Chromatin and RNA splicing

BACKGROUND

Alternative splicing is a highly regulated RNA process used by the cell to increase its protein diversity despite a limited coding genome. By generating different mature mRNAs from the same transcribed gene, the cell can easily gain new functions that will impact its phenotype. For instance, changes in alternative splicing are necessary for the sex determination in flies, for light and temperature signalling in plants, and for brain and muscular development in mammals. However, its dysregulation can also lead to disease, as in the case of cancer cells that have taken advantage of alternative splicing to gain new phenotypic traits essential for their adaptation to the new tumour microenvironment and dissemination into metastatic sites (Figure 1).

Our aim is to understand the mechanisms orchestrating this cancer-specific splicing reprogramming by looking into an unsuspected novel regulatory layer: the chromatin.

In the early 2000s, it was shown that contrary to well-established assumptions, splicing is rather a co-transcriptional process, in which transcriptional regulators, chromatin binding proteins, histone and DNA methylation marks, nucleosome positioning play a role in the final splicing outcome by regulating splicing factors recruitment to the pre-mRNA (Figure 2). 

However the molecular mechanisms linking chromatin to the splicing machinery, nor the physiological impact of this novel regulatory layer have remained poorly understood.

Combining state-of-the-art genome-wide approaches (like RNA-seq, ChIP-seq, ATAC-seq, microC) with gene-targeting approaches based on innovative CRISPR editing tools (dCasRx for RNA splicing and dCas9 for epigenomic editing), we are studying the mechanisms of chromatin-mediated splicing regulation to assess their impact in splicing dynamics during the epithelial-to-mesenchymal transition (EMT), a cell reprogramming involved in tumour cell dissemination and resistance to treatment.

Our team has already identified a new role for H3K27 marks in driving the changes in alternative splicing necessary for the gain in cell motility and invasiveness characteristic of EMT (Figure 3).

We want now to fully understand how these marks regulate splicing and what chromatin-mark exons have in common.

For this we are:

• Developing novel computational tools to cluster alternatively spliced exons dependent on shared chromatin regulators to better understand the role of chromatin in splicing regulation. 
• Addressing the role of 3D chromatin organization in splicing regulation and coordination of functionally-related genes.
• Using CRISPR genomic screens to identify novel regulators of alternative splicing, such as R-loop binders.
• Setting up innovative antibody-based proximity labelling approaches to identify the chromatin proteins connecting H3K27ac to the splicing machinery.
• Address the role of G4 quadruplexes in alternative splicing regulation during EMT.
• Address the role of PTMs in splicing factors function.
• Using single cell approaches to identify a splicing signature characteristic of pro-invasive cells.

Results from these projects will open new perspectives in alternative splicing regulation and its role in tumour progression and invasiveness for the development of better targeting therapies.