IEA DREEMI
Dissecting the role of distal regulatory elements in the initiation of the epithelial-to-mesenchymal transition using single-cell sequencing technologiesIEA DREEMI
2024 – 2025
Contact:
French Partner :
Andrew Oldfield
Australian partner:
Pengyi Yang
NEWS
Introduction
Promoter-distal regulatory elements (DREs), such as enhancers, regulate spatio-temporal control of gene expression and are critical determinants of development and disease [1]. Their dysregulation can lead to transcriptome re-wiring and the establishment of detrimental cellular programs, such as cancer [2],[3]. Understanding the mechanisms that lead to enhancer activation during cell-identity changes is thus imperative if we are to prevent such phenomena [4].
To observe the dynamics of enhancer activation and decommission during a cellular identity change, we have chosen to use the epithelial-to-mesenchymal transition (EMT) as my cellular model. This reprogramming process is defined by epithelial cells acquiring a less differentiated, more apoptosis-resistant, migratory and invasive state. The EMT is crucial during embryogenesis, and tumour metastasis [5] and is also a source of cancer stem cells [6], suggesting that understanding mechanisms of cell reprogramming during this transition may be crucial to efficiently treat cancer recurrence and resistance to treatment.
Enhancer reprogramming has recently been shown to be essential for the EMT to take place [7],[8]. Yet, the mechanisms underlying the activation of mesenchymal-specific enhancers during the EMT and how they participate in tumor metastasis remain completely unknown.
Main objectives of research
Concretely, the objectives of this project are (Fig. 1):
– Aim 1: Identification of the EMT regulatory landscape at cell-type specific DREs.
Briefly, using multi-omics approaches, we investigated CTCF and RAD21 (a Cohesin complex subunit) recruitment to our cell-type-specific DREs during the EMT. CTCF and Cohesin are the main actors in eutherian enhancer-promoter (E-P) interactions. In our cells, although CTCF is surprisingly absent at both epithelial and mesenchymal-specific enhancers in either cell type, Cohesin was found to robustly bind cell type specific enhancers, but only when in their active states. This could indicate that M-spe DREs need mesenchymal-specific factors to recruit Cohesin and produce E-P contacts.
Thus, the next step is to identify these DREs’ target genes. For this, I have established a collaboration with Tom Sexton (IGBMC, Strasburg, France), to perform Capture-Hi-C [9] using all E-spe DREs and M-spe DREs as baits at all 3 EMT stages (T0, T1, T7). This will allow us to identify the DREs responsible for regulating EMT-specific gene expression and directly correlate their expression changes with enhancer activity during the EMT.
– Aim 2: Identification and mechanistic dissection of candidate transcription factors.
Briefly, using both genome-wide and locus-specific approaches, we will undertake the validation of our protein candidates, identified thanks to our bioinformatic pipeline, as regulators of cell-type-specific DRE activity during the EMT. Employing state of the art molecular and biochemical approaches, we will also identify their interacting partners and dissect their regulatory kinetics.
Additionally, we will attempt to block EMT initiation by promoting epithelial-specific enhancer activation mechanisms while inhibiting the identified mechanisms of mesenchymal-specific enhancer activation. This should result in the imposition of the epithelial pattern of enhancer activity while staving off the mesenchymal gene expression program.
– Aim 3: Validate our findings in primary breast tumor-derived xenografts and reconstitute gene regulatory networks.
Because MCF10a-ER-SNAIL cells are non-cancerous and have undergone genetic engineering their physiological relevance can be considered limited. To address this issue, the team of C. Ginestier and E.Charafe-Jauffret (CRCM/IPC, Marseille) will investigate whether they can confirm the existence of our mesenchymal-specific ATAC-seq and RNA-seq profiles in subpopulations of primary breast tumor-PDXs.
Expected results
By obtaining direct chromatin accessibility and transcriptomic measurements from the same cell, we will be able to identify different subpopulations from the breast cancer PDXs. Thanks to the identification of M-spe DREs based on bulk ATAC-seq data in our EMT model and to the coupling of each M-spe DRE with a specific target gene (Aim 1), we will compare these ATAC-seq and RNA-seq profiles to those of the various subpopulations identified from the single-cell experiments. We expect that at least one subpopulation of single cells will demonstrate similar chromatin accessibility and gene expression profiles to what we observe at the onset of EMT induction. Furthermore, by reconstructing a global gene regulatory network, we will be able to compare the regulatory function of our cell-type-specific DREs between our cellular model and the in vivo PDXs. These GRNs also have the potential to inform us of the different type of regulatory combinations between enhancers and promoters in the different subsets of cells and therefore at different stages of the EMT process.
This experiment will demonstrate that this specific subset of active enhancers is physiologically relevant, plays an important role in EMT and could be used for downstream treatments focusing on EMT-repression and the inhibition of tumor metastasis.
Institutions and laboratories involved
France
UMR9002, Institut de Génétique Humaine (IGH)
Australia
Charles Perkins Centre – The University of Sydney
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