The cohesin modifier ESCO2 is stable during DNA replication

Chromosome Res (2023) 31:6 https://doi.org/10.1007/s10577-023-09711-1 RESEARCH The cohesin modifier ESCO2 is stable during DNA replication Allison M. Jevitt · Brooke D. Rankin Jingrong Chen · Susannah Rankin · Received: 25 August 2022 / Revised: 1 November 2022 / Accepted: 13 December 2022 © The Author(s) 2023 Abstract Cohesion between sister chromatids by the cohesin protein complex ensures accurate chromosome segregation and enables recombinational DNA repair. Sister chromatid cohesion is promoted by acetylation of the SMC3 subunit of cohesin by the ESCO2 acetyltransferase, inhibiting cohesin release from chromatin. The interaction of ESCO2 with the DNA replication machinery, in part through PCNA-interacting protein (PIP) motifs in ESCO2, is required for full cohesion establishment. Recent reports have suggested that Cul4-dependent degradation regulates the level of ESCO2 protein following replication. To follow up on these observations, we have characterized ESCO2 stability in Xenopus egg extracts, a cell-free system that recapitulates cohesion establishment in vitro. We found that ESCO2 was stable during DNA replication in this system. Indeed, further challenging the system Supplementary Information The online version contains supplementary material available at https://doi. org/10.1007/s10577-023-09711-1. Responsible Editor: Beth Sullivan A. M. Jevitt · J. Chen · S. Rankin Cell Cycle and Cancer Biology Program, Oklahoma Medical Research Foundation, Oklahoma City, OK 73104, USA B. D. Rankin · S. Rankin (*) Department of Cell Biology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA e-mail: by inducing DNA damage signaling or increasing the number of nuclei undergoing DNA replication had no significant impact on the stability of ESCO2. In transgenic somatic cell lines, we also did not see evidence of GFP-ESCO2 degradation during S phase of the cell cycle using both flow cytometry and live-cell imaging. We conclude that ESCO2 is stable during DNA replication in both embryonic and somatic cells. Keywords Chromosome cohesion · DNA replication · ESCO2 · E3 ubiquitin ligase · Xenopus laevis egg extract · Cell cycle Abbreviations APC Anaphase promoting complex Cdh1 Cdc20 homolog 1 Cdt1 Chromatin licensing and DNA replication factor 1 Chk1 Checkpoint kinase 1 CRL4 Cullin-RING ubiquitin ligase complex 4 CSF Cytostatic factor CUL4 Cullin 4 DCAF1 DDB1 and CUL4 associated factor 1 DDB1 Damage specific DNA binding protein 1 Dox Doxycycline Eco1p Establishment of cohesion protein 1 ESCO1 Establishment of cohesion 1 ESCO2 Establishment of cohesion 1 homolog 2 FoxM1 Forkhead box M1 MCM Minichromosome maintenance NF2 Neurofibromatosis type 2 Vol.: (0123456789) 13 6 Page 2 of 15 ORC2 Origin recognition complex subunit 2 PCNA Proliferating cell nuclear antigen PIP PCNA-interacting protein SAMHD1 SAM and HD domain containing deoxynucleoside triphosphate triphosphohydrolase 1 SMC3 Structural maintenance of chromosomes 3 VprBP Viral protein R binding protein WAPL Wings apart-like protein homolog Introduction The tethering together of sister chromatids during DNA replication depends in part on acetylation of the SMC3 subunit of cohesin, which renders the complex resistant to removal from chromatin by the WAPL protein (Unal et al. 2008; Zhang et al. 2008; Sutani et al. 2009). In vertebrates, SMC3 acetylation is achieved by one of two related acetyltransferase enzymes, ESCO1 and ESCO2 (Hou and Zou 2005). Using the Xenopus egg extract system, we previously showed that ESCO1 is developmentally regulated and not present at functional levels until after zygotic transcription begins (Lafont, Song, and Rankin 2010). In egg extracts, therefore, ESCO2 is the sole cohesin acetyltransferase required for cohesion between sister chromatids, and depletion of ESCO2 from egg extract results in significant loss of cohesion (Song et al. 2012; Lafont, Song, and Rankin 2010). Multiple reports suggest cell cycle-dependent fluctuations in ESCO2 protein levels, although there are conflicting reports about the precise timing. Some reports indicate that ESCO2 levels peak during S phase (Minamino et al. 2018) and are thus low prior to mitotic entry, while others have suggested that ESCO2 is degraded during M phase (Lelij et al. 2009; Hou and Zou 2005). ESCO2 has also been reported to be stabilized by interaction with the MCM helicase during replication licensing, suggesting a third, perhaps indirect, level of stability control (Minamino et al. 2018; Bender et al. 2019; Ivanov et al. 2018). ESCO2 protein levels are controlled at least in part by ubiquitin-dependent proteolysis (Lafont, Song, and Rankin 2010). The anaphase-promoting complex (APC) is an E3 ubiquitin ligase that has numerous substrates, including some that are degraded at mitotic exit, and others that continue to be recognized through G1 Vol:. (1234567890) 13 Chromosome Res (2023) 31:6 (Davey and Morgan 2016). As in other APC targets, a degron sequence in ESCO2 mediates recognition and modification by the APC when it is bound to the G1 specificity factor called Cdh1 (Visintin, Prinz, and Amon 1997; Davey and Morgan 2016; Lafont, Song, and Rankin 2010). Mutation of this sequence stabilizes ESCO2, preventing its degradation in the presence of active APCCdh1 (Lafont, Song, and Rankin 2010). It has been suggested that degradation of ESCO2 is also controlled by a second E3 ubiquitin ligase, the CUL4-DDB1 complex via the specificity factor DCAF1 (DDB1 and CUL4 associated factor 1, also called VprBP), resulting in post-replicative degradation prior to M phase (Minamino et al. 2018). Together, these reports suggest an interesting dual regulation of ESCO2 by proteolysis: in G1 by the APC, and during S phase by the CUL4-DDB1-DCAF1VprBP complex. To better understand the regulation of ESCO2 protein turnover, we set out to identify the degron that might mediate recognition of ESCO2 by CUL4DDB1-DCAF1VprBP. To this end, we analyzed ESCO2 stability, utilizing the Xenopus egg extract system, which is a powerful tool to investigate CUL4-dependent mechanisms (Jin et al. 2006; Arias and Walter 2005; Arias and Walter 2006; Havens et al. 2012; Havens and Walter 2009). Our results indicate that ESCO2 is stable during DNA replication in the egg extract system. We also tested ESCO2 stability in cultured somatic cells, where we saw no evidence of degradation after G1 phase of the cell cycle. Our data suggest that accumulation of ESCO2 in the absence of CUL4-DDB1-DCAF1VprBP seen previously may occur through indirect mechanisms. Results Extracts prepared from the eggs of the frog Xenopus laevis are stockpiled with sufficient proteins for the replication of thousands of nuclei per microliter, making this system ideal for the study of DNA replication-dependent events in vitro (Jevitt and Rankin 2022; Rankin 2019). Demembranated sperm heads added to the extract are assembled into nuclei through the recruitment of me (...truncated)