Biology subjects

Trauernicht, M.

Publications and source records attributed to Trauernicht, M..

3 recordsLinked to original sources

bioRxiv2024

Optimized reporters for multiplexed detection of transcription factor activity

In any given cell type, dozens of transcription factors (TFs) act in concert to control the activity of the genome by binding to specific DNA sequences in regulatory elements. Despite their considerable importance in determining cell identity and their pivotal role in numerous disorders, we currently lack simple tools to directly measure the activity of many TFs in parallel. Massively parallel reporter assays (MPRAs) allow the detection of TF activities in a multiplexed fashion; however, we lack basic understanding to rationally design sensitive reporters for many TFs. Here, we use an MPRA to systematically optimize transcriptional reporters for 86 TFs and evaluate the specificity of all reporters across a wide array of TF perturbation conditions. We thus identified critical TF reporter design features and obtained highly sensitive and specific reporters for 60 TFs, many of which outperform available reporters. The resulting collection of "prime" TF reporters can be used to uncover TF regulatory networks and to illuminate signaling pathways. HIGHLIGHTSO_LISystematic design and optimization of transcriptional reporters for 86 TFs C_LIO_LICharacterization of TF-specific reporter design optimization rules C_LIO_LIEvaluation of reporter TF-specificity across a wide array of TF perturbations C_LIO_LIIdentification of a collection of 60 "prime" TF reporters with optimized performance C_LI

genomics↗

bioRxiv2023

Optimisation of TP53 reporters by systematic dissection of synthetic TP53 response elements

TP53 is a transcription factor that controls multiple cellular processes, including cell cycle arrest, DNA repair, and apoptosis. The relation between TP53 binding site architecture and transcriptional output is still not fully understood. Here, we systematically examined in three different cell lines the effects of binding site affinity and copy number on TP53-dependent transcriptional output, and also probed the impact of spacer length and sequence between adjacent binding sites, and of core promoter identity. Paradoxically, we found that high-affinity TP53 binding sites are less potent than medium-affinity sites. TP53 achieves supra-additive transcriptional activation through optimally spaced adjacent binding sites, suggesting a cooperative mechanism. Optimally spaced adjacent binding sites have a [~]10-bp periodicity, suggesting a role for spatial orientation along the DNA double helix. We leveraged these insights to construct a log-linear model that explains activity from sequence features, and to identify new highly active and sensitive TP53 reporters.

genomics↗

bioRxiv2023

Chromatin context-dependent effects of epigenetic drugs on CRISPR-Cas9 editing

The efficiency and outcome of CRISPR/Cas9 editing depends on the chromatin state at the cut site. It has been shown that changing the chromatin state can influence both the efficiency and repair outcome, and epigenetic drugs have been used to improve Cas9 editing. However, because the target proteins of these drugs are not homogeneously distributed across the genome, the efficacy of these drugs may be expected to vary from locus to locus. Here, we systematically analyzed this chromatin context-dependency for 160 epigenetic drugs. We used a human cell line with 19 stably integrated reporters to induce a double-stranded break (DSB) in different chromatin environments. We then measure Cas9 editing efficiency and repair pathway usage by sequencing the mutational signatures. We identified 67 drugs that modulate Cas9 editing efficiency and/or repair outcome dependent on the local chromatin environment. For example, we find a subset of histone deacetylase inhibitors that improve Cas9 editing efficiency throughout all types of heterochromatin (e.g., PCI-24781), while others were only effective in H3K27me3-marked regions (e.g., Vorinostat). In summary, this study reveals that most epigenetic drugs alter CRISPR editing in a chromatin-dependent manner, and provides a detailed guide to improve Cas9 editing more selectively at the desired location. HIGHLIGHTSO_LIA screen identifies dozens of drugs that alter Cas9 editing in a chromatin context-dependent manner C_LIO_LIMany HDAC inhibitors boost Cas9 editing efficiency throughout all types of heterochromatin C_LIO_LIThe DNMT inhibitor Decitabine completely blocks resection-dependent repair across the genome C_LI

genomics↗