The EMBL Conference ‘Quantitative biology to molecular mechanisms‘ took place last month at EMBL Heidelberg and virtually. Formerly known as ‘From functional genomics to systems biology’, this meeting series aims to bridge the gap between technologies and scales, serving as a platform for the emergence of ideas and concepts to model complex biological systems. Leaders in genomics, functional proteomics, quantitative imaging, microscopy, and theory convene to discuss their data and models, transcending their respective fields of expertise. The meeting tackles three interconnected themes where systems level approaches in molecular biology research are transforming our understanding of biological systems.

For this year’s edition of this conference, we had 175 people attending on-site and 80 participants attending remotely. There were 13 fellowships provided by the EMBL Corporate Partnership Programme and EMBO. With the total of a round 100 of posters to view, we held two poster sessions during which the presenters could discuss their research — their work was then voted for by all participants. There were five poster prizes awarded during the meeting. We are pleased to share with you four of the winners’ abstracts.

Optimized reporters for multiplexed detection of transcription factor activity

Presenter: Max Trauernicht

Authors: Teodora Filipovska, Max Trauernicht, Chaitanya Rastogi, Bas van Steensel

Abstract:

Transcription factors (TFs) play a crucial role in nearly all biological processes by translating signals from signaling pathways into the regulation of target genes. Direct measurements of the activity of TFs are critical to understand how TFs interpret incoming signals and how they drive the downstream changes in gene expression. For this, TF reporter constructs have been widely used in studies of cell signaling, developmental biology, and disease models. However, for many mammalian TFs no reliable reporters are available; and for many others it has been unclear whether the reporters could be improved in terms of sensitivity and specificity. In this study, to enable reliable and scalable detection of TF activity, we have optimized reporters for 86 TFs in parallel. We systematically designed a library of 35,500 TF reporter variants to explore how various design features, such as motif spacing, spacer sequence composition, and core promoter identity, affect reporter performance. To rigorously evaluate the sensitivity and specificity of all TF reporters, we probed this library across nine human and mouse cell lines, and almost 100 TF perturbation conditions. Our findings revealed that precise reporter design significantly influences reporter quality. For instance, we found that HNF4A reporters change their activity 8 fold just by altering the spacing between the binding motifs. Similarly, we identified a spacer sequence that renders GATA reporters specific to only one of the GATA factors. These insights provide valuable information on how TFs drive transcription and how to design TF specific reporters. Finally, by reviewing TF activities across cell types and perturbation responses, we have identified a collection of ‘prime’ reporters for 62 TFs. Most of these prime reporters outperform currently available TF reporters, or represent TFs for which no reporters were previously available. We recently assembled a mini prime TF reporter library, which contains only the 62 identified prime reporters. Given the low complexity of this library, accurate multiplexed TF activity detection may be achieved even from hard to transfect cell models such as organoids, and enable detection from a small number of cells, making it suitable for applications such as 96 well screens.

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Identifying determinants of inter-allelic heterogeneity at the Xist locus at the onset of X-chromosome inactivation

Presenter: Ingrid Pelaez Conde

Authors: Ingrid Pelaez Conde, Jana Tünnermann, Luca Giorgetti, Edda Schulz

Abstract:

X chromosome inactivation (XCI) is the process by which female mammals inactivate one of their two X chromosomes to compensate the dosage imbalance of X linked genes. The long non coding RNA (lncRNA) Xist is the master regulator of XCI and it is upregulated from one of the two X chromosomes promoting the silencing of the whole chromosome in cis. Even though a wide range of Xist regulators has already been identified, it remains unclear how the symmetry between the two X chromosomes is broken to adopt two opposite states within the same cell. To address this question, we aim to quantify the dynamics of Xist upregulation using live cell imaging techniques to describe the process with high temporal resolution. To shed light on the possible mechanisms for symmetry breaking between the two X chromosomes, we are developing a cell sorting system with allelic specificity which will allow the discrimination between the active and inactive X chromosome. Due to the non coding nature of Xist, it is not feasible to directly tag it with a fluorescent protein. Therefore, we will tag each allele of an X linked protein coding gene, which is highly expressed and rapidly silenced upon Xist upregulation with different fluorescent proteins as a proxy for Xist expression. Subsequently, the cells will be sorted through fluorescence activated cell sorting (FACS) based on their fluorescent protein expression. Allele specific molecular profiling will allow us to determine the molecular differences between the Xist expressing and Xist silent alleles at the Xist locus at the onset of XCI, such as the presence or absence of DNA binding proteins, distinctive histone marks, DNA methylation patterns or 3D genome interactions. Overall, this study will provide insights into the molecular mechanisms governing the early stages of X chromosome inactivation in mammals and contribute to a better understanding of how monoallelic gene expression is established.

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Negative autoregulation and Hox-mediated activation maintain terminal selector gene expression in post-mitotic neurons

Presenter: Honorine Destain

Authors: Honorine Destain

Abstract:

Across species, terminal selectors are transcription factors that establish and maintain the identity of specific neuron types throughout life. Emerging evidence suggests that, in post mitotic neurons, the expression levels of terminal selectors must be kept within a critical range over time, yet the underlying mechanisms remain unclear. Here, we study UNC 3 (Collier/Olf/EBF), the terminal selector for cholinergic motor neurons in C. elegans. Studies of a human ortholog of UNC 3, EBF3, implicate the deviation of EBF3 expression levels from a critical range in an EBF3 neurodevelopmental syndrome. This suggests a conserved importance, from C. elegans to humans, for regulation of critical UNC 3/EBF levels. Using quantitative fluorescence microscopy, we find that UNC 3 protein is continuously expressed throughout life, but in a dynamic fashion; UNC 3 expression is high early in postmitotic neuronal life, lowers, and rises again in adulthood. We find that two antagonistic forces regulate UNC 3 levels: Hox proteins activate unc 3 transcription and UNC 3 negatively regulates its own transcription. We further demonstrate evidence for negative autoregulation via a direct transcriptional mechanism, as tested by mutation of UNC 3 binding sites in the unc 3 locus and supported by ChIP sequencing. We show that both the DNA binding domain and the lesser understood IPT (Immunoglobulin/Plexin/Transcription factor) domain are necessary for mediating UNC 3 negative autoregulation. Using temporally controlled protein degradation, we also demonstrate that negative autoregulation occurs continuously. Finally, we show that impairing negative autoregulation results in increased adult worm swim speeds, presumably due to misregulation of motor neuron identity genes and thereby modified motor neuron function. Altogether, we uncover a molecular mechanism for the precise regulation of terminal selectors with a consequence on locomotory behavior. Because terminal selectors are conserved regulators of neuronal identity, the mechanisms described here may be broadly applicable.

Funding sources: T32 GM139782, NSF GRFP

Due to the confidentiality of the unpublished data, we cannot share the poster.

SON modulates cell-to-cell variability in gene expression with functional implications in cellular state switching and fate choice

Presenter: Óscar García Blay

Authors: Óscar García Blay, Xinyu Hu, Christin Wassermann, Tom van Bokhoven, Frèderique Struijs, Maike Hansen

Abstract:

Cell to cell variability in gene expression is a driving force in population dynamics that impact physiology and pathology, such as symmetry breaking during development or the evolution of cancerous cell populations. A relevant fraction of this variability is stochastic in nature—stemming from transcriptional bursting and propagating downstream in gene expression. While this variability plays a role in physiology, it can be detrimental when in excess. Therefore, evolution has shaped mechanisms that control cell to cell variability to maintain it at homeostatic levels while also allowing dynamic tuning when necessary. Our understanding of these mechanisms and how to identify them is limited. To discover novel regulators of cell to cell variability, we implemented a proteome wide perturbation screen combining single cell RNA sequencing, mass spectrometry proteomics, and a regulator target association analysis. We identified SON, a protein component of nuclear speckles, as a regulator of cell to cell variability in mouse embryonic stem cells (mESCs). While investigating the mechanisms behind SON’s function, we found that SON regulates the mean expression of a subset of genes by modulating their splicing efficiency. However, the mechanistic link between SON and the regulation of cell to cell variability appeared to be related to intronic length. Furthermore, many of the genes regulated in terms of cell to cell variability were associated with differentiation. This prompted us to investigate the role of this regulation from a functional perspective. We explored the role of SON in mouse embryonic stem cell differentiation and fate choice and found evidence of both decreased fate exploration and decreased state switching upon SON depletion. Overall, SON exemplifies a crucial aspect of cell to cell variability as a controlled feature of gene expression with functional mechanistic roles.

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The EMBL Conference ‘Quantitative biology to molecular mechanisms‘ took place from 19 – 22 November 2024 at EMBL Heidelberg and virtually.