Thèse Étude des Fonctions Spécifiques au Domaine du Récepteur de la Progestérone dans la Dynamique Transcriptionnelle et la Progression 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 : 2027-10-01 Date limite de candidature : 2027-01-24T23:59:59 Les facteurs de transcription (FT) régulent l'expression des gènes par leurs interactions avec les éléments régulateurs de la chromatine et le complexe de transcription, notamment l'ARN polymérase II (ARNPII). Le récepteur de la progestérone (RP) joue un rôle important dans le développement de la glande mammaire et la progression du cancer du sein, tandis qu'une expression réduite du RP est fréquemment associée à des phénotypes agressifs de cancer du sein et à un mauvais pronostic. Malgré des données suggérant le potentiel thérapeutique de la modulation de la signalisation du RP, les mécanismes moléculaires sous-jacents à son activité transcriptionnelle restent mal compris. Si le rôle de la signalisation du RP dans la régulation transcriptionnelle est bien établi, la contribution de ses différents domaines protéiques demeure peu caractérisée. Nos travaux récents ont démontré que le domaine N-terminal intrinsèquement désordonné (NTD) et le domaine structuré de liaison à l'ADN (DBD) contribuent tous deux à la dynamique spatio-temporelle du RP et à la transcription hormono-dépendante dans les cellules cancéreuses du sein. Cependant, la manière dont ces domaines régulent différemment l'organisation de la chromatine et les programmes transcriptionnels reste encore à élucider. Sur la base de nos résultats antérieurs et de données préliminaires, nous formulons l'hypothèse que les domaines N-terminal (NTD) et de liaison à l'ADN (DBD) du récepteur de la progestérone (PR) exercent des fonctions distinctes mais complémentaires dans la coordination de l'organisation de la chromatine et de la dynamique transcriptionnelle, modulant ainsi l'expression des gènes sensibles aux hormones et le comportement cellulaire oncogène. Ce projet de doctorat vise à analyser les contributions spécifiques de chaque domaine des domaines NTD et DBD du PR à l'organisation du génome et à la régulation transcriptionnelle en réponse aux hormones stéroïdiennes. Grâce à des approches complémentaires de biologie moléculaire, cellulaire, génomique et d'imagerie, nous étudierons comment ces domaines influencent les interactions de la chromatine, l'activité des activateurs de transcription, le recrutement des régulateurs de transcription et les programmes d'expression des gènes sensibles aux hormones. À terme, ce travail permettra d'établir les mécanismes moléculaires par lesquels les différents domaines du PR coordonnent les programmes transcriptionnels et contribuent aux phénotypes oncogènes et à la progression du cancer du sein.

Cancer progression is fundamentally driven by the ability of tumor cells to rewire transcriptional programs that sustain proliferation, survival, and adaptation. Emerging evidence shows that this transcriptional plasticity is regulated not only by DNA alterations or transcription factor abundance, but also by the spatial organization of transcriptional machinery (1,2). The spatiotemporal organization of transcription machinery forms multivalent interactions among transcription factors, coactivators, RNA polymerase II (RNAPII), and chromatin-associated regulators, serving as dynamic hubs that concentrate regulatory factors and fine-tune gene expression (3-5). Cancer cells exploit genome dynamics to reinforce oncogenic transcription, promote tumor plasticity, and drive therapy resistance. Understanding how genome organization reshapes transcriptional programs therefore offers a new framework for uncovering mechanisms of tumor aggressiveness and therapeutic vulnerability.

In this context, the progesterone receptor (PR), encoded by the PGR gene, represents an important model for investigating how transcription factors organize and regulate gene expression. PR is a ligand-dependent nuclear transcription factor that mediates progesterone-responsive gene expression and plays essential roles in mammary gland development and breast cancer progression (6). Upon hormone binding, PR undergoes conformational changes that promote receptor activation, dimerization, and engagement with chromatin, ultimately leading to the regulation of hormone-responsive transcriptional programmes. These processes rely on the coordinated activities of distinct PR functional domains and their interactions with transcriptional coregulators and chromatin-associated proteins (CAPs, 6,7). The N-terminal domain (NTD), an intrinsically disordered region containing the transcriptional activation function, provides interaction surfaces for regulatory proteins. In contrast, the DNA-binding domain (DBD) mediates sequence-specific recognition of progesterone response elements, while the ligand-binding domain mediates hormone recognition, contributes to receptor dimerization, and provides the interface for coregulator recruitment (6). Together, these domains enable PR to integrate hormonal signals with the molecular machinery governing transcription.

Importantly, PR-mediated transcriptional regulation extends beyond direct interactions with DNA and involves dynamic changes in chromatin organization and the spatial relationships between genomic regulatory elements (8-11). Chromatin is organized at multiple structural levels, including active and inactive compartments, topologically associating domains, and long-range enhancer-promoter interactions. These architectural features contribute to the coordination of transcriptional programmes by facilitating communication between distal regulatory elements and target gene promoters. In hormone-responsive breast cancer cells, PR activation has been shown to induce changes in chromatin organization and long-range genomic interactions, highlighting the contribution of genome architecture to progesterone-dependent gene regulation. Thus, understanding how PR coordinates its interactions with chromatin and the transcriptional machinery is essential for deciphering the mechanisms underlying hormone-dependent transcriptional regulation.

The importance of PR-mediated transcriptional regulation is particularly relevant in hormone receptor-positive breast cancer, which accounts for approximately 70% of all breast cancer cases and comprises predominantly Luminal A and Luminal B subtypes (12). Although both subtypes are characterized by estrogen receptor (ER)-dependent transcriptional activity, they differ substantially in their molecular profiles and clinical outcomes. Luminal B tumors generally retain high ESR1 (Estrogen Receptor 1) expression, but exhibit reduced PGR (Progesterone Receptor) expression compared with Luminal A tumors. Despite this reduced PGR expression, Luminal B tumors display a more proliferative molecular profile, characterized by elevated expression of proliferation-associated genes, including MKI67 (Marker of Proliferation Ki-67), AURKA (Aurora Kinase A), and CCNB1 (Cyclin B1) (Figure 1A-B, unpublished data, Rabaaoui A et al., submitted). Consistent with this molecular profile, Luminal B tumors exhibit more aggressive clinical behaviour, greater susceptibility to endocrine resistance, and poorer survival outcomes than Luminal A tumors (Figure 1C, unpublished data, Rabaaoui A et al., submitted). These differences underscore the importance of understanding how alterations in transcriptional regulatory mechanisms contribute to breast cancer heterogeneity and progression.

Building on this concept, recent findings from our team (13) provide a new mechanistic perspective by demonstrating that both the intrinsically disordered NTD and the structured DBD contribute to PR transcriptional condensate formation and function. Using single-particle tracking and advanced trajectory analysis, we showed that disruption of either domain alters PR dynamics and is associated with distinct transcriptional programmes and functional consequences for breast cancer cell proliferation and migration (13). These findings extend the conventional understanding of PR function beyond sequence-specific DNA recognition and transcriptional activation, revealing that both structured and intrinsically disordered domains contribute to the dynamic organization of transcriptional regulatory assemblies. Importantly, this work identifies PR domain-dependent condensate organization as an additional layer of transcriptional regulation, providing a new framework for investigating how the molecular properties of transcription factors influence gene expression and breast cancer cell behaviour.

Scientific challenges
Despite the clinical benefits of endocrine therapy for estrogen receptor-positive (ER+) breast cancer, acquired or intrinsic resistance remains a major clinical challenge, with approximately 40% of patients developing resistance after five years of treatment, ultimately contributing to metastatic disease (14,15). The response to endocrine therapy is influenced by complex transcriptional programmes integrating steroid hormone signalling, epigenetic mechanisms, and chromatin organization (15). In particular, steroid hormone receptor-mediated transcription plays a central role in breast cancer progression (6,8-11). As discussed above, Luminal B tumors display more aggressive clinical behaviour and greater endocrine resistance than Luminal A tumors (Figure 1, in attached file). However, the molecular mechanisms underlying these differences, particularly how hormone receptor-dependent transcriptional programmes are established and maintained in these tumors, remain incompletely understood.
A major unresolved question is how alterations in PR abundance and domain-specific functions influence transcriptional regulation and genome organization in breast cancer. We hypothesize that distinct PR domains make complementary contributions to hormone-dependent transcriptional regulation through their differential effects on chromatin engagement, transcriptional complex assembly, and interactions with chromatin-associated proteins. In particular, we propose that the NTD and DBD contribute distinct molecular properties to the organization of transcriptional regulatory assemblies, thereby influencing gene expression and higher-order genome architecture. Although the contribution of individual PR domains to transcriptional activation has been investigated (6), their specific roles in coordinating transcriptional machinery and shaping genome organization remain largely unexplored.
To address this knowledge gap, we have generated engineered MCF7 breast cancer cell lines expressing distinct GFP-tagged PR domain variants and obtained strong preliminary evidence that disruption of individual PR domains significantly alters progestin-induced gene expression programmes (13). These findings suggest that PR domain-specific functions extend beyond conventional transcriptional activation and may contribute to the organization of genomic regulatory elements. Given that three-dimensional interactions between enhancers, promoters, and other regulatory elements are fundamental to transcriptional control, we propose that PR domains may differentially influence the establishment and organization of these interactions.
At the chromatin level, topologically associating domains (TADs) provide structural frameworks that constrain and facilitate interactions between genomic regulatory elements, thereby contributing to the coordination of transcriptional programmes. Hormone-dependent changes in chromatin architecture, including interactions between ER- and PR-mediated signalling pathways, have been implicated in breast cancer progression and metastasis (8-11). Nevertheless, whether distinct PR domains differentially regulate hormone-induced TAD organization and how these effects contribute to transcriptional outputs remain unknown. Furthermore, the molecular interactions between PR domains and chromatin-organizing protein complexes, and their potential contribution to transcriptional plasticity and stem-like properties of breast cancer cells, have not been systematically characterized.
We therefore propose to investigate how distinct PR domains influence progestin-induced genome organization, with particular emphasis on TAD architecture, long-range regulatory interactions, and the recruitment of chromatin-organizing protein complexes. By integrating the characterization of PR domain-dependent transcriptional programmes with genome architecture and molecular interaction analyses, this project will establish how the structural and functional properties of PR domains contribute to hormone-dependent genome regulation. Ultimately, these studies will provide new mechanistic insights into the relationship between PR domain-specific functions, transcriptional plasticity, and breast cancer progression.
1- Project Overview

2.1 - Project position relative to the state of the art and preliminary data
The spatial organization of the genome is a fundamental determinant of transcriptional regulation, enabling coordinated interactions between promoters, enhancers, and other distal regulatory elements. At the three-dimensional level, chromatin is organized into topologically associating domains (TADs), which provide structural frameworks that constrain and facilitate interactions between genomic regulatory regions (8). These domains contribute to the establishment of specific transcriptional programmes by regulating the communication between regulatory elements and their target genes. Importantly, genome organization is not static but undergoes dynamic changes in response to cellular signals, including steroid hormone stimulation. Previous studies have established that hormone receptor activation can reshape chromatin interactions and influence the transcriptional landscape of breast cancer cells (8-11). However, how individual functional domains of steroid hormone receptors contribute to these changes in genome architecture remains poorly understood.
In this context, PR provides an interesting model to investigate the molecular mechanisms connecting transcription factor organization, chromatin architecture, and gene expression. Our previous work demonstrated that the intrinsically disordered NTD and the structured DBD contribute to PR transcription machinery and function (13). Building on these findings, our unpublished preliminary data suggest that these two domains have distinct contributions to progesterone-dependent transcriptional regulation, controlling partially distinct gene regulatory networks associated with breast cancer progression. Notably, our results indicate that PR-NTD- and PR-DBD-dependent activities are not functionally interchangeable, suggesting that individual PR domains may influence the organization of transcriptional regulatory assemblies and, consequently, the genomic regulatory landscape. Given the close relationship between transcriptional regulation and three-dimensional genome organization, we hypothesize that PR domains may differentially coordinate chromatin interactions and the recruitment of chromatin-organizing protein complexes in response to hormonal stimulation.
PR NTD- and DBD-dependent transcriptional regulation orchestrates distinct oncogenic gene regulatory networks driving breast cancer progression
To investigate the distinct contributions of PR functional domains to transcriptional regulation, we generated engineered MCF7 breast cancer cell models in which endogenous PR was replaced with either (i) wild-type PR (WT-PR), (ii) PR lacking the intrinsically disordered N-terminal domain (NTD), or (iii) a DNA-binding-deficient PR mutant carrying key point mutations in the DNA-binding domain (DBD). These models provide a unique experimental system to dissect the individual contributions of PR domains while maintaining a common cellular background. We first performed RNA sequencing in biological triplicates to characterize the transcriptional responses of these models before and after stimulation with the synthetic progestin R5020. Comparative transcriptomic analyses revealed distinct gene expression profiles depending on the integrity of the PR functional domains (Figure 2A, attached file). In WT-PR cells, progestin stimulation preferentially regulated gene programmes associated with cell migration, proliferation, cellular organization, and metabolism. Strikingly, these transcriptional responses were substantially altered or attenuated following disruption of either the NTD or DBD, demonstrating that both domains are required for the full transcriptional response to progesterone signalling (Figure 2B). Furthermore, comparison of the transcriptional profiles revealed domain-dependent differences, suggesting that the NTD and DBD contribute distinct regulatory properties rather than functioning exclusively as interchangeable components of PR-mediated transcription.
To validate these transcriptomic observations, we performed RT-qPCR analysis of selected progestin-responsive genes associated with cancer-related regulatory programmes. Notably, the induction of SNAIL1, PAX6, SOX18, and PHF19 was strongly dependent on the integrity of both the NTD and DBD (Figure 2C-D). In contrast, other target genes displayed distinct domain-dependent responses, further supporting the existence of complementary and potentially gene-specific regulatory functions of the PR NTD and DBD. These observations indicate that PR functional domains contribute not only to the magnitude of hormone-induced transcription but also to the selection and regulation of specific gene expression programmes. Importantly, these molecular differences were also reflected at the cellular level. Disruption of either the NTD or DBD significantly impaired progestin-induced breast cancer cell proliferation and migration compared with WT-PR cells (Figure 2E-F). These functional observations establish a link between PR domain integrity, hormone-dependent transcriptional regulation, and cancer-associated cellular phenotypes. Together, our preliminary findings demonstrate that the NTD and DBD are both essential for establishing the full transcriptional and functional responses to progestin stimulation, while contributing distinct regulatory properties to PR-mediated gene expression.
Collectively, these findings, together with our previous demonstration of PR domain-dependent transcriptional regulation and oncogenic function, provide a strong conceptual and experimental foundation for the proposed thesis. We propose that the NTD and DBD contribute complementary molecular properties to the organization of PR-containing transcriptional assemblies, thereby influencing their interactions with chromatin and the establishment of hormone-dependent genomic regulatory landscapes. However, whether these domain-specific functions directly influence three-dimensional genome organization, including TAD architecture and long-range chromatin interactions, remains unknown.
The overarching objective of this thesis is therefore to elucidate how distinct PR functional domains coordinate transcriptional machinery organization, chromatin interactions, and hormone-dependent genome architecture in breast cancer cells. By combining our established engineered cellular models with transcriptomic, genome-wide chromatin interaction, and molecular characterization approaches, this project will investigate the mechanistic relationship between PR domain-specific functions, genome organization, and transcriptional regulation. Ultimately, these studies will provide new insights into how transcription factor domain architecture contributes to the dynamic regulation of the cancer genome and may uncover previously unrecognized mechanisms underlying breast cancer cell plasticity and progression.
The main goal of the project is to define how distinct PR domains contribute to chromatin organization and transcription regulation in response to progestin stimulation in breast cancer cells.

The project comprises three complementary aims.

- Aim 1. Define the contribution of distinct PR domains to genome-wide chromatin architecture.
Generate high-resolution, genome-wide chromatin contact maps to characterize changes in chromatin organization, including topologically associating domains, in engineered MCF7 breast cancer cells expressing wild-type (WT), N-terminal domain-depleted (NTD), or DNA-binding domain-deficient (DBD) PR, before and after progestin stimulation.

- Aim 2. Characterize the PR domain-dependent interactome of chromatin-associated proteins.
Identify and characterize the interactions between PR and chromatin-associated proteins (CAPs) in engineered MCF7 breast cancer cells expressing WT, NTD, or DBD PR, under basal and progestin-stimulated conditions, to determine how distinct PR domains influence the recruitment and association of chromatin regulatory factors.

- Aim 3. Functionally validate the contribution of selected chromatin-associated proteins to PR-mediated chromatin organization and transcriptional regulation.
Perform loss-of-function experiments targeting selected CAPs identified in Aims 1 and 2 to determine their functional contributions to PR domain-dependent chromatin organization and transcriptional regulation, and to identify key regulatory hubs mediating these processes.

Unraveling the function of PR distinct domains on genome dynamics under progesterone hormone regulation in breast cancer cells.

The potential candidate will employ a combination of complementary molecular and genomic approaches to address the three specific aims of the project. These methods will enable the characterization of PR domain-dependent changes in chromatin organization, the identification of interactions between PR and chromatin-associated proteins, and the functional validation of key regulatory factors involved in progesterone-dependent genome regulation.

- Aim 1: Generate high-resolution, genome-wide chromatin contact maps to characterize changes in chromatin organization, including topologically associating domains, in engineered MCF7 breast cancer cells expressing wild-type (WT), N-terminal domain-depleted (NTD), or DNA-binding domain-deficient (DBD) PR, before and after progestin stimulation.
- Aim 2: Identify and characterize the interactions between PR and chromatin-associated proteins (CAPs) in engineered MCF7 breast cancer cells expressing WT, NTD, or DBD PR, under basal and progestin-stimulated conditions, to determine how distinct PR domains influence the recruitment and association of chromatin regulatory factors.
- Aim 3: Perform loss-of-function experiments targeting selected CAPs identified in Aims 1 and 2 to determine their functional contributions to PR domain-dependent chromatin organization and transcriptional regulation, and to identify key regulatory hubs mediating these processes.