Periodic Reporting for period 4 - SYNC_DEV (The importance of transcriptional coordination during development)
Okres sprawozdawczy: 2021-07-01 do 2022-12-31
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.
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.
Task1) Impact of promoter sequences on transcriptional bursting- We found that, in contrast to TATA promoters, captured by a single two state model, the INR promoter motif leads to an extra checkpoint of regulation associated with stochastic pausing, imposed on only a subset of polymerases [Pimmett*, Dejean*, et al., 2021]. This novel view of non-obligatory pausing and its instruction by core promoter motifs has fascinating implications for the control of gene expression variation during development and disease.
Task2) Transcriptional memory- By directly monitoring transcription in living developing embryos, we showed, that the transcriptional status of mother nuclei biases that of their daughters after division, a phenomenon referred to as ‘transcriptional memory’ [Ferraro et al., 2016]. We then showed that the pioneer factor Zelda, is not essential for this memory [Dufourt et al., 2018] and developed a modelling framework [Bellec et al., 2018]. In contrast to Zelda, we discovered that the transcriptional activator GAF acts as a mitotic bookmarker with key functions during early development [Bellec et al., 2022].
TASK 3: Imaging translation dynamics
We deployed the SunTag method to image twi translation dynamics in the embryo. We discovered a spatial heterogeneity in translation [Dufourt*, Bellec*, et al., 2021]. This technique was also implemented in adult Drosophila brains [Formicola et al., 2021].
This work leverages the power of live imaging (with the SunTag/MS2 method) and represents an important technological breakthrough that allowed the characterization of exciting features of translation during development, summarized below:
1-live imaging single molecules of cytoplasmic mRNA (for the first time in a developing organism), with the simultaneous detection of their translation and their co-movement. 2-quantification of translation kinetics of a developmental gene (elongation rate and ribosome turnover). Such measurements have, so far, never been performed in single cells of a living embryo. Indeed, our knowledge concerning translation efficiencies during development is solely based on bulk measurements (ribosome profiling) which cannot capture the inherent dynamics in space and time. 3-discovery a translation heterogeneity, where the efficiency of translation of two identical mRNA depends on their localization. 4-unmasking a potential mechanism for formation of basal peri-nuclear mRNA clusters
This work is now published in Dufourt*, Bellec* et al., Science 2021.
Task2) Transcriptional memory- By directly monitoring transcription in living developing embryos, we showed, that the transcriptional status of mother nuclei biases that of their daughters after division, a phenomenon referred to as ‘transcriptional memory’ [Ferraro et al., 2016]. We then showed that the pioneer factor Zelda, is not essential for this memory [Dufourt et al., 2018] and developed a modelling framework [Bellec et al., 2018]. In contrast to Zelda, we discovered that the transcriptional activator GAF acts as a mitotic bookmarker with key functions during early development [Bellec et al., 2022].
TASK 3: Imaging translation dynamics
We deployed the SunTag method to image twi translation dynamics in the embryo. We discovered a spatial heterogeneity in translation [Dufourt*, Bellec*, et al., 2021]. This technique was also implemented in adult Drosophila brains [Formicola et al., 2021].
This work leverages the power of live imaging (with the SunTag/MS2 method) and represents an important technological breakthrough that allowed the characterization of exciting features of translation during development, summarized below:
1-live imaging single molecules of cytoplasmic mRNA (for the first time in a developing organism), with the simultaneous detection of their translation and their co-movement. 2-quantification of translation kinetics of a developmental gene (elongation rate and ribosome turnover). Such measurements have, so far, never been performed in single cells of a living embryo. Indeed, our knowledge concerning translation efficiencies during development is solely based on bulk measurements (ribosome profiling) which cannot capture the inherent dynamics in space and time. 3-discovery a translation heterogeneity, where the efficiency of translation of two identical mRNA depends on their localization. 4-unmasking a potential mechanism for formation of basal peri-nuclear mRNA clusters
This work is now published in Dufourt*, Bellec* et al., Science 2021.
To integrate the dynamics of transcription, my team has and continues to optimize current live imaging (MS2-MCP), single molecule labelling (smiFISH) and multiplex genomic DNA painting (Hi-M) techniques to adapt them to a multicellular organism. Technological developments allowed us to characterize key features of transcriptional regulation during development summarized below.
1-We have been invited to contribute to a chapter dedicated to imaging gene expression (Book ‘Imaging Gene Expression’ edited by Pr. Y.Shav-Tal Springer editions)[Fernandez and Lagha 2019].
2-We have contributed to setting up the Hi-M technique in Drosophila embryos [Gizzi et al 2019] and then employed this single cell spatial genomics approach to decipher the impact of 3D contacts on transcriptional control [Espinola et al, 2021].
3-To extract quantitative data from these imaging-based approaches, we implemented specific image analysis software, able to segment nuclei and transcriptional spots and track them even during divisions [Trullo et al., 2019].
4-With these tools, we showed, for the first time in a multicellular developing organism, that the transcriptional status of mother nuclei biases that of their daughters after division, a phenomenon referred to as ‘transcriptional memory’ [Ferraro et al., 2016].
5-We showed that the pioneer factor Zelda is not essential for this memory and developed [Dufourt, Trullo et al., 2018] a modelling framework for transcriptional memory [Bellec et al., 2018].
6-We discovered that the transcriptional activator and pioneer factor GAF acts as a mitotic bookmarker with key functions during early development [Bellec et al., 2022] . For this study, we developed a protocol to perform ChIP-Seq from a pure population of mitotic embryos; embryonic mitotic ChIP has never been performed.
7-By imaging Zelda dynamic behaviour in living embryos, we discovered that Zelda proteins accumulate in nuclear hubs [Dufourt et al., 2018]. We then questioned the impact of these hubs on 3D genome organization during zygotic genome activation. Surprisingly, we found that 3D organization of cis-regulatory hubs is similar between cell types and occurs prior to the emergence of TADs in the embryo [Espinola et al., 2021].
8-We revealed the contribution of specific core promoter motifs on promoter state dynamics and how this creates coordinated gene expression in the developing drosophila embryo. In this study, we employed fast live imaging and an innovative machine-learning approach that allowed us to replace each individual polymerase initiation event for each nucleus in vivo. This analysis framework revealed that the INR promoter motif leads to an extra checkpoint of regulation associated with stochastic pausing, imposed on only a subset of polymerases. This novel view of non-obligatory pausing and its instruction by core promoter motifs, has fascinating implications for the control of gene expression variation in both development (during ZGA) and disease [Pimmett, Dejean et al., 2021] .
9-We implemented the Suntag labelling method in Drosophila, which allowed for the first time to visualize and quantify translational dynamics in a multicellular living embryo. By focusing on the translation of the conserved major epithelial-mesenchymal transition (EMT)-inducing transcription factor Twist, we identified spatial heterogeneity in mRNA translation efficiency and revealed the existence of translation factories, where clustered mRNAs are co-translated preferentially at basal perinuclear regions [Dufourt, Bellec et al., 2021].
1-We have been invited to contribute to a chapter dedicated to imaging gene expression (Book ‘Imaging Gene Expression’ edited by Pr. Y.Shav-Tal Springer editions)[Fernandez and Lagha 2019].
2-We have contributed to setting up the Hi-M technique in Drosophila embryos [Gizzi et al 2019] and then employed this single cell spatial genomics approach to decipher the impact of 3D contacts on transcriptional control [Espinola et al, 2021].
3-To extract quantitative data from these imaging-based approaches, we implemented specific image analysis software, able to segment nuclei and transcriptional spots and track them even during divisions [Trullo et al., 2019].
4-With these tools, we showed, for the first time in a multicellular developing organism, that the transcriptional status of mother nuclei biases that of their daughters after division, a phenomenon referred to as ‘transcriptional memory’ [Ferraro et al., 2016].
5-We showed that the pioneer factor Zelda is not essential for this memory and developed [Dufourt, Trullo et al., 2018] a modelling framework for transcriptional memory [Bellec et al., 2018].
6-We discovered that the transcriptional activator and pioneer factor GAF acts as a mitotic bookmarker with key functions during early development [Bellec et al., 2022] . For this study, we developed a protocol to perform ChIP-Seq from a pure population of mitotic embryos; embryonic mitotic ChIP has never been performed.
7-By imaging Zelda dynamic behaviour in living embryos, we discovered that Zelda proteins accumulate in nuclear hubs [Dufourt et al., 2018]. We then questioned the impact of these hubs on 3D genome organization during zygotic genome activation. Surprisingly, we found that 3D organization of cis-regulatory hubs is similar between cell types and occurs prior to the emergence of TADs in the embryo [Espinola et al., 2021].
8-We revealed the contribution of specific core promoter motifs on promoter state dynamics and how this creates coordinated gene expression in the developing drosophila embryo. In this study, we employed fast live imaging and an innovative machine-learning approach that allowed us to replace each individual polymerase initiation event for each nucleus in vivo. This analysis framework revealed that the INR promoter motif leads to an extra checkpoint of regulation associated with stochastic pausing, imposed on only a subset of polymerases. This novel view of non-obligatory pausing and its instruction by core promoter motifs, has fascinating implications for the control of gene expression variation in both development (during ZGA) and disease [Pimmett, Dejean et al., 2021] .
9-We implemented the Suntag labelling method in Drosophila, which allowed for the first time to visualize and quantify translational dynamics in a multicellular living embryo. By focusing on the translation of the conserved major epithelial-mesenchymal transition (EMT)-inducing transcription factor Twist, we identified spatial heterogeneity in mRNA translation efficiency and revealed the existence of translation factories, where clustered mRNAs are co-translated preferentially at basal perinuclear regions [Dufourt, Bellec et al., 2021].