Glutamine methylation and its role in regulating FACT binding to chromatin

Most cells that make up our body contain approximately two metres of our genetic material - DNA. In order to accommodate this huge molecule within a cell's nucleus it must be tightly compacted. As a first step in its compaction, DNA is wrapped around a set of proteins, called histones. Just under two helical turns of DNA are wrapped around a histone octamer, comprising two copies each of histones H2A, H2B, H3 and H4. This structure is termed a nucleosome and it is the fundamental unit of chromatin. The compaction of DNA within a nucleosome constitutes a fundamental problem for the cell since histones create a barrier for other proteins that need to access the DNA for processes such as transcription, replication or repair. However, cells have evolved a number of mechanisms to control this barrier so that all DNA processes can be tightly regulated. For example, enzymes have evolved that chemically modify histone proteins. Research into the modification of histone proteins is part of a biological field, termed epigenetics. We are particularly interested in this process because 1) it encompasses a basic biological problem of how cells convert environmental stimuli into processes that trigger, for example, a transcriptional output and 2) many histone modifying enzymes are mis-regulated in diseases such as cancer. Recent years have seen the development of drugs that target enzymes regulating histone modifications and they are nowadays used very successfully in the clinic in the fight against cancer. Here, we propose a line of research that is based on the discovery of a novel type of histone modification -methylation of glutamine 105 in histone H2A. The enzyme catalyzing this methylation is part of a well-studied biological pathway responsible for the generation of ribosomes, macromolecular machines that are at the heart of protein production. We have gained preliminary insight into this pathway and can show that the modification regulates the accessibility of a key factor in chromatin biology, FACT. FACT is responsible for the removal of histones from DNA to allow processes like transcription and DNA repair to occur. Indeed, recently it was suggested that inhibiting FACT might be beneficial to cancer patients because FACT normally promotes repair of Cisplatin (a common chemotherapy compound)-induced DNA damage. Following on from our initial work we would like to better understand the molecular mechanism of the interplay between FACT and glutamine methylation. In turn, we hope that this detailed information might lead to the development of compounds that modulate the binding of FACT to chromatin. These might then be used to modulate FACT activity in order to gain an even better understanding of this molecule, but perhaps more importantly they may show efficacy in the treatment of certain diseases such as cancer.

We have identified a novel type of histone modification - glutamine methylation on
histone H2AQ105. Importantly, we have identified the enzyme performing this modification as Nop1/fibrillarin, a methyltransferase previously implicated in ribosome biogenesis.. Finally, we have found that methylation of H2AQ105 inhibits FACT binding to nucleosomes. FACT is a key player in chromatin biology and is responsible for transition of polymerases through chromatinized DNA. We want to better understand the influence of H2A Q105 methylation on the recruitment of FACT to chromatin and the effect on the cell by modifying the chromatin structure. To address the mechanistic aspects of this project, we will will employ a combination of biochemical assays (in vitro assays usingrecombinantly purified components), biophysical approaches (Biacore and potentially crystallization) and yeast genetics. To gain an understanding into the impact of this pathway on cells, we will use mammalian tissue culture for e.g. immunoprecipitation and immunofluorescence studies.
We will integrate these classical approaches with high-throughput sequencing of chromatin immunoprecipitation experiments. This will enable us to better understand the consequences of the modification on the underlying chromatin, e.g. the genomic localization of H2AQ105 methylation and its effect on chromatin structure and chromatin-related processes. Finally, biochemistry coupled with mass spectrometry will provide insight in to the regulation of Nop1. The enzyme has roles in at least two different biological pathways and we would like to understand 1) how modification of Nop1/fibrillarin controls different pathways and 2) identify co-factors (proteins and RNA) of Nop1/fibrillarin.

Since the proposed research will investigate a novel biological pathway, it is difficult to
predict with any accuracy whether there will be other beneficiaries outside of academia. However, there are several indications that a detailed understanding of a biological pathway can have tremendous benefits for the wider community. Furthermore, these can lead to benefits in public health. Even though research into epigenetic pathways is a relatively young field that has only developed over the last 15 years, it has brought about a series of highly promising new compounds such as 5-azacytidine, followed by compounds inhibiting histone deacetylases (HDACs), such as Vorinostat. Both show great potential in chemotherapy. Also several start-up companies (e.g. Chroma Therapeutics, Constellation Pharmaceuticals) have been set up to exploit insights into epigenetic research and established multinational pharmaceutical companies such as GSK have established their own independent epigenetic programs. Therefore, we believe that our findings will be absorbed by many sectors outside academia. It is difficult to provide exact timescales for the benefits to be realized, but our recent paper on BET inhibitors indicates that a decade of dedicated research is necessary from discovery of an epigenetic pathway to a promising drug. Our laboratory has performed textbook research over the last two decades and reviews written by Professor Kouzarides and his laboratory members are highly cited. Once more, this highlights the fact that research in epigenetics (by us and many other laboratories) generates real interest and is still placed at the forefront of biomedical research. The researcher on this project, Dr. Peter Tessarz, will develop a number of useful skills that could later be applied in different employment sectors. Dr. Tessarz will become increasingly independent on this project - he will plan, conduct and analyze experiments, will liaise with collaboration partners within and outside the laboratory and will write manuscripts resulting from the proposed work. All of these are transferrable skills and the success will be guaranteed by the close mentorship of Professor Kouzarides.