Chromatin, epigenetics and alternative splicing

IGNACIO E. SCHOR; MARIANO ALLO; ANA FISZBEIN; BERTUCCI P; VALERIA BUGGIANO; LUCIANA GOMEZ ACUÑA; ALBERTO KORNBLIHTT

Granada

Congreso; Second International EURASNET Conference on Alternative Splicing; 2011

EU Network of Excellence on Alternative Splicing (EURASNET)

Chromatin changes affecting alternative splicing can be triggered by physiological signals and cell differentiation. We had reported that membrane depolarization of neuronal cells promotes skipping of exon 18 from the neural cell adhesion molecule (NCAM) by increasing transcription elongation through chromatin opening at NCAM via intragenic H3K9 hyperacetylation. In addition, we observed that depolarization of N2a cells causes general increase in histones H3 and H4 acetylation, and that chromatin relaxation induced both by depolarization or trichostatin A treatment of N2a cells results in redistribution of SR proteins SF2/ASF and SC35, decreasing their localization in the nucleoplasm and concentrating them in the granular compartment. Differentiation of N2a cells into post-mitotic, neurite-containing, cells is associated to an apparently inversed process that involves methylation of H3K9 at the same NCAM intragenic region affected by depolarization, and higher inclusion levels of exon 18. In an attempt to target specific chromatin regions, we found that small interfering RNAs (siRNAs) control alternative splicing (AS) by promoting changes in chromatin structure. When targeting promoter regions, siRNAs trigger transcriptional gene silencing (TGS), by promoting heterochromatin formation. We showed that siRNAs targeting intronic or exonic sequences located close to an alternative exon regulate its splicing. The effect requires RNA:RNA hybridization with endogenous target transcripts, is AGO1-dependent and is counterbalanced by factors favoring chromatin opening or transcriptional elongation. The promotion of heterochromatin marks (H3K9me2 and H3K27me3) at the target site, the need for HP1alpha, and a reduction in pol II processivity suggest a mechanism involving the kinetic coupling, that we called TGS-AS for AS regulated by transcriptional gene silencing. Using ChIp-seq we identified 24,000 target regions for AGO1 in the human genome. AGO1 is enriched in exons and promoters and shows preferential overlapping with H3K9me2, H3K27me3 and H3K36me2 histone marks. Among several candidates we identified an alternative splicing event of the gene encoding SYNE2 (spectrin repeat containing nuclear envelope 2) to be regulated by AGO1 binding at an intronic region that coincides with an internal transcriptional promoter.