosome dynamics, Communications in Theoretical Physics, 62, e1003456
4. Cortini, R.; Caré, B.; Victor, JM & Barbi, M. (2015), Theory and simulations of toroidal and rod-like structures in single-molecule DNAcondensation, J. Chem. Phys. 142
5. 1. Wang R, Mozziconacci J, Bancaud A, Gadal O., Principles of chromatin organization in yeast: relevance of polymer models to describe nuclear organization and dynamics, Curr Opin Cell Biol. 2015 May 5;34:54-60. doi: 10.1016/j.ceb.2015.04.004 review

The spatial organization of the eukaryotic nucleus is among the most intriguing intellectual challenges in biology. Much remains to be understood on the conformation of chromosomes and on their dynamics, which both appear to play key roles in epigenetic gene regulation. Our commitment is to develop a rational framework based on polymer physics to describe transcription-induced chromatin reorganization in living yeast.

The project relies on a cross-disciplinary consortium involving cell biologists, biophysicists, and theoretical physicists with a unique expertise in the biophysical modelling of chromosome structure and dynamics. Our researches are driven by experiments, which will be conducted with innovative fluidic technologies and using advanced optics to track single genes in living yeast. Specifically, we will devise generic microfluidic systems to perform high-throughput live cell imaging under precisely controlled conditions in order to monitor the kinetics of chromosome reorganization induced by transcription activation. Our data will be analyzed with polymer physics models that quantitatively reproduce the nuclear organization of the yeast, and we aim to provide new insights on the principles of chromatin folding and on the physical parameters governing its movements. The expected outcomes will significantly increase our understanding of the organization of chromosomes and its functional role.
Our objectives for this project are to address the following questions thanks to a strong technological background:

- Do gene relocalization events involve global or local chromosome reorganizations?
- How do chromosome structural properties contribute to or evolve during gene activation?
- Is chromosome reorganization the cause or a consequence of transcription activation?
- How do chromatin movements correlate with gene expression at the mRNA level?

Chromatin is highly organized in the interphase nucleus, and the position of particular genetic loci can influence its transcriptional state. In the yeast Saccharomyces cerevisiae, the localization at the nuclear periphery is traditionally seen as a hallmark of gene silencing. The interplay between molecular complexes responsible for transcription and chromosome architecture remains unclear, and recent analyses of chromosome structural data obtained by chromosome conformation capture techniques hinted to the central role of volume exclusion rather than molecular interactions to guide the folding of chromosomes. Our knowledge of the mechanisms involved in these rearrangements is still very limited, and few studies have addressed the relationship between chromatin mobility and transcription activation. There is a critical lack of methods to monitor transient relocalization events associated with transcription induction. In this proposal we aim to devise innovative technologies to fill this gap, and we wish to provide a picture based on quantitative modeling within a realistic description of the yeast nucleus. We emphasize that gene spatial reorganization provides a clear biological read-out that can be connected to a polymer-physics model, a situation therefore ideal for biophysical studies. We will set up a novel platform combining advanced microfluidics and high-throughput imaging to elucidate the molecular mechanisms of chromatin reorganization upon transcription activation.