Hells Promotes Transcription in Lymphoma By Resolving Topological Conflicts and By Preventing DNA Damage

T cell lymphomas (TCLs) are a rare and heterogeneous group of non Hodgkin Lymphoma with aggressive courses and prognoses. TCLs are characterized by genomic instability and massive transcriptional activity that is required to sustain cancer cell proliferation. Indeed, high rate of transcription increases DNA topological tension resulting in accumulation of DNA-RNA hybrid structures called R-Loops, in which the nascent RNA pairs with its DNA-template, favouring structural alterations and DNA-lesions.

DNA helicases are a class of enzyme which major function is to alleviate DNA topological stresses by solving R-Loops and protecting DNA from damage. DNA helicases also facilitate RNA-PolymeraseII (RNAPII) progression along the gene body during transcription thus being the major determinant of gene expression.

We recently identified the DNA-helicase HELLS - a member of the SWI/SNF2 family- as a new genetic vulnerability of TLCs. HELLS orchestrates a transcriptional program essential to the survival and proliferation of TCLs and its genetic ablation profoundly impairs mitosis and cell proliferation.

To understand how HELLS manages and coordinates transcription and to define the HELLS-associated gene expression program in TCLs, we performed RNA-seq in TLBR-2 after the depletion of HELLS (HELLS ^KD). Gene expression profiling showed that HELLS ^KD affects the expression of 728 genes (413 downregulated and 315 upregulated genes). These genes were mainly involved in the regulation of cytoskeleton, cell cycle, cytokinesis, chromatin remodelling and DNA repair as indicated by a gene ontology analysis. Next, to evaluate the distribution of chromatin markers after HELLS ^KD, we performed Chromatin Immunoprecipitation (ChIP) sequencing against active (H3K4me3) and repressive (H3K9me3) histone markers. Depletion of HELLS results in a modest but significant change in the H3K4me3 level at promoters and distal intergenic regions. Differential analysis identified 1,571 bound sites corresponding to 1,278 genes deregulated after HELLS ^KD. Notably, 59/1,278 genes resulted concordantly deregulated in RNA-seq analysis. Accordingly with the role of HELLS in T lymphocytes development, gene ontology analysis on identified 59 genes reveals enrichment in T cell activation, T-helper 17 differentiation and lineage commitment processes. Instead, no significant changes in H3K9me3 level at examined regulatory regions were observed after HELLS ^KD, suggesting that HELLS does not modify the distribution of this marker.

To deeply study the transcriptional function of HELLS, we investigated its ability to promote transcription by solving DNA topological tension. By performing S9.6 antibody staining on a panel of TCL cells, we showed that loss of HELLS leads to an accumulation of R-Loops that co-localized with the active form of RNAPII (S2-CTD), suggesting that HELLS alleviates RNAPII stall upon collision with R-loops during elongation. Co-immunoprecipitation (Co-IP) experiments showed that HELLS interacts with RNAPII and Chromatin Immunoprecipitation (ChIP) assays in TLBR-2 cells demonstrating that the loss of HELLS leads to changes in the distribution of active form of RNAPII that accumulates on transcriptional starting sites of selective target genes. By immunofluorescence staining, we detected that the decreased RNAPII activity after HELLS ^KD was associated with a significant decrease in the incorporation of 5-ethynyl uridine (EU) into nascent RNA confirming that transcription process is attenuated across all transcriptome.

As R-loops accumulation and persistence is strictly associated with DNA damage, we assessed by immunofluorescences, the level of yH2AX (marker of DNA-damage) upon depletion of HELLS. We observed a significant increase in the intensity of nuclear yH2AX signal and a formation of yH2AX foci in TCL HELLS ^KD cell lines. Noticeably, accumulating yH2AX foci were observed in correspondence of R-loops and in co-localization with the active form RNAPII (S2-CTD).

Collectively, our results indicate that HELLS plays an essential function in supporting TCLs progression promoting gene expression by resolving DNA topological conflicts, easing RNAPII progression and protecting DNA from damaging events simultaneously. These key functions qualify HELLS as a new dependency of TCLs and therefore as potential vulnerability of these lymphomas.