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Cryo-Electron Microscopy of Histone Deacetylase Complexes

Principal Supervisor: Prof John Schwabe - Department of Molecular and Cell Biology

Co-supervisor: Dr. Shaun Cowley

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

University of Registration: University of Leicester

Project outline:

1

Crystal structure of HDAC1:MTA1 with InsP6 bound at the interface between the two proteins. The HDAC inhibitor, a modified histone H4 peptide H4K16Hx (pink), is bound to the active site (Watson 2016).

2Negative stain EM model of the NuRD complex (Millard 2016).

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.
They are also promising therapeutics for a number of other
diseases such as spinal muscular atrophy, Friedrich’s ataxia,
Alzheimer’s disease and HIV infection. 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 theHDAC3/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.

Recent publications:

  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. Itoh T, Fairall L, Muskett FW, Milano CP, Watson PJ, Arnaudo N, Saleh A, Millard CJ, El-Mezgueldi M, Martino F & Schwabe JWR (2015) Structural and functional characterization of a cell cycle associated HDAC1/2 complex reveals the structural basis for complex assembly and nucleosome targeting. Nucleic Acids Res 43: 2033–2044
  4. Hudson GM, Watson PJ, Fairall L, Jamieson AG & Schwabe JWR (2015) Insights into the Recruitment of Class IIa Histone Deacetylases (HDACs) to the SMRT/NCoR Transcriptional Repression Complex. J Biol Chem 290: 18237–18244

BBSRC Strategic Research Priority: Molecules Cells and Systems

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