Laura Hartmann’s research while affiliated with University of Stuttgart and other places

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Publications (4)


Transcriptional regulators ensuring specific gene expression and decision-making at high TGFβ doses
  • Article
  • Full-text available

November 2024

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32 Reads

Life Science Alliance

Laura Hartmann

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Stefan Legewie

TGFβ-signaling regulates cancer progression by controlling cell division, migration, and death. These outcomes are mediated by gene expression changes, but the mechanisms of decision-making toward specific fates remain unclear. Here, we combine SMAD transcription factor imaging, genome-wide RNA sequencing, and morphological assays to quantitatively link signaling, gene expression, and fate decisions in mammary epithelial cells. Fitting genome-wide kinetic models to our time-resolved data, we find that most of the TGFβ target genes can be explained as direct targets of SMAD transcription factors, whereas the remainder show signs of complex regulation, involving delayed regulation and strong amplification at high TGFβ doses. Knockdown experiments followed by global RNA sequencing revealed transcription factors interacting with SMADs in feedforward loops to control delayed and dose-discriminating target genes, thereby reinforcing the specific epithelial-to-mesenchymal transition at high TGFβ doses. We identified early repressors, preventing premature activation, and a late activator, boosting gene expression responses for a sufficiently strong TGFβ stimulus. Taken together, we present a global view of TGFβ-dependent gene regulation and describe specificity mechanisms reinforcing cellular decision-making.

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Zellen sind Individualisten — Entscheidungsprozesse auf Einzelzellebene

June 2024

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28 Reads

BIOspektrum

Decision-making is a fundamental aspect of life. However, our understanding of how cells encode and decode information to enable reliable fate decisions remains limited. Employing live cell imaging and automated analysis, our research unveils substantial heterogeneity in the cellular response to TGFβ and sheds light on the intricate link between the dynamics of SMAD signaling, the state of individual cells and their fate decisions.


CRISPR gene and transcriptome engineering (CRISPRgate) improves loss-of-function genetic screening approaches

May 2024

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10 Reads

The CRISPR/Cas9 technology has revolutionized genotype-to-phenotype assignments through large-scale loss-of-function (LOF) screens. However, limitations like editing inefficiencies and unperturbed genes cause significant noise in data collection. To address this, we introduce CRISPR Gene and Transcriptome Engineering (CRISPRgate), which uses two specific sgRNAs to simultaneously repress and cleave the target gene within the same cell, increasing LOF efficiencies and reproducibility. CRISPRgate outperforms conventional CRISPRko, CRISPRi, or CRISPRoff systems in suppressing challenging targets and regulators of cell proliferation. Additionally, it efficiently suppresses modulators of EMT and impairs neuronal differentiation in a human iPSC model. In a multiplexed chromatin-focused phenotypic LOF screen, CRISPRgate exhibits improved depletion efficiency, reduced sgRNA performance variance, and accelerated gene depletion compared to individual CRISPRi or CRISPRko, ensuring consistency in phenotypic effects and identifying more significant gene hits. By combining CRISPRko and CRISPRi, CRISPRgate increases LOF rates without increasing genotoxic stress, facilitating library size reduction for advanced LOF screens. Motivation The CRISPR technology (CRISPRko/CRISPRi) enables the specific depletion of target genes with fewer off-target effects, facilitating precise investigations of gene function. Despite its benefits, CRISPR applications have limitations. Residual active protein expression mediated by in-frame DNA repair or alternative splicing 1–8 as well as strong epigenetic regulation and difficulties in sgRNA design to the transcription start site (TSS) 9–12 hinder the full potential of loss-of-function studies using CRISPRko or CRISPRi. We aimed to achieve robust target gene reduction in order to improve the reproducibility of the CRISPR technology by integrating the widely used CRISPRko and CRISPRi approaches into a single application.


Figure 1
Figure 2
Figure 4: Evidence for feedforward regulation in SMAD-dependent gene expression
Figure 5: TFs shaping TGFβ target gene expression and cell fates
Transcriptional regulators ensuring specific gene expression and decision making at high TGFβ doses

April 2024

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43 Reads

TGFβ-signaling regulates cancer progression by controlling cell division, migration and death. These outcomes are mediated by gene expression changes, but the mechanisms of decision making towards specific fates remain unclear. Here, we combine SMAD transcription factor imaging, genome-wide RNA sequencing and morphological assays to quantitatively link signaling, gene expression and fate decisions in mammary epithelial cells. Fitting genome-wide kinetic models to our time-resolved data, we find that the majority of TGFβ target genes can be explained as direct targets of SMAD transcription factors, whereas the remainder show signs of complex regulation, involving delayed regulation and strong amplification at high TGFβ doses. Knockdown experiments followed by global RNA sequencing revealed transcription factors interacting with SMADs in feedforward loops to control delayed and dose-discriminating target genes, thereby reinforcing the specific epithelial-to-mesenchymal transition at high TGFβ doses. We identified early repressors, preventing premature activation, and a late activator, boosting gene expression responses for a sufficiently strong TGFβ stimulus. Taken together, we present a global view of TGFβ-dependent gene regulation and describe specificity mechanisms reinforcing cellular decision making.