The role of chromatin structure in splicing factor function

SCHOR IE; LLÈRES D; ULE J; LAMOND AI; KORNBLIHTT AR

Iguazú

Simposio; Gene Expression and RNA Processing; 2011

ICGEB - ANPCYT - CONICET

Recent studies show that chromatin structure is connected to the exon-intron architecture of genes, including specific nucleosome positioning in exons as well as histone and DNA modifications. Considering a kinetic model of coupling between transcription and splicing, we reasoned that a closed chromatin conformation on exons can help recruitment of splicing factors and help exon definition. We decided to test this model using an imaging approach to analyze distribution of splicing factors in different chromatin conditions. Our previous work shows that depolarization of the membrane potential of neuronal cells triggers an increase in intragenic H3 acetylation along the NCAM gene, which results in skipping of the alternative exon 18 from the mature mRNA. We also detected an increase of total histone acetylation after depolarization treatment, suggesting a more general role of histone acetylation. Now we report that both depolarization and the hyper-acetylating drug trichostatin A (TSA) cause a similar redistribution of SC35 and SF2/ASF splicing factors to nuclear speckles. The same is seen in HeLa cells for several alternative and constitutive splicing factors, suggesting a general effect on splicing rather than on the function of individual factors. Immunofluorescence analysis of endogenous SC35 shows that this splicing factor is depleted from the nucleoplasm after TSA treatment or HP1α knockdown, consistent with less efficient recruitment after relaxation of intragenic chromatin structure. To address this possibility we performed iCLIP of U2AF65-bound transcripts. Preliminary results suggest that indeed a significant proportion of the genes analyzed  have lower U2AF65 binding to the intron-exon junctions.