Biology subjects

Oudelaar, M.

Publications and source records attributed to Oudelaar, M..

7 recordsLinked to original sources

bioRxiv2026

Analysis of multi-way chromatin interactions at sub-nucleosome resolution identifies synergistic nano-scale interactions within super-enhancers

In-depth understanding of the 3D organization of the genome and its role in the regulation of gene expression requires techniques to measure complex chromosome conformations at single alleles at high resolution. Here, we present multi-way Micro-Capture-C (mwMCC), a method to detect concurrent interactions between multiple chromatin regions at single alleles at sub-nucleosome resolution. mwMCC resolves the internal 3D structure of super-enhancers and identifies synergistic multi-way interactions between their constituent elements and gene promoters, providing a structural basis for cooperation within super-enhancers in nano-scale chromatin hubs. The dynamic formation and dissolution of these hubs during cellular differentiation correlates with up- and down-regulation of gene expression. Although large-scale chromatin hubs are dependent on the architectural protein CTCF, nano-scale hubs are robust to CTCF depletion. Instead, integration of footprinting analysis and protein occupancy data indicates a role for transcription factors and the co-activator Mediator in the formation of synergistic enhancer-promoter interactions within nano-scale chromatin hubs.

genomics↗

bioRxiv2026

Self-organization of Drosophila chromatin architecture in a cell-free system

Metazoan genomes are organized by folding of the nucleosome fiber into loops and domains that support long-range regulatory interactions. Although cohesin-mediated loop extrusion and architectural DNA-binding proteins are central to current models of genome organization, how these mechanisms integrate to generate higher-order structure remains incompletely understood. Early Drosophila melanogaster embryogenesis provides a unique window into the emergence of chromatin architecture, as rapid syncytial nuclear divisions occur largely in the absence of transcription. However, probing the mechanisms underlying this primordial folding in vivo is technically challenging. Here, we establish an in vitro system that reconstitutes complex chromatin using extracts from syncytial embryos. Nucleosome mapping and Micro-C analyses reveal that long-range interactions, including loops and topologically associating domains (TADs), emerge spontaneously from soluble extract components. While some structures resemble those observed in early embryos, others represent latent interaction potentials that are constrained in vivo. Focusing on the eve locus, we find that TAD formation is incompatible with a simple loop extrusion model and instead requires direct pairing of boundary elements mediated by the insulator protein Suppressor-of-hairy-wing Su(Hw). Together, our work demonstrates that key features of 3D genome organization can be reconstituted in a cell-free system and provides a tractable platform for mechanistic dissection of chromatin folding in Drosophila.

genomics↗

bioRxiv2024

Senescent cells cluster CTCF on nuclear speckles to sustain their splicing program

Senescence --the endpoint of replicative lifespan for normal cells-- is established via a complex sequence of molecular events. One such event is the dramatic reorganization of CTCF into senescence-induced clusters (SICCs). However, the molecular determinants, genomic consequences, and functional purpose of SICCs remained unknown. Here, we combine functional assays, super-resolution imaging, and 3D genomics with computational modelling to dissect SICC emergence. We establish that the competition between CTCF-bound and non-bound loci dictates clustering propensity. Upon senescence entry, cells repurpose SRRM2 --a key component of nuclear speckles-- and BANF1 --a molecular glue for chromosomes-- to cluster CTCF and rewire genome architecture. This CTCF-centric reorganization in reference to nuclear speckles functionally sustains the senescence splicing program, as SICC disruption fully reverts alternative splicing patterns. We therefore uncover a new paradigm, whereby cells translate changes in nuclear biochemistry into architectural changes directing splicing choices so as to commit to the fate of senescence. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/603680v1_ufig1.gif" ALT="Figure 1"> View larger version (85K): org.highwire.dtl.DTLVardef@cfb561org.highwire.dtl.DTLVardef@8cfdf3org.highwire.dtl.DTLVardef@7485d5org.highwire.dtl.DTLVardef@1fe9051_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LIHMGB2-bound loci compete with CTCF-bound ones for nuclear speckle association C_LIO_LISenescent cells repurpose SRRM2 and BANF1 to cluster CTCF on speckles C_LIO_LIBANF1 is essential, but not sufficient for CTCF clustering C_LIO_LIThe SRRM2 RNA-binding domain directs CTCF clustering C_LIO_LISICCs rewire chromatin positioning to sustain the senescence splicing program C_LI

cell biology↗

bioRxiv2023

Active regulatory elements recruit cohesin to establish cell-specific chromatin domains.

As the structure of the genome is analysed at ever increasing resolution it is becoming clear that there is considerable variation in the 3D chromatin architecture across different cell types. It has been proposed that this may, in part, be due to increased recruitment of cohesin to activated cis-elements (enhancers and promoters) leading to cell-type specific loop extrusion underlying the formation of new subTADs. Here we show that cohesin correlates well with the presence of active enhancers and this varies in an allele-specific manner with the presence or absence of polymorphic enhancers which vary from one individual to another. Using the alpha globin cluster as a model, we show that when all enhancers are removed, peaks of cohesin disappear from these regions and the erythroid specific subTAD is no longer formed. Re-insertion of the major alpha globin enhancer (R2) is associated with the appearance of a new peak of cohesin at the site of insertion. In complementary experiments insertion of R2 into a "neutral" region of the genome recruits cohesin, induces transcription and creates a new large (75kb) erythroid specific domain. Together these findings support the proposal that active enhancers recruit cohesin, stimulate loop extrusion and promote the formation of cell specific subTADs.

genomics↗

bioRxiv2022

Multipartite super-enhancers function in an orientation-dependent manner

Transcriptional enhancers regulate gene expression in a developmental-stage and cell-specific manner. They were originally defined as individual regulatory elements that activate expression regardless of distance and orientation to their cognate genes. Genome-wide studies have shown that the mammalian enhancer landscape is much more complex, with different classes of individual enhancers and clusters of enhancer-like elements combining in additive, synergistic and redundant manners, possibly acting as single, integrated regulatory elements. These so-called super-enhancers are largely defined as clusters of enhancer-like elements which recruit particularly high levels of Mediator and often drive high levels of expression of key lineage-specific genes. Here, we analysed 78 erythroid-specific super-enhancers and showed that, as units, they preferentially interact in a directional manner, to drive expression of their cognate genes. Using the well characterised -globin super-enhancer, we show that inverting this entire structure severely downregulates -globin expression and activates flanking genes 5 of the super-enhancer. Our detailed genetic dissection of the -globin locus clearly attributes the clusters functional directionality to its sequence orientation, demonstrating that, unlike regular enhancers, super-enhancers act in an orientation-dependent manner. Together, these findings identify a novel emergent property of super-enhancers and revise current models by which enhancers are thought to contact and activate their cognate genes.

molecular biology↗

bioRxiv2019

Enhancers predominantly regulate gene expression in vivo via transcription initiation.

Gene transcription occurs via a cycle of linked events including initiation, promoter proximal pausing and elongation of RNA polymerase II (Pol II). A key question is how do transcriptional enhancers influence these events to control gene expression? Here we have used a new approach to quantify transcriptional initiation and pausing in vivo, while simultaneously identifying transcription start sites (TSSs) and pause-sites (TPSs) from single RNA molecules. When analyzed in parallel with nascent RNA-seq, these data show that differential gene expression is achieved predominantly via changes in transcription initiation rather than Pol II pausing. Using genetically engineered mouse models deleted for specific enhancers we show that these elements control gene expression via Pol II recruitment and/or initiation rather than via promoter proximal pause release. Together, our data show that enhancers, in general, control gene expression predominantly by Pol II recruitment and initiation rather than via pausing.

genomics↗

bioRxiv2017

A tissue-specific self-interacting chromatin domain forms independently of enhancer-promoter interactions

A variety of self-interacting domains, defined at different levels of resolution, have been described in mammalian genomes. These include Chromatin Compartments (A and B)1, Topologically Associated Domains (TADs)2,3, contact domains4,5, sub-TADs6, insulated neighbourhoods7 and frequently interacting regions (FIREs)8. Whereas many studies have found the organisation of self-interacting domains to be conserved across cell types389, some do form in a lineage-specific manner6710. However, it is not clear to what degree such tissue-specific structures result from processes related to gene activity such as enhancer-promoter interactions or whether they form earlier during lineage commitment and are therefore likely to be prerequisite for promoting gene expression. To examine these models of genome organisation in detail, we used a combination of high-resolution chromosome conformation capture, a newly-developed form of quantitative fluorescence in-situ hybridisation and super-resolution imaging to study a 70 kb self-interacting domain containing the mouse -globin locus. To understand how this self-interacting domain is established, we studied the region when the genes are inactive and during erythroid differentiation when the genes are progressively switched on. In contrast to many current models of long-range gene regulation, we show that an erythroid-specific, decompacted self-interacting domain, delimited by convergent CTCF/cohesin binding sites, forms prior to the onset of robust gene expression. Using previously established mouse models we show that formation of the self-interacting domain does not rely on interactions between the -globin genes and their enhancers. As there are also no tissue-specific changes in CTCF binding, then formation of the domain may simply depend on the presence of activated lineage-specific cis-elements driving a transcription-independent mechanism for opening chromatin throughout the 70 kb region to create a permissive environment for gene expression. These findings are consistent with a model of loop-extrusion in which all segments of chromatin, within a region delimited by CTCF boundary elements, can contact each other. Our findings suggest that activation of tissue-specific element(s)within such a self-interacting region is sufficient to influence all chromatin within the domain.

cell biology↗