Venus trap in the mouse embryo reveals distinct molecular dynamics underlying specification of first embryonic lineages

Mammalian development begins with the segregation of embryonic and extra-embryonic lineages in the blastocyst. Recent studies revealed cell-to-cell gene expression heterogeneity and dynamic cell rearrangements during mouse blastocyst formation. Thus, mechanistic understanding of lineage specification requires quantitative description of gene expression dynamics at a single-cell resolution in living embryos. However, only a few fluorescent gene expression reporter mice are available and quantitative live image analysis is limited so far. Here, we carried out a fluorescence gene-trap screen and established reporter mice expressing Venus specifically in the first lineages. Lineage tracking, quantitative gene expression and cell position analyses allowed us to build a comprehensive lineage map of mouse pre-implantation development. Our systematic analysis revealed that, contrary to the available models, the timing and mechanism of lineage specification may be distinct between the trophectoderm and the inner cell mass. While expression of our trophectoderm-specific lineage marker is upregulated in outside cells upon asymmetric divisions at 8- and 16-cell stages, the inside-specific upregulation of the inner-cell-mass marker only becomes evident at the 64-cell stage. This study thus provides a framework toward systems-level understanding of embryogenesis marked by high dynamicity and stochastic variability.

Single live cell monitoring of protein turnover reveals intercellular variability and cell cycle dependence of degradation rates

Andrea Brigitta Alber

Cells need to be able to precisely control their proteome composition in order to maintain homeostasis. This is of particular relevance in fast dividing cells, such as embryonic stem cells, since their global protein levels need to be doubled at each division to ensure reliable growth and pluripotency maintenance. However, how protein synthesis and degradation interplay to regulate global protein expression levels throughout the cell cycle remains unclear. In addition, while cell-to-cell variability at the level of transcription and protein expression is relatively well studied, it remains unknown to what extent protein degradation rates vary between single cells. Previous studies focusing on protein turnover have mainly relied on bulk biochemical approaches, thus only providing population averages and a low temporal resolution. In addition, many approaches to study the changes in protein turnover throughout the cell cycle have been dependent on cell cycle synchonizing drugs, which might interfere with protein synthesis and also reduce cell viability. Therefore, we further developed and applied two fluorescence time-lapse imaging strategies to study the cell cycle dependence and cell-to-cell variability of protein synthesis and degradation in unperturbed dividing mouse embryonic stem (ES) cells: 1) a Mammalian Cell-optimized Tandem Fluorescent Timer (MCFT), consisting of the fusion of the fast maturing green fluorescent protein superfolder GFP (sfGFP) and the slower maturing red fluorescent protein mOrange2, which in combination with computational modeling allows for disentangling changes in protein synthesis from changes in protein degradation in single living cells, and 2) fluorescent pulse labeling of protein tags (SNAP/Halo-tags) to monitor protein half-lives, as well as transient changes in protein degradation rates in single cells without the use of protein synthesis inhibitors. We discovered a substantial cell cycle-dependence in both protein synthesis and degradation. In particular, a large fraction (40%) of proteins was stabilized around cytokinesis, an effect that has not been described previously. Interestingly, protein degradation rates were highly variable between single cells and were positively correlated with synthesis rates in individual cells to buffer protein expression level variability. Furthermore, degradation rates of different proteins co-varied between single cells, both in ES cells and fibroblasts, suggesting variability in proteasome activity as a source of global extrinsic noise in gene expression. We identified the proteasome component ADRM1 as a potential limiting factor that contributes to generating such noise, as its expression levels were negatively correlated with protein half-lives in single cells. Our work demonstrates the importance of single cell live imaging approaches to study dynamic processes and cell-to-cell variability and sheds more light on the complex interplay of protein synthesis and degradation in determining protein expression levels in single mammalian cells.

EPFL2018

Developement of a gateway compatible system for speciﬁc DNA integration in mouse embryonic stem cells and evaluation of the effect of nucleosome positioning on gene expression

Julien Bryois

An emerging model views gene regulation as a competition for regulatory sequences between transcription factors and nucleosomes at thermodynamic equilibrium. A computational model of the DNA affinity of nucleosomes was recently developed allowing to predict the nucleosome occupancy of DNA sequences. The aim of this project was to investigate the effect of different predicted nucleosome occupancies of an important transcription factor binding site on mammalian gene expression. To this end, a two site and three site gateway compatible system allowing integration of DNA at a speciﬁc locus of two mouse embryonic stem cell lines was developed. An important POU5F1 (previously known as OCT4)/SOX2 binding site located in the Nanog promoter was chosen to be the transcription factor binding site of interest as it was shown to play a major role for Nanog expression. A 2kb sequence from the Nanog proximal promoter (-1585 to +415) was cloned upstream of two different minimal promoters and a GFP reporter using three site gateway cloning. These constructs were integrated in both cell lines and expressed low to medium levels of GFP. Next, several variants of a 1kb sequence from the Nanog promoter (-933 to +53), previously shown to be functional, were constructed. 35bp surrounding the POU5F1/SOX2 binding site were altered resulting in differential predicted nucleosome occupancy of the binding site. These promoters were linked to GFP with or without the addition of a kozak consensus sequence and a SV40 polyadenylation signal. Surprisingly, all these variants showed low GFP expression after transfection. This was not caused by biases introduced by the gateway system as a gateway construct containing the CMV promoter linked to GFP was shown to be functional. Furthermore, direct cloning of the 1kb Nanog promoter in a GFP expressing vector without gateway showed similar GFP expression as gateway cloning. Results indicate that both 2kb and 1kb Nanog promoters express GFP at low levels and that the constructs produced may be useful to measure differences in GFP expression using ﬂow cytometry

2010

TRIM28 repression of retrotransposon-based enhancers is necessary to preserve transcriptional dynamics in embryonic stem cells

Andrea Corsinotti, Johan Jakobsson, Adamantia Kapopoulou, Helen Mary Rowe, Didier Trono

TRIM28 is critical for the silencing of endogenous retroviruses (ERVs) in embryonic stem (ES) cells. Here, we reveal that an essential impact of this process is the protection of cellular gene expression in early embryos from perturbation by cis-acting activators contained within these retroelements. In TRIM28-depleted ES cells, repressive chromatin marks at ERVs are replaced by histone modifications typical of active enhancers, stimulating transcription of nearby cellular genes, notably those harboring bivalent promoters. Correspondingly, ERV-derived sequences can repress or enhance expression from an adjacent promoter in transgenic embryos depending on their TRIM28-sensitivity in ES cells. TRIM28-mediated control of ERVs is therefore crucial not just to prevent retrotransposition, but more broadly to safeguard the transcriptional dynamics of early embryos.