UNIVERSITY OF WISCONSIN River Falls

Emma Brandt

Emma Brandt

Emma BrandtMajor: Biology - Biomedical

Minors: Chemistry & Music

Anticipated Graduation Date: May, 2013

Research Supervisor: Dr. Liewei Wanglink, Molecular Pharmacology & Experiemental Therapeutics

Faculty Mentor: Dr. Scott Ballantyne, Biology, University of Wisconsin-River Falls

Research Location: Mayo Graduate School, Summer Undergraduate Research Fellowship (SURF), Summer 2012

Research Topic: Pharmacoepigenomics of Cytidine Analogue: A Genome Wide Association of CpG Methylation Using a Lymphoblastoid Cell Line System

Abstract: Background/Aims: Two cytidine analogues, gemcitabine (dFdC) and cytosine arabinoside (AraC), are widely used in the treatment of a variety of types of cancer. However, both drugs show a large individual variation in treatment response.  Pharmacogenomics is the study of the role of inheritance in individual variation in drug response phenotypes. Beyond the DNA sequence, epigenetic factors including CpG methylation might also be responsible for variation in gene regulation. In this present study, we used a human lymphoblastoid cell line (LCL) model system with extensive genomic data to perform genome wide association studies (GWAS) with gemcitabine and AraC cytotoxicity phenotypes and to identify potential epigenetic biomarkers that might contribute to response to these two cytidine analogues.  Methods: Our cell line model system consists of 300 genomic-data rich human lymphoblastoid cell lines (LCLs) from multiple ethnicities including 100 Caucasian-American (CA), 100 African-American (AA) and 100 Han Chinese-American (HCA). The extensive genomic data including 54,000 basal mRNA expression and 470,000 DNA CpG methylation sites for all of those 300 LCLs were obtained. We also obtained the IC50 values for both gemcitabine and AraC. Genome-wide association studies (GWAS) were performed for both drugs using IC50 values, CpG methylation and mRNA expression levels. We then performed functional studies with selected candidate genes using siRNA knockdown, followed by qRT-PCR and MTS Assay.Results: We identified 10 genes with 22 CpG methylation sites associated with gemcitabine cytotoxicity and 8 genes with 24 CpG methylation sites for AraC. Three are common for both AraC and gemcitabine, including MGMT, COL5A1, and GHR. Methylation levels of these three genes were also significantly associated with their gene expression levels, so called a "cis-regulation".  In addition, knockdown of GHR desensitized drug cytotoxicity to both AraC and gemcitabine, consistent with the observation from our GWAS.  The GHR expression in a human breast cancer T47D cells was significantly induced by the treatment of a demethylation reagent, 5-azacytidine indicating that GHR expression might be regulated through CpG methylation.  There were two methylation sites in the 5' UTR of the GHR gene that were associated with GHR gene expression (P = 1.67 x 10 -10), as well as drug cytotoxicity. These two sites are undergoing further investigation. Conclusions: Our methylation GWAS performed in the LCLs would help to study the role of epigenetic regulation in drug response.  Follow-up studies will be pursued to find the underlying mechanism of transcription of gene expression by CpG methylation and how this might contribute to variation in drug response.



Faculty Mentor & Research Supervisor: Dr. Scott Ballantyne, Biology, University of Wisconsin-River Falls

Research Location: University of Wisconsin-River Falls, 2011

Research Topic: Mothers are Important: control of polyadenylate binding protein by siah1 in vertebrate embryos

Abstract: When a baby is first born they are completely dependent on their mother for everything.  The same concept also applies to the embryo since vertebrates begin life without transcription.  The developing embryo relies upon a dowry of mRNA that are provided by the mother and stored in the egg.  These maternal mRNA are selectively activated upon fertilization, often by recruiting the embryonic polyadenylate binding protein (ePAB).  Embryos eventually use their own DNA.  This switch to zygotic transcription coincides with the disappearance of ePAB.  We are using the frog, Xenopus laevis,as a model to study maternal gene regulation during embryogenesis.  Our approach is to study Xenopusproteins that control maternal mRNA using a molecular genetic screening technique in yeast known as the two-hybrid system.  We discovered a novel interaction between ePAB and seven in absentia homolog 1 (SIAH1), a ubiquitin ligase that promotes protein decay.  We propose that SIAH1 is responsible for ePAB degradation during embryogenesis.  We show that the ePAB:SIAH1 two-hybrid interaction is specific and that SIAH1 interacts with C-terminal sequences in ePAB.  Interestingly, the C-terminal ePAB sequence contains a consensus SIAH1 binding site (PxAxVxP).  We are currently using site-directed mutagenesis to test whether or not this consensus sequence is required for the ePAB:SIAH1 interaction. We are also using deletion mapping to identify the sequences in SIAH1 that are needed for binding ePAB.  Our work will provide the foundation for future functional studies in Xenopusthat test whether SIAH1 controls ePAB during embryogenesis.  PAB and SIAH1 are found in nearly all eukaryotic creatures and cell-types.  We have begun to examine if these other PAB bind SIAH1.  Our preliminary data indicates that several other PAB contain putative SIAH1 binding sequences that are near or perfect matches to the PxAxVxP consensus, suggesting that SIAH1 may control PAB in the soma. This work may have clinical significance, as both SIAH1 and PAB are linked to several human diseases including cancer.