Biology subjects

Giehr, P.

Publications and source records attributed to Giehr, P..

4 recordsLinked to original sources

bioRxiv2025

Epitranscriptomic control of stress adaptations in Escherichia coli

Bacterial stress responses have been studied at the phenotypic, transcriptional, and translational levels, demonstrating the presence of an "alarm" phase immediately after stress exposure. However, the contributions of RNA modifications during stress adaptation remain largely unexplored. Here, we map the epitranscriptomic changes of Escherichia coli after exposure to oxidative and acid stress using direct RNA sequencing of mRNA, rRNA, and tRNA, combined with mass spectrometry, deletion mutant phenotyping, and single-nucleotide PCR. We identified widespread, dynamic RNA modifications that include central metabolism transcripts and increased levels of rRNA methylations (m4Cm and m5C) under both stresses, with potential consequences for translation. In uncharged tRNAs, stress-specific modifications via the Mnm and Q pathways accumulated at the wobble position; these modifications proved crucial for survival. Together, these findings reveal a multifaceted layer of post-transcriptional regulation, establishing the first comprehensive view of the bacterial epitranscriptome during the alarm phase of stress adaptation.

microbiology↗

bioRxiv2025

Biochemical engineering of 5hmdC-DNA using a Tet3 double-mutant

5-Hydroxymethyl-2-deoxycytidine (5hmdC) is an important epigenetic marker involved in gene regulation and DNA demethylation. It has potential use as a biomarker for cancer and other diseases due to its significant depletion in various cancers and disease models. This research aimed to develop a reliable and efficient method for generating 5hmdC-containing DNA, addressing limitations in existing techniques. We created a Tet3 stalling mutant that converts 5-methyl-2'-deoxycytidine (5mdC) into a mixture of 5hmdC and 5-formyl-2'-deoxycytidine (5fdC), followed by a reduction step to convert 5fdC to 5hmdC, ensuring a pure 5hmdC state within the CpG context. This method can convert any PCR product, synthetic oligos, and entire genomes into 5hmdC-modified DNA. The principal results demonstrate high specificity and efficiency, providing a robust tool for epigenetic research, cancer diagnostics, and protein binding assays. Additionally, our technique offers 5hmdC-DNA for functional studies and as standards for diagnostic assays.

biochemistry↗

bioRxiv2024

Decoding the Escherichia coli epitranscriptome

Modifications of RNA, known as the epitranscriptome, affect gene expression, translation, and splicing in eukaryotes, with implications for developmental processes, cancer, and viral infections. In prokaryotes, regulation at the level of the epitranscriptome is still poorly understood. Here, we used Nanopore direct RNA sequencing of Escherichia coli to study RNA modifications and their changes under heat stress. With a single sequencing reaction, we detected most known modification types in rRNA, tRNA, and mRNA. RNA sequencing was complemented by a multifaceted approach that included mass spectrometry, deletion mutants, single-nucleotide PCR, and in vitro methylation. Known m5C and m6A sites in the rRNA were confirmed, but these types of modifications could not be localized in the mRNA. In response to heat stress, levels of m5C, m6A, and m6,6A increased in the 16S rRNA. Sequencing and mass spectrometry data demonstrated a decrease in tRNA modification abundance in the anticodon loop at 45 {degrees}C. In general, mRNA modifications at 37 {degrees}C were enriched in the coding regions of genes associated with general metabolism and RNA processing, which shifted to genes involved in cell wall synthesis and membrane transport under heat stress. This study provides new insights into the complexity of post-transcriptional regulation in bacteria. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/602490v2_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@11b9bc4org.highwire.dtl.DTLVardef@18f77feorg.highwire.dtl.DTLVardef@efdeaorg.highwire.dtl.DTLVardef@402859_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2020

Genome wide efficiency profiling reveals modulation of maintenance and de novo methylation by Tets

A precise understanding of DNA methylation dynamics on a genome wide scale is of great importance for the comprehensive investigation of a variety of biological processes such as reprogramming of somatic cells to iPSCs, cell differentiation and also cancer development. To date, a complex integration of multiple and distinct genome wide data sets is required to derive the global activity of DNA modifying enzymes. We present GwEEP - Genome-wide Epigenetic Efficiency Profiling as a versatile approach to infer dynamic efficiency changes of DNA modifying enzymes at base pair resolution on a genome wide scale. GwEEP relies on genome wide oxidative Hairpin Bisulfite sequencing (HPoxBS) data sets, which are translated by a sophisticated hidden Markov model into quantitative enzyme efficiencies with reported confidence around the estimates. GwEEP in its present form predicts de novo and maintenance methylation efficiencies of Dnmts, as well as the hydroxylation efficiency of Tets but its purposefully flexible design allows to capture further oxidation processes such as formylation and carboxylation given available data in the future. Applied to a well characterized ES cell model, GwEEP precisely predicts the complex epigenetic changes following a Serum-to-2i shift i.e., (i) instant reduction in maintenance efficiency (ii) gradually decreasing de novo methylation efficiency and (iii) increasing Tet efficiencies. In addition, a complementary analysis of Tet triple knock-out ES cells confirms the previous hypothesized mutual interference of Dnmts and Tets. GwEEP is applicable to a wide range of biological samples including cell lines, but also tissues and primary cell types. MOTIVATIONDynamic changes of DNA methylation patterns are a common phenomenon in epigenetics. Although a stable DNA methylation profile is essential for cell identity, developmental processes require the rearrangement of 5-methylcytosine in the genome. Stable methylation patterns are the result of balanced Dnmts and Tets activities, while methylome transformation results from a coordinated change in Dnmt and Tet efficiencies. Such transformations occur on a global scale, for example during the reprogramming of maternal and paternal methylation patterns and the establishment of novel cell type specific methylomes during embryonic development in vivo, but also in vitro during (re)programming of induced pluripotent stem cells, as well as somatic cells. In addition, local (de)methylation events are key for gene regulation during cell differentiation. A detailed understanding of Dnmt and Tet cooperation is essential for understanding natural epigenetic adaptation as well as optimization of in vitro (re)programming protocols. For this purpose, we developed a pipeline for quantitative and precise estimation of Dnmt and Tet activity. Using only double strand methylation information, GwEEP infers accurate maintenance and de novo methylation efficiency of Dnmts, as well as hydroxylation efficiency of Tets at single base resolution. Thus, we believe GwEEP provides a powerful tool for the investigation of methylome rearrangements in various systems.

genomics↗