EASTBIO Biophysical properties of a nucleo-protein mesh

In mammalian cells DNA is packaged with proteins to form chromatin – a complex macromolecular structure. Many of the components required for packaging chromatin are known, but how they assemble and function in health and disease are still a mystery. In this project you will investigate the biophysical properties of a nucleo-protein mesh that is important for regulating the packaging of mammalian DNA and has been linked to the DNA damage response and neurological disorders.
In mammalian cells DNA is packaged with proteins to form chromatin – a complex macromolecular structure. Many of the components required for packaging chromatin are known, but how they assemble and function in health and disease are still a mystery. In this project you will investigate the biophysical properties of a nucleo-protein mesh that is important for regulating the packaging of mammalian DNA and has been linked to the DNA damage response and neurological disorders.


Figure 1 Formation of a nucleo-protein mesh

The use of next generation sequencing has enabled us to identify mutations responsible for many human diseases. A surprising result showed that mutations in an abundant nuclear protein called Scaffold attachment factor A (SAF-A) or HNRNPU is frequently mutated in patients with epilepsy and neurodevelopmental disorders [1]. From our recent studies [2] we have shown that HNRNPU is able to form a nuclear mesh (Figure 1) in the presence of RNA that can regulate chromatin structure; altered levels of HNRNPU cause genome instability and a DNA damage response. HNRNPU seems to function by oligomerising in the presence of RNA forming chains that creates a gel or mesh creating phase-separated structures and partitioning different nuclear environments.

In mammalian cells there are two proteins homologous to HNRNPU: HNRNPUL1 and HNRNPUL2 [3]. Little is known about them but as they have specific sequence variants in important domains we speculate they may have a role in regulating the formation of a HNRNPU dependent mesh. In this project we therefore hypothesise that HNRNPUL1 and HNRNPUL2 regulate the size and properties of phase-separated structures in the nucleus. This in turn will affect gene regulation, genome stability and the DNA damage response. To investigate this hypothesis our aim is to use structural biology to investigate the formation of HNRNPU-dependent phase separated structures and to understand how these are altered in disease such as cancer or neurodevelopmental disorders.

To achieve our aim individual objectives will involve expressing and purifying the HNRNPU variant proteins and analysing their conformations using approaches such as X-ray crystallography, light scattering and small angle x-ray scattering. We will then build on methods we have already developed to reconstitute an HNRNPU / RNA mesh to understand how its properties are regulated by the HNRNPUL1 and HNRNPUL2 accessory proteins. Depending on the nature of the structures formed we will then use physical and biophysical approaches to investigate their properties such as electron microscopy, single molecule imagine and FRET. Finally, we will re-capitulate genetic mutations identified from next generation sequencing studies to understand how altered versions of these proteins influence the formation and function of phase separated structures in the nucleus.