Understanding emergent physical properties of chromatin using synthetic nuclei

Información del proyecto

SynthNuc

Identificador del acuerdo de subvención: 101045468

DOI

10.3030/101045468

Fecha de la firma de la CE 17 Mayo 2022

Fecha de inicio 1 Septiembre 2023

Fecha de finalización 31 Agosto 2028

Financiado con arreglo a

• European Research Council (ERC)

Coste total

€ 1 999 550,00

Aportación de la UE

€ 1 999 550,00

Coordinado por

TECHNISCHE UNIVERSITAET DRESDEN
Germany

Periodic Reporting for period 1 - SynthNuc (Understanding emergent physical properties of chromatin using synthetic nuclei)

Período documentado: 2023-09-01 hasta 2026-02-28

Resumen del contexto y de los objetivos generales del proyecto

In humans, a two-meter-long DNA polymer is physically folded into a micron-sized nucleus. Fundamental biological processes such as transcription or condensation of chromatin during mitosis require to physically bring distant DNA sequences together in space. For this to happen, the local material properties of chromatin need to be properly tuned in space and time. The physical properties of chromatin, however, are emergent and result from the molecular activities that are in turn regulated by those properties. Molecular processes make chromatin and active material. For example, DNA can be actively extruded into loops, a process thought to regulate gene expression by bringing together enhancer and promoters or packaging chromatin into stacked loops during chromatid formation. Phase separation regulated by the transcription machinery is thought to physically bring distant regulatory sequences together and to segregate chromatin into active and inactive compartments at large scales. However, the physics of these molecular processes and how they contribute to the emergence of the large-scale organization of chromatin and its material state is not well understood. In this project, we aim to understand how the physics of molecular-scale activities result in the emergent material properties of chromatin and how those contribute to chromatin organization and function. One of the major limitations in this field is the lack of methods to quantitatively explore chromatin across scales. To achieve this goal, we will bridge the gap in scales and biochemistry between previous pure in vitro assays and measurements in intact cells by reconstituting chromatin processes in Xenopus laevis egg extracts across scales.

Trabajo realizado desde el comienzo del proyecto hasta el final del período abarcado por el informe y los principales resultados hasta la fecha

Over the past two years, our research has mainly focused on establishing assays to measure chromatin material properties in vivo, and in isolated full chromosomes in cell free extracts. We have successfully adapted protocols to isolate biotinylated chromosomes from tissue culture cells. We have established an optical tweezer set up in the lab that allows us to grab the individual chromosomes and perform rheological measurements in buffer and in cell free extracts. We have optimised the preparation of cell free extracts from Xenopus leavis egg extracts arrested in metaphase and interphase, as well as autonomously cycling. We have optimised filtering protocols to remove most of free membranes in these extracts (which perturbs the optical tweezer measurements) while testing for extract activity (formation of nuclei). We have also developed an assay to directly measure force between chromosomes. This assay consists of assembling synthetic chromosomes around beads using lambda phage DNA and incubating in extracts to chromatinize them. Using optical tweezers we can bring two synthetic chromosomes together and quantify the attraction/repulsion force between chromosomes in various conditions, including the presence of molecules (for example Ki-67) that have been suggested to act as surfactants or glue depending on the cell cycle. These assays allow measurements in physiological conditions that were currently not possible. Our data has already allowed us to quantify the softening of chromatin as it transitions from metaphase to interphase and will be the basis to understand how these emergent material properties arise from molecular activities during the cell cycle. Our synthetic chromosome assay has already demonstrated that chromatin self-interacts and that Ki-67 does act as a glue in interphase.

Finally, towards evaluating the functional role of chromatin material properties, we have established a protocol to inject beads into early zebrafish embryos, move those beads inside of nuclei, and measure rheology in vivo as a function of developmental stage. The goal of this assay is to test whether mechanical changes of chromatin during early development is what drives the zygotic to genome activation transition. Our preliminary data suggests mechanical changes of chromatin as the embryo develops.

Avances que van más allá del estado de la técnica e impacto potencial esperado (incluida la repercusión socioeconómica y las implicaciones sociales más amplias del proyecto hasta la fecha)

In this first phase of the project, we have established protocols to be able to understand how chromatin material properties emerge from molecular activities and how those impact nuclear function. The biggest achievement is the establishment of such assays, in particular the synthetic chromosomes and the measurements of individual isolated chromosomes in cell free extracts. Our assays are the first ones that allow to directly measure chromatin material properties in a cellular context. Previous work has successfully measured rheological properties of metaphase chromosomes in buffer. However, in these conditions chromatin lacks any activity and it is essentially dead. In our assays, we can study both those properties in physiological conditions (intact cytoplasm) and more importantly monitor how these properties are modulated during the cell cycle. Specifically, the transition from metaphase condensed chromatin to interphase loose chromatin has never been studied. Our assay provides this opportunity.