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.

CD4+ T cells harbor a disproportionate enrichment of immune disease risk loci and represent the primary cellular context for immune disease biology, yet the genes and regulatory programs these variants affect remain largely unknown. We combined targeted Perturb-seq of 1,032 cis -regulatory elements (CREs) overlapping 4,724 variants across 14 immune diseases with genome-wide Perturb-seq of all expressed genes in primary human CD4+ T cells, spanning 4.1 million cells. We identified 626 CRE-gene pairs, and connected CRE targets to downstream regulatory cascades. At the TYK2 and DEXI /CLEC16A loci, we resolved target genes and linked noncoding variants to inflammatory and metabolic programs. Across diseases, we revealed that dispersed variants converged on shared and disease-specific programs. Our work provides a framework for tracing variant-to-CRE-to-gene-to-network in disease-relevant primary cells.

The influenza A virus hijacks host cell machinery by “snatching” molecular caps from cellular RNAs to prime its own transcription. While this process is typically associated with the beginning of genes, this study reveals that the viral RNA polymerase exerts a far-reaching influence on host gene expression. Using genome-wide mapping and comparative pathway analyses, we show that viral polymerase (FluPol) recruitment is decoupled from general host transcriptional activity; instead, FluPol is markedly enriched at a distinct subset of promoters and enhancers associated with defense-related pathways. Beyond the initiation site, FluPol remains associated with host transcripts throughout elongation, extending into downstream-of-gene (DoG) regions. It further accumulates at gene ends, potentially contributing to widespread defects in transcription termination. By remaining linked to host transcripts across the entire gene body, FluPol could extend its opportunity for cap-snatching and impair the recycling of cellular transcription machinery. Together, these findings demonstrate how FluPol accumulation across key cellular defense loci spans from initiation through to termination, driving host transcriptional attenuation.

The influenza A virus (IAV) RNA polymerase (FluPol) primes the viral transcription by using capped 5 prime-ends snatched from host nascent RNAs. However, the exact localization of the FluPol on the genome or the timing of its snatching activity remains poorly characterized. Here, we have monitored IAV infection from the perspective of the FluPol interaction with the host chromatin template. Quantification of chromatin-bound RNAs shows a significant perturbation in host transcription that correlates with a relocalization of RNA polymerase II (RNAPII) from the gene bodies to downstream intergenic regions. This extended transcription leading to the production of RNA downstream of genes (DoGs) was previously linked to the NS1-mediated inhibition of the transcriptional termination. However, immunoprecipitation of FluPol-bound RNA in the chromatin fraction revealed that FluPol remains linked to nascent host transcripts during phases of transcriptional elongation and termination, thereby extending the window of opportunity in which cap-snatching may occur. In addition, chromatin-associated FluPol was enriched at transcription termination sites, suggesting that it may participate in the virus-induced termination defects. Finally, we observed that, rather than targeting just highly expressed genes, the FluPol was preferentially recruited to promoters activated by the viral infection and enhancers. Together, these observations suggest the FluPol uses the early immune response to target regulatory elements of defense-related genes at which it interferes with the fate of the transcripts, and possibly also limits RNAPII re-initiation by impairing the termination process.