Histone marks are drivers of the splicing changes necessary for an epithelial-to-mesenchymal transition
Alexandre Segelle, Yaiza Nunez-Alvarez, Andrew J Oldfield, Kimberly M Webb, Philipp Voigt, Reini F Luco
Chromatin and Splicing
Alternative splicing is one of the most general and important biological processes in the eukaryotic cell. It affects more than 90% of human genes, it is essential for protein diversity and any misregulation of the highly tissue-specific alternative splicing programs can lead to disease, such as cancer. However, the mechanisms of cell-specific alternative splicing regulation are still largely unknown.
Unexpectedly, in the past 20 years, chromatin and epigenetic modifications have been shown to play an important role in the regulation of alternative splicing. In particular, we have shown that non-coding RNAs and histone modifications can communicate with the splicing machinery via recruitment of chromatin/splicing-adaptor complexes.
Our objectives now are to better understand the role of chromatin, enhancers and long non-coding RNAs in the onset and maintenance of a cell-specific splicing program, using as an inducible cell reprogramming model system the epithelial-to-mesenchymal transition (EMT), a process involved in early development and cancer metastasis. For that purpose, we will use state-of-the-art deep sequencing approaches (RNA-seq, ChIP-seq, 4C-seq), combined with molecular and cell biology tools (CRISPR/dCas9), to depict the molecular mechanisms of cell-specific alternative splicing regulation in a cancer-relevant model system, the EMT.
The regulation of alternative splicing is a multilayer process in which chromatin (in blue), long non-coding RNAs (in purple) and RNA binding factors (in orange) work in an integrated way to establish the final splicing outcome.
Publications of the team
A cell-to-patient machine learning transfer approach uncovers novel basal-like breast cancer prognostic markers amongst alternative splice variants
Jean-Philippe Villemin, Claudio Lorenzi, Marie-Sarah Cabrillac, Andrew Oldfield, William Ritchie & Reini F. Luco
Splicing-associated chromatin signatures: a combinatorial and position-dependent role for histone marks in splicing definition.
Agirre E, Oldfield AJ, Bellora N, Segelle A, Luco RF
Retrotransposons jump into alternative-splicing regulation via a long noncoding RNA
Luco RF
A lncRNA regulates alternative splicing via establishment of a splicing-specific chromatin signature
Gonzalez I, Munita R, Agirre E, Dittmer TA, Gysling K, Misteli T, Luco RF.
Epigenetics in alternative pre-mRNA splicing
Luco RF, Allo M, Schor IE, Kornblihtt AR, Misteli T.
Regulation of alternative splicing by histone modifications
Luco RF, Pan Q, Tominaga K, Blencowe BJ, Pereira-Smith O, Misteli T.
Thèses et hdr
Thèses soutenues 24/11/2020
A large-scale approach to detect novel regulators of alternative splicing during epithelial-mesenchymal transition.
Defended by Jean-Philippe Villemin on 24/11/2020 under the supervision of Reini Fernandez de luco and William Ritchie
The role of histone modifications in the regulation of alternative splicing during the epithelial-to-mesenchymal transition
Defended by Alexandre Segelle on 28/09/2020 under the supervision de Reini Fernandez de Luco
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Projects
A histone code for alternative splicing
(Eneritz Agirre and Jean-Philippe Villemin)
We have found that histone modifications differentially mark alternatively spliced exons in a combinatorial way. Taking advantage of available genome-wide ChIP-seq and RNA-seq data from the ENCODE and ROADMAP Epigenomics project, we are now identifying the chromatin signatures that differentially mark included and excluded exons, to study what these events have in common in order to depict novel regulatory mechanisms of alternative splicing and improve the current prediction tools by adding to the splicing code the information embedded at the chromatin level.
Histone modifications differentially mark, in a combinatorial way, alternatively spliced exons, which in turn has an impact on the recruitment of the splicing regulators to the pre-mRNA via chromatin-adaptor complexes and/or modulation of RNA Polymerase II elongation rate.
A dynamic role for histone marks in EMT-dependent alternative splicing
(Alexandre Segelle and Jean-Phillipe Villemin)
During EMT, there are well-known changes in alternative splicing that can happen early in the EMT, or later at more final stages of the reprogramming process. Moreover, we have found many of these alternatively spliced genes to be regulated by chromatin modifications, raising the question of what is the dynamic interplay between the two regulatory layers. In this project, we will combine genome-wide epigenomics and transcriptomics data with more classical molecular biology methodologies, to identify and correlate in time the changes in chromatin and lncRNAs with changes in splicing during EMT. As a final proof of the role of these histone marks in EMT-dependent splicing, we will adapt the CRISPR/dCas9 system to edit the epigenome exon-specifically and test the effect on alternative splicing.
By correlating in time (T0, T1, T6, T21 days) the genome-wide changes in alternative splicing, lncRNAs expression levels and histone modification enrichment levels during the establishment and maintenance of a new EMT-specific splicing program, we will identify novel regulators of splicing that will be further studied with more mechanistic approaches.
The role of enhancers in alternative splicing regulation
(Andrew Oldfield)
Recently, alternatively spliced exons have been shown to physically interact with distal regulatory sequences via chromatin looping. However a true functional study addressing the role of these physical interactions in alternative splicing is still missing. We aim to address this question by using conformation capture assays coupled to CHIP-seq to first identify all the splicing events that physically interact with enhancers during EMT and then to modify these enhancers to test the effect on alternative splicing.
Recently, enhancers have been shown to physically interact with alternatively spliced exons, but with no prove of a functional link. We propose that enhancers can target to the regulated exons, chromatin regulators that by protein-protein interaction can favor the recruitment of the splicing factors to the pre-mRNA, modulating like this the final splicing outcome.
