PhD student (M/F) in developmental biology and quantitative imaging

Job Description

Thesis Subject

How do transcription factor-RNA interactions shape nuclear organization and developmental robustness in a changing world?

Background
Precise gene expression during development requires coordination between transcription and co-transcriptional RNA processing, including splicing. Co-transcriptional regulation is a key interface connecting genome activity and developmental robustness. Yet, how these mechanisms are modulated in vivo remains poorly understood. While transcription factors (TFs) are defined by their DNA binding properties, emerging evidence suggests that many TFs also possess RNA binding functions, contributing directly to RNA maturation. This dual activity remains underexplored, especially in physiological contexts.

The Carnesecchi lab (IGMM) has shown that the Hox TF Ultrabithorax (Ubx) binds RNA and interacts with splicing factors in a tissue-specific manner in Drosophila embryo. Transcriptomic profiling of Ubx-depleted muscle nuclei revealed widespread alternative splicing events independent of transcriptional changes. We identified a point mutant (UbxK58A) that disrupts RNA binding alone without affecting DNA binding. This mutant fails to regulate splicing, confirming that Ubx-RNA binding is required for its function in splicing. Furthermore, genetic interactions between Ubx and splicing factors become lethal under temperature stress, revealing the environmental sensitivity of this regulation. Thus, Ubx lies at the interface between transcription, splicing, and environmental responsiveness. They underscore the need to understand how co-transcriptional regulation adapts, or fails, under different perturbations in the context of environmental challenges during development.

In parallel, spatial organization within the nucleus is also linked to the robust regulation of embryo development. TFs can be localized into transcriptional hubs at their target genes regulating cell fate. The Tsai lab (CRBM) showed that Ubx is enriched in these hubs with specific cofactors, promoting regulatory specificity. Furthermore, these environments buffer gene regulation against external environmental stresses, ensuring embryo development remains resilient. These multi-component hubs may provide a place for distinct processes surrounding gene expression to communicate. For TFs that interact with RNA, these hubs can be the spatial scaffold coupling transcriptional and co-transcriptional processes.

However, it remains unclear how these processes coexist within a hub, if its organization differs between cell lineages, how Ubx-RNA interactions affect this organization, and how all these elements contribute to gene regulation and phenotype development. Resolving these knowledge gaps will clarify how TFs may be uniquely positioned to coordinate precise gene expression in an organism exposed to environmental perturbations. It could also reveal how transcriptional and co-transcriptional regulation interacts with and adapts to environmental parameters, allowing a robust biological system to emerge from stochastic molecular interactions.

Objectives
Working together with the Tsai lab and the Carnesecchi lab, this PhD project will integrate genome editing in Drosophila, -omics techniques, super-resolution imaging in whole embryos and analysis from biophysics for the following main goals:

-Generate and validate a CRISPR-based RNA binding mutant (K58A) of Ubx in Drosophila

-Map the nuclear organization of Ubx with cofactors, splicing factors, and its target genes in the ectoderm using spatial and topological metrics.

-Determine how temperature stress and altered transcriptional kinetics (slow Pol II mutant) affect the nuclear organization of Ubx, Ubx-dependent splicing, Ubx-RNA binding targets, and Ubx-associated protein networks.

-Evaluate how disrupting Ubx-RNA interactions affects the spatial organization of Ubx, its interactional networks, and its ability to respond to temperature stress, including developmental outcomes.

-Extend this characterization to the mesoderm to identify how nuclear organization differs between cell lineages.

-Develop a mechanistic framework to explain how the nuclear organization of TFs confers regulatory robustness against biological and environmental stresses.

Methods
-CRISPR/Cas9 engineering of Ubx-RNA binding mutant and knock-in of tagged splicing factors.

-Environmental and genetic perturbations (temperature shift cold-stress 14°C, homeostasis 25°C, heat-stress 32°C, genetic background mutant of slow Pol II)

-Biotin-based proximity labeling coupled with mass spectrometry (BioID, proteomics)

-xRIP (UV-crosslinked RNA-Immunoprecipitation of Ubx followed by sequencing or qPCR)

-Bulk RNA-seq and differential splicing analysis (transcriptomics)

-Phenotypic assays (viability, tissue patterning with confocal imaging)

-3D AiryScan imaging and Expansion microscopy + lattice lightsheet imaging for quantitative mapping of the organization of (co-)transcriptional hubs.

-HCR-RNA FISH to identify and examine transcription sites of Ubx target genes.

-Analysis using geometric and topological metrics to extract the organizational features of Ubx/partner associations.

Competencies
The person recruited must be highly motivated and autonomous, in addition to being familiar with Drosophila melanogaster, its genetics and embryo development. The person must also have prior experience with confocal microscopy and computational image analysis. The person recruited must be proficient with English to communicate professionally and to assimilate the knowledge contained in state-of-the-art scientific literature (B2 level).

Your Work Environment

The recruited person will be co-directed by Dr. Julie Carnesecchi at the IGMM and Dr. Albert Tsai at the CRBM. The person will thus work in both labs throughout the course of the thesis research.

The Institut de Génétique moléculaire de Montpellier (IGMM) UMR 5535 and the Centre de Recherche en Biologie cellulaire de Montpellier (CRBM) UMR 5237 are located in the CNRS campus at Route de Mende, which also hosts the Institut de Recherche en Infectiology à Montpellier (IRIM) and the Pôle Chimie Balard. The IGMM consists of more than 200 and the CRBM more than 160 researchers, post-docs, doctoral students, and technical staff. The IGMM conducts fundamental research relevant to molecular and cellular medicine, with a particular emphasis on cancer and specific infections and genetic diseases. The CRBM covers a wide range of topics in cellular biology, exploring how internal cellular processes and external environmental cues shape cell behavior and function.

The project benefits from the comprehensive scientific environment of the BioCampus Montpellier and its technological platforms (proteomics, sequencing, imaging; www.biocampus.cnrs.fr).

Constraints and risks

This research project involves working with small insects (Drosophila melanogaster) and their embryos, as well as working on stereo-microscopes and super-resolution light microscopes.

Possibility of coming in on certain weekends and public holidays to maintain the living samples (1 to 2 hours maximum per weekend).

Compensation and benefits

Compensation

2300 € gross monthly

Annual leave and RTT

44 jours

Remote Working practice and compensation

Pratique et indemnisation du TT

Transport

Prise en charge à 75% du coût et forfait mobilité durable jusqu’à 300€

About the offer

About the CNRS

The CNRS is a major player in fundamental research on a global scale. The CNRS is the only French organization active in all scientific fields. Its unique position as a multi-specialist allows it to bring together different disciplines to address the most important challenges of the contemporary world, in connection with the actors of change.

CNRS

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