Biology subjects

Hoerl, D.

Publications and source records attributed to Hoerl, D..

4 recordsLinked to original sources

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

Rapid and synchronous chemical induction of replicative-like senescence via a small molecule inhibitor

Cellular senescence is now acknowledged as a key contributor to organismal ageing and late-life disease. Although popular, the study of senescence in vitro can be complicated by the prolonged and asynchronous timing of cells committing to it and its paracrine effects. To address these issues, we repurposed the small molecule inhibitor inflachromene (ICM) to induce senescence to human primary cells. Within six days of treatment with ICM, senescence hallmarks, including the nuclear eviction of HMGB1 and -B2, are uniformly induced across IMR90 cell populations. By generating and comparing various high throughput datasets from ICM-induced and replicative senescence, we uncovered significant similarity of the two states. Notably though, ICM suppresses the proinflammatory secretome associated with senescence, thus alleviating most paracrine effects. In summary, ICM induces a senescence-like phenotype rapidly and synchronously thereby allowing the study of its core regulatory program without any confounding heterogeneity.

genomics↗

bioRxiv2021

Differences in nanoscale organization of DNase I hypersensitive and insensitive chromatin in single human cells

Methodological advances in conformation capture techniques have fundamentally changed our understanding of chromatin architecture. However, the nanoscale organization of chromatin and its cell-to-cell variance are less studied. By using a combination of high throughput super-resolution microscopy and coarse-grained modelling we investigated properties of active and inactive chromatin in interphase nuclei. Using DNase I hypersensitivity as a criterion, we have selected prototypic active and inactive regions from ENCODE data that are representative for K-562 and more than 150 other cell types. By using oligoFISH and automated STED microscopy we systematically measured physical distances of the endpoints of 5kb DNA segments in these regions. These measurements result in high-resolution distance distributions which are right-tailed and range from very compact to almost elongated configurations of more than 200 nm length for both the active and inactive regions. Coarse-grained modeling of the respective DNA segments suggests that in regions with high DNase I hypersensitivity cell-to-cell differences in nucleosome occupancy determine the histogram shape. Simulations of the inactive region cannot sufficiently describe the compaction measured by microscopy, although internucleosomal interactions were elevated and the linker histone H1 was included in the model. These findings hint at further organizational mechanisms while the microscopy-based distance distribution indicates high cell-to-cell differences also in inactive chromatin regions. The analysis of the distance distributions suggests that direct enhancer-promoter contacts, which most models of enhancer action assume, happen for proximal regulatory elements in a probabilistic manner due to chromatin flexibility.

cell biology↗