van den Berg, A. A. | Search bioRxiv

bioRxiv2025

Nucleosome unwrapping and PARP1 allostery drive affinities for chromatin and DNA breaks

Poly[ADP-ribose] polymerase 1 (PARP1) detects DNA strand breaks that occur in duplex DNA and chromatin. We employed correlative optical tweezers and fluorescence microscopy to quantify how single molecules of PARP1 identify single-strand breaks (i.e., nicks), undamaged nucleosome core particles (NCP) and NCPs containing DNA nicks. Fluorescently-tagged PARP1 or PARP2 from nuclear extracts bound nicks with nanomolar affinity but did not engage undamaged dsDNA regions. In contrast, PARP1 avidly bound undamaged NCPs, and partial NCP unwrapping induced by DNA tension significantly increased the on rate and affinity. Catalytically dead PARP1 or EB-47 inhibition greatly increased PARP1 affinity to DNA nicks and undamaged NCP, implicating a mechanism where PARP1 reverse allostery regulates PARP1 retention to undamaged chromatin. We also monitored ADP-ribosylation in real time upon PARP1 binding undamaged or nicked NCPs. These results provide key mechanistic insights into domain allostery and how pharmacological intervention alters PARP1 binding dynamics for therapeutic impacts.

biophysics↗

bioRxiv2022

Transcription shapes 3D chromatin organization by interacting with loop-extruding cohesin complexes

Cohesin folds mammalian interphase chromosomes by extruding the chromatin fiber into numerous loops. "Loop extrusion" can be impeded by chromatin-bound factors, such as CTCF, which generates characteristic and functional chromatin organization patterns. It has been proposed that transcription relocalizes or interferes with cohesin, and that active promoters are cohesin loading sites. However, the effects of transcription on cohesin have not been reconciled with observations of active extrusion by cohesin. To determine how transcription modulates extrusion, we studied mouse cells in which we could alter cohesin abundance, dynamics, and localization by genetic knockouts of the cohesin regulators CTCF and Wapl. Through Hi-C experiments, we discovered intricate, cohesin-dependent contact patterns near active genes. Chromatin organization around active genes exhibited hallmarks of interactions between transcribing RNA polymerases (RNAPs) and extruding cohesins. These observations could be reproduced by polymer simulations in which RNAPs were "moving barriers" to extrusion that obstructed, slowed, and pushed cohesins. The simulations predicted that preferential loading of cohesin at promoters is inconsistent with our experimental data. Additional ChIP-seq experiments showed that the putative cohesin loader Nipbl is not predominantly enriched at promoters. Therefore, we propose that cohesin is not preferentially loaded at promoters and that the barrier function of RNAP accounts for cohesin accumulation at active promoters. Altogether, we find that RNAP is a new type of extrusion barrier that is not stationary, but rather, translocates and relocalizes cohesin. Loop extrusion and transcription might interact to dynamically generate and maintain gene interactions with regulatory elements and shape functional genomic organization. Significance StatementLoop extrusion by cohesin is critical to folding the mammalian genome into loops. Extrusion can be halted by CTCF proteins bound at specific genomic loci, which generates chromosomal domains and can regulate gene expression. However, the process of transcription itself can modulate cohesin, thus refolding chromosomes near active genes. Through experiments and simulations, we show that transcribing RNA polymerases (RNAPs) act as "moving barriers" to loop-extruding cohesins. Unlike stationary CTCF barriers, RNAPs actively relocalize cohesins, which generates characteristic patterns of spatial organization around active genes. Our model predicts that the barrier function of RNAP can explain why cohesin accumulates at active promoters and provides a mechanism for clustering active promoters. Through transcription-extrusion interactions, cells might dynamically regulate functional genomic contacts.

genomics↗

bioRxiv2019

Chromosome organization by one-sided and two-sided loop extrusion

SMC complexes, such as condensin or cohesin, organize chromatin throughout the cell cycle by a process known as loop extrusion. SMC complexes reel in DNA, extruding and progressively growing DNA loops. Modeling assuming two-sided loop extrusion reproduces key features of chromatin organization across different organisms. In vitro single-molecule experiments confirmed that yeast condensins extrude loops, however, they remain anchored to their loading sites and extrude loops in a "one-sided" manner. We therefore simulate one-sided loop extrusion to investigate whether "one-sided" complexes can compact mitotic chromosomes, organize interphase domains, and juxtapose bacterial chromosomal arms, as can be done by "two-sided" loop extruders. While one-sided loop extrusion cannot reproduce these phenomena, variants can recapitulate in vivo observations. We predict that SMC complexes in vivo constitute effectively two-sided motors or exhibit biased loading and propose relevant experiments. Our work suggests that loop extrusion is a viable general mechanism of chromatin organization. Impact statementWe reconcile seemingly contradictory findings of single-molecule and in vivo experiments on a major mechanism of chromosome organization by computationally investigating mechanisms of loop extrusion that are consistent with both.

biophysics↗