The overarching goal of this proposal is to uncover a previously uncharacterizedmolecular function of c-Myc (hereafter referred to as Myc), a proto-oncoprotein that is frequently amplified inbreast cancer and many other types of human cancer. Recently, we found that Myc overexpression leads toelevated expression of glutamate-ammonia ligase (GLUL) and interestingly, this Myc-induced GLUL is notthrough the direct transactivation by Myc, rather it involves promoter demethylation of the GLUL gene. Wefurther found that the demethylation is dependent on increased expression of thymine DNA glycosylase (TDG),which is a direct Myc transcriptional target. These results suggest an unexpected role of Myc in promotingglutamine synthesis, and intriguingly, suggest a previously unidentified molecular function of Myc in activatinggene expression by regulating DNA methylation. This prompts us to form the hypothesis that Myc can regulategene expression via the modulation of DNA methylation. We propose two Specific Aims to study thishypothesis. In Aim 1, we plan to identify Myc-induced DNA methylation and gene expression profiles bywhole genome bisulfite sequencing (WGBS) and RNA-Seq using various breast cancer cell lines with stable orinducible expression or knock-down of Myc. We will first prepare cell cultures with different treatments andextract genomic DNA and total cellular RNA for the next-generation sequencing (NGS). After NGS sequencereads are obtained, we will perform the bioinformatics analysis to (1) identify and annotate differentiallymethylated regions (DMR) in WGBS, with focus on gene promoters; (2) identify differentially expressed genes(DEG) in RNA-seq, using the same samples for DMR; and (3) rank Myc “epigenetic targets” using integratedbioinformatics analysis of DMR and DEG, and identify potential biological pathways preferentially affected byMyc through epigenetic regulation. We expect to discover specific “epigenetic targets” of Myc in various breastcancer cell lines. In Aim 2, we plan to validate the identified methylation profiles using traditional moleculartechniques and examine their biological relevance. We will first use the quantitative PCR and focal bisulfatesequencing on specific gene promoters to validate the “epigenetic targets” targets of Myc to be found in Aim 1.We will also examine the expression of TDG and the Myc epigenetic targets in various breast cancer cell lines.Furthermore, we will examine the expression patterns of TDG and validated epigenetic Myc targets by IHCusing de-identified breast cancer clinical tissue samples, and correlate them with histopathologicalcharacteristics and clinical outcomes. If successful, this project will uncover DNA demethylation as a novelmechanism for Myc regulated gene expression and oncogenesis. In the long run, the knowledge gained fromthis study will help with the understanding of cancer etiology and shed light on the development of noveltherapeutics.
|Effective start/end date||8/18/16 → 7/31/18|
- National Institutes of Health (NIH)
Thymine DNA Glycosylase
RNA Sequence Analysis
Cell Culture Techniques