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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - ESEMT (DETERMINING THE FUNCTIONAL CONSEQUENCES OF TRANSCRIPTIONAL ENHANCER ACTIVATION ON ALTERNATIVE SPLICING DURING EMT)

Teaser

Summary

Alternative splicing is a finely regulated process which is essential to create protein diversity within the cell. Dysregulation of this phenomenon can lead to numerous cellular and developmental problems and contribute to emergence of diseases, such as cancer. Understanding the manner in which alternative splicing is regulated during development and in pathologies is therefore of the utmost importance.
Recent findings have demonstrated that the regulation of alternative splicing is not only orchestrated by the recruitment of specific proteins (splicing factors) at the messenger-RNA level in a co-transcriptionnal manner but also by other biological phenomena which are more closely linked to epigenetic, such as histone post-translational modifications or long non-coding RNAs.
My research project is based on the hypothesis that yet another level of regulation of alternative splicing exists, which is governed by activity modification of transcriptional enhancer elements. Cell-type- and cell-state-specific variation in gene expression patterns are mediated by specialized cis-regulatory elements called enhancers. One of the mechanisms through which enhancers exert their regulatory effect on gene expression is through transcription factor recruitment by chromatin looping. We propose that this differential recruitment of various protein factors and chromatin tridimensional structure remodeling can influence alternative splicing patterns. These enhancer elements would therefore be essential for the establishment and the maintenance of cell-type-specific alternative splicing programs.
With this project, I investigate the role of enhancer activity modification in the regulation of alternative splicing during the Epithelial-to-Mesenchymal Transition (EMT) of human mammary cells. The EMT is an important physiological process which has been implicated in early embryogenesis, wound-healing, establishment of tumor metastasis and cancer recurrence.
We anticipate that this study will provide results with important ramifications pertaining to the understanding of how cancer-specific alternative splicing programs are established and maintained. Furthermore, this work has the potential to discover novel therapeutic targets that would allow us to reverse the EMT process and therefore reduce tumor metastasis and cancer progression.

Work performed

Mapping enhancers undergoing activity changes to EMT-specific alternatively spliced genes.
In order to establish a direct functional link between enhancer activity and AS patterns, we first needed to map both alternatively spliced axons and enhancers undergoing activity changes during the EMT.
We therefore started by determining which axons are alternatively spliced during the EMT at 3 different time points, T0 (untreated cells), T1 (EMT induction for 1 day) and T7 (EMT induction for 7 days). This was done using RNA-seq generated in the laboratory. Using stringent criteria, we have found over 300 skipped or mutually exclusive exons being significantly alternatively spliced during the EMT.
The next step was to map all enhancers with altered activity states during EMT. To do this, we undertook ChIP-seq (of 8 post-translational modifications, 2 proteins) and ATAC-seq experiments as well as collaborated with the Adelman laboratory (Harvard University, USA) in order to observe enhancer transcription. Preliminary analysis of the genome-wide sequencing data shows that over 2000 potential distal regulatory elements were deactivated while over 3500 were activated during the 7 day EMT.
In collaboration with the Sexton lab (IGBMC, Strasbourg, France), we are now currently undertaking a 4C-seq analysis of specific candidate regions in order to physically link the alternatively spliced axons to potential distal regulatory elements whose activity changes during EMT.
Being that, after 4 months, this project is still in its preliminary phase, the results have yet to be disseminated, aside from during internal seminars at the host institution.

Final results

To date, the mechanical workings of the relationship between tridimensional chromatin structure and gene regulation mostly remain a mystery. Our efforts to undertake such a study over the course of a detrimental cellular transition, such as the epithelial-mesenchymal transition puts together novel concepts and pioneering approaches in the field of epigenetics and cancer biology that will bring, conceptually and mechanistically, new insights into alternative splicing regulation and how cancer-specific splicing programs are established and maintained. Although it has previously been shown (for only a few specific examples) that enhancers can modulate alternative splicing regulation, neither the mechanism, nor the genome-wide prevalence of this phenomenon has been demonstrated. Similarly, the role of chromatin tri-dimensional structure in AS regulation is a completely virgin field of study. Long-term, this study has the potential to discover unexpected and innovative new molecular mechanisms of regulation of alternative splicing that can be applied as novel and maybe more efficient therapeutic strategies against tumor metastasis and recurrence.