Exposure to ultraviolet (UV) light is the principal etiological agent for melanoma and other skin cancers. UV light induces damage to the cellular DNA, primarily cyclobutane pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PPs). Sequencing of DNA from human skin cancers have revealed high levels of UV- induced mutations that are heterogeneously distributed across the genome. It has recently been discovered that UV-induced mutations are particularly enriched at the DNA binding sites of transcription factors. While this has been attributed to less efficient repair at such binding sites, it is also possible that variations in initial damage formation due to transcription factor binding could stimulate mutation rates. To test this hypothesis, we have used our newly developed CPD-seq method to map CPD formation across the human genome immediately following UV irradiation. Our preliminary data indicate that initial UV damage formation is significantly elevated at many transcription factor binding sites (TFBS), particularly binding sites for the E26 transformation-specific (ETS) family of transcription factors (TFs). Importantly, stimulation of CPD formation by ETS binding correlates with significantly higher mutation rates in melanoma tumors, indicating that variations in initial DNA damage formation are an important contributor to the mutational 'landscape' in human cancers. The overall objective of this proposal is to elucidate the mechanism by which ETS binding stimulates UV damage formation and determine whether ETS-induced CPD 'hotspots' drive recurrent mutagenesis at individual binding sites in melanoma tumors. To investigate the mechanism by which ETS protein binding stimulates UV damage formation, we will characterize the effects of selected ETS transcription factors on UV damage formation and repair in vitro (Aim I). In parallel, we will use molecular dynamics simulations to model how DNA binding by different ETS transcription factors predisposes dipyrimidine sequences to form UV photoproducts. Finally, we will examine whether ETS TF binding inhibits repair of CPD lesions in vitro.
In Aim II, we will develop the CPD-capture-seq method to map the formation and repair of CPD lesions with high sequencing depth and single nucleotide resolution at specific genomic regions of interest, including sites of recurrent promoter mutations in melanoma and ETS binding sites. In parallel, we will map ETS binding sites in human melanocytes using the ChIP-exo method. Comparison of CPD-capture-seq data with UV-induced mutations identified in human melanomas will allow us determine with high resolution whether ETS-induced CPD 'hotspots' and repair inhibition are associated with recurrent mutations in skin cancer. These data should provide new insights into the etiology of some of the most recurrent mutations in melanoma, which occur at ETS binding sites. ETS transcription factors are known oncogenes that regulate many genes involved in cell differentiation, migration, proliferation, and apoptosis; hence, recurrent mutations at ETS binding sites likely contribute to carcinogenesis in skin cancer and could be exploited in future therapeutics.

Public Health Relevance

Mutations that underlie carcinogenesis are heterogeneously distributed across human genomes. This proposal will use in vitro biochemistry and whole-genome sequencing techniques to investigate the mechanism(s) by which DNA binding by E26 transformation- specific (ETS) family transcription factors stimulate UV damage formation, inhibit subsequent repair, and thus promote cancer-driving mutations in human skin cancers.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21ES029655-02
Application #
9904652
Study Section
Cancer Etiology Study Section (CE)
Program Officer
Humble, Michael C
Project Start
2019-04-01
Project End
2021-03-31
Budget Start
2020-04-01
Budget End
2021-03-31
Support Year
2
Fiscal Year
2020
Total Cost
Indirect Cost
Name
Washington State University
Department
Veterinary Sciences
Type
Schools of Veterinary Medicine
DUNS #
041485301
City
Pullman
State
WA
Country
United States
Zip Code
99164