Transcription-Driven Translocation of Cohesive and Non-Cohesive Cohesin In Vivo

Abstract

Cohesin is a central architectural element of chromosomes that regulates numerous DNA-based events. The complex holds sister chromatids together until anaphase onset and organizes individual chromosomal DNAs into loops and self-associating domains. Purified cohesin diffuses along DNA in an ATP-independent manner but can be propelled by transcribing RNA polymerase. In conjunction with a cofactor, the complex also extrudes DNA loops in an ATP-dependent manner. In this study we examine transcription-driven translocation of cohesin under various conditions in yeast. To this end, obstacles of increasing size were tethered to DNA to act as roadblocks to complexes mobilized by an inducible gene. The obstacles were built from a GFP-lacI core fused to one or more mCherries. A chimera with four mCherries blocked cohesin passage in late G1. During M phase, the threshold barrier depended on the state of cohesion: non-cohesive complexes were also blocked by four mCherries whereas cohesive complexes were blocked by as few as three mCherries. Furthermore cohesive complexes that were stalled at obstacles, in turn, blocked the passage of non-cohesive complexes. That synthetic barriers capture mobilized cohesin demonstrates that transcription-driven complexes translocate processively in vivo. Together, this study reveals unexplored limitations to cohesin movement on chromosomes.

Keywords:

• cohesin
• transcription
• sister chromatid cohesion
• loop extrusion
• Eco1
• Wpl1

SUPPLEMENTAL MATERIAL

Supplemental data for this article can be accessed online at https://doi.org/10.1080/10985549.2023.2199660.

ACKNOWLEDGEMENTS

We thank the following individuals for reagents: Jason Brickner (pAFS144-FFAT), Michael Knop (pMAM12, pMAM44 and pMAM88), Takehiko Kobayashi (strain YSI142 with MCD1-6xHIS-10xFLAG::kanMX), Kiran Madura (anti-Rad23), Adam Rudner (pTIR5), and Frank Uhlmann (YIplac128-GAL1p-Scc1(R180D,R268D)-3xHA (LEU2) and strain K5833 with scc1-73).

DATA AVAILABILITY STATEMENT

A book of Excel spreadsheets with the primary data for the figures in this study is available at Figshare https://doi.org/10.6084/m9.figshare.22551586.

Additional information

Funding

This work was funded by United States Public Health Service Grant [NIGMS 51402 (to M.R.G.)], New Jersey Commission on Cancer Research Predoctoral Research Fellowship [COCR22PRF006 (to P.K.)] and a Busch Biomedical Grant from Rutgers University (to M.R.G.). The Flow Cytometry Core Facility of Robert Wood Johnson Medical School and The Rutgers Cancer Institute of New Jersey is supported by grants from the National Cancer Institute [Grant P30ES005022] and NIH [Grant S10OD026876].