A chromatin code for alternative splicing involving a putative association between CTCF and HP1alpha proteins

BACKGROUND: Alternative 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. RESULTS:Using chromatin immunoprecipitation sequencing (ChIP-Seq) data for CTCF, AGO1, HP1a, 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 HP1a, CTCF, AGO1, RNAPII and histone marks around regulated exons and can differentiate patterns of skipping and inclusion. Additionally, we found a significant association of HP1a and CTCF activities around the regulated exons and a putative DNA binding site for HP1alpha. CONCLUSIONS:Our results show that a considerable number of alternative splicing events could have a chromatin-dependent regulation involving the association of HP1a and CTCF near regulated exons. Additionally, we find further evidence for the involvement of HP1a and AGO1 in chromatin-related splicing regulation.This work was partly supported by the European Alternative Splicing Network (EURASNET). Additionally, E.A, N.B, A.P. and E.E were supported by grants BIO2011-23920 and CSD2009-00080 from MINECO (Spanish Government) and by a grant from the Sandra Ibarra Foundation for Cancer; A.R.K. was supported by grants from the Agencia Nacional de Promoción de Ciencia y Tecnología of Argentina, the University of Buenos Aires, the Howard Hughes Medical Institute; and M.A. by a short term fellowships from EMBO, the Journal of Cell Science and UICC