Evidence for a chromatin code of splicing involving AGO1 and CTCF

NICOLAS BELLORA; ENERITZ AGIRRE; EDUARDO EYRAS; ALBERTO KORNBLIHTT

Long Beach - California

Congreso; Intelligent Systems for Mollecular Biology (ISMB), Alternative Splicing -sig; 2012

INTELLIGENT SYSTEMS FOR MOLLECULAR BIOLOGY (ISMB)

BACKGROUNDThe regulation of alternative splicing has been generally thought of being primarily controlled by the interaction of splicing factors and by the elongation rate of the RNA polymerase II. However, in the last few years a new picture has emerged whereby various mechanisms of regulation are coupled in a network of interactions between RNA, chromatin and protein factors [1,2]. Recent experiments have shown that the argonaute protein AGO1 localizes in the nucleus [3] and can trigger heterochromatin formation and affect splicing by affecting RNAPII elongation [4]. Likewise, the CCCTC-binding factor (CTCF) has been shown to regulate the splicing by affecting the elongation of RNAPII [5]. These results raise the question of whether AGO1 and CTCF could have a coordinated role in the regulation of splicing.RESULTSUsing a novel statistical approach, we model the role of AGO1 and CTCF, together with chromatin marks, in the regulation of splicing, comparing MCF-10A cells and its cancer-derived counterpart, MCF-7. Using a combination of measurable features for histone marks and protein factors, obtained by a systematic analysis of deep-sequencing data, we are able to describe Alternative Splicing changes of relative signal changes at specificlocations around regulated exons. Quantifying the relevance of the data attributes we are able to build a chromatin RNA-map that can explain about 70% of the regulated events. Additionally, we find that a potential association of AGO1 and CTCF in the chromatin-dependent regulation of alternative splicing. We find that both overlap more than expected in intragenic regions, especially in events overlapping internal promoters. Moreover,they bind to similar DNA motifs in exonic and intronic regions and their coordinate relative change between the two cell lines alone can explain 60% of the regulated events.CONCLUSIONSOur results suggest a chromatin code for splicing regulation involving AGO1 and CTCF for approximately 60-70% of the regulated events. Our method has the advantage over previous methods that we can relate the change of the chromatin signal between two conditions to the actual regulation of an exon, thereby providing a better mechanistic hypothesis for splicing regulation. The strong association found between AGO1 and CTCF at internal promoters gives an indication that AGO1 may affect splicing co-transcriptionally in a mechanism dependent of CTCF.REFERENCES1. Luco RF, Allo M, Schor IE, Kornblihtt AR, Misteli T. Epigenetics in alternative pre-mRNA splicing. Cell. 2011 Jan7;144(1):16-26.2. Kornblihtt AR. CTCF: from insulators to alternative splicing regulation. Cell Res. 2012 22(3):450-2.3. Kim, D. H., Villeneuve, L. M., Morris, K. V., and Rossi, J. J. (2006). Argonaute-1 directs siRNA-mediatedtranscriptional gene silencing in human cells. Nat Struct Mol Biol 13, 793-797.4. Alló M, Buggiano V, Fededa JP, Petrillo E, Schor I, de la Mata M, Agirre E, Plass M, Eyras E, Elela SA, Klinck R,Chabot B, Kornblihtt AR. Control of alternative splicing through siRNA-mediated transcriptional gene silencing. NatStruct Mol Biol. 2009 Jul;16(7):717-24.5. Shukla S, Kavak E, Gregory M, Imashimizu M, Shutinoski B, Kashlev M, Oberdoerffer P, Sandberg R, Oberdoerffer S.CTCF-promoted RNA polymerase II pausing links DNA methylation to splicing. Nature. 2011 Nov 3;479(7371):74-9