Abstract
Functional genomics studies have unraveled many of the instructions encoded in the human genome. Taking
advantage of significant advances in parallel sequencing and a wide diversity of other protocols, studies are
proposed in this application to resolve gene-environment interactions that determine variations in the
susceptibility to lung cancer. These studies are timely in light of the worsening levels of environmental pollution
in many US cities that contribute to the development of respiratory disease and to disparities across populations.
Of concern is the persistence of polycyclic aromatic hydrocarbon (PAH) contaminants, such as benzo(a)pyrene
(BaP), and their ability to damage the DNA of lung epithelial cells. While the mutagenicity of BaP has been
extensively characterized, questions remain about the degree to which DNA damage intersects with epigenetic
disruption in driving the overwhelming carcinogenic response of BaP. Genome-wide assessments of BaP toxicity
in the Ramos Laboratory revealed that the parent hydrocarbon and its metabolites activate LINE-1
retrotransposons in lung epithelial cells via epigenetic mechanisms. LINE-1 (Long Interspersed Element-1)
activation is associated with oncogenic signaling, changes in chromatin structure, disruption of epigenetic
control, and malignant transformation. LINE-1 is a family of mobile elements with the ability to copy their own
DNA and use these complementary sequences to randomly insert at other locations within the genome. This
process can be highly mutagenic and associated with genomic instability. We posit that alterations in the lifecycle
of LINE-1 retroelements is a key missing link in our understanding of the complex genetic and epigenetic deficits
associated with BaP carcinogenicity. Of ~100 full-length LINE-1 elements that remain competent for
retrotransposition, nine are recognized as “hot” LINE-1s responsible for the vast majority of pathogenic events.
These hot elements are polymorphic in humans and may therefore contribute to heterogeneity in genetic
susceptibility to environmental PAH toxicity. In response to RFA-ES-20-018, we propose to use in vitro functional
genomics for discovery and validation to test the hypothesis that polymorphic LINE-1 elements differentially
regulate oncogenic signaling in lung epithelial cells and account for differences in susceptibility to BaP. In Aim
1, we will examine hot LINE-1 elements and their constitutive and inducible expression in normal lung and
cancerous epithelial cells by targeted DNA sequencing and single cell RNA sequencing (scRNA-seq). In Aim 2,
we will use CRISPR/dCas9m to edit the LINE-1 promoter to either enhance or silence retrotransposition and its
impact on chromatin structure and transcriptomic landscapes. In Aim 3, we will use computational approaches
to study genetic relationships across variable networks defined by polymorphic LINE-1 variants, with a focus on
TP53, AHR, and RB. These studies will reveal important mechanistic insights into the activation of LINE-1 by
PAH carcinogens and illuminate our understanding of the human variability in carcinogen susceptibility. This
knowledge will lead to better biomarker designs and novel therapies to combat environmental toxicities.