Profiling the methylome targets of histone lysine methyltransferases
File(s)
Author(s)
Addison, Katie
Type
Thesis
Abstract
It is widely understood that dysregulation of epigenetic events such as post-translational modifications (PTMs) of histone proteins, are associated with a plethora of diseases, such as cancer. A prevalent and regularly studied PTM is the methylation of lysine residues on histones, mediated by the family of enzymes – histone lysine methyltransferases (HKMTs). However, to date there are considerable challenges in defining unambiguously the methylation targets of a single HKMT. The major reasons for this are: 1) there is considerable redundancy in histone methylation and multiple HKMTs can ‘write’ the same target methylation modification, 2) HKMTs also often have many non-histone substrates.
The majority of HKMTs contain the SET (Su(var)3-9, Enhancer-of-zeste and Trithorax) binding domain which binds the co-factor S-adenosyl methionine (SAM) enabling the transfer of the methyl donor group to the intended substrate. Through computational studies, the group has located an endogenous ‘hole’ in the SET binding domain of SETD7 making it structurally different to other HKMTs (e.g. G9a). The work presented here shows the synthesis of a small library of SAM analogues to be utilised by SETD7. The analogues have various groups appended at the C-2 position of the adenine ring to compliment the endogenous ‘hole’ in SETD7. Additionally, these cofactors include an isotopically labelled CD3 reporter group so newly deposited ‘heavy’ methyl marks can be readily distinguished from endogenous methylation using LC-MS/MS analysis. Initial in vitro and in vivo studies have been performed to demonstrate the validity of this approach for histone targets with the intention of extending to a library of non-histone targets.
The majority of HKMTs contain the SET (Su(var)3-9, Enhancer-of-zeste and Trithorax) binding domain which binds the co-factor S-adenosyl methionine (SAM) enabling the transfer of the methyl donor group to the intended substrate. Through computational studies, the group has located an endogenous ‘hole’ in the SET binding domain of SETD7 making it structurally different to other HKMTs (e.g. G9a). The work presented here shows the synthesis of a small library of SAM analogues to be utilised by SETD7. The analogues have various groups appended at the C-2 position of the adenine ring to compliment the endogenous ‘hole’ in SETD7. Additionally, these cofactors include an isotopically labelled CD3 reporter group so newly deposited ‘heavy’ methyl marks can be readily distinguished from endogenous methylation using LC-MS/MS analysis. Initial in vitro and in vivo studies have been performed to demonstrate the validity of this approach for histone targets with the intention of extending to a library of non-histone targets.
Version
Open Access
Date Issued
2018-09
Date Awarded
2019-02
Copyright Statement
Creative Commons Attribution NonCommercial Licence
License URL
Advisor
Fuchter, Matthew
DiMaggio, Peter
Sponsor
Engineering and Physical Sciences Research Council
Grant Number
CHBBC
Publisher Department
Chemistry
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)