The chromatin remodeler hells regulates transcription-replication conflicts and genome integrity in ALK-negative anaplastic large cell lymphoma Free

Transcription and replication both utilize genomic DNA as a template, creating the potential for frequent interference between the two machineries. Such transcription–replication conflicts (TRCs) can compromise genome stability due to the abnormal accumulation and persistence of R-loops—three-stranded nucleic acid structures comprising a DNA:RNA hybrid and a displaced single-stranded DNA strand (Bhowmick R., et al., Mol Cell. 2023). To safeguard genome integrity, cells deploy multiple R-loop resolution factors that prevent R-loop-associated replication stress (Brickner J. R., et al., Mol Cell. 2022). Persistent R-loops are also associated with the stalling of RNA polymerase II (RNAPII) on chromatin, which further impedes replication fork progression (Zardoni L., et al., NAR 2021).

Our observation of cells depleted of the chromatin remodelerHELLS, which displayed an accumulation of R-loops and stalled RNAPII on highly transcribed genes (Tameni A., Mallia S., et al., NAR 2024) in aggressive anaplastic large cell lymphoma (ALCL), led us to hypothesize that HELLS-dependent R-loop accumulation can induce replication stress and TRCs. To test replication forks progression, we performed DNA fiber analysis by sequentially labeling the cells with two thymidine analogs, 5-chloro-2′-deoxyuridine (CldU, red) and 5-iodo-2′-deoxyuridine (IdU, green). While control cells showed a CIdU/ldU ratio close to 1, indicating the absence of stalled forks, TLBR-2 and MAC2A HELLS-KD cells displayed an CIdU/ldU ratio close to 2, strongly indicating the presence of stalled forks in absence of HELLS. Then, we performed proximity-ligation assays (PLA) for measuring the presence of TRCs. Specifically, we used antibodies against phospho-forms of RNAPII, pSer5-RNAPII and pSer2-RNAPII, corresponding to initiation and elongation transcriptional complexes respectively and PCNA, as key component of DNA replicative machinery. We found that PLA signal between PCNA and pSer5-RNAPII, but not pSer2 RNAPII, increased in HELLS-KD cells compared to control, suggesting that the depletion of HELLS increases the collision between RNAPII and replicative complex at promoter-proximal sites before pause release. Importantly, ectopic expression of RNaseH1 in HELLS-KD cells reduced R-loop-dependent TRCs, confirming the functional involvement of R-loops in this process.

Despite the large number of genes demonstrated or proposed to regulate R-loop homeostasis and, therefore, to impact TRCs and in turn on genome integrity, our knowledge of specific HELLS-regulated factors is still limited. To explore the mechanism contributing to TRCs and thus to genome instability, we performed gene set enrichment analysis focusing on genes belonging to functional categories related to chromatin regulation previously identified as HELLS transcriptional direct genes. Among them, mini chromosome maintenance 5 (MCM5) emerged as a potential candidate. Inducible knockdown of MCM5 significantly reduced cell proliferation, increased TRCs levels in lymphoma cells, as measured by pSer5-RNAPII–PCNA PLA foci, led to forks stall and to enhanced nuclear R-loop accumulation, phenocopying HELLS depletion. Notably, the ectopic overexpression of MCM5 in TLBR-2 HELLS-KDcells fully rescues cell proliferation reducing R-loop formation and the presence of TRCs. Additionally, perturbations observed upon MCM5-KD were dramatically reduced when R-loops were suppressed by overexpression of RNaseH1 indicating that, in absence of MCM5, the persistence of R-loop disrupts the replication fork process through TRCs, mimicking HELLS. These data highligh MCM5 as the downstream target of HELLS in replicative processes.

Integrative analysis of MCM5, and RNAPII chromatin occupancy (ChIP-seq), alongside chromatin accessibility profiles (ATAC-seq) in TLBR-2 cells, further revealed that MCM5 was excluded from accessible, actively transcribed loci, confirming the not-transcriptional role of MCM5.

Collectively, these findings position the axis HELLS/MCM5 as a critical regulator of replicative processes in aggressive ALCL. Disruption of HELLS/MCM5 activity induces aberrant TRCs and R-loop accumulation, ultimately compromising genome integrity and highlighting a potential therapeutic vulnerability in lymphoma cells.