An SMN2 splicing-correcting antisense oligonucleotide promotes gene looping

We have recently found that by promoting transcriptional elongation, histone deacetylase inhibitors (HDACi) cooperate with the antisense oligonucleotide nusinersen (ASO1) to upregulate exon 7 (E7) inclusion into SMN2 mRNA (Marasco et al., 2022). In parallel, ASO1 also elicits the deployment of the silencing histone mark H3K9me2 on the SMN2 gene, creating a roadblock to RNAPII elongation that downregulates E7 inclusion. By removing the roadblock, HDACi counteract the undesired chromatin effects of the ASO, resulting in higher E7 inclusion, which allowed us to propose a combined therapy for spinal muscular atrophy (SMA). We show here that the histone methyl transferase G9a is involved in the ASO1-elicited deployment of H3K9me2 not only at the SMN2 E7 region, located towards the 3’ end of the gene, but also at its promoter. Furthermore, using a CRISPR/dCas-based (dead-Cas9-based) strategy, we show that targeting H3K27 histone acetylation at the SMN2 E7 region duplicates the HDACi effect, which overcomes potentially pleiotropic effects. Most noticeably, when acetylation is targeted to the E7 region alone, the 30-kbp-distant promoter becomes acetylated and activated. These cross-talks between the two ends of the SMN2 gene prompted us to use chromosome conformation analysis (3C) to find that ASOs can promote gene looping. This novel property of ASOs depends on cohesin and may explain promoter activation by distant alternative splicing events.