Elucidating the molecular basis of gene silencing by an ORC-HP1 interaction and their contribution to human health disorders

Some parts of our genome contain important information that represent a molecular building plan of the human body, while other regions contain little information or repetitive DNA sequences that do not appear to have any information associated with themselves. These less-informative regions of our genome are packed away and exist as highly compacted 'heterochromatin' in the nucleus of the cell. These 'heterochromatic' regions are covered by a protein called HP1, which helps the compaction process. HP1 is known to form oligomers, which act as a molecular glue, and therefore restrict access of other proteins to the compacted parts of the genome. Moreover, work over the last few years has shown that HP1 is not only found in condensed regions of the genome, but that HP1 can also compact smaller patches in accessible regions of the genome with important functions for gene regulation and genome stability. Thus HP1 acts as a global factor controlling access of other regulatory factors to the DNA. This way HP1 can affect many important processes in the cell - in the absence of HP1 the organism cannot survive. Misregulation of 'heterochromatin' and HP1 is involved in several diseases, including epigenetic diseases such as Friedreich's Ataxia or Facioscapulohumeral muscular dystrophy. Furthermore, HP1 has also been linked to aging, e.g. cells from older people or patients with an accelerated aging disease (Hutchinson-Gilford Progeria Syndrome) are characterized by a loss of key chromatin proteins, like HP1. In cancer, HP1 is frequently down regulated, which promotes tumorigenesis. It is not fully understood how HP1 is recruited to DNA, but this question is central for the normal function of HP1 and its role in disease. Recently it was shown that the origin-recognition complex (ORC), which is involved in DNA replication, is very important for HP1 recruitment to the genome. We want to explore the molecular mechanism that ORC employs to promote HP1 recruitment to chromatin. We will analyse the effect of ORC-HP1 interactions for its normal role in cells and in the context of the epigenetic disease, Friedreich's Ataxia, to understand the functional relevance of the complex. Finally, we will test if we can alter the activity of HP1 in cells with a peptide, which may spur the development of HP1 therapeutics. This project has not only the potential to identify a crucial mechanism of heterochromatin formation, the formation of the 'molecular glue', that regulates many processes in the cell, but also has additional implications for our understanding of cancer, epigenetic- and age related-diseases.

HP1 is a key constituent of heterochromatin. HP1 promotes chromatin compaction and restricts access of regulatory factors to DNA. Misregulation of the HP1 chromatin interaction is associated with epigenetic disease, cancer and aging. How HP1 binds to chromatin is only partially understood, but of significant biological relevance. HP1 is made up of two conserved domains, the chromo domain and the chromoshadow domain, which are linked by a hinge region. HP1 recruitment to chromatin depends on a chromo domain interaction with a histone H3 that carries a trimethylation mark on lysine 9. However, recently it was found that HP1 recruitment to chromatin also requires the origin recognition complex (ORC) proteins, which usually function in DNA replication. However, how ORC promotes HP1 recruitment to heterochromatin and its molecular function in transcription and role in disease is unknown. We will investigate how ORC proteins influence HP1 recruitment to chromatin and if ORC alters HP1 dimerisation and oligomerisation on chromatin. We will identify structural changes in HP1 in response to ORC binding and in context of the nucleosome. Furthermore, we will analyse the genome wide location of the HP1-ORC complexes and analyse the role of the complex in transcriptional regulation. Finally, we will explore if ORC-HP1 is involved in heterochromatin formation during oncogene induced senescence or in Friedreich's ataxia, a triplet expansion illness where hetero-chromatin induced down-regulation of frataxin gene (FXN) expression leads to a neurodegenerative phenotype in patients. These experiments address a key and largely unanswered question in chromatin biology: How is the spreading of heterochromatin regulated? By providing answers to these questions we will obtain a wide ranging understanding of many biological processes including the basis of epigenetic memory, which is disrupted during aging and disease.

The academic sector will benefit largely from the proposed project, through knowledge gain (CS, RF,TB), development of new methods (CS,TB) and technologies (CS). Indeed TB is leading the development of chemical modified-nucleosome and the PDRA will transfer some of this cross-disciplinary knowledge and technology to the group of CS - leading to enhanced research capacities. Similarly, RF has developed very powerful assays to measure HP1 functionality in cells and the joint efforts will allow knowledge gain by the PDRA and the Speck group. Furthermore, as the new chemical modified-nucleosome has not been studied by cryo-EM, this project will also benefit an international collaboration with a structural electron microscopy group in the US. Application of this cutting edge technology will have a positive impact within the fields of chromatin biology and DNA replication and other associated research areas; foremost it will allow the formulation of new research questions and generating of new results. The project will offer an opportunity for career development and training for the PDRA, which will be readily transferrable to other related fields across the spectrum of Molecular Biology and Biochemistry. Previous group members (CS and RF) have been readily employed within world-leading research institutes, universities and industry.
This project could have both short- and long-term socio-economic impacts for the United Kingdom. As with every research project the impact depends on the results obtained and is therefore difficult to predict. In the near-term to medium-term this project may spur the development of ORC-HP1 interaction inhibitors, thereby addressing a potential way to treat epigenetic diseases or cancer cell proliferation. This could spur the commercialisation of scientific knowledge and products in the long-term. As 1 in 3 people in the UK will develop some form of cancer, any new cancer therapeutic would be of great benefit to the society. Lessening the disease burden will help to maintain high productivity levels of workers and increase health and wellbeing. Importantly, the employment of a PDRA will contribute to the national economy, while the interdisciplinary nature of the project will greatly enhance the skill sets of the PDRA to include high-end modern techniques ranging from biochemical, structural, FRET to mass-spectrometry based methods. The statistical analysis and modelling of structural and biochemical data featured in the proposed research, and the integration of the data from various data sets, demonstrate the power of this interdisciplinary approach. Such a trained PDRA (and associated PhD, technician, masters and undergraduate students) are likely to benefit the UK biotechnology and pharmaceutical companies, as well as the academic sector at both national and international levels. Finally, the general public will benefit from this work by becoming informed about our science in general and the project specifically.