Accumulation of senescent cells is an important contributor to chronic inflammation upon aging. The inflammatory phenotype of senescent cells was previously shown to be driven by cytoplasmic DNA. Here, we propose that cytoplasmic double-stranded RNA has a similar effect. We find that several cell types driven into senescence by different routes share an accumulation of long promoter RNAs and 3′ gene extensions rich in retrotransposon sequences. Accordingly, these cells display increased expression of genes involved in response to double stranded RNA of viral origin downstream of the interferon pathway. The RNA accumulation is associated with evidence of reduced RNA turnover, including in some cases, reduced expression of RNA exosome subunits. Reciprocally, depletion of RNA exosome subunit EXOSC3 accelerated expression of multiple senescence markers. A senescence-like RNA accumulation was also observed in cells exposed to oxidative stress, an important trigger of cellular senescence. Altogether, we propose that in a subset of senescent cells, repeat-containing transcripts stabilized by oxidative stress or reduced RNA exosome activity participate in driving and maintaining the permanent inflammatory state characterizing cellular senescence.

DNA methylation (meDNA) is a modulator of alternative splicing, and splicing perturbations are involved in tumorigenesis nearly as frequently as DNA mutations. However, the impact of meDNA on tumorigenesis via splicing-mediated mechanisms has not been thoroughly explored. Here, we found that HCT116 colon carcinoma cells inactivated for the DNA methylases DNMT1/3b undergo a partial epithelial to mesenchymal transition associated with increased CD44 variant exon skipping. These skipping events are directly mediated by the loss of intragenic meDNA and the chromatin factors MBD1/2/3 and HP1γ and are also linked to phosphorylation changes in elongating RNA polymerase II. The role of meDNA in alternative splicing was confirmed by using the dCas9/DNMT3b tool. We further tested whether the meDNA level could have predictive value in the MCF10A model for breast cancer progression and in patients with acute lymphoblastic leukemia (B ALL). We found that a small number of differentially spliced genes, mostly involved in splicing and signal transduction, are correlated with the local modulation of meDNA. Our observations suggest that, although DNA methylation has multiple avenues to affect alternative splicing, its indirect effect may also be mediated through alternative splicing isoforms of these meDNA sensors.

Promoters and enhancers are sites of transcription initiation (TSSs) and carry specific histone modifications, including H3K4me1, H3K4me3, and H3K27ac. Yet, the principles governing the boundaries of such regulatory elements are still poorly characterized. Alu elements are good candidates for a boundary function, being highly abundant in gene-rich regions, while essentially excluded from regulatory elements. Here, we show that the interval ranging from TSS to first upstream Alu, accommodates all H3K4me3 and most H3K27ac marks, while excluding DNA methylation. Remarkably, the average length of these intervals greatly varies in-between tissues, being longer in stem- and shorter in immune-cells. The very shortest TSS-to-first-Alu intervals were observed at promoters active in T-cells, particularly at immune genes, where first-Alus were traversed by RNA polymerase II transcription, while accumulating H3K4me1 signal. Finally, DNA methylation at first-Alus was found to evolve with age, regressing from young to middle-aged, then recovering later in life. Thus, the first-Alus upstream of TSSs appear as dynamic boundaries marking the transition from DNA methylation to active histone modifications at regulatory elements, while also participating in the recording of immune gene transcriptional events by positioning H3K4me1-modified nucleosomes.

As an important immune stimulator and modulator, IFNγ is crucial for gut homeostasis and its dysregulation links to diverse colon pathologies, such as colitis and colorectal cancer (CRC). Here, we demonstrated that the epigenetic regulator, CBX3 (also known as HP1γ) antagonizes IFNγ signaling in the colon epithelium by transcriptionally repressing two critical IFNγ-responsive genes: STAT1 and CD274 (encoding Programmed death-ligand 1, PD-L1). Accordingly, CBX3 deletion resulted in chronic mouse colon inflammation, accompanied by upregulated STAT1 and CD274 expressions. Chromatin immunoprecipitation indicated that CBX3 tethers to STAT1 and CD274 promoters to inhibit their expression. Reversely, IFNγ significantly reduces CBX3 binding to these promoters and primes gene expression. This antagonist effect between CBX3 and IFNγ on STAT1/PD-L1 expression was also observed in CRC. Strikingly, CBX3 deletion heightened CRC cells sensitivity to IFNγ, which ultimately enhanced their chemosensitivity under IFNγ stimulation in vitro with CRC cells and in vivo with a syngeneic mouse tumor model. Overall, this work reveals that by negatively tuning IFNγ-stimulated immune genes’ transcription, CBX3 participates in modulating colon inflammatory response and CRC chemo-resistance.

Influenza virus transcription is catalyzed by the viral RNA-polymerase (FluPol) through a cap-snatching activity. The snatching of the cap of cellular mRNA by FluPol is preceded by its binding to the flexible C-terminal domain (CTD) of the RPB1 subunit of RNA-polymerase II (Pol II). To better understand how FluPol brings the 3′-end of the genomic RNAs in close proximity to the host-derived primer, we hypothesized that FluPol may recognize additional Pol II subunits/domains to ensure cap-snatching. Using binary complementation assays between the Pol II and influenza A FluPol subunits and their structural domains, we revealed an interaction between the N-third domain of PB2 and RPB4. This interaction was confirmed by a co-immunoprecipitation assay and was found to occur with the homologous domains of influenza B and C FluPols. The N-half domain of RPB4 was found to be critical in this interaction. Punctual mutants generated at conserved positions between influenza A, B, and C FluPols in the N-third domain of PB2 exhibited strong transcriptional activity defects. These results suggest that FluPol interacts with several domains of Pol II (the CTD to bind Pol II), initiating host transcription and a second transcription on RPB4 to locate FluPol at the proximity of the 5′-end of nascent host mRNA.

HP1α/CBX5 is an epigenetic regulator with a suspected role in multiple sclerosis (MS). Here, using high-depth RNA sequencing on monocytes, we identified a subset of MS patients with reduced CBX5 expression, correlating with progressive stages of the disease and extensive transcriptomic alterations. Examination of rare non-coding RNA species in these patients revealed impaired maturation/degradation of U snRNAs and enhancer RNAs, indicative of reduced activity of the Integrator, a complex with suspected links to increased MS risk. At protein-coding genes, compromised Integrator activity manifested in reduced pre-mRNA splicing efficiency and altered expression of genes regulated by RNA polymerase II pause–release. Inactivation of Cbx5 in the mouse mirrored most of these transcriptional defects and resulted in hypersensitivity to experimental autoimmune encephalomyelitis. Collectively, our observations suggested a major contribution of the Integrator complex in safeguarding against transcriptional anomalies characteristic of MS, with HP1α/CBX5 emerging as an unexpected regulator of this complex’s activity. These findings bring novel insights into the transcriptional aspects of MS and provide potential new criteria for patient stratification.