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

Author: 
Date created: 
2017-07-10
Identifier: 
etd10238
Keywords: 
O-linked N-acetylglucosamine (O-GlcNAc)
O-GlcNAc transferase (OGT)
Chemical biology
Chromatin immunoprecipitation followed by sequencing (ChIP-seq)
Time course ChIP-seq analysis software
Bioinformatics
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 type: 
Thesis
Rights: 
This thesis may be printed or downloaded for non-commercial research and scholarly purposes. Copyright remains with the author.
File(s): 
Senior supervisor: 
David Vocadlo
Department: 
Science: Department of Molecular Biology and Biochemistry
Thesis type: 
(Thesis) M.Sc.
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