Gene transactivation is coupled to DNA double-strand (DSB) formation on local chromatin. While emerging evidence indicates that these topoisomerase II (TOP2)-dependent DSBs underlie full-blown transcriptional output, it remains unclear how transcription-induced DSBs are regulated, and how perturbing the temporal dynamics of local DSB formation and processing affects maintenance of gene expression programmes and chromosome stability.

ARIP4 (RAD54L2) was recently identified as a component of TOP2-protein complexes(1-3). ARIP4 resolves transcription intermediates ie R-loops, promotes TOP2-dependent DSB formation at transcription start sites (TSSs), and enforces hormone-induced gene transactivation(3). Despite the working hypothesis wherein ARIP4 licenses transcription-associated DSBs to facilitate gene induction, it is not known how ARIP4 is dynamically assembled on transcribed chromatin, and how ARIP4 dosage imbalance may perturb transcription-associated DSB formation and processing, a premise that may lead to and explain how chromosomal translocations are most prevalent on transcribed chromatin.

Built on our ongoing efforts to uncover broader roles of ARIP4 in gene transactivation, we found that ARIP4 exists as biomolecular condensates, and exhibits liquid-liquid phase separation (LLPS) properties in manners that relies on its intrinsically-disorder C-terminal region. ARIP4 assembles readily at gene promoter regions, and upon gene induction, colocalises with transcriptional and epigenetic regulator Brd4. Further analysis of the ARIP4 interactome also uncovered that ARIP4 interacts with PARP1 and enforces gene transactivation in a PARP1-dependent manner. Together, these observations suggest that ARIP4 plays an early role in transcription induction, and that this may be accomplished in part via its ability to undergo LLPS to assemble at TSSs.

To appreciate how ARIP4 coordinates TOP2-dependent DSB formation and transcription induction, we will define the molecular determinants that promote its LLPS properties and accumulation at TSSs, and will study its choreographic assembly at gene promoters for full-blown gene transactivation. We will determine how forced expression of ARIP4 effects transcription-associated DSBs and chromosomal translocations, and will study the epistastic relationship of ARIP4 and PARP1 in transcription regulation. Finally, we will examine how ARIP4-dependent resolution of transcription-associated R-loops underlies TOP2 dynamics and sensitivity to TOP2-trapping chemotherapeutics.

Our proposed experimentations will advance current understanding of R-loop resolution in transcription regulation, and will provide a framework to further decipher how TOP2-dependent DSBs are tightly regulated to suppress chromosome translocations. That ARIP4 is over-expressed in prostate cancers and is important in androgen-dependent gene expression also raise the exciting possibility that targeting the ARIP4 helicase may represent a feasible therapeutic intervention for the treatment of ARIP4-dysregulated human diseases.