The lncRNA HOTAIR is a well-known scaffold for two key epigenetic regulators: the PRC2 member EZH2, responsible of the main repressive H3K27 chromatin mark, and LSD1, able to selectively catalyze the removal of mono- and dimethylated groups from H3K4 and H3K9 (with H3K4 trimethylation generally associated with gene activation and H3K9 dimethylation with gene repression). LSD1 is also responsible of the transcriptional mark H3K36 trimethylation.
HOTAIR acts as an oncogene, overexpressed in several epithelial cancers and strongly correlated to invasion. Furthermore, this lncRNA exerts a key role in the Epithelial-to-Mesenchymal transition (EMT), a transdifferentiation process that is required to metastasis of tumor cells, by allowing migration and invasion. Our recent evidence, indeed, demonstrated that the repressive role of the "master" transcriptional factor Snail, sufficient to induce and maintain EMT, requires the assembly of a HOTAIR/Snail/EZH2 complex on Snail binding sites on the promoters of epithelial genes.
LSD1 function has been previously correlated to EMT regulation. However, while a role in epithelial gene repression has been attributed to this chromatin modifier, its possible function in mesenchymal gene induction is still unexplored. Poorly understood are also whether and how LSD1 contributes to HOTAIR function.
Building on this body of evidence, this project aims to: i) the study of the role of HOTAIR and LSD1 in mesenchymal genes activation in EMT; ii) the identification of LSD1/HOTAIR partners by proteomics approaches and the study of their mechanism of action, specifically focusing on positive regulators of gene expression.
These goals will further clarify the regulatory circuitries in which HOTAIR participates; they will also contribute to the knowledge of how the deregulation of this lncRNA impacts on cancer metastasis.
The proposal builds on literature data and our previous results that characterized the role of Snail/HOTAIR/EZH2 complex in EMT and the transcriptional regulation of HOTAIR in epithelial cells [13, 15]. Moreover, our hypothesis is corroborated by further unpublished data of the group that attribute to HOTAIR a function in gene expression activation in epithelial cells transdifferentiation.
The functional role of LSD1/HOTAIR complex is poorly explored and current evidence limited to a cooperation between LSD1 and EZH2 in controlling gene repression. We here propose the innovative investigation of the LSD1-mediated role in mesenchymal gene activation and the study of the requirement of HOTAIR for the regulation of the same genes, as a scaffold for this key chromatin modifier.
The mechanisms that, in both physiological and/or pathological EMT cell reprogramming, confer specificity to LSD1 recruitment to specific genes are still largely unknown. It seems likely that LSD1 can be recruited at each gene through combinatorial interactions with specific factors, imparting sequence-specific DNA binding. In this scenario, an attractive hypothesis is that transcriptional factors, able to bind both ncRNA molecule platforms and specific DNA sequences, could be implicated (as previously demonstrated for the Snail/HOTAIR/Ezh2 complex [15]).
Moreover, we propose the identification by proteomic approaches of LSD1/HOTAIR partners and the study of their mechanism of action hold promise in the further clarification of the regulatory circuitries in which HOTAIR participates.
The proposed goals will also contribute to the knowledge of how the deregulation of this lncRNA impacts on cancer. Furthermore, these challenges pave the way for the possible design, in future, of innovative lncRNA-based strategies to counteract LSD1 specific targeting in EMT.
Both aims hold promise of success being in line with previous researches [15, 31]; moreover, with respect to the feasibility, the proponent has a large experience in the characterization of molecular events underlying the EMT/MET dynamics of the epithelial cells, in physiological as well as pathological conditions, and this is highlighted by the relative and highly cited publications. Notably, she designed the research plan, coordinated the experimental work, interpreted the results and wrote the recent manuscripts, on which large part of the proposed research builds, describing the role of HOTAIR in EMT (Battistelli et al., Oncogene 2017, IF 2018 6,6; 78 Citations by Scopus, 8 June 2020) and the topological regulation of its expression (Battistelli et al, Celle Death and Differentiation 2019, IF 2018 8,1, 26 Citations by Scopus, 8 June 2020). Furthermore, the proponent set up all the innovative protocols used in these works to in vivo study of RNA/DNA/protein interactions in the chromatin context.
We also believe that the expertise of the rearch group in cellular and molecular biology will expedite successful advances in the proposed task.
All the techniques included in the experimental procedure are currently in use in the laboratory. The here proposed proteomic protocol has been successfully used by the group to explore RNA-binding proteins in the exosome machinery ([33] and unpublished results collected in a manuscript in preparation). With respect to proteomic studies, they will rely to the external collaboration with Dr. Claudia Montaldo (Proteomic Unit, National Institute for Infectious Diseases L. Spallanzani, IRCCS, Rome, Italy).