Biomedical Center, Molecular Biology Department

Project description

Background & Rationale: R-loops (RNA:DNA hybrids) arise during transcription and, when persistent, stall replication forks, provoke transcription–replication conflicts, and trigger DNA damage responses. How chromatin-coupled signaling tunes R-loop helicases remains unclear. Our work in Drosophila has unveiled a chromatin-associated axis comprising the kinase JIL-1, the chromatin reader JASPer, and the DEAD-box helicase MLE. Depleting MLE, JIL-1 or JASPer increases R-loops and DNA damage (notably in S phase), and the C-terminal G-box of MLE is phosphorylated by JIL-1 and is required for efficient R-loop resolution in vitro.

Central hypothesis: Phosphorylation modulates MLE activity at active genes to prevent transcription–replication conflicts and safeguard genome integrity.

Objectives: (1) Define how MLE’s helicase/R-loop–resolution activities maintain genome stability in Drosophila cells. (2) Determine how phosphorylation by JIL-1 and DNA-damage kinases (ATM/ATR) regulates MLE function. (3) Develop single-molecule R-loop footprinting combined with Oxford Nanopore long-read sequencing and integrate R-loop landscapes with chromatin features (e.g., H3K36me3, H3K79me2, JIL-1/JASPer).

Approach & Methods:

• Cells & genetics: Create an MLE allelic series using CRISPR/Cas9 (null, N-terminal dsRNA-binding domain deletions, C-terminal G-box deletion); establish inducible rescue lines. Quantify R-loops by RNase H–controlled assays (DRIP-qPCR) and DNA damage via γH2Av; profile cell cycle and induce R-loops/replication stress with short CPT/DRB treatments.
• Biochemistry: Express/purify wild-type and phospho-variant MLE. Compare JIL-1– versus ATM/ATR-driven phosphorylation effects on ATPase/helicase activities using phosphomimetic and non-phosphorylatable mutants (collaboration with the Sebesta group, Brno).
• Genomics: Integrate R-loop maps with MNase–ChIP-seq/CUT&RUN features. Build targeted single-molecule R-loop footprinting using methylation footprinting or non-denaturing enzymatic conversion followed by Nanopore sequencing; the compact Drosophila genome (~170 Mb) may enable genome-wide scaling after pilot loci.

Expected outcomes & impact: The project will deliver mechanistic insight into how chromatin signaling (JIL-1 and damage-response kinases) enables an helicase to prevent conflicts at coding regions, alongside reusable single-molecule R-loop datasets in Drosophila. Given the conservation of helicases and R-loop biology, principles are expected to extend to other metazoans.