Biology subjects

Elsässer, S. J.

Publications and source records attributed to Elsässer, S. J..

12 recordsLinked to original sources

bioRxiv2025

G-quadruplex profiling in complex tissues using single-cell CUT&Tag

G-quadruplexes (G4) are non-canonical DNA structures that gained increasing attention for their potential roles in gene regulation, with implications in neurodegenerative diseases and cancer. Despite their biological significance, G4 structures have not been studied systematically across tissues and cell types. In this study, we employ G4 single-cell CUT&Tag (G4 scCUT&Tag) to characterize G4 landscapes in postnatal mouse brain cells, leveraging single-cell analytical approaches commonly used in scRNA-Seq and scATAC-Seq datasets. Using conventional single-cell omics workflows to process and explore our data, we distinguished different cell lineages based on G4 heterogeneity and established that a subset of lineage-specific genes show unique promoter G4s. Multi-omics integration with scRNA-Seq gene expression profiles, using both a covariance-based technique (canonical correlation analysis) and a transfer learning-based approach, enabled a more detailed annotation of cell types. These integrations not only revealed significant correlation of G4 and gene expression signals, but demonstrated that G4 scCUT&Tag enables detailed examination of G4 heterogeneity in complex tissues and supports integrative analysis of G4 profiles with other omics layers, offering new insights into the epigenomic landscapes of the developing brain.

developmental biology↗

bioRxiv2024

The Role of BAZ2-dependent Chromatin Remodeling in Suppressing G4 DNA Structures and Associated Genomic Instability

DNA G-quadruplexes (G4s) are secondary structures with significant roles in regulating genome function and stability. Dysregulation of the dynamic formation of G4s is linked to genomic instability and disease, but the underlying mechanisms are not fully understood. In this study, we conducted a screen of chromatin-modifying enzymes and identified nine potential inhibitors of G4 formation, including seven that were not previously characterized. Among these, we highlight the role of BAZ2 chromatin remodelers as key suppressors of G4 DNA and G4-related genome instability. Depletion of BAZ2 subunits led to increased G4 formation, especially at transcriptional regulatory elements. BAZ2B was found to associate with G4 loci, suggesting that it plays a direct role in suppressing G4s. While BAZ2-deficient cells exhibited modest genomic instability, treatment with the G4-stabilizing ligand BRACO19 exacerbated double-strand breaks (DSBs), highlighting its utility as a tool to study G4-dependent genome instability. DSB profiling using INDUCE-seq uncovered distinct breakage patterns around G4s, further underscoring the impact of G4s on genome integrity. Notably, we found that within G4s, G repeats were more susceptible to DSBs than loops. These results establish BAZ2 chromatin remodeling complexes as direct regulators of G4 dynamics and provide new insights into G4-dependent genome instability.

molecular biology↗

bioRxiv2024

Pooled overexpression screening identifies PIPPI as a novel microprotein involved in the ER stress response

Microproteins encoded by short open reading frames (sORFs) of less than 100 codons have been predicted to constitute a substantial fraction of the eukaryotic proteome. However, relevance and roles of the majority of microproteins remain undefined because only a small fraction of these intriguing cellular players have been in-depth characterized so far. Here we use pooled overexpression screens with a library of 11338 sORFs to overcome the challenge of elucidating which of the thousands of putative translated sORFs are biologically functional. As a proof-of-concept, we performed a phenotypic screen to identify sORFs protecting cells from treatment with the nucleotide analogue 6-thioguanine. With this approach, we identified two cytoprotective microproteins: altDDIT3 and PIPPI. PIPPI is encoded as part of the LC16a core duplicon/Morpheus gene cluster, a highly duplicated region of the human genome, which is undergoing rapid positive selection in primates. Our data show that PIPPI interacts with proteins of the endoplasmic reticulum, including protein disulfide isomerase ERp44. Besides providing mechanistic insights on a new microprotein, this study highlights the power of using pooled overexpression screens to identify functional microproteins.

cell biology↗

bioRxiv2023

A large-scale sORF screen identifies putative microproteins and provides insights into their interaction partners, localisation and function

The human genome contains thousands of potentially coding short open reading frames (sORFs). A growing set of microproteins translated from these sORFs are known to have important cellular functions. However, the majority remains uncharacterised. Thus, larger screens to find functional microproteins have become more vital. Here, we performed a high-throughput CRISPR/Cas9 knock-out screen with a customised library of 11,776 sORFs, curated from literature and databases to identify microproteins essential for cancer cell line growth. 16/17 tested candidates displayed a reproducible knockout phenotype. We selected our top six hits, consisting of 11 to 63 amino acids. Various of these candidates localised to distinct subcellular compartments and the majority showed specific interaction partners. Endogenous tagging demonstrated translation of an sORF in the CENPBD2P pseudogene that bears no resemblance to the CENPBD2P name-giving CENPB DNA binding domains. For two candidates, uORFs in the DSE and NUTF2 genes, the microprotein supplied in trans ameliorated the growth defect of the respective knock-out. RNA-seq analysis revealed however that gene expression changes in the knock-out could only partially be rescued. Overall, we identified various putative microproteins and a microprotein-producing pseudogene that might be involved in cancer cell growth, but also illustrate the limitations and caveats of sORF functional screening and characterisation.

molecular biology↗

bioRxiv2023

Dual stop codon suppression in mammalian cells with genomically integrated genetic code expansion machinery

Genetic code expansion via stop codon suppression is a powerful strategy to engineer proteins. Pyrrolysyine-tRNA (tRNAPyl)/pyrrolysyl-tRNA synthetase (PylRS) pairs from methanogenic archaea and engineered bacterial tRNA/aminoacyl-tRNA synthetases (aaRS) pairs are used for site-specific incorporation of noncanonical amino acids (ncAAs) in response to stop codons in mammalian cells. Routinely, ncAA incorporation is achieved by transient expression of the tRNA/aaRS pair leading to heterogeneous suppression. Genomic integration of tRNA/aaRS expression cassettes for more homogenous, adjustable and reproducible levels of protein, containing one or more ncAA, will greatly benefit protein engineering, chemical control and imaging applications in mammalian cells. Here, we demonstrate that piggyBac-mediated genomic integration of archaeal tRNAPyl/PylRS or bacterial tRNA/aaRS pairs, using a modular plasmid design with multi-copy tRNA arrays, allows for homogeneous and efficient, genetically encoded ncAA incorporation in diverse mammalian cell lines. We assess opportunities and limitations of using ncAAs for fluorescent labeling applications in stable cell lines. We explore simultaneous suppression of ochre and opal stop codons and finally incorporate two distinct ncAAs with mutually orthogonal click chemistries for site-specific, dual fluorophore labeling of a cell surface receptor on live mammalian cells.

cell biology↗

bioRxiv2022

minute: A MINUTE-ChIP data analysis workflow

Quantitative ChIP-seq methods are essential for accurately characterizing and comparing genome-wide DNA-protein interactions across samples. Procedures that enable such quantitative comparisons involve addition of spike-in chromatin or recombinant nucleosome material, or a multiplexed process using barcoding of chromatin fragments. ChIP-seq analysis workflows typically require a number of computational steps involving multiple tools in order to reach interpretable results, and quantitative analyses require additional steps that ensure scaling of the processed output according to the quantitative measurements. Crucially, the different quantitative approaches have unique analysis requirements reflecting the disparate experimental workflows, hence no universal analysis pipeline exists for quantitative ChIP-seq. Here, we developed minute, a user-friendly computational workflow to easily process multiplexed ChIP data that handles the specific needs of quantitative ChIP. minute enables transformation of raw, multiplexed FASTQ files into a set of normalized, scaled bigWig files that can serve as a basis for a quantitative, comparative downstream analysis. minute is implemented in Python and Snakemake and paired with a Conda environment, to facilitate usability and reproducibility in different platforms. Source code of minute is available on GitHub: https://github.com/NBISweden/minute

bioinformatics↗

bioRxiv2021

Polycomb Repressive Complex 2 shields naïve human pluripotent cells from trophectoderm differentiation

The first lineage choice made in human embryo development separates trophectoderm from the inner cell mass, which proceeds to form the pluripotent epiblast and primitive endoderm. Trophectoderm on the other hand gives rise to the placenta. Naive pluripotent stem cells are derived from the pluripotent epiblast of the blastocyst and offer possibilities to explore how lineage integrity is maintained. Here, we discover that Polycomb repressive complex 2 (PRC2) restricts an intrinsic capacity of naive pluripotent stem cells to give rise to trophectoderm. Through quantitative epigenome profiling, we find that broad histone H3 lysine 27 trimethylation (H3K27me3) hypermethylation is a common feature of naive pluripotency across species. We define a previously unappreciated, naive-specific set of bivalent promoters, featuring PRC2-mediated H3K27me3 concomitant with H3K4me3. Naive bivalency maintains key trophectoderm transcription factors in a transcriptionally poised state that is resolved to an active state upon depletion of H3K27me3 via inhibition of the enzymatic subunits of PRC2, EZH1/2. Conversely, primed human embryonic stem cells cannot be driven towards trophectoderm development via PRC2 inhibition. While naive and primed hESCs share the majority of bivalent promoters, PRC2 contributes to the repression of largely non-overlapping subsets of these promoters in each state, hence H3K27me3-mediated repression provides a highly adaptive mechanism to restrict lineage potential during early human development.

developmental biology↗

bioRxiv2021

Engineered human induced pluripotent cells enable genetic code expansion in brain organoids

Human induced pluripotent stem cell (hiPSC) technology has revolutionized human biology. A wide range of cell types and tissue models can be derived from hiPSCs to study complex human diseases. Here, we use PiggyBac mediated transgenesis to engineer hiPSCs with an expanded genetic code. We demonstrate that genomic integration of expression cassettes for a pyrrolysyl-tRNA synthetase (PylRS), pyrrolysyl-tRNA (PylT) and the target protein of interest enables site-specific incorporation of a non-canonical amino acid (ncAA) in response to amber stop codons. Neural stem cells, neurons and brain organoids derived from the engineered hiPSCs continue to express the amber suppression machinery and produce ncAA-bearing reporter. The incorporated ncAA can serve as a minimal bioorthogonal handle for further modifications by labeling with fluorescent dyes. Site-directed ncAA mutagenesis will open a wide range of applications to probe and manipulate proteins in brain organoids and other hiPSC-derived cell types and complex tissue models. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=176 SRC="FIGDIR/small/449576v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@4e1fa6org.highwire.dtl.DTLVardef@fff373org.highwire.dtl.DTLVardef@1c839e6org.highwire.dtl.DTLVardef@5150e1_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2021

Newly synthesized RNA Sequencing Characterizes Transcription Dynamics in Three Pluripotent States

Unique transcriptomes define naive, primed and paused pluripotent states in mouse embryonic stem cells. Here we perform transient transcriptome sequencing (TT-seq) to de novo define and quantify coding and non-coding transcription units (TUs) in different pluripotent states. We observe a global reduction of RNA synthesis, total RNA amount and turnover rates in ground state naive cells (2i) and paused pluripotency (mTORi). We demonstrate that elongation velocity can be reliably estimated from TT-seq nascent RNA and RNA polymerase II occupancy and observe a transcriptome-wide attenuation of elongation velocity in the two inhibitor-induced states. We also discover a relationship between elongation velocity and termination read-through distance. Our analysis suggests that steady-state transcriptomes in mouse ES cells are controlled predominantly on the level of RNA synthesis, and that signaling pathways governing different pluripotent states immediately control key parameters of transcription.

systems biology↗

bioRxiv2021

Genome-wide mapping of G-quadruplex structures with CUT&Tag

Single-stranded genomic DNA can fold into G-quadruplex (G4) structures or form DNA:RNA hybrids (R loops). Recent evidence suggests that such non-canonical DNA structures affect gene expression, DNA methylation, replication fork progression and genome stability. When and how G4 structures form and are resolved remains unclear. Here we report the use of Cleavage Under Targets and Tagmentation (CUT&Tag) for mapping native G4 in mammalian cell lines at high resolution and low background. Mild native conditions used for the procedure retain more G4 structures and provide a higher signal-to-noise ratio than ChIP-based methods. We determine the G4 landscape of mouse embryonic stem cells (mESC), observing widespread G4 formation at active promoters, active and poised enhancers. We discover that the presence of G4 motifs and G4 structures distinguishes active and primed enhancers in mESCs. Further, performing R-loop CUT&Tag, we demonstrate the genome-wide co-occurence of single-stranded DNA, G4s and R loops, suggesting an intricate relationship between transcription and non-canonical DNA structures.

genomics↗

bioRxiv2019

Quantitative multiplexed ChIP reveals global alterations that shape promoter bivalency in ground state embryonic stem cells

To understand the epigenetic foundation of naive pluripotent cells, we implement a quantitative multiplexed ChIP-Seq method (MINUTE-ChIP) comparing mouse ESC grown in 2i versus Serum conditions. Combined with quantitative western blot and mass spectrometry, we find compelling evidence for a broad H3K27me3 hypermethylation of the genome, concomitant with the widespread loss of DNA CpG methylation. We show that opposing action of EZH2 and JMJD3/UTX shape the H3K27me3 landscape, with almost all bivalent promoters stably retaining high H3K27me3 levels in 2i. In parallel, we show a mechanistically uncoupled, global decrease of H3K4me3 and strong reduction at bivalent promoters, suggesting that low H3K4me3 and high H3K27me3 levels at bivalent promoters safeguard naive pluripotency.

molecular biology↗

bioRxiv2018

Methanomethylophilus alvus Mx1201 provides basis for mutual orthogonal pyrrolysyl tRNA/aminoacyl-tRNA synthetase pairs in mammalian cells

Genetic code expansion via stop codon suppression is a powerful technique for engineering proteins in mammalian cells with site-specifically encoded non-canonical amino acids (ncAAs). Current methods rely on very few available tRNA/aminoacyl-tRNA synthetase pairs orthogonal in mammalian cells, the pyrrolysyl tRNA/aminoacyl-tRNA synthetase pair from Methanosarcina mazei (Mma PylRS/PylT) being the most active and versatile to date. We found a previously uncharacterized pyrrolysyl tRNA/aminoacyl-tRNA synthetase pair from the human gut archaeon Methanomethylophilus alvus Mx1201 (Mx1201 PylRS/PylT) to be active and orthogonal in mammalian cells. We show that the new PylRS enzyme can be engineered to expand its ncAA substrate spectrum. We find that due to the large evolutionary distance of the two pairs, Mx1201 PylRS/PylT is partially orthogonal to Mma PylRS/PylT. Through rational mutation of Mx1201 PylT, we abolish its non-cognate interaction with Mma PylRS, creating two mutually orthogonal PylRS/PylT pairs. Combined in the same cell, we show that the two pairs can site-selectively introduce two different ncAAs in response to two distinct stop codons. Our work expands the repertoire of mutually orthogonal tools for genetic code expansion in mammalian cells and provides the basis for advanced in vivo protein engineering applications for cell biology and protein production.

synthetic biology↗