Biology subjects

Bergauer, K.

Publications and source records attributed to Bergauer, K..

4 recordsLinked to original sources

bioRxiv2026

BARe-seq enables high-throughput dissection of cis-regulatory control of transcriptional bursting

Transcriptional bursts determine RNA output through two kinetic parameters: burst size and burst frequency. How cis-regulatory DNA encodes these kinetic parameters remains unclear, in part because existing approaches do not combine scalable sequence perturbation with allele-resolved burst inference. Here, we developed Bulk Allele Resolution Sequencing (BARe-seq), an allele-resolved massively parallel reporter assay that enables inference of transcriptional burst parameters from bulk sequencing. Applying BARe-seq to libraries of 1000 promoters and 1000 enhancers in Drosophila S2 cells revealed distinct kinetic properties of promoters and enhancers. Promoter-dependent mean expression was driven by both burst size and burst frequency: TATA-box promoters showed larger bursts, whereas DPE promoters showed higher burst frequency. In contrast, enhancer strength was primarily driven by burst frequency, although specific transcription factor motifs were also associated with burst size. Thus, BARe-seq dissects cis-regulatory control of transcriptional bursting and extends allele-resolved measurements to pooled reporter assays in bulk sequencing experiments.

molecular biology↗

bioRxiv2025

Comparison of picolyl azide-based BONCAT and microautoradiography for assessing the heterotrophic prokaryotic activity in the deep ocean

Prokaryotes play a central role in marine biogeochemical cycles, yet quantifying their activity requires sensitive methods due to low biomass and metabolic rates, particularly in the deep ocean. One recent method to determine single-cell activity of prokaryotes is bioorthogonal non-canonical amino acid tagging (BONCAT), which offers a non-radioactive approach to measure protein synthesis. However, direct comparisons between BONCAT and radioisotope-based techniques across ocean depth gradients remain limited, particularly for low-activity prokaryotic communities. To address this knowledge gap, we tested an optimised BONCAT protocol using picolyl azide fluorophores (BONCAT-pic) to assess single-cell heterotrophic activity in prokaryotic communities from surface to bathypelagic depths (1000-4000 m) in the Southern Ocean near the Kerguelen Islands. The method was first optimised using aged coastal and open-ocean seawater, and then compared to microautoradiography with 3H-methionine uptake. Statistical analysis shows that BONCAT-pic significantly improved detection sensitivity compared to standard azide reagents. BONCAT-pic consistently detected active cells in profiles over the open ocean water column, with cell proportions and fluorescence signals closely correlating with both microautoradiography (R2 = 0.9, p < 0.001) and bulk methionine incorporation (R2 = 0.6, p < 0.001). Our results demonstrate that BONCAT-pic is a reliable, fluorescence-based method for quantifying heterotrophic activity at the single-cell level, extending its applicability to prokaryotic communities in the deep ocean.

microbiology↗

bioRxiv2022

Functionally distinct promoter classes initiate transcription via different mechanisms reflected in focused versus dispersed initiation patterns

Recruitment of RNA polymerase II (Pol II) to promoter regions is essential for transcription. Despite conflicting evidence, the Pol II Pre-Initiation Complex (PIC) is often thought to be of uniform composition and assemble at all promoters via an identical mechanism. Here, we show using Drosophila melanogaster S2 cells as a model that promoter classes with distinct functions and initiation patterns function via PICs that display different compositions and dependencies: developmental promoter DNA readily associates with the canonical Pol II PIC, whereas housekeeping promoter DNA does not and instead recruit different factors such as DREF. Consistently, TBP and DREF are required by distinct sets of promoters, and TBP and its paralog TRF2 function at different promoter types, partly exclusively and partly redundantly. In contrast, TFIIA is required for transcription from all promoters, and we identify factors that can recruit and/or stabilize TFIIA at housekeeping promoters and activate transcription. We show that promoter activation by these factors is sufficient to induce the dispersed transcription initiation patterns characteristic of housekeeping promoters. Thus, different promoter classes direct distinct mechanisms of transcription initiation, which relate to different focused versus dispersed initiation patterns.

molecular biology↗

bioRxiv2022

Identification and characterization of repressive domains in Drosophila transcription factors

All multicellular life relies on differential gene expression, determined by regulatory DNA elements and DNA-binding transcription factors that mediate activation and repression via cofactor recruitment. While activators have been extensively characterized, repressors are less well studied and their repressive domains (RDs) are typically unknown, as are the RDs properties and the co-repressors (CoRs) they recruit. Here, we develop the high-throughput next-generation-sequencing-based method Repressive-Domain (RD)-seq to systematically identify RDs in complex libraries. Screening more than 200,000 fragments covering the coding sequences of all transcription-related proteins in Drosophila melanogaster, we identify 195 RDs in known repressors and in proteins not previously associated with repression. Many RDs contain recurrent short peptide motifs that are required for RD function, as demonstrated by motif mutagenesis, and are conserved between fly and human. Moreover, we show that RDs which contain one of five distinct repressive motifs interact with and depend on different CoRs, including Groucho, CtBP, Sin3A or Smrter. Overall, our work constitutes an invaluable resource and advances our understanding of repressors, their sequences, and the functional impact of sequence-altering mutations.

molecular biology↗