Chemical tools for the study of epigenetic mechanisms

Thesis

The overall goal of my work was to develop and apply new chemical methods for the study of epigenetic DNA and protein modifications.

In Chapter 3 the development of Suzuki-Miyaura cross coupling (SMcc) for the post-synthetic modification of DNA is described. DNA modification by SMcc is efficient (4-6h) and proceeds under mild conditions (37°C, pH 8.5). The incorporation of various groups useful for biological investigations is demonstrated using this methodology. Using a photocrosslinker, introduced into the DNA by SMcc capture experiments are performed to identify potential binding partners of modified DNA.

In Chapter 4 a dehydroalanine (Dha) based chemical protein modification method is described that enables the introduction of posttranslational modification (PTM) mimics into histones. The PTM mimics introduced by this method are tested using western- and dot-blot and binding and enzymatic assays, confirming they function as mimics of the natural modifications.

Chapter 5 describes the use of a generated PTM mimics to elucidate the function of O-linked β-Nacetylglucosamine (GlcNAc) of histones in transcriptional regulation. It is shown that GlcNAcylation of Thr-101 on histone H2A can destabilize nucleosome by modulating the H2A/B dimer – H3/H4 tetramer interface.

N- and C-terminal histone tails play an important role in transcriptional regulation. In Chapter 6, nuclear magnetic resonance is used to investigate the structure of the histone H3 N-terminal tail in a nucleosome. The H3 tail, while intrinsically disordered, gains some α-helical character and adopts a compact conformation in a nucleosome context. This H3 tail structure is shown to be modulated by Ser-10 phosphorylation.

The effect of a new covalent DNA modification, 5- hydroxymethylcytosine (5hmC), on transcription factor binding is investigated in Chapter 7. 5hmC influences HIF1α/β, USF and MAX binding to their native recognition sequence, implying involvement of this modification in epigenetic regulation.

Authors: Lercher, L

• Publication date: 2014
• DOI: 10.5287/ora-g7b0ow79d
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: Oxford University, UK
• Language: English
• Keywords: Epigenetic regulation; protein NMR; DNA modification; posttranslational modifications
• Subjects: Organic chemistry; Chemical biology; NMR spectroscopy; Molecular biophysics (biochemistry); Protein chemistry