Towards the development of a novel proteomic tool to map trimethyllysine marks on histones via a Hofmann-type chemical modification.
File(s)
Author(s)
Ekinci, Melis Eda
Type
Thesis
Abstract
One of the key epigenetic processes for transcriptional control is dynamic post-translational modification (PTM) of histone proteins. Dysregulation of these epigenetic mechanisms have been linked to the aetiology of human diseases such as cancer and neurological disorders. To decipher the epigenetic pathways leading to the development of disease and to gain insight into the function of post-translational modifications of histones, a great interest was taken on the genome-wide mapping of epigenetic marks. The most commonly used method to profile PTMs on histones is the ChIP-seq technique (chromatin immunoprecipitation followed by DNA sequencing), which is based on the use of antibodies to target and enrich specific epigenetic marks. However, the dependence of ChIP-seq on antibodies constitutes a significant limitation due to possible cross-reactivity and poor specificity of those antibodies towards the targeted epigenetic marks. In addition, the ChIP-seq technique only allows the mapping of single epigenetic marks to genomic loci. Here we present a novel antibody-free chemical biological tool ‘CLICK-seq’ to map combinatorial histone lysine trimethylation marks on intact mono-nucleosomes. It is based on the selective modification of trimethyllysine residues on histone proteins to obtain an alkene functionality on these substrates, which enables the introduction of affinity tags via a thiol-ene click chemistry for selective enrichment. This work demonstrates diverse chemical approaches taken towards the elimination of trimethylamine in quaternary ammonium substrates to achieve the formation of an alkene under mild and ‘protein-compatible’ conditions. With the development of a novel reaction - Pd-catalysed Hofmann-type elimination – the removal of trimethylamine was achieved at low temperature on model small molecules (albeit in a low yield).
In the second project, a novel design for acid-cleavable cross-linkers was developed to simultaneously achieve the cross-linking of histone protein complexes and the subsequent chemical labelling of histone lysine residues upon acid-catalysed cleavage. This work portrays our efforts towards the synthesis of a small library of acid-cleavable cross-linkers.
In the second project, a novel design for acid-cleavable cross-linkers was developed to simultaneously achieve the cross-linking of histone protein complexes and the subsequent chemical labelling of histone lysine residues upon acid-catalysed cleavage. This work portrays our efforts towards the synthesis of a small library of acid-cleavable cross-linkers.
Version
Open Access
Date Issued
2019-10
Date Awarded
2020-03
Copyright Statement
Creative Commons Attribution NonCommercial Licence
License URL
Advisor
Fuchter, Matthew
DiMaggio, Peter
Bartke, Till
Publisher Department
Chemistry
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)