#	Pagina
attuale pagina	/open-h2020/projects/206112/results.html

Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - SYNC_DEV (The importance of transcriptional coordination during development)

Teaser

Summary

The proposed project centers on the question of temporal control of gene expression during development. While numerous studies have established how enhancers integrate spatial information, little is known about the temporal aspects of transcription. The main goal of my ERC_SyncDev proposal is to integrate the dynamic aspects of transcription to understand how coordination is achieved and whether it is required during development. Transcriptional coordination refers to the inter-nuclear temporal coordination in gene activation (synchrony) and homogeneity in mRNA distribution across a field of coordinately developing cells. My team improves newly available live imaging techniques to address fundamental questions about transcriptional dynamics in a multicellular developing embryo, the Drosophila embryo, with three main objectives:
TASK 1: examine the effects of promoter sequences and enhancer priming on
transcriptional coordination.
TASK 2: analyse the inheritance of transcriptional states from mother to daughter cells and
identify the bookmarking mechanisms responsible for this memory.
TASK 3:explore the functional role of transcriptional coordination for cell fate
specification during cardiogenesis.

Work performed

TASK1: UNCOVER THE MECHANISMS RESPONSIBLE FOR TRANSCRIPTIONAL COORDINATION

Subtask1.1: Impact of promoter sequences on transcriptional dynamics
During the course of development, patterns of gene expression are progressively established. The spatio-temporal precision of these patterns is often attributed to enhancers. While the effect of enhancer location and sequences has been extensively studied, promoter sequences have so far received little attention in the context of multicellular developing organisms. In particular the impact of core promoter motifs on particular step of transcriptional initiation remains elusive.
To investigate the role of minimal promoter sequences on transcriptional dynamics, we employed quantitative live imaging (MS2/MCP RNA labeling combined with high speed confocal microscopy) and single molecule labeling to extract transcriptional bursting properties of various Drosophila developmental promoters. We have developed an image analysis pipeline, for nuclei tracking and 3D detection of the associated mRNA molecules. Using an innovative machine-learning approach, we are able to deconvolve MS2/MCP fluorescent traces to position each individual polymerase initiation event for each nucleus in vivo. By combining data from hundreds of transcribing nuclei located in a defined spatial domain within an embryo, we can infer promoter-switching rates for each promoter sequence, distinguishing Pol II firing rates from burst size

Subtask1.2 Transcriptional dynamics of developmental genes
To monitor transcription from endogenous loci, we introduced MS2 repeats in intronic or 3â€™UTR sequences by Crispr/Cas9 technology.
We quantified transcriptional dynamics in living embryos at various developmental stages to specifically question: 1) the impact of enhancer priming on transcriptional bursting 2) the Impact of core promoter elements on transcriptional bursting.

TASK2: EXPLORE THE EXISTENCE AND FUNCTION OF TRANSCRIPTIONAL MEMORY

Temporal control of transcription by the pioneer factor Zelda through transient interactions in hubs
During the awakening of the zygotic Drosophila genome, thousands of enhancers get accessibility to ensure their precise spatio-temporal activation. In this context, the pioneer transcription activator Zelda plays an essential role. To investigate its impact on the timing of transcriptional activation and on memory, we employed quantitative live imaging and mathematical modeling from synthetic MS2 containing transgenes with varying number of Zelda binding sites.
We demonstrate that increasing the number of Zelda binding sites accelerates the kinetics of nuclei transcriptional activation (Figure 4) regardless of their transcriptional past. Despite its known pioneering activities, we show that Zelda does not remain detectably associated with mitotic chromosomes and is neither necessary nor sufficient to foster memory. We discussed these results and a mathematical model that we developed in collaboration with O.Radulescu (Univ.Montpellier) in the context of mitotic bookmarking in a recent review that we were invited to write for Current Opinions in Systems Biology:
Bellec M, Radulescu O and Lagha M (2018) Remembering the past: Mitotic bookmarking in a developping embryo. Current Opinions in Systems Biology 11:41â€“49

We further reveal that Zelda forms sub-nuclear dynamic hubs (Figure 5) where Zelda binding events are transient, ranging from hundreds of milliseconds (likely unspecific binding) to seconds (likely specific binding) and this occurs regardless of its local concentration.
This work is published in: Dufourt J, Trullo A, Hunter J, Fernandez C, Lazaro J, Dejean M, Morales L, Nait-Amer S, Schulz KN, Harrison MM, Favard C, Radulescu O, Lagha M (2018) Temporal control of gene expression by the pioneer factor Zelda through transient interactions in hubs. Nat Commun. 2018 Dec 5;9(1):5194. doi: 10.1038/s41467-018-07613-z.

Final results

1-Deciphering the mechanisms underlying gene regulation of endogenous loci in a developing embryo requires a constant improvement of mRNA labeling techniques as well as their imaging. Taking advantage of the technological improvements developed by the team of E.Bertrand (IGMM), we tested new MS2 repeats (128X) and new fluorescent detectors in the fly embryo. This labeling method significantly improves the signal, crucial to the detection of lowly expressed developmental genes.We also tested a simplified Inexpensive single molecule FISH protocol, which successfully detects single molecules in fixed Drosophila embryos and is significantly cheaper than purchasing fluorescent-conjugated DNA probes (e.g Stellaris).

2-During development, progenitor cells undergo multiple rounds of cellular divisions during which transcriptional programs must be faithfully propagated. To study mitotic inheritance of these programs, it is essential to be able to track transcriptional activities and their transmission from mother to daughter cells in living embryos.
We developed a user-friendly software, MitoTrack, which tracks nuclei and their lineage in consecutive cell cycles in Drosophila embryos. For each nucleus, MitoTrack records the precise timing of transcriptional activation while keeping its lineage tree history. The software works automatically but provides the possibility to easily supervise, correct and validate each step (Trullo et al, submitted).

3-We contributed to the development of a new microscopy-based technology, called Hi-M, that simultaneously reveals 3D chromatin organization and transcriptional activity.
This methodology was recently published in Cardozo Gizzi AM, Cattoni DI, Fiche J-B, Espinola S, Gurgo J, Messina O, Houbron C, Ogiyama Y, Papadopoulos G, Cavalli G, Lagha M and Nollmann M (2019) Microscopy-based chromosome conformation capture enables simultaneous visualization of genome organization and transcription in intact organisms. DOI:10.1016/j.molcel.2019.01.011 Molecular Cell.