Structural Basis of Eco1-Mediated Cohesin Acetylation

www.nature.com/scientificreports OPEN received: 28 September 2016 accepted: 06 February 2017 Published: 14 March 2017 Structural Basis of Eco1-Mediated Cohesin Acetylation William C. H. Chao1,*, Benjamin O. Wade1,*, Céline Bouchoux2, Andrew W. Jones3, Andrew G. Purkiss4, Stefania Federico5, Nicola O’Reilly5, Ambrosius P. Snijders3, Frank Uhlmann2 & Martin R. Singleton1 Sister-chromatid cohesion is established by Eco1-mediated acetylation on two conserved tandem lysines in the cohesin Smc3 subunit. However, the molecular basis of Eco1 substrate recognition and acetylation in cohesion is not fully understood. Here, we discover and rationalize the substrate specificity of Eco1 using mass spectrometry coupled with in-vitro acetylation assays and crystallography. Our structures of the X. laevis Eco2 (xEco2) bound to its primary and secondary Smc3 substrates demonstrate the plasticity of the substrate-binding site, which confers substrate specificity by concerted conformational changes of the central β hairpin and the C-terminal extension. The topological entrapment of chromosomes by cohesin (Smc1, Smc3, Scc1, and Scc3) is central to genome integrity1–3. Cohesin is loaded onto chromatin by the Scc2-Scc4 loader complex4–6, however persistent cohesion cannot be established without the acetylation of two conserved tandem lysines on the Smc3 subunit by the Eco1 acetyltransferase (ACT)7–10. Cohesin release from DNA relies on the interaction between DNA and these conserved lysines11. Acetylation of these lysines antagonizes Wapl-mediated cohesin release by blocking DNA-induced ATP hydrolysis and the dissociation of the Smc3/Scc1 interface, thus locking cohesin onto chromatin11–15. Eco1 is an acetyltransferase that belongs to the Gcn5-related N-acetyltransferase (GNAT) family16,17. Unlike other GNAT family members, Eco1 contains a zinc-finger (ZnF) domain that is similar to those found in the Moz/Ybfs/Sas2/Tip60 (MYST) family of histone acetyltransferases (HATs). The ZnF is responsible for HATs recognizing nucleosome in histone tail acetylation18–20, while in Eco1, the ZnF enhances its acetyltransferase activity during sister chromatid cohesion21. Its S-phase localization to the replication fork is thought to be via a direct interaction with PCNA22–24. As well as its function in establishing replication-coupled cohesion, Eco1 has also been proposed to act during double-strand break repair. Break-induced phosphorylation of Scc1 is thought to trigger its Eco1-mediated acetylation, which also antagonizes Wapl releasing activity 25. Furthermore, Eco1 is regulated through Cdk1-mediated phosphorylation, which promotes its SCFCdc4-dependent ubiquitination and degradation after S phase26. Interestingly, the recruitment of xEco2, the primary cohesin ACT in Xenopus laevis, onto chromatin was dependent on the pre-RC assembly but independent of cohesin loading and DNA synthesis27. Like cohesin and its regulatory subunits, Eco1 homologues also have important implications in human developmental disorders28,29. Human Esco1 (hEsco1) is enriched at sites occupied by cohesin and CTCF, whereas human Esco2 is targeted to genes controlled by RE-1 silencing transcription factor (REST), implying the cohesin ACTs’ roles in gene regulation30. In fact, the loss of human Esco2 results in SC Phocomelia and Roberts Syndrome (RBS)31,32, while in yeast a RBS mutant equivalent of Eco1 reduces ribosomal DNA (rDNA) transcription33. Despite the importance of acetylation in cohesion establishment and human diseases, little is known about how Eco1 targets its canonical substrate Smc3 and how this acetylation stabilizes cohesin on DNA. To provide further insights into substrate recognition by Eco1, we reconstitute an in-vitro acetylation assay and coupled it with mass spectrometry (MS) analyses to determine both the dynamics and substrate specificity of Eco1-mediated acetylation. In our assays, we observe a fast acetylation event of K112 occurring prior to the acetylation of K113. By aligning the sequences of different acetylated cohesin peptides, we show that the target lysine of acetylation is 1 Structural Biology of Chromosome Segregation Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK. 2Chromosome Segregation Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK. 3Protein Analysis and Proteomics Platform, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK. 4 Structural Biology Platform, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK. 5Peptide Chemistry Laboratory, The Francis Crick Institute, 1 Midland Road, London NW1 1AT, UK. *These authors contributed equally to this work. Correspondence and requests for materials should be addressed to M.R.S. (email: martin.singleton@ crick.ac.uk) Scientific Reports | 7:44313 | DOI: 10.1038/srep44313 1 www.nature.com/scientificreports/ favoured by the flanking of an aliphatic residue and an acidic/polar residue, and that this motif can be recognized in a bidirectional fashion. Furthermore, we present the crystal structures of Xenopus laevis Eco2 (xEco2) ACT domain bound to two different substrate peptides, which reveal that Eco1 substrate specificity is determined by the concerted conformational changes of the conserved central βhairpin and the C-terminal extension (C extension). Results and Discussion S. cerevisiae Eco1 Acetylates Smc3 K112 Prior to K113. Previous studies in yeast have shown that Eco1-mediated acetylation of the conserved tandem lysine motif (S. cerevisiae K112/K113; human K105/106) in Smc3 (Fig. 1A) is important in establishing sister-chromatid cohesion during DNA replication7,9. In human Smc3, a non-acetylatable human K106R mutation, but not K105R, of the tandem lysine motif was the primary cause of sister chromatid cohesion loss8. To study the relative importance of the two conserved lysines, we reconstituted an in-vitro Eco1-mediated acetylation time-course assay using recombinantly purified S. cerevisiae cohesin and Eco1 (Fig. 1B). Reaction products were separated by SDS-PAGE, with Smc3-containing gel bands subsequently subjected to an in-gel reductive di-methylation step34. As a result of the enzymatic and chemical reactions, K112 and K113 were either acetylated or di-methylated at all time points. This strategy facilitated our analysis since K112 and K113 are blocked for trypsin proteolysis and hence the peptide TVGLKKDDYQLNDR could be consistently extracted from the gel prior to mass spectrometry analysis. A Parallel Reaction Monitoring (PRM) mass spectrometry experiment was used to target three different peptide masses corresponding to the four different variants of the target peptide. To distinguish the isobaric variants acK112-meK113 and meK112-acK113 the intensities of the y9 and b5 ion were monitored in the Higher Energy Collisional Dissociation (HCD) fragmentation spectra (Supplementary Fig. S1B and S1C)35. In the same run, we also measured the peptide sign (...truncated)