A promoter G-quadruplex recruits Hrb27C to regulate NEMO and immune defense against Lyme disease pathogens in the deer tick.

Genomic DNA can adopt noncanonical secondary structures, such as G-quadruplexes (G4s), which serve as critical regulatory elements beyond the traditional double helix. Whether promoter G4s also participate in controlling innate immune signaling and host-pathogen interactions, however, remains unknown in arthropods. In this study, we identify a G4 structure within the promoter of the NEMO (NF-κB essential modulator) gene in the deer tick Ixodes scapularis. Using a combination of circular dichroism, isothermal difference spectra, and fluorescence-based assays, we demonstrate that this sequence forms a stable, parallel G4 topology in vitro and in vivo. Functional characterization via luciferase reporter assays reveals that this G4 scaffold is essential for proper NEMO transcriptional output. Importantly, disruption of the G4 structure significantly reduces NEMO expression and increases the burden of the Lyme disease spirochete Borrelia burgdorferi. Genome-wide expression profiling further reveals that perturbation of the NEMO G4 produces transcriptional changes that overlap with those observed following NEMO silencing. These shared transcriptional responses are enriched for immune-associated genes and ribonucleoprotein-related pathways. We further identify the heterogeneous nuclear ribonucleoprotein Hrb27C as a structural binding protein that specifically recognizes the NEMO G4 through its RNA recognition motif domains and facilitates NEMO expression. Collectively, our findings establish promoter G4 architecture as an important regulatory layer in tick immunity, linking DNA topology to pathogen proliferation and providing a structural framework for understanding host-pathogen dynamics in disease vectors.