Lysine acetylation (Lys-Ac) is an abundant post-translational modification (PTM) occurring on thousands of proteins. However, our understanding of ‘acetylome’ regulation and its functional consequences remains well behind other common PTMs such as phosphorylation and ubiquitination. Analogous to these latter PTMs, acetylation alters the charge on the amino acid, potentially changing the conformation or association of other proteins, and it is the docking site for other factors containing a Bromodomain. A land-mark paper in 2009 by Choudhary et al., used SILAC labelling, immuno-affinity purification of Lys-Ac peptides coupled with mass-spectrometry to identify ~3,000 sites on 1,750 proteins1. Other groups have taken a similar approach in numerous cell types2 and identified thousands of additional sites, but these have largely been global Lys-Ac surveys, mapping sites rather than studying regulation or downstream function (reviewed in 3). The goals of this project are designed to address the major outstanding questions in the field: how these networks of Lys-Ac sites are regulated, the mechanisms by which Lys-Ac alters protein function, to what purpose, and whether these pathways can be targeted therapeutically to treat disease.
In mammalian cells the acetylome is regulated by the opposing activities of 22 KAT (Lys acetyl-transferase) and 18 KDAC (Lys deacetylase) enzymes, often found as part of multi-protein complexes. Recently published data has shown that the highly related KATs, p300 and CBP (hence referred to jointly as p300) catalyse a large fraction (30%) of cellular acetylation4. Equivalent data from the Cowley lab has shown that the sister proteins, KDAC1 and KDAC2 (KDAC1/2; 82% identical and found within the same co-repressor complexes), contribute approximately 50% of total deacetylase activity5 and regulate >800 Lys-Ac sites in embryonic stem cells (Fig 1). Both p300 and KDAC1 are essential for embryogenesis, cause a loss of cell growth and/or promote differentiation when inhibited, and are mutated or over-expressed in numerous disease states. We propose that these critical enzymes form a p300/KDAC1 axis in cells, which dynamically regulates hundreds (possibly thousands) of Lys-Ac sites, during the cell cycle, development and homeostasis of primary cells. By examining Lys-Ac from the perspective of both KATs and KDACs (something not previously reported in the same study) we are more likely to select for functionally consequential sites among a back-ground of several thousand sites.
Objective 1: Regulating the Acetylome: defining a network of dynamic Lys-Ac sites controlled by the p300/KDAC1 axis.
We recently performed a SILAC-based analysis of the acetylome in ES cells deleted for KDAC1/25 (of 7,668 unique sites, 883 (12%) have >1.5 fold increase in Lys-Ac) (Fig 1). Having demonstrated the sensitivity of the assay and our own proficiency in acetylome analysis we feel that we are in an excellent position to address acetylome regulation on a wider scale. We will examine the role of the p300/KDAC1 axis in regulating the acetylome using SILAC-labelled primary cells treated with either p300 or KDAC1 inhibitors.
Objective 2: Characterisation of specific Lys-Ac regulated by the p300/KDAC1 axis: does modification equate with function?
With a few notable exceptions (p53, SMC3, core histones, etc.), the mechanistic consequences of Lys-Ac for protein function remain largely unstudied. By examining specific Lys-Ac sites, either up- or down- regulated by perturbation of p300/KDAC1 axis (Objective 1), we will be able to focus on a group of dynamically regulated residues.
We propose to study the regulatory mechanisms of a major, but largely unstudied and under-exploited, PTM in primary human cells. The successful completion of these objectives would have a transformative impact on the field of study and our ability to target these pathways therapeutically.
Techniques that will be undertaken during the project
Cell culture, SILAC labelling and preparation of samples for mass-spectrometry analysis. Bioinformatics analysis of large datasets to find physiologically relevant changes in Lys-Ac in a range of signalling pathways. Individual Lys-Ac sites of high interest (approx. 5-8) will be examined by mutating LysArg in individual genes using CRISPR/Cas9. All of these techniques are currently being used in the Cowley and Panne laboratories.
Available to UK/EU applicants only
Application information https://www2.le.ac.uk/research-degrees/doctoral-training-partnerships/bbsrc
1 Choudhary, C. et al. Lysine acetylation targets protein complexes and co-regulates major cellular functions. Science 325, 834-840, doi:1175371 [pii] 10.1126/science.1175371 (2009).
2 Lundby, A. et al. Proteomic analysis of lysine acetylation sites in rat tissues reveals organ specificity and subcellular patterns. Cell reports 2, 419-431, doi:10.1016/j.celrep.2012.07.006 (2012).
3 Choudhary, C., Weinert, B. T., Nishida, Y., Verdin, E. & Mann, M. The growing landscape of lysine acetylation links metabolism and cell signalling. Nat Rev Mol Cell Biol 15, 536-550, doi:10.1038/nrm3841 (2014).
4 Weinert, B. T. et al. Time-Resolved Analysis Reveals Rapid Dynamics and Broad Scope of the CBP/p300 Acetylome. Cell 174, 231-244 e212, doi:10.1016/j.cell.2018.04.033 (2018).
5 Jamaladdin, S. et al. Histone deacetylase (HDAC) 1 and 2 are essential for accurate cell division and the pluripotency of embryonic stem cells. Proc Natl Acad Sci U S A 111, 9840-9845, doi:10.1073/pnas.1321330111 (2014).