Thèse Impact de la Citrullination sur la Dynamique Transcriptionnelle et la Progression Oncogène du Cancer du Sein H/F

Doctorat_Gouv

Établissement : Université de Toulouse
École doctorale : BSB - Biologie, Santé, Biotechnologies
Laboratoire de recherche : IPBS - Institut de Pharmacologie et Biologie Structurale
Direction de la thèse : Priyanka SHARMA ORCID 0000000338903590
Début de la thèse : 2026-10-01
Date limite de candidature : 2026-11-30T23:59:59

La citrullination des protéines est la modification post-traductionnelle de la peptidyl-arginine en peptidyl-citrulline, catalysée par une famille d'enzymes appelées peptidyl arginine déiminases (PADI). La citrullination augmente l'hydrophobicité et la conformation des protéines, ce qui peut potentiellement affecter la formation de liaisons hydrogène, la structure des protéines, les interactions protéine-protéine et les interactions protéine-acide nucléique (1-4). Il a été démontré que la citrullination influence les interactions histone-ADN, l'expression des gènes et l'agrégation des protéines (1,5,6). Compte tenu de l'importance de ces interactions pour la régulation physiologique essentielle, la citrullination peut moduler de manière unique plusieurs processus cellulaires. Parmi la famille des PADI, PADI2 est le plus fréquemment surexprimée dans plusieurs tumeurs dont le cancer du sein (2,9,11). Nos travaux récents mettent en lumière la citrullination de l'arginine1810 (Cit1810) de l'ARN polymérase II, médiée par PADI2, en tant qu'acteur clé de la transcription et de la prolifération dans les cellules cancéreuses du sein (14,15). Étant donné que la surexpression de PADI2 est associée à un mauvais pronostic chez les patientes atteintes d'un cancer du sein, et que les hormones stéroïdes et leurs récepteurs jouent également un rôle important dans la progression des cancers du sein, nous avons émis l'hypothèse que PADI2 joue un rôle dans la modulation de la transcription induite par les hormones dans les cellules cancéreuses du sein. Nos données, non publiées à ce jour, montrent également que PADI2 affecte principalement la transcription régulée par les hormones stéroïdes dans les cellules cancéreuses du sein. Par conséquent, un projet de doctorat multidisciplinaire est proposé dans notre équipe pour étudier la fonction régulatrice de PADI2 dans la transcription du génome et l'organisation de la chromatine afin de moduler le réseau de régulation du génome associé aux processus de transcription dans le cancer du sein.

Arginine citrullination or deimination is a post-translational conversion of peptidyl-arginine to non-coded peptidyl-citrulline catalyzed by Ca2+ dependent peptidyl arginine deiminase (PADI) enzymes (1-3). Citrullination produces a loss of a positive charge and also increases the hydrophobicity and conformation of proteins, which can potentially affect hydrogen bond formation, protein structure, protein-protein interactions, and protein-nucleic acid interactions (1-4). Given the significance of these interactions for essential cellular functions, citrullination may uniquely modulate critical cellular processes involved in breast cancer progression and drug resistance (1,2). In this regard, citrullination of core histones known to associate with transcription regulation (5,6), DNA damage (7), and pluripotency (8), suggesting the potential link of citrullination to the essential physiological process.

Among the five PADI family members, PADI2 is the most conserved and widely expressed (2,3). Importantly, an elevated level of PADI2 affects several cellular processes and is associated with diverse pathological disorders including autoimmune diseases, neurological disorders, and several cancers including breast and ovarian cancers (2,7-11). PADI2 is broadly found to be associated with cancer progression and with the acquisition of drug resistance (2,12-13). However, the molecular mechanisms by which PADI2-mediated citrullination affects cellular processes at multiple levels and contributes to cancer progression remain poorly understood.

Our recent work spotlights that among PADI family members, PADI2 citrullinates the arginine at position 1810 (R1810) present in the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase 2 (RNAP2) (14, highlighted in 15). The low complexity CTD of RNAP2 is a landing platform for the recruitment of key proteins of the transcription machinery which drive gene regulation (15). In breast cancer cells, we demonstrated that the conversion of R1810 to citrulline (R>Cit1810) at CTD is crucial for RNAP2 to overcome the pausing barrier close to the transcription start site allowing the efficient transcription of highly expressed genes needed for cell cycle progression, metabolism, and cell proliferation (14). We found that the Cit1810 enhances the association of active RNAP2 with the positive transcription elongation factor b (P-TEFb) kinase complex (14). Therefore, Cit1810 enables paused RNAP2 to overcome pausing and facilitates transcription of genes needed for cell proliferation. This pioneering study highlights a mechanism by which PADI2 overexpression might contribute to tumorigenesis.

Among the family, PADI2 is the most frequently overexpressed in primary breast tumors and is upregulated in luminal breast cancer cell lines (2,11). Since PADI2 overexpression is associated with poor prognosis in breast cancer patients, and steroid hormones and their receptors play important role in the progression of breast cancers, we hypothesized that PADI2 plays a role in modulating hormone-induced transcription in breast cancer cells. Indeed, our unpublished data show that PADI2 impairs the transcription of the majority of progesterone-regulated genes (in response to progestin analog R5020) in T47D breast cancer cells. Notably, PADI2 regulated genes affect predominantly involved in chromatin structural organization (unpublished data). This observation suggests the potential function of PADI2 in chromatin-associated transcriptional changes in T47D breast cancer cells in response to progesterone induction.

Considering the regulatory connection between gene expression and genome organization and its functional impact to fine-tune the genome dynamics in cancer progression (16,17), we proposed to investigate the potential function of PADI2 to regulate the genome organization in response to a steroid hormone. Therefore, we proposed to analyze the three-dimensional spatial organization of chromatin in cells using in-situ Hi-C experiments. Indeed, this raises an intriguing question in the field to investigate the molecular mechanism by which PADI2 could tether with the genome architectural proteins to fine-tune the gene regulation in breast cancer cells. Therefore, this is critical to analyze PADI2 interaction with the essential architectural proteins to get mechanistic insights regarding the profound impact of PADI2 on genome dynamics. We expect this work will unveil a key regulatory function of PADI2 in genome organization in breast cancer cells through an impact on progesterone-induced chromatin organization. Furthermore, we planned to confirm our fundamental findings in loss of function experiments, and hence we aim to perform single cell omics in breast cancer cells. We envisage that this knowledge will be useful to design experiments in patient samples to provide better therapeutic strategies.

The main goal of the project is to define the function of PADI2 on chromatin organization and transcription regulation under progesterone hormone regulation in breast cancer cells. The proposed project objective comprises 3 main aims.

-Aim 1. Generation of high-resolution genome-wide topological associated domains contacts maps after specific depletion of PADI2 prior and after exposure progestin in T47D breast cancer cells.

-Aim 2. Characterization of PADI2 interaction with chromatin-associated proteins (CAPs) in T47D breast cancer cells.

-Aim 3. The next objective is to confirm obtained results (from Aim 1 & 2) by applying the loss of function of specific CAPs to define the chromatin organization regulatory hub in breast cancer cells.

Unraveling the function of PADI2 on genome dynamics under progesterone hormone regulation in breast cancer cells. The potential candidate will use the following methods to achieve each of the aims.

-Aim 1. Generation of high-resolution genome-wide contact maps using in situ Hi-C after specific depletion of PADI2 prior and after exposure progestin in T47D breast cancer cells.

-Aim 2. To characterize the PADI2 interaction with chromatin-associated protein complexes, we will adopt biochemical methods including PADI2 specific immunoprecipitation followed by mass spectroscopy assays in T47D breast cancer cells.

-Aim 3. Considering the obtained result from Aim 1 & 2, the next objective to validate basic concepts to define the chromatin organization and expression at single cell level using the single-cell multi-omics assay and RNA sequencing on breast cancer cells.