Biological physics of protein clustering in epigenetic memory and transcriptional control

In recent years it has become clear that many proteins act collectively inside single cells by teaming-up in large numbers into dense molecular clusters. However, how these clusters form and what biological function they perform often remains a mystery. In this proposal, we aim to unlock these mysteries by investigating two types of clustering at a single target, a gene called FLOWERING LOCUS C in the plant Arabidopsis.

The first type of clustering is caused by proteins gathering together in an ordered way, such that close association of a critical number of proteins will then stimulate further feedback to recruit more proteins into the cluster. This phenomenon is called oligomerization. Our preliminary evidence indicates that this type of clustering occurs at FLC and has a critical function in establishing a memory of how active the gene is. Specifically, the presence of this cluster of proteins attached to the FLC DNA can cause transcription to be switched off, a state that is then inherited through DNA replication, cell division and through many subsequent cell cycles. Such memory is vitally important in controlling how cells behave and is sometimes called epigenetic memory.

The second type of protein clustering has a different physical origin: many proteins undergo what is called liquid-liquid phase separation, where they will spontaneously separate themselves from the surrounding medium and form a self-assembling compartment. This process is analogous to the spontaneous separation of oil in water into droplets. Our preliminary evidence demonstrates that this type of clustering is also present at FLC, though at a different time period in plant development to the oligomeric clustering. We believe that these phase-separated clusters are also critical regulators of gene expression.

In this project, we aim to mechanistically understand the formation and biological function of both types of cluster. To do this will require a wide diversity of techniques and expertise from both biology and physics. Physics thinking is specifically needed because the mechanisms by which the clusters are believed to form, oligomerization and phase separation, are intrinsic physics phenomena.

We will use molecular biology and genetics to perturb the components of the clusters and examine their effects on gene expression. We will use advanced single-molecule imaging techniques to observe the clusters, measure their dynamics and count the number of molecules involved. Finally, we will develop detailed theoretical physics models of the two types of clusters incorporating the results from the experiments. These experiments and models may potentially reveal how new kinds of biological physics have been exploited by biology to provide the exquisite control needed for transcriptional regulation and memory.

The project will investigate the biophysical origin, functional importance and interactions of different sets of protein clusters regulating a model plant gene system, the key floral regulator FLC. The primary impact of this research will be academic, with our new insights into epigenetic memory and transcriptional control immediately important for focused applications to flowering time. However, our results will also be much more widely relevant, as protein clustering is prevalent in numerous aspects of sub-cellular organisation. In the long-term, fundamental concepts emerging from this project will, we believe, have benefits to human health. Accordingly, we will constantly monitor our results for commercially exploitable output. Nevertheless, the time horizon for translating these results into directly exploitable commercial outcomes may be rather long. In the shorter term, we will also exploit any novel microscopy techniques through engagement with relevant industrial contacts, including Nikon, Photometrics and the software firm Laboratory Imaging (LIM).

We will use the project to enhance the status of interdisciplinary collaboration, including theoretical modelling and single molecule biophysical tools. This research project itself will provide training for the experimental and theoretical postdocs in a truly interdisciplinary environment, mixing theoretical biological physics with advanced imaging and genetics/cell biology. This combination of skills and ability to think broadly is greatly in demand in both academic and commercial environments. We will also emphasise the importance of mixing disciplines in uncovering fundamental mechanisms to undergraduate and graduate physical and biological science students. In particular, theoretical modelling approaches are still hugely under-utilised in biology as compared to bioinformatics methods.

We will also continue our outreach activities in schools by delivering an in-class teaching aid illustrating protein clustering and its fundamental role in gene expression. This will be developed together with the Norfolk Teacher Scientist Network (TSN), which specialises in one-to-one teacher-scientist partnerships. In a similar vein, we will also develop on-line teaching content for undergraduate and graduate courses for both physics and biology audiences that will introduce the novel concepts emerging from the project. These materials will describe how clustering can regulate epigenetic memory and transcriptional output, and how fundamental physics principles are exploited by the biology to generate the clusters. Protein clustering is an exciting area of research, both intrinsically and as an example of the close interaction and cross-fertilisation between physics and biology. We therefore believe that such teaching material will be widely used.