Biology subjects

Pustova, I.

Publications and source records attributed to Pustova, I..

3 recordsLinked to original sources

bioRxiv2018

An RNA-binding region regulates CTCF clustering and chromatin looping

Mammalian genomes are folded into Topologically Associating Domains (TADs), consisting of cell-type specific chromatin loops anchored by CTCF and cohesin. Since CTCF and cohesin are expressed ubiquitously, how cell-type specific CTCF-mediated loops are formed poses a paradox. Here we show RNase-sensitive CTCF self-association in vitro and that an RNA-binding region (RBR) mediates CTCF clustering in vivo. Intriguingly, deleting the RBR abolishes or impairs almost half of all chromatin loops in mouse embryonic stem cells. Disrupted loop formation correlates with abrogated clustering and diminished chromatin binding of the RBR mutant CTCF protein, which in turn results in a failure to halt cohesin-mediated extrusion. Thus, CTCF loops fall into at least 2 classes: RBR-independent and RBR-dependent loops. We suggest that evidence for distinct classes of RBR-dependent loops may provide a mechanism for establishing cell-specific CTCF loops regulated by RNAs and other RBR partner.

biophysics↗

bioRxiv2018

Architectural Features of 3D Genome Organization Revealed by Counting CTCF and Cohesin Molecules

Achieving a quantitative and predictive understanding of 3D genome architecture remains a major challenge, as it requires quantitative measurements of the key proteins involved. Here we report the quantification of CTCF and cohesin, two causal regulators of topologically associating domains (TADs) in mammalian cells. Extending our previous imaging studies (Hansen et al., 2017), we estimate bounds on the density of putatively DNA loop-extruding cohesin complexes and CTCF binding site occupancy. Furthermore, co-immunoprecipitation studies of an endogenously tagged subunit (Rad21) suggest the presence of cohesin dimers and/or oligomers. Finally, based on our cell lines with accurately measured protein abundances, we report a method to conveniently determine the number of molecules of any Halo-tagged protein in the cell. We anticipate that our results and the established tool for measuring cellular protein abundances will advance a more quantitative understanding of 3D genome organization, and facilitate protein quantification, key to comprehend diverse biological processes.

biophysics↗

bioRxiv2016

CTCF and Cohesin Regulate Chromatin Loop Stability with Distinct Dynamics

Abstract/summaryFolding of mammalian genomes into spatial domains is critical for gene regulation. CTCF and cohesin control domain location by folding domains into loop structures, which are thought to be highly stable. Combining genomic, biochemical and single-molecule imaging approaches, we show that although CTCF and cohesin can physically interact, CTCF binds chromatin much more dynamically than cohesin (~1 min vs. ~22 min residence time). Moreover, after unbinding, CTCF quickly rebinds another cognate site unlike cohesin (~1 min vs. ~33 min). Thus, CTCF and cohesin form a rapidly exchanging \"dynamic complex\" rather than a typical stable complex. Since CTCF and cohesin are required for loop domain formation, our results suggest that chromatin loops constantly break and reform throughout the cell cycle.

biophysics↗