I did my graduate studies in Professor Philip Batterham‘s lab in the Department of Genetics at The University of Melbourne in Australia. It was a wonderful and productive period of my life. I was fortunate enough to be co-advised by Dr. Phillip Daborn, who taught me all the molecular techniques and fly pushing skills that I know. Under the leadership of both Phils and collaborations with other graduate students, I had a productive period producing many papers that contribute to the understanding of the regulation and functions of cytochrome P450s, a group of enzymes with roles in both development and xenobiotic resistance.
Project 1: Overexpression of a P450 involved in insecticide resistance was due to insertion of a transposable element in the 5′ region of this gene
(Chung et al. 2007 Genetics)
I showed that widespread resistance to the insecticide DDT in the fruitfly Drosophila melanogaster, was due to tissue specific cis-regulatory element (CRE) carried on a transposable element inserted in the regulatory region of a P450 encoding gene known as Cyp6g1. This CRE-bearing allele drove constitutive tissue specific overexpression of Cyp6g1 in the midgut, Malpighian tubules and fat body in resistant populations.
Fig 1. Cyp6g1 alleles with Accord inserted into the 5′ region has increased expression of Cyp6g1 in the midgut, Malpighian tubules and fat body.
Fig 2. The Accord element carries cis-regulatory elements which drives GFP in the midgut, Malpighian tubules and fat body.
Project 2: Xenobiotic induction of cytochrome P450s is tissue specific and requires a novel class of cis-regulatory element
(Willoughby et al. 2006, IBMB),(Chung et al. 2011 IBMB)
Together with Lee Willoughby, we showed that induction of P450s by xenobiotics such as Phenobarbital and caffeine is tissue specific and and requires synergistic interaction between tissue specific CREs and a novel class of CRE, which we called a modulator, to modulate the induction response.
Fig 3. Induction by Phenobarbital is tissue specific.
Fig 4. Tissue specific induction requires a novel xenobiotic response element called a modulator that interacts with tissue specific enhancers.
Project 3: Evolutionary changes in gene expression, coding sequence and copy-number at the Cyp6g1 locus contribute to resistance to multiple insecticides in Drosophila
(Harrop et al. 2014 PLoS ONE)
Together with Tom Harrop, we showed that changes in tissue specific gene expression, coding sequence evolution and copy number variation have taken place at the Cyp6g1 locus across multiple species of Drosophila.
Fig 5. Overexpression of different Cyp6g1 orthologs confers resistance to different insecticides.
Fig 6. Evolution of Cyp6g1 tissue -specific gene expression in Drosophila species (3rd instar larvae)
Project 4: Characterization of Drosophila melanogaster Cytochrome P450s
(Chung et al. 2009 PNAS), (Sztal et al. 2011 PLoS ONE)
Cytochrome P450s form a large and diverse family of heme-containing proteins capable of carrying out many different enzymatic reactions. There are 85 P450s in the annotated D. melanogaster genome but not much is known about the functions of most of these genes. Together with Tamar Sztal, we characterized Drosophila melanogaster P450 expression patterns in embryos and 2 stages of third instar larvae. We identified numerous P450s expressed in the fat body, Malpighian (renal) tubules, and in distinct regions of the midgut, consistent with hypothesized roles in detoxification processes, and other P450s expressed in organs such as the gonads, corpora allata, oenocytes, hindgut, and brain. Combining expression pattern data with an RNA interference lethality screen of individual P450s, we identify candidate P450s essential for developmental processes and distinguish them from P450s with potential functions in detoxification. One such P450, Cyp301a1, is conserved in insects and play a role in cuticle formation.
Fig 7. Venn diagram representing unique and overlapping tissue expression of P450 genes expressed in the 3 key metabolic tissues of third instar larvae. FB, fat body; MG,midgut; MT, Malpighian tubules.
Fig 8. P450s with specific expression patterns in third instar larvae as determined by in situ hybridization.
Fig 9. RNAi of Cyp301a1 lead to cuticle malformation