Methods for chemical mapping of O-GlcNAc in the Drosophila genome

Resource type
Thesis type
(Thesis) M.Sc.
Date created
2017-07-10
Authors/Contributors
Abstract
O-linked N-acetylglucosamine (O-GlcNAc) is an important protein modification installed onto hundreds of nucleocytoplasmic proteins by O-GlcNAc transferase (OGT). Here, I discuss the development of an antibody-free metabolic feeding approach, which enables unbiased mapping of O-GlcNAcylated proteins in a genome-wide manner. This mapping method is detailed in Drosophila and compared to other O-GlcNAc mapping methods related to chromatin immunoprecipitation followed by sequencing (ChIP-seq), in order to demonstrate its overall efficacy. Using a combination of experimental and bioinformatics methods, I define new genes regulated by OGT. I also report on the development of robust software used to process and analyse time course ChIP-seq data, and prove its versatility and proficiency using both simulated and published data sets. This software is then applied to the analysis of a time course O-GlcNAc chemical mapping experiment in Drosophila larvae, generating the first ever time course ChIP-seq experiment performed on both a protein modification and in a living organism. Using this approach I am able to distinguish between loci that are more sensitive to O-GlcNAc cycling and those that are affected more by protein turnover. These studies provide an improved understanding of the regulation of gene expression by O-GlcNAc, while providing the wider community with new computational tools for time resolved analysis of genome-wide binding by proteins.
Document
Identifier
etd10238
Copyright statement
Copyright is held by the author.
Permissions
This thesis may be printed or downloaded for non-commercial research and scholarly purposes.
Scholarly level
Supervisor or Senior Supervisor
Thesis advisor: Vocadlo, David
Attachment Size
etd10238_MMyschyshyn.pdf 6.81 MB