Abstract
BackgroundAlternative splicing is primarily controlled by the activity of splicing factors and by the elongation of the RNA polymerase II (RNAPII). Recent experiments have suggested a new complex network of splicing regulation involving chromatin, transcription and multiple protein factors. In particular, the CCCTC-binding factor (CTCF), the Argonaute protein AGO1, and members of the heterochromatin protein 1 (HP1) family have been implicated in the regulation of splicing associated with chromatin and the elongation of RNAPII. These results raise the question of whether these proteins may associate at the chromatin level to modulate alternative splicing.ResultsUsing chromatin immunoprecipitation sequencing (ChIP-Seq) data for CTCF, AGO1, HP1α, H3K27me3, H3K9me2, H3K36me3, RNAPII, total H3 and 5metC and alternative splicing arrays from two cell lines, we have analyzed the combinatorial code of their binding to chromatin in relation to the alternative splicing patterns between two cell lines, MCF7 and MCF10. Using Machine Learning techniques, we identified the changes in chromatin signals that are most significantly associated with splicing regulation between these two cell lines. Moreover, we have built a map of the chromatin signals on the pre-mRNA, that is, a chromatin-based RNA-map, which can explain 606 (68.55%) of the regulated events between MCF7 and MCF10. This chromatin code involves the presence of HP1α, CTCF, AGO1, RNAPII and histone marks around regulated exons and can differentiate patterns of skipping and inclusion. Additionally, we found a significant association of HP1α and CTCF activities around the regulated exons and a putative DNA binding site for HP1α.ConclusionsOur results show that a considerable number of alternative splicing events could have a chromatin-dependent regulation involving the association of HP1α and CTCF near regulated exons. Additionally, we find further evidence for the involvement of HP1α and AGO1 in chromatin-related splicing regulation.Electronic supplementary materialThe online version of this article (doi:10.1186/s12915-015-0141-5) contains supplementary material, which is available to authorized users.
Highlights
Alternative splicing is primarily controlled by the activity of splicing factors and by the elongation of the RNA polymerase II (RNAPII)
In this work, we have derived a chromatin code for splicing that involves binding signals for HP1α and CCCTC-binding factor (CTCF), as well as argonaute 1 protein (AGO1), RNAPII and histone marks, activity around regulated exons
Feature selection and crossvalidation shows that this regulatory code is predictive for nearly 70% of the alternative splicing events regulated between two cell lines, MCF7 and MCF10, providing further evidence for a role of chromatin in the regulation of alternative splicing
Summary
Alternative splicing is primarily controlled by the activity of splicing factors and by the elongation of the RNA polymerase II (RNAPII). The CCCTC-binding factor (CTCF), the Argonaute protein AGO1, and members of the heterochromatin protein 1 (HP1) family have been implicated in the regulation of splicing associated with chromatin and the elongation of RNAPII. The main function of Argonaute proteins is traditionally described to be performed in the cytoplasm in relation to the post-transcriptional gene silencing mechanism [29], there is increasing evidence for a nuclear role [27,28,30,31,32,33,34,35,36,37] In this regard, we have recently shown by ChIP-Seq that AGO1 binds to active transcriptional enhancers in mammalian cells and that through this binding it regulates the constitutive and alternative splicing of neighboring genes [37]. These results raise two interesting questions: whether the CTCF, AGO1 and HP1 proteins associate together at the chromatin level in human cells, and whether these associations play any role in alternative splicing regulation
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