Enzymatic tools for studying the epigenome

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Ubych, Krystian Benedykt (2021). Enzymatic tools for studying the epigenome. University of Birmingham. Ph.D.

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Abstract

In cells, methyltransferase enzymes catalyse the methylation reaction of a numerous of biomolecules such as DNA, RNA, proteins and small molecules. Most methyltransferases require the presence and binding of a cofactor. These small molecules are methyl group donors of which the most common in nature is S-adenosyl-ʟ-methionine (AdoMet). DNA methylation plays a crucial role in epigenetic gene regulation and protein function modulation. Abnormal levels of CG methylation are found in cancer cells. Hence, methyltransferase enzymes are potential therapeutic targets and their inhibitors are of a high demand. Synthetic analogues of S-adenosyl-ʟ-homocysteine (AdoHcy), including N6-modified derivatives, show promise as inhibitors of AdoMet/AdoHcy dependent enzymes. However, current synthetic methods of N6-functionalisation is limited to inert modifications or require protecting groups in the process.
Here we present an efficient and protection-group-free synthesis of N6-substituted AdoHcy/AdoMet analogues. A broad range of functional groups was introduced including carboxyl, amine, alkyne and azide groups. We also show that such modified AdoHcy analogues can be used in the synthesis of AdoMet analogues which are recognised by two tested methyltransferases (M.TaqI and M.MpeI).
In cancer, hypermethylation is found in regions of the genome rich in cytosine and guanine, so-called CpG islands; yet, across the entire genome hypomethylation is the dominant trait. Even though the methylated cytosine residues can be detected with single nucleotide resolution using sequencing methods, little is known about the impact of DNA methylation in chromatin organization in nucleus.
Herein, we report a novel approach for the three-dimensional visualisation the architecture of accessible unmethylated DNA in mammalian cells. We employ methyltransferase-directed labelling of the epigenome using synthetic cofactor analogues for these enzymes bearing a clickable moiety for fluorescent labelling of DNA We combine this with expansion microscopy and inverted selective plane illumination microscopy (iSPIM) to reveal the organized structures of unmethylated regions of chromatin in healthy and cancerous cells.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

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