Title: Dissecting NFkB clusters with single-molecule fluorescence microscopy
Funder: The Academy of Medical Sciences

Abstract: Aberrantly active transcription factor, NF-kB, is a driver of inflammatory disease and cancer and is consequently an attractive drug target. However, NF-kB and other transcription factors (TFs) have proven difficult to drug. Using single-molecule fluorescence microscopy, I showed that TFs in model yeast cells formed clusters and now lightsheet microscopy has revealed similar TF clusters in human cells. Clustering speeds up TF search time, increases binding and alters transcription and so disrupting it has therapeutic potential. Preliminary evidence from my lab suggests NF-kB subunit, RelA, also forms clusters, offering a potentially new way to target the pathway. This project aims to test this hypothesis by building a new single-molecule fluorescence microscope optimised for imaging clusters in human cells. Using new oblique plane microscopy, the advantages of lightsheet imaging can be combined with the quantitative imaging techniques that I have developed to observe TF clusters in live cells. On top of this, key interventions will be tested targeting the disordered transactivation domain of RelA, which mediates interactions with other proteins and may drive clustering. This project will generate fundamental insight into the basic mechanisms of gene regulation through the new paradigm of TF clustering, benefiting the whole biomedical research community, as well as specific new knowledge of disease associated NF-kB, potentially benefiting sufferers of cancer and inflammatory disease. Finally, by seeking to disrupt TF clustering, this work may lead to a new class of therapeutic directly targeting transcriptional misregulation in disease, benefiting patients with a wide range of disorders.

Lay abstract: A family of proteins called NF-kB help cells respond quickly to harmful stresses by switching specific genes on and off but in many cancers and inflammatory diseases, such as arthritis, it is overactive and may actually be driving disease. Drugs to reduce its activity would be very beneficial to these and other diseases but designing drugs forNF-kB and other gene regulating transcription factor proteins has remained an intractable health problem. This is because the molecular arrangement of transcription factors in cells has not been well understood. Recently, myself and others have developed new microscopes which can see individual molecules inside cells and shown that transcription factors arrange themselves into clusters. However, exactly how clusters regulate genes and how and why they form is still unknown. Understanding clusters might lead to the development of new drugs to disrupt them. Imaging from my lab suggests NF-B also forms clusters but a more sensitive microscope is required, specifically designed to image TF clusters in cells at high speed, to observe cluster formation and movement. I plan to develop this microscope to investigate whether NF-kB, forms clusters and also test if they can be disrupted. Perturbing these clusters has the potential to define a new approach to drug design for a wide range of different diseases, with many resulting benefits to global health and society.

Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS
Health categories: INFLAMMATORY AND IMMUNE SYSTEM