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Cryo-Electron Microscopy of Histone Deacetylase Complexes

Primary Supervisor: Professor John Schwabe, Department of Molecular and Cell Biology

Secondary Supervisor: Professor Shaun Cowley

PhD project title: Cryo-Electron Microscopy of Histone Deacetylase Complexes.

University of Registration: University of Leicester

Project outline:

Histone deacetylase complexes play a role in many cellular processes, such as cancer, cell cycle progression and DNA repair. Investigating how these HDAC complexes act to control gene expression and their interactions with other proteins should lead to an understanding of their influence in the cell. HDAC inhibitors are currently used to treat some cancers. An understanding of the structure and function of the various histone deacetylase complexes should mean that inhibitors can be designed to specific complexes to reduce side effects.

Class-1 histone deacetylases (HDACs 1, 2, 3) are essential enzymes present in the nucleus of all mammalian cells, where they help regulate chromatin structure as the catalytic component of large multi-protein co-repressor complexes such as Sin3A, NuRD, CoREST, MIDAC and NCOR/SMRT. Each of these complexes is recruited to target genes by specific transcription factors to regulate transcription. Incorporation into specific complexes is fundamental to HDAC 1, 2 and 3 function since this directs both substrate specificity as well as regulating the enzymatic activity of the HDAC. Histone deacetylase enzymes (HDACs) are generally thought to regulate gene expression by removing acetyl groups from lysine residues in histone tails resulting in chromatin condensation and gene repression, although gene profiling has shown HDACs are predominately located at active genes, suggesting a role in resetting chromatin between rounds of transcription.

The Schwabe group have been successful in expressing and purifying the core of many HDAC complexes in HEK293F cells in sufficient quantity for structural studies. This has led to a number of structures of HDAC complexes using X-ray crystallography and negative stain electron microscopy. In addition the structure of the HDAC3/SMRT complex led to the discovery that class I histone deacetylases are activated by inositol phosphates.

There has recently been a revolution in Cryo-Electron Microscopy, which means that large protein and protein complex structures that previously could not be crystallised can now be solved at atomic resolution. The aim of this PhD project is to solve the structure of histone deacetylase complexes using Cryo-electron microscopy and to fully understand the structure of the different HDAC complexes, their interaction with chromatin and their interaction with other components involved in transcription. An understanding of the structure of the different HDAC complexes may lead to the design of more specific histone deacetylase inhibitors.

References:

  1. Millard CJ, Varma N, Saleh A, Morris K, Watson PJ, Bottrill AR, Fairall L, Smith CJ & Schwabe JW (2016) The structure of the core NuRD repression complex provides insights into its interaction with chromatin. eLife 5: e13941
  2. Watson PJ, Millard CJ, Riley AM, Robertson NS, Wright LC, Godage HY, Cowley SM, Jamieson AG, Potter BVL & Schwabe JWR (2016) Insights into the activation mechanism of class I HDAC complexes by inositol phosphates. Nature Communications 7: 11262
  3. Kalin, J.H., Wu, M., Gomez, A.V., Song, Y., Das, J., Hayward, D., Adejola, N., Wu, M., Panova, I., Chung, H.J., Kim, E., Roberts, H.J., Roberts, J.M., Prusevich, P., Jeliazkov, J.R., Roy Burman, S.S., Fairall, L., Milano, C., Eroglu, A., Proby, C.M., Dinkova-Kostova, A.T., Hancock, W.W., Gray, J.J., Bradner, J.E., Valente, S., Mai, A., Anders, N.M., Rudek, M.A., Hu, Y., Ryu, B., Schwabe, J.W.R., Mattevi, A., Alani, R.M., Cole, P.A., (2018). Targeting the CoREST complex with dual histone deacetylase and demethylase inhibitors. Nature Communications 9, 53.
  4. Zhang, T., Wei, G., Millard, C.J., Fischer, R., Konietzny, R., Kessler, B.M., Schwabe, J.W.R., Brockdorff, N., (2018). A variant NuRD complex containing PWWP2A/B excludes MBD2/3 to regulate transcription at active genes. Nature Communications 9, 3798.

 

BBSRC Strategic Research Priority: Understanding the Rules of Life: Structural Biology

Techniques that will be undertaken during the project:

This project will involve protein cloning, protein expression, purification, western blots, mass spectrometry, proteomics approaches and cryo-electron microscopy. Proteomics approaches and cryo-electron microscopy are techniques that are at the forefront of biological science research.

Contact: Professor John Schwabe, University of Leicester