Thèse Compréhension Mécanistique de l'Impact de la Citrullination sur la Régulation Transcriptionnelle et la Progression Oncogénique du Cancer du Sein H/F

Doctorat.Gouv.Fr

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 : 2026-05-20T23:59:59 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 désiminases (PADI). La citrullination augmente l'hydrophobicité et modifie la conformation des protéines, ce qui peut potentiellement affecter la formation de liaisons hydrogène, la structure protéique, les interactions protéine-protéine et les interactions protéine-acide nucléique (1-4). Il a été démontré qu'elle influence les interactions histone-ADN, l'expression génique et l'agrégation protéique (1,5,6). Compte tenu de l'importance de ces interactions pour la régulation physiologique essentielle, la citrullination pourrait moduler de manière spécifique plusieurs processus cellulaires. Parmi les membres de cette famille, la PADI2 est la plus fréquemment surexprimée dans plusieurs tumeurs, notamment le cancer du sein (2,9,11). Nos travaux récents mettent en lumière la citrullination de l'arginine 1810 (Cit1810) de l'ARN polymérase II, catalysée par PADI2, comme un acteur clé de la transcription et de la prolifération des cellules cancéreuses du sein (14,15). L'expression excessive de PADI2 étant associée à un mauvais pronostic chez les patientes atteintes d'un cancer du sein, et les hormones stéroïdiennes et leurs récepteurs jouant un rôle important dans la progression de ces cancers, nous avons émis l'hypothèse que PADI2 intervient dans la modulation de la transcription induite par les hormones dans les cellules cancéreuses du sein. Nos données non publiées montrent également que PADI2 affecte principalement la transcription régulée par les hormones stéroïdiennes dans ces cellules. Par conséquent, nous proposons de développer un projet multidisciplinaire pour un(e) candidat(e) potentiel(le) au doctorat afin d'étudier le mécanisme moléculaire par lequel PADI2 influence la régulation de la transcription et l'organisation de la chromatine 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 an 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 are 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 mechanism by which PADI2 regulates transcription regulation and chromatin organization in response to a steroid hormone. Therefore, we proposed to analyze the PADI2 interaction with protein chromatin landscape proteins in cells using Rapid immunoprecipitation mass spectrometry of endogenous proteins (RIME, 18) experiments. Indeed, this raises an intriguing question in the field to investigate the molecular mechanism by which PADI2-interacting proteins could tether with the genome architectural proteins to fine-tune the gene regulation in breast cancer cells. Therefore, it is critical to validate PADI2 interaction with the essential architectural proteins to get mechanistic insights regarding the profound impact of PADI2 on the transcriptional landscape. Also, we will get the preclinical data on the best identified PADI2 inhibitor with patient samples. We expect this work to unveil key regulatory mechanistic insights on PADI2 in transcriptional landscapes and chromatin organization in breast cancer cells through an impact on progesterone-induced chromatin organization. Furthermore, we planned to confirm our fundamental findings by using immunostaining on the patients' samples in our ongoing collaboration with Oncopole, Toulouse. 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 molecular mechanism by which PADI2 affects chromatin organization and transcription regulation under progesterone hormone regulation in breast cancer cells. The proposed project objective comprises 3 main aims.

- Aim 1. Characterization of PADI2 interaction with chromatin-associated proteins (CAPs) in T47D breast cancer cells.

- Aim 2. To confirm the results obtained in Aim 1, an engineered breast cancer cell line will be generated using the CRISPR-Cas9 system by targeting specific CAPs to define the chromatin organization and transcription regulatory hub.

- Aim 3. Preclinical data of PADI2-dependent identified the CAPs regulation in breast cancer patients.


Unraveling the mechanistic insight into the PADI2 function on transcription regulation and chromatin organization under progesterone hormone regulation in breast cancer cells. The potential candidate will use the following methods to achieve each of the aims.

- Aim 1. Characterization of PADI2 interaction with chromatin-associated proteins (CAPs) in T47D breast cancer cells prior and after exposure to progestin in T47D breast cancer cells.

- Aim 2. To validate the results obtained in Aim 1, an engineered breast cancer cell line will be generated using the CRISPR-Cas9 system by targeting specific CAPs to define the chromatin organization and transcription regulatory hub

- Aim 3. Preclinical data of PADI2-dependent identified the CAPs regulation in breast cancer patients.