Stress-triggered satellite RNA HSAT3 shapes replication restart and TOP2A localization

Long non-coding RNAs transcribed from pericentromeric Satellite 3 repeats, known as HSAT3, are induced by a range of cellular stressors and assemble into nuclear stress bodies that recruit numerous RNA-binding and regulatory proteins. A new study published in Epigenetics & Chromatin now reports that these transcripts accumulate together with topoisomerase IIα (TOP2A) in nuclear stress bodies, that HSAT3 RNA can associate with TOP2A in biochemical assays, and that depletion of HSAT3 delays replication re-initiation in cultured cells. The work also shows that reduced HSAT3 levels delay tumor growth in xenograft models and that epigenetic compounds capable of suppressing HSAT3 expression enhance cellular sensitivity to the TOP2A inhibitor etoposide.

The research team, led by Julian Kanne and Michal R. Schweiger, built on earlier findings from their own group showing that increased HSAT3 expression confers resistance to etoposide in lung cancer models. TOP2A binds double-stranded DNA and introduces strand breaks to resolve topological problems arising during transcription and replication, making it the direct target of etoposide. How HSAT3 might influence DNA replication dynamics and its mechanistic relationship with TOP2A had remained largely unclear, and the new study was designed to address this gap.

To characterize the interaction spatially, the researchers subjected HeLa cells to heat stress at 44 degrees Celsius for one hour, followed by recovery periods ranging from zero to 24 hours, and combined TOP2A immunofluorescence with HSAT3 RNA fluorescence in situ hybridization. Under non-stress conditions, HSAT3 and TOP2A foci were rarely detected. Immediately after heat stress, both types of foci were induced and showed partial colocalization, which persisted for up to approximately 16 hours of recovery. At later time points of 20 hours or more, HSAT3 foci became less frequent and TOP2A colocalization decreased. The team also observed colocalization of HSAT3 and TOP2A in the non-small cell lung cancer cell line A549 following heat stress at 42 degrees Celsius, although immunofluorescence analysis in HCC827 cells was precluded because those cells detached from coverslips during the heat stress procedure.

To test whether TOP2A directly associates with HSAT3 RNA, the researchers performed in vitro RNA pulldown assays using a biotinylated HSAT3 RNA oligonucleotide consisting of GGAAU repeats, a major component of HSAT3 transcripts. TOP2A was consistently detected in the pulldown fractions from HeLa cell lysates under non-stress, heat stress, and recovery conditions, as well as in lysates from HCC827 and A549 cells under non-stress and heat stress conditions. The RNA-binding protein ALYREF, used as a positive control, was efficiently co-precipitated, consistent with previous reports. The authors note that these findings indicate TOP2A can associate with HSAT3 RNA sequences, while the precise molecular nature of the interaction and its impact on TOP2A enzymatic activity remain to be defined.

Because heat stress induces transient cell cycle arrest at the G1/S and G2/M checkpoints, and nuclear stress body formation has been reported to occur predominantly in late S and early G2 phases, the team next examined whether HSAT3 depletion affects cell cycle progression. Using quantitative imaging-based cell cycle analysis in HeLa cells transfected with control or HSAT3-targeting siRNA, they found that heat stress alone enriched cells in G1 phase, a distribution not markedly altered by HSAT3 knockdown. Etoposide treatment caused accumulation of cells in late S and early G2 phases, consistent with replication-associated checkpoint activation. At 20 micromolar etoposide, HSAT3 depletion led to a reduction of cells in G2 phase and a concomitant increase in G1 phase compared with control siRNA-treated cells, whereas at 5 micromolar only minor changes were observed. HSAT3 signals following heat stress were strongly enriched in late S and early G2/M phases, an induction further enhanced by combined heat stress and etoposide treatment and efficiently suppressed by HSAT3 knockdown.

Given that HSAT3 expression is regulated by BET proteins including BRD4, the researchers tested whether BET inhibition phenocopies HSAT3 depletion. Treatment with the BET inhibitors JQ1 or CPI-203 alone resulted in a predominance of cells in G1 phase, similar to DMSO controls. Importantly, combined treatment of etoposide with either inhibitor shifted the cell population toward G1 and reduced S and G2 fractions compared with etoposide alone. Both inhibitors also markedly reduced HSAT3 foci formation under heat stress conditions, either alone or in combination with etoposide.

The central functional result came from single-molecule DNA fiber analyses in HeLa cells. Replication was monitored by sequential incorporation of IdU and CldU to assess ongoing replication and replication restart, respectively. HSAT3 knockdown alone, as well as treatment with low-dose etoposide at 1 micromolar, did not significantly affect ongoing replication as indicated by IdU track length. However, combined HSAT3 depletion and etoposide treatment resulted in a reduction of track length, suggesting impaired replication progression. Strikingly, HSAT3 knockdown significantly reduced replication re-initiation efficiency, reflected by decreased CldU track length, and this defect was further exacerbated by combined treatment with etoposide, whereas etoposide alone had no significant impact on replication restart. In the assay setup, higher IdU to CldU ratios represent delayed re-initiation. The authors caution that their data do not yet distinguish whether the delayed re-initiation phenotype primarily reflects altered origin firing, impaired fork restart, reduced fork progression, or a broader defect in replication-associated stress recovery.

To assess functional relevance in vivo, the team used a cell line-derived xenograft model in immunodeficient mice injected subcutaneously with HCC827 lung cancer cells carrying stable HSAT3 knockdown or a GFP control. While both groups formed tumors and no difference in proliferation was seen in vitro, HSAT3 knockdown resulted in a marked delay in tumor progression, with tumors in the knockdown group taking significantly longer to reach a volume of 100 cubic millimeters. Notably, after tumors reached that size, the growth curves of the two groups were similar, and quantitative PCR two weeks later showed no difference in HSAT3 expression between groups. Immunohistochemical analysis of five tumors per group revealed no significant differences in Ki67 or cleaved caspase-3 staining. The authors interpret these findings as evidence that HSAT3 contributes to efficient tumor expansion in vivo, consistent with its role in supporting replication re-initiation and cell cycle progression under stress conditions, while noting that future studies will be needed to determine the underlying mechanism.

The study also included a targeted screen of a curated library of 178 epigenetic compounds in HeLa cells, in which cells were pre-treated with individual compounds followed by heat stress to induce HSAT3 expression, with levels quantified by RT-qPCR and high-content RNA-FISH analysis. A subset of compounds suppressed HSAT3 induction upon heat stress, with 11 compounds showing both decreased HSAT3 expression and reduced nuclear stress body formation. Among the most prominent hits were inhibitors targeting BET proteins, consistent with the known role of BRD4 in HSAT3 transcriptional regulation, and compounds affecting chromatin modifiers and cell cycle regulators also reduced HSAT3 levels. Quantitative imaging confirmed that these compounds reduced not only overall HSAT3 transcript levels but also the formation of nuclear stress bodies. Several HDAC inhibitors identified in the screen enhanced etoposide-induced cytotoxicity in proliferation assays, indicating a synergistic interaction. The authors emphasize that these data do not yet establish that all compound effects are mediated specifically through HSAT3, but they support the principle that repression of HSAT3 can be exploited in combination-treatment settings.

Overall, the findings support a functional link between stress-induced repetitive RNAs, nuclear compartmentalization, and replication-associated processes, and provide a framework for understanding how pericentromeric lncRNAs may contribute to genome stability and drug response in cancer cells. The authors highlight several limitations: the molecular nature of the HSAT3-TOP2A association and its impact on TOP2A enzymatic activity remain undefined, further work is required to dissect the relative contributions of origin firing, fork progression, and replication stress signaling to the re-initiation phenotype, and additional studies are needed to determine the specificity and mechanistic basis of the compound screen results. Even so, the study expands the functional landscape of HSAT3 beyond its established role in stress-dependent RNA processing and splicing regulation, positioning stress-induced satellite RNAs as potentially actionable regulators at the interface of chromatin organization, genome maintenance, and cancer therapy.

Subject of Research: The role of the stress-induced lncRNA HSAT3 in TOP2A localization, replication re-initiation, and etoposide response in cancer cells

Article Title: HSAT3 lncRNA regulates replication re-initiation and colocalizes with TOP2A under stress conditions

Article References: Kanne, J., Heß, F., Min, W., Isensee, J., Wasserburger-Zichel, E., Tschernoster, N., Dartenne, L., Hoppe, S., Altmüller, J., Odenthal, M., Hucho, T., Grimm, C., & Schweiger, M. R. (2026). HSAT3 lncRNA regulates replication re-initiation and colocalizes with TOP2A under stress conditions. Epigenetics & Chromatin, 19(1), Article 46. https://doi.org/10.1186/s13072-026-00707-9

Image Credits: AI Generated

DOI: 10.1186/s13072-026-00707-9

Keywords: HSAT3, lncRNA, nuclear stress bodies, TOP2A, replication re-initiation, etoposide, pericentromeric satellite RNA, DNA fiber assay, xenograft, epigenetic compound screen, BET inhibitors, lung cancer