Understanding emergent physical properties of chromatin using synthetic nuclei

Introduction

The main aim of this proposal is to resolve how the physics of molecular-scale activities result in the emergent material properties of chromatin and how those contribute to chromatin organization and function. Mounting evidence suggests that the material properties of chromatin regulate essential nuclear processes.

Current Approaches

Chromatin has been studied with two disconnected approaches:

1. In vitro studies: These are perfectly suited for careful biophysical measurements on single DNA molecules but lack the complexity of a cell.
2. Intact cell measurements: These provide limited access to measure material properties and small-scale chromatin dynamics.

The physical properties of chromatin, however, are emergent and result from the molecular activities that are in turn regulated by those properties.

Need for New Experimental Assays

As a consequence, it is crucial to establish new experimental assays that connect these two scales and levels of complexity.

Proposed Methodology

Here, I will bridge the gap in scales and biochemistry between pure in vitro assays and measurements in intact cells by reconstituting chromatin processes in Xenopus laevis egg extracts across scales.

Techniques to be Used

I will combine:

• Quantitative microscopy
• Optical tweezer measurements
• Theory

These techniques will be used to biophysically characterize the self-organization of protein-DNA co-condensation and loop extrusion, as well as single chromatin molecules of increasing complexity.

Synthetic Nuclei Assembly

To bridge the microscopic and macroscopic scales, I will assemble synthetic nuclei made of pre-engineered DNA sequences, which allows for exquisite control of DNA length, amount, and chromatin activities.

Investigation of Collective Behavior

In combination with:

• Microrheology
• Micropipette aspiration
• Magnetic tweezers

I will unravel how the collective behavior of chromatin activities gives rise to the emergence of large-scale material properties of chromatin.

Conclusion

This project will provide a physical description of the material state of chromatin across scales and contribute to revealing the basic physical principles that govern nuclear organization and function.