Cryo-Electron Microscopy of Histone Deacetylase Complexes.

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 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. HDAC inhibitors have increasingly been shown to be useful in cancer, and diseases such HIV and Friedrichs Ataxia.

Of the class I HDAC's HDAC3 is uniquely incorporated into the NCOR/SMRT complex. However HDAC's 1 and 2 interact with Sin3A/B and at least 13 co-repressor proteins with ELM2-SANT domains. MTA1, 2 and 3 are in the NURD complex, CoREST 1, 2 and 3 are in the CoREST complex, MIDEAS is in the MIDAC complex, ZNF541 and TRERF1 are in homologous complexes to the MIDAC complex. The RERE and the MIER1, 2 and 3 co-repressor proteins have not been well characterised.

RERE is proposed to be in a complex with HDAC1/2, ATN1 and WDR5. The aims of this project are to characterise the domains of the proteins that interact in the RERE complex. An understanding of the interactome of this complex will also be important to understand its function since ATN1 and RERE are large proteins which will interact with other proteins such as transcription factors to recruit the RERE complex to chromatin.

In order to test for interactions there will be a candidate approach and a fishing approach. The candidate approach will involve co-expressing candidate proteins with the RERE complex and using a FLAG tag attached to one of the components to pull out complexes. The fishing approach will use a technique known as BioID, which uses a promiscuous biotin ligase tag, to tag the various components of the complex at the N and C terminal. The idea is that the biotin ligase will label proximal proteins with biotin which can be purified on streptavidin agarose and identified using mass spectrometry. There are two advantages of the BioID technique. One is that it can be used to look for transient interactions, the other is that because the streptavidin:biotin interaction is very high affinity, harsh conditions can be used that dissociate the proteins from chromatin.

This project will involve bioinformatics to design constructs for RERE, ATN1 and WDR5, cloning the proteins, transient transfections to express the proteins in HEK293F mammalian cells, western blots, mass spectrometry and proteomics approaches. Cryo-electron microscopy will be used to solve the structure of the RERE complex.

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. An understanding of the structure of the different HDAC complexes may lead to the design of more specific histone deacetylase inhibitors.