A chromatin code for alternative splicing involving a putative association between CTCF and HP1α 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, 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.


CONCLUSIONS
Our 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.
Universitat Pompeu Fabra, E08003, Barcelona, Spain. eneritz.agirre@igh.cnrs.fr.|Present address: Institute of Human Genetics, CNRS UPR 1142, Montpellier, France. eneritz.agirre@igh.cnrs.fr.
Eneritz
E
Agirre
Universitat Pompeu Fabra, E08003, Barcelona, Spain. nbellora@comahue-conicet.gob.ar.|Present address: INIBIOMA, CONICET-UNComahue, Bariloche, Río Negro, Argentina. nbellora@comahue-conicet.gob.ar.
Nicolás
N
Bellora
IFIBYNE-UBA-CONICET, Departamento de Fisiología, Biología Molecular y Celular, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, (C1428EHA), Buenos Aires, Argentina. mariano.allo@embl.de.|Present address: European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117, Heidelberg, Germany. mariano.allo@embl.de.
Mariano
M
Alló
Universitat Pompeu Fabra, E08003, Barcelona, Spain. amadis.pages@upf.edu.|Centre for Genomic Regulation, E08003, Barcelona, Spain. amadis.pages@upf.edu.
Amadís
A
Pagès
IFIBYNE-UBA-CONICET, Departamento de Fisiología, Biología Molecular y Celular, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, (C1428EHA), Buenos Aires, Argentina. bertucci@embl.de.|Present address: European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117, Heidelberg, Germany. bertucci@embl.de.
Paola
P
Bertucci
IFIBYNE-UBA-CONICET, Departamento de Fisiología, Biología Molecular y Celular, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, (C1428EHA), Buenos Aires, Argentina. ark@fbmc.fcen.uba.ar.
Alberto R
AR
Kornblihtt
Universitat Pompeu Fabra, E08003, Barcelona, Spain. eduardo.eyras@upf.edu.|Catalan Institution of Research and Advanced Studies (ICREA), E08010, Barcelona, Spain. eduardo.eyras@upf.edu.
Eduardo
E
Eyras
D017398:Alternative Splicing / Q000235:genetics*; D060565:Argonaute Proteins / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D000076246:CCCTC-Binding Factor; D002460:Cell Line; D002843:Chromatin / Q000378:metabolism*; D000090266:Chromobox Protein Homolog 5; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism*; D039642:Eukaryotic Initiation Factors / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D059372:Nucleotide Motifs / Q000235:genetics; D011485:Protein Binding; D012313:RNA / Q000235:genetics / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism*
C115848:AGO1 protein, human; D060565:Argonaute Proteins; C000715594:CBX5 protein, human; D000076246:CCCTC-Binding Factor; C000619697:CTCF protein, human; D002843:Chromatin; D002868:Chromosomal Proteins, Non-Histone; D039642:Eukaryotic Initiation Factors; D012097:Repressor Proteins; D000090266:Chromobox Protein Homolog 5; D012313:RNA