Biology subjects

Steinek, C.

Publications and source records attributed to Steinek, C..

5 recordsLinked to original sources

bioRxiv2026

Combinatorial DNA-PAINT

State-of-the-art super-resolution microscopy enables nanometer-resolution imaging of proteins, but its multiplexing capacity has been fundamentally constrained. Here, we present Combi-PAINT, a combinatorial DNA barcoding strategy based on DNA-PAINT, that facilitates superlinear scaling of multiplexing with the number of imaging rounds. Instead of assigning one unique docking strand per target, Combi-PAINT encodes targets as combinations of orthogonal sequences, allowing nonlinear scaling of target number with imaging rounds. Using just six imaging rounds, we resolve 41 targets in a field-of-view of 100 x 100 m2 with [~]2.5 nm localization precision and [~]90% decoding accuracy in under 30 minutes, representing the fastest sub-10 nm super-resolution microscopy acquisition to date. We benchmark decoding fidelity and demonstrate robust in situ performance in mammalian cells, achieving 97% decoding accuracy. Combi-PAINT is compatible with existing DNA-PAINT workflows and speed-enhancing techniques, offering a scalable, accessible platform for high-content, single-molecule spatial proteomics.

biophysics↗

bioRxiv2025

Nanoscale Dynamics of Enhancer-Promoter Interactions during Exit from Pluripotency

While there is compelling genetic evidence for the role of enhancers in regulating promoter activity even over large genomic distances, it is unclear to what extent physical proximity with the respective promoters is involved. Here we combine DNA fluorescence in situ hybridization (FISH) with confocal and stimulated emission depletion (STED) super resolution microscopy to investigate enhancer-promoter (E-P) distances at selected loci (Dppa3, Nanog, Dnmt3a, Sox2, Prdm14) that contain multiple enhancers and undergo transcriptional changes at the transition from naive to primed pluripotency in mouse embryonic stem cells. Automated distance measurements in thousands of cells revealed that both pairwise and multiway E-P conformations undergo only small ({Delta} median distance = [~]22 nm) changes, despite large, up to 1500-fold changes in transcription, arguing against lasting changes in genome architecture at these loci. As transcription often occurs in transient bursts in a small fraction of cells, we performed RNA FISH to identify actively transcribed alleles. We found that in actively transcribed Dppa3 alleles (<10%) the median E-P distances are significantly shorter ({Delta} median distance = [~]90 nm) than in non-transcribed ones. These data are consistent with a transient spatial interaction of enhancers and promoter during the initiation of transcription. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=83 SRC="FIGDIR/small/633941v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@1d3d529org.highwire.dtl.DTLVardef@4f904dorg.highwire.dtl.DTLVardef@18ad558org.highwire.dtl.DTLVardef@1bc9bc3_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2024

Generation of densely labeled oligonucleotides for the detection of small genomic elements

The genome contains numerous regulatory elements that may undergo complex interactions and contribute to the establishment, maintenance, and change of cellular identity. Three-dimensional genome organization can be explored with fluorescence in situ hybridization (FISH) at the single-cell level, but the detection of small genomic loci remains challenging. Here, we provide a rapid and simple protocol for the generation of bright FISH probes suited for the detection of small genomic elements. We systematically optimized probe design and synthesis, screened polymerases for their ability to incorporate dye-labeled nucleotides and streamlined purification conditions to yield nanoscopy-compatible oligonucleotides with dyes in variable arrays (NOVA-probes). With these probes, we detect genomic loci ranging from genome-wide repetitive regions down to non-repetitive loci below the kilobase scale. In conclusion, we introduce a simple workflow to generate densely labeled oligonucleotide pools that facilitate detection and nanoscopic measurements of small genomic elements in single cells.

cell biology↗

bioRxiv2023

Altered enhancer-promoter interaction leads to MNX1 expression in pediatric acute myeloid leukemia with t(7;12)(q36;p13)

Acute myeloid leukemia (AML) with the t(7;12)(q36;p13) translocation occurs only in very young children and has a poor clinical outcome. The expected oncofusion between breakpoint partners (MNX1 and ETV6) has only been reported in a subset of cases. However, a universal feature is the strong transcript and protein expression of MNX1, a homeobox transcription factor that is normally not expressed in hematopoietic cells. Here, we map the translocation breakpoints on chromosomes 7 and 12 in affected patients to a region proximal to MNX1 and either introns 1 or 2 of ETV6. The frequency of MNX1 overexpression in pediatric AML (n=1556, own and published data) is 2.4% and occurs predominantly in t(7;12)(q36;p13) AML. Chromatin interaction assays in a t(7;12)(q36;p13) iPSC cell line model unravel an enhancer-hijacking event that explains MNX1 overexpression in hematopoietic cells. Our data suggest that enhancer-hijacking is a more common and overlooked mechanism for structural rearrangement-mediated gene activation in AML. Key pointsO_LIExpression analysis of over 1500 pediatric AML samples demonstrates MNX1 expression as a universal feature of t(7;12)(q36;p13) AML as well as in rare cases without t(7;12)(q36;p13) C_LIO_LIMNX1 is activated by an enhancer-hijacking event in t(7;12)(q36;p13) AML and not, as previously postulated, by the creation of a MNX1::ETV6 oncofusion gene. C_LI

cancer biology↗

bioRxiv2022

Probing protein ubiquitination in live cells

The reversible attachment of ubiquitin governs the interaction, activity and degradation of proteins whereby the type and target of this conjugation determine the biological response. The investigation of this complex and multi-faceted protein ubiquitination mostly relies on painstaking biochemical analyses. Here, we employ recombinant binding domains to probe the ubiquitination of proteins in living cells. We immobilize GFP-fused proteins of interest at a distinct cellular structure and detect their ubiquitination state with red fluorescent ubiquitin binders. With this ubiquitin fluorescent three-hybrid (ubiF3H) assay we identified HP1{beta} as a novel ubiquitination target of UHRF1. The use of linkage specific ubiquitin binding domains enabled the discrimination of K48 and K63 linked protein ubiquitination. To enhance signal-to-noise ratio, we implemented fluorescence complementation (ubiF3Hc) with split YFP. Using in addition a cell cycle marker we could show that HP1{beta} is mostly ubiquitinated by UHRF1 during S phase and deubiquitinated by the protease USP7. With this complementation assay we could also directly detect the ubiquitination of the tumor suppressor p53 and monitor its inhibition by the anti-cancer drug Nutlin-3. Altogether, we demonstrate the utility of the ubiF3H assay to probe the ubiquitination of specific proteins and to screen for ligases, proteases and small molecules controlling this posttranslational modification.

cell biology↗