Biology subjects

Dostatni, N.

Publications and source records attributed to Dostatni, N..

5 recordsLinked to original sources

bioRxiv2024

Both the transcriptional activator, Bcd, and transcriptional repressor, Cic, form small mobile oligomeric clusters in early fly embryo nuclei

Transcription factors play an essential role in pattern formation during early embryo development, generating a strikingly fast and precise transcriptional response that results in sharp gene expression boundaries. To characterize the steps leading up to transcription, we performed a side-by-side comparison of the nuclear dynamics of two morphogens, a transcriptional activator, Bicoid (Bcd), and a transcriptional repressor, Capicua (Cic), both involved in body patterning along the anterior-posterior axis of the early Drosophila embryo. We used a combination of fluorescence recovery after photobleaching, fluorescence correlation spectroscopy, and single particle tracking to access a wide range of dynamical timescales. Despite their opposite effects on gene transcription, we find that Bcd and Cic have very similar nuclear dynamics, characterized by the co-existence of a freely diffusing monomer population with a number of oligomeric clusters, which range from low stoichiometry and high mobility clusters to larger, DNA-bound hubs. Our observations are consistent with the inclusion of both Bcd and Cic into transcriptional hubs or condensates, while putting constraints on the mechanism by which these form. These results fit in with the recent proposal that many transcription factors might share a common search strategy for target genes regulatory regions that makes use of their large unstructured regions, and may eventually help explain how the transcriptional response they elicit can be at the same time so fast and so precise. SIGNIFICANCEBy conducting a comparative study of the nuclear dynamics of Bicoid (a transcriptional activator) and Capicua (a transcriptional repressor) in the Drosophila embryo, we have uncovered a striking similarity in their behaviours. Despite their divergent roles in transcription, both proteins have a propensity to form oligomeric species ranging from highly mobile, low stoichiometry clusters to larger, DNA-bound hubs. Such findings impose new constraints on the existing models of gene regulation by transcription factors, particularly in aspects related to target search and oligomeric binding to gene regulatory regions needed to explain the rapid and precise transcriptional response observed in developmental processes.

biophysics↗

bioRxiv2021

Synthetic deconstruction of hunchback regulation by Bicoid

For over 40 years, the Bicoid-hunchback (Bcd-hb) system in the fruit fly embryo has been used as the model to study how positional information in morphogen concentration gradients is robustly translated into step-like responses. A body of quantitative comparisons between theory and experiment have since questioned the initial paradigm that the sharp hb transcription pattern emerges solely from diffusive biochemical interactions between the Bicoid transcription factor and the gene promoter region. Several alternative mechanisms have been proposed, such as additional sources of positional information, positive feedback from Hb proteins or out-of-equilibrium transcription activation. By using the MS2-MCP RNA-tagging system and analysing in real time, the transcription dynamics of synthetic reporters for Bicoid and/or its two partners Zelda and Hunchback, we show that all the hb expression pattern features and temporal dynamics are compatible with an equilibrium model with a short decay length Bicoid activity gradient as a sole source of positional information. Meanwhile, Bicoids partners speed-up the process by different means: Zelda lowers the Bicoid concentration threshold required for transcriptional activation while Hunchback reduces burstiness and increases the polymerase firing rate.

developmental biology↗

bioRxiv2019

Constraints and limitations on decoding positional information: the Bicoid case-study

The regulation of the hunchback promoter expression by the maternal Bicoid gradient has been studied as a model system in development for many years. Yet, at the level of quantitative agreement between data and theoretical models, even the first step of this regulation, transcription, continues to be challenging. This situation is slowly progressing, thanks to quantitative live-imaging techniques coupled to advanced statistical data analysis and modelling. Here we outline the current state of our knowledge of this apparently "simple" step, highlighting the newly appreciated role of bursty transcription dynamics and its regulation.

developmental biology↗

bioRxiv2018

Precision in a rush: trade-offs between positioning and steepness of the hunchback expression pattern

Fly development amazes us by the precision and reproducibility of gene expression, especially since the initial expression patterns are established during very short nuclear cycles. Recent live imaging of hunchback promoter dynamics shows a stable steep binary expression pattern established within the three minute interphase of nuclear cycle 11. Considering expression models of different complexity, we explore the trade-o between the ability of a regulatory system to produce a steep boundary and minimize expression variability between different nuclei. We show how a limited readout time imposed by short developmental cycles affects the genes ability to read positional information along the embryos anterior posterior axis and express reliably. Comparing our theoretical results to real-time monitoring of the hunchback transcription dynamics in live flies, we discuss possible regulatory strategies, suggesting an important role for additional binding sites, gradients or non-equilibrium binding and modified transcription factor search strategies.

biophysics↗

bioRxiv2018

3 minutes to precisely measure morphogen concentration

Morphogen gradients provide concentration-dependent positional information along polarity axes. Although the dynamics of establishment of these gradients is well described, precision and noise in the downstream activation processes remain elusive. A simple paradigm to address these questions is the Bicoid morphogen gradient that elicits a rapid step-like transcriptional response in young fruit fly embryos. Focusing on the expression of the main Bicoid target, hunchback (hb), at the onset of zygotic transcription, we used the MS2-MCP approach which combines fluorescent labeling of nascent mRNA with live imaging at high spatial and temporal resolution. Removing 36 putative Zelda binding sites unexpectedly present in the original MS2 reporter, we show that the 750 bp of the hb promoter are sufficient to recapitulate endogenous expression at the onset of zygotic transcription. After each mitosis, in the anterior, expression is turned on to rapidly reach a plateau with all nuclei expressing the reporter. Consistent with a Bicoid dose-dependent activation process, the time period required to reach the plateau increases with the distance to the anterior pole. Remarkably, despite the challenge imposed by frequent mitoses and high nuclei-to-nuclei variability in transcription kinetics, it only takes 3 minutes at each interphase for the MS2 reporter loci to measure subtle differences in Bicoid concentration and establish a steadily positioned and steep (Hill coefficient ~ 7) expression boundary. Modeling based on cooperativity between the 6 known Bicoid binding sites in the hb promoter region and assuming rate limiting concentrations of the Bicoid transcription factor at the boundary is able to capture the observed dynamics of pattern establishment but not the steepness of the boundary. This suggests that additional mechanisms are involved in the steepness of the response.

developmental biology↗