Biology subjects

Hutin, S.

Publications and source records attributed to Hutin, S..

3 recordsLinked to original sources

bioRxiv2026

EARLY FLOWERING 3 (ELF3): a novel role in integrating environmental stimuli with root stem cell niche maintenance

Maintaining stem cell niche (SCN) homeostasis in the root apical meristem (RAM) is essential for proper root growth and thus for overall plant development. In Arabidopsis thaliana, a group of slowly dividing cells at the SCN center known as the quiescent center (QC) maintain the surrounding stem cells, including the distally located columella stem cells (CSCs) which give rise to the differentiated columella cells. Many actors, including the PLETHORA (PLT) family transcription factors, regulate the QC quiescence and CSC fate. However, little is known about the integration of external and/or internal cues into regulating SCN homeostasis. In this study, we report for the first time the interaction between PLT3 and the thermosensor and circadian clock related transcriptional regulator, EARLY FLOWERING 3 (ELF3), which are both expressed in the root SCN. We show that ELF3 localizes, similar to PLT3, to subcellular condensates and sustains the QC and CSC fate. We demonstrate that ELF3 forms condensates in vitro and in vivo, in the cytoplasm, as well as in the nucleus, where it then co-localizes with PLT3. We reveal that the interaction of ELF3 and PLT3 is mediated by their intrinsically disordered prion-like domains (PrDs). Furthermore, transient expression in human epithelial cells (HEp-2) cells and in Nicotiana benthamiana shows that PHYTOCHROME INTERACTING FACTORS 3 and 4 (PIF3/4) function as nuclear shuttles for ELF3, recruiting it to nuclear condensates, where it co-localizes with PLT3, PIF3, and PIF4. Accordingly, we propose a model where the co-localization and interactions of ELF3, PLT3, PIF3, and PIF4 represent a dynamic mechanism to integrate environmental signals into SCN maintenance and cell fate decisions.

plant biology↗

bioRxiv2023

Structure and flexibility of the DNA polymerase holoenzyme of vaccinia virus

The year 2022 was marked by the mpox outbreak caused by human monkeypox virus (MPXV), which is about 98 % identical to vaccinia virus (VACV) at the sequence level regarding the proteins involved in DNA replication. We present the strategy for the production of the VACV DNA polymerase holoenzyme composed of the E9 polymerase associated with its co-factor, the A20-D4 heterodimer, which led to the 3.8 [A] cryo-electron microscopy (cryo-EM) structure of the DNA-free form of the holoenzyme. Model building used high-resolution structures of components of the complex and the A20 structure predicted by AlphaFold 2. The structure of E9 does not change in context of the holoenzyme compared to the crystal structure. As for the MPXV holoenzyme, a contact between E9 and D4 is mediated by a cluster of hydrophobic residues. The holoenzyme structure is quite compact and surprisingly similar to the MPXV holoenzyme in presence of a DNA template, with the exception of a movement of the finger domain and the thumb domain, which becomes ordered in presence of DNA. Even in absence of DNA, the VACV holoenzyme structure is too compact for an agreement with SAXS data. This suggests the presence of more open conformations in solution, which are also predicted by Alphafold 2 indicating hinge regions located within A20. Using biolayer interferometry we showed that indeed, the E9-D4 interaction is weak and transient although very important as it has not been possible to obtain viable viruses carrying mutations of key residues in the E9-D4 interface. Author SummaryThe 2022 outbreak of mpox is caused by monkeypox virus closely related to the best studied model, vaccinia virus. Genome replication, which takes place largely autonomously in the cytosol of the infected cell, is still not really understood. Viral DNA synthesis involves a DNA repair enzyme, the uracil-DNA glycosylase D4 linked to the structural protein A20 forming the processivity factor, which in turn binds to E9 forming the complex required for processive DNA synthesis. Here we present the first structure of the vaccinia virus polymerase holoenzyme E9-A20-D4 at 3.8 [A] obtained by cryo-electron microscopy. This structure, together with several recent structures from monkeypox virus, provide a static view of the complex with a previously undescribed contact between E9 and D4. Our small-angle scattering data show that other conformations, taking advantage of 2 hinge regions in A20, exist in solution. Using site-directed mutagenesis and binding studies we show that the contact between E9 and D4, which serves to encircle the template strand, is important, but transient. Thus the current model of the orientation of the holoenzyme on the replication fork may not be the only one possible.

molecular biology↗

bioRxiv2023

Phase separation and molecular ordering of the prion-like domain of the thermosensory protein EARLY FLOWERING 3

Liquid-liquid phase separation (LLPS) is an important mechanism enabling the dynamic compartmentalisation of macromolecules, including complex polymers such as proteins and nucleic acids, and occurs as a function of the physicochemical environment. In the model plant, Arabidopsis thaliana, LLPS by the protein EARLY FLOWERING3 (ELF3) occurs in a temperature sensitive manner and controls thermoresponsive growth. ELF3 contains a largely unstructured prion-like domain (PrLD) that acts as a driver of LLPS in vivo and in vitro. The PrLD contains a poly-glutamine (polyQ) tract, whose length varies across natural Arabidopsis accessions. Here, we use a combination of biochemical, biophysical and structural techniques to investigate the dilute and condensed phases of the ELF3 PrLD with varying polyQ lengths. We demonstrate that the dilute phase of the ELF3 PrLD forms a monodisperse higher order oligomer that does not depend on the presence of the polyQ sequence. This species undergoes LLPS in a pH and temperature-sensitive manner and the polyQ region of the protein tunes the initial stages of phase separation. The liquid phase rapidly undergoes aging and forms a hydrogel as shown by fluorescence and atomic force microscopies. Furthermore, we demonstrate that the hydrogel assumes a semi-ordered structure as determined by small angle X-ray scattering, electron microscopy and X-ray diffraction. These experiments demonstrate a rich structural landscape for a PrLD protein and provide a framework to describe the structural and biophysical properties of biomolecular condensates.

biophysics↗