Date of Award


Document Type

Thesis - ECU Access Only


Edith Cowan University

Degree Name

Doctor of Philosophy


School of Medical and Health Sciences

First Supervisor

Dr Robert B White

Second Supervisor

Dr Meghan G Thomas

Third Supervisor

Professor Sarah Dunlop

Fourth Supervisor

Professor Ken Nosaka


The Paired box gene 6 (PAX6) is a tissue-specific transcription factor, which controls proliferation and differentiation processes of embryonic- and adult progenitor cells. Consequently, dysregulation of PAX6 can cause cancer and a tight regulation is essential. The present thesis focuses on two mechanisms for regulation of PAX6, which are microRNAs (Study 1) and DNA methylation (Study 2 and Study 3), respectively.

Study 1 examines three predicted microRNA-7 (miR-7) target sites within the 3'untranslated region (3'UTR) of the human PAX6 gene. A Luciferase reporter assay demonstrates that two of the predicted miR-7 target sites are functionally active and transient transfection of miR-7 mimics reduces PAX6 protein levels in a human cell line.

Study 2 examines the DNA methylation profile of the PAX6 gene, specifically focusing on promoter CpG islands (Study 2a), exons (Study 2b) and intronic CpG islands (Study 2c). In neural induction of human pluripotent stem cells, PAX6 gene expression is first activated, and then repressed. Using targeted bisulfite sequencing and methylation-specific PCR it is revealed that the PAX6 gene is differentially methylated in a region-specific and differentiation stagedependent manner. Promoter CpG islands and 5'located exons remain predominantly non-methylated, whilst 3'located exons are constitutively methylated. In contrast, central exons and intronic CpG islands are differentially methylated with the highest level of DNA methylation at the neuroectoderm stage when PAX6 expression also peaks.

Study 3 focuses on a potential link between DNA methylation and differential exon usage (DEU). The alternative exon 5a of PAX6 contains a noncanonical CpG dinucleotide (a common target site for DNA methylation) at its 5'donor splice site and a high CpG content on immediate up- and downstream exons. Further based on previous genome-wide studies suggesting that the DNA methylation status of alternative exons may influence exon inclusion and that exon 5a of PAX6 does not contain a single CpG site, it is hypothesized that the DNA methylation status of gene-specific CpG profiles on- and around alternative splice sites is associate with DEU. A novel approach for detection of DEU from whole-transcriptome sequencing (RNA-seq) data is developed and future integrative studies aim to examine a potential association with DNA methylation.