Loss of lactate dehydrogenase A disrupts R-loop homeostasis and induces DNA damage in breast cancer cells.

Introduction: R-loops, three-stranded nucleic acid structures composed of an RNA-DNA hybrid and displaced single-stranded DNA, are tightly regulated by single-stranded DNA-binding proteins. However, the role of lactate dehydrogenase A (LDHA), an atypical DNA-binding protein and key glycolytic enzyme, in R-loop regulation remains unclear. Methods: Cleavage Under Targets and Tagmentation sequencing (CUT&Tag-seq) was utilized to map R-loop and LDHA genomic landscapes in wild-type (WT) and LDHA knockout (KO) 4T1 cells. RNA-seq was employed for transcriptomic profiling. Immunofluorescence was used to evaluate the co-localization of LDHA and R-loops. Western blotting was performed to assess the protein levels of LDHA, RNase H1, and γ-H2AX. Results: LDHA knockout disrupted R-loop homeostasis, resulting in altered S9.6-enriched peaks, with 199 peaks increased and 81 decreased in 4T1 cells. Exogenous lactate failed to rescue these R-loop perturbations, suggesting that LDHA regulates R-loops through a mechanism beyond its metabolic activity. Genomic analysis revealed partial overlap between LDHA-binding sites and R-loop-prone regions, indicating a potential direct role of LDHA in R-loop formation. Transcriptomic profiling showed widespread gene expression changes following LDHA loss, although only four genes were associated with both LDHA binding and R-loop peaks. Importantly, LDHA knockout induced accumulation of R-loop foci and DNA damage, both of which were reversed by RNase H1 expression. Discussion: These findings identify LDHA as a novel regulator of R-loops and suggest that it may influence R-loop homeostasis through direct DNA binding rather than solely through its enzymatic role in glycolysis. Further studies are needed to elucidate the molecular mechanism underlying LDHA-mediated R-loop regulation.