Epigenetics in alternative splicing : links between chromatin structure, transcription and non-coding RNA mediated regulation

Generalment s'ha pensat que la regulació de l'splicing alternatiu està controlada principalment per la interacció entre els factors reguladors de l'splicing i la taxa d'elongació de la ARN polimerasa II (RNAPII). Hi ha un evidencia emergent de la complexitat de la regulació de l'splicing alternatiu, que ara també inclou l'activitat d'ARNs no codificants i l'estat de la cromatina. Diverses experiments han demostrat que modificacions en les histones poden regular la inclusió d'exons alternatius, i que la taxa d'elongació de la RNAPII pot estar influenciada pels diferents estats de la cromatina. Els ARNs petits (sRNAs) són una família d'ARNs no codificants associats amb membres de la família de proteïnes Argonauta i són efectors de la via de silenciació gènica. Alguns sRNAs participen en una via alternativa anomenada via de silenciació gènica transcripcional (TGS). Evidències experimentals ha mostrat que els sRNAs interferents que s'uneixen a introns poden promoure l'aparició de modificacions en les histones que alteren la taxa de elongació de la transcripció provocant canvis en l'splicing alternatiu. Aquesta via és coneguda com via de silenciació gènica transcripcional acoplada a splicing alternatiu (TGS-AS). Tenint aixó en compte, nosaltres vam proposar que la proteïna Argonauta 1 (AGO1), podria induir la formació d'heterocromatina i canviar l'splicing alternatiu alterant l'elongació de la RNAPII. Per tal de realitzar una análisi a escala genómica de la regulació de l'splicing alternatiu, hem utilitzat dades provinents de noves tècniques de seqüenciació a gran escala, com ChIP-Seq i RNA-Seq. Hem trobat que hi ha regulació d'splicing alternatiu depenent d'AGO1. Els nostres resultats suggereixen que ARNs interferents endógens podrien estar relacionats amb aquesta regulació. A més, a la part final de la tesi demostrem que hi ha un codi de cromatina que requereix AGO1 que regula l'splicing alternatiu i que és específic per diferents tipus cel·lulars. Adicionalment hem trobat que altres efectors, com CTCF i HP1 alpha, també sòn importants per explicar els canvis en l'splicing dels pre-ARNs. Conjunatment amb altres treballs, aquesta tesis demostra que la regulació de l'splicing alternatiu implica la funció de molts components nuclears i probablement de molts altres que encara han de ser descoberts.The regulation of alternative splicing has been generally thought of being primarily controlled by the interaction of splicing factors with the RNA molecule and by the elongation rate of the RNA polymerase II (RNAPII). There is an emerging understanding of the complexity of how alternative splicing is regulated which now involves the activity of non-coding RNAs and the chromatin state. Different experiments have shown that histone modifications can regulate the inclusion of alternative exons and that the elongation rate of the RNAPII could be influenced by different chromatin states. In this sense, small RNAs (sRNAs), which are a family of non-coding RNAs associated with members of the Argonaute family of proteins, that are effectors of the silencing pathway, which can participate in an alternative pathway known as transcriptional gene silencing (TGS). Experimental evidence shows that siRNAs targeting introns can induce chromatin marks that affect the rate of transcriptional elongation, affecting the splicing of pre-mRNAs, which is called transcriptional gene silencing alternative splicing (TGS-AS) \citep{Allo2009}. Thus, we proposed that the Argonaute protein (AGO1) could trigger heterochromatin formation and affect splicing by affecting RNAPII elongation. In order to perform a genome-wide analysis of the regulation of alternative splicing we used new highthroughput sequencing technologies as ChIP-Seq and RNA-Seq. We found that there is AGO1 dependent alternative splicing regulation, and our results suggest that endogenous sRNAs could be involved. Additionally, in the last part of the thesis we show a cell specific alternative splicing chromatin code, which also involves AGO1. Even though AGO1 regulation of alternative splicing was related to some specific cases, we found that other effectors, CTCF and HP1$\alpha$ were also important for the splicing changes decisions. This thesis and other recent reports show the regulation of alternative splicing as an integrated process

Databases and resources for human small non-coding RNAs

Recent advances in high-throughput sequencing have facilitated the genome-wide studies of small non-coding RNAs (sRNAs). Numerous studies have highlighted the role of various classes of sRNAs at different levels of gene regulation and disease. The fast growth of sequence data and the diversity of sRNA species have prompted the need to organise them in annotation databases. There are currently several databases that collect sRNA data. Various tools are provided for access, with special emphasis on the well-characterised family of micro-RNAs. The striking heterogeneity of the new classes of sRNAs and the lack of sufficient functional annotation, however, make integration of these datasets a difficult task. This review describes the currently available databases for human sRNAs that are accessible via the internet, and some of the large datasets for human sRNAs from high-throughput sequencing experiments that are so far only available as supplementary data in publications. Some of the main issues related to the integration and annotation of sRNA datasets are also discussed

Databases and resources for human small non-coding RNAs

Recent advances in high-throughput sequencing have facilitated the genome-wide studies of small non-coding RNAs (sRNAs). Numerous studies have highlighted the role of various classes of sRNAs at different levels of gene regulation and disease. The fast growth of sequence data and the diversity of sRNA species have prompted the need to organise them in annotation databases. There are currently several databases that collect sRNA data. Various tools are provided for access, with special emphasis on the well-characterised family of micro-RNAs. The striking heterogeneity of the new classes of sRNAs and the lack of sufficient functional annotation, however, make integration of these datasets a difficult task. This review describes the currently available databases for human sRNAs that are accessible via the internet, and some of the large datasets for human sRNAs from high-throughput sequencing experiments that are so far only available as supplementary data in publications. Some of the main issues related to the integration and annotation of sRNA datasets are also discussed

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