The structural basis of transcription factor 3C recruitment by N-myc

The uncontrolled proliferation that is characteristic of cancer cells is driven by the overexpression or aberrant regulation of proteins whose normal physiological role is to drive cellular growth in response to growth signals. One such protein is the transcription factor N-myc, one member of a family of three closely related proteins (c-myc, N-myc, and L-myc) all of which alter the expression of many genes. Overexpression of myc proteins leads to increased levels of cellular proliferation and is a frequent feature of cancer cells. Myc induces senescent cells to re-enter the cell cycle, subverts key checkpoints in the cell cycle, and facilitates the rewiring of cellular metabolism to favour both cell cycle progression and biomass accumulation.
N-myc was recently found to increase gene expression via an interaction with a general transcription factor complex, TFIIIC. These molecules acting together were found to activate the production of hundreds of genes. This activated set of genes disproportionally involved in functions of N-myc known to drive cancer; functions such as cell cycle progression, DNA replication, nucleotide metabolism, and telomere biology. However, it is currently unknown how N-myc and TFIIIC complex recognise each other, and how their interaction impacts on the many other proteins they interact with.
The first aim of our research proposal is to characterise the molecular basis of the N-myc:TFIIIC interaction at the level of the individual amino acids in the proteins. This will be done using orthogonal methods of X-ray crystallography, cryo-electron microscopy, and cross-linking mass spectrometry. The second aim is to validate the structure in vitro and in cells by using mutations at the binding interface. The final aim is to use these mutations to begin to understand what other proteins form part of the N-myc:TFIIIC complex and to begin to understand the mechanism by which N-myc interacts with TFIIIC over other molecules which are known to bind the same, or nearby, myc sequences.
A potential benefit of this research will be to help validate transcriptional activation of myc as a target for cancer drug discovery. Drug discovery against Myc has proven very difficult and we believe the problem can be solved by targeted the protein-protein interactions that underpin Myc's oncogenic activity. However, it is not clear which these are, despite many years of research. Mutations which are validated in this work can be by us and others to determine the effect of disrupting the N-myc:TFIIC interaction on cancer cell proliferation, checkpoint integrity and cancer cell metabolism. If disruption of this interaction has significant effect on cancer cell proliferation it validates this interaction as a potential approach to develop new therapeutics that target Myc and this would be a big development in the field. Moreover, an additional benefit of our research is that structures and structural knowledge generated by this work will be available as a template for the design of inhibitors.

Myc proteins function as a transcriptional regulators that recruit other proteins such as transcription factors (e.g. TFIIIC), protein kinases (e.g. CDK9) and chromatin-modifying adaptors and enzymes (e.g. STAGA), which act at multiple regulatory steps to activate transcription. This proposal focuses on the interaction between N-myc and TFIIIC, which leads to activation of RNA Polymerase II (Pol II) transcription of a subset of genes important for cellular proliferation.

In preliminary work we have identified a small region of the N-myc sequence that interacts directly with a subcomplex of TFIIIC. We have also demonstrated that we can express and purify this minimal interaction module. Most of the work outlined in the proposal is dedicated to understanding the N-myc:TFIIIC interaction at a structural level using three orthogonal approaches: X-ray crystallography, cryo-electron microscopy, and structural mass spectrometry. Additionally, we aim to validate the structure using mutation and protein:protein interaction assays, initially in vitro using biophysical approaches (ITC, FP), then in cultured cells (co-IP, PLA). Finally, we aim to extend the validated structural observations and mutations to determine how the complex relates to other N-myc interactions involved in transcriptional activation and its regulation. We will use mutation combined with affinity tag proteomics to determine which interactors are dependent on the N-myc:TFIIIC interaction. This will enable us to identify other proteins that associate with the complex. Additional analysis by cell XL-MS will help us understand the basic architecture of the higher-order complex.
Results can be applied to work by molecular oncologists performing synthetic lethality screens of important oncogenic pathways. They can also be exploited by the cancer drug discovery community, using structural and computational methods to block protein:protein interactions.