The Myc protooncogene is one of the most frequently dysregulated genes in human cancer. Even small changes in Myc expression levels can support Myc-driven tumorigenesis, thus the mechanisms that control Myc levels have been extensively scrutinized and cataloged. Less studied is the regulation of Myc activity. It is very clear that Myc does not function in isolation, but operates within a constellation of related transcription factors: the extended Myc network. The basic functions of each member of the extended Myc network have been known for more than 15 years, yet how they influence Myc function is largely speculative. Further, this important question has not been addressed using state-of-the-art molecular and genomic approaches. In this R03 application, we propose to develop the tools to assess the genomic occupancy of each member of the extended Myc network. This tool building exercise and subsequent pilot studies are a perfect fit for the R03 funding mechanism because they are well defined, address a critical question in cancer biology and can be completed in the 2-year funding period with a limited budget. The extended Myc network comprises two branches, one anchored by Max and the other anchored by Mlx. Max can interact with each Myc paralog, c-, N- and L- Myc. Generally, Myc:Max complexes bind to genomic CANNTG E-box elements to activate transcription of genes involved in progrowth pathways. Max can also form heterodimers with each member of the Mxd/Mad family of transcriptional repressors and a related factor called Mnt. Mxd:Max and Mnt:Max complexes also bind CANNTG E-box elements, but instead of activating gene expression, these complexes repress gene expression. Because Myc:Max and Mxd(Mnt):Max complexes all bind CANNTG E-box elements, the simple model is that they occupy similar genomic sites, but regulate expression of linked genes in the opposite direction. This model has held true at a handful of selected binding sites and target genes, but has not been tested comprehensively. Mlx interacts with members of the MondoA family: MondoA and ChREBP. MondoA:Mlx and ChREBP:Mlx complexes also bind CANNTG sites and generally activate gene expression in response to changing glucose levels. The Max- centered and Mlx-centered networks are speculated to be intertwined because Mlx can interact with a subset of the Mxd family. How each heterocomplex of the extended Myc network identifies its genomic binding sites in the context of the other network complexes and what dictates the functional outcome of each binding event is largely unknown. To address the first question, we propose to perform a comprehensive ChIP-seq analysis for each member of the extended Myc network. Because ChIP-seq is limited by the availability, specificity and affinity of the antibodies for each factor under study, we propose to use CRISPR/Cas9 editing to fuse the same epitope tag to the endogenous allele of each member of the extended Myc network. We will use ChIP-seq to examine the genomic binding of each network member during the G0-G1 transition where the levels and/or activity of many members of the extended Myc network are dynamically regulated.
The Myc protooncogene is a transcription factor and is among the most frequently dysregulated oncogenes in human cancer. Myc functions within a network of related transcription factors, the extended Myc network, to control gene expression programs that support cell growth and division. In this application we propose to develop new genomic tools to understand how crosstalk and redundant interactions between members of the extended Myc network contribute to tumorigenesis.
