Chromatin affinity purification coupled with mass spectrometry indetifies novel histone ubiquitylation interactors

Abstract

Histone ubiquitylation is the post-translational covalent attachment of ubiquitin to histones. Ubiquitylation of histones regulates chromatin-templated processes through a combination of structural and functional effects. To date, several ubiquitylation sites have been mapped on all histones, including residues from the flexible histone tails and residues from the structured core domains. It is not yet clear if the different ubiquitylation marks are read separately of each other or if they act in synchrony. How ubiquitylation of histones is recognised by the nuclear proteome remains a standing question in the field of chromatin biochemistry. To address this question, we prepared site-specifically ubiquitylated histones by native chemical ligation and incorporated them into homogeneous nucleosomal arrays. Histone H2B ubiquitylated at lysine 120 (H2BK120ub), H2BK34ub and H3K23ub nucleosomal arrays were used in chromatin affinity purification experiments coupled with mass spectrometry to find the proteins that were enriched by ubiquitylation. We showed that the different ubiquitylation marks generally recruited unique proteins and protein complexes and that ubiquitylated histones needed to be embedded within nucleosomes for recognition by the nuclear proteome. Three separate ubiquitylated constructs were prepared for the N-terminal ubiquitylation of histone H3: H3K18ub, H3K23ub and H3K18/23ub2. On all templates, DNA methyltrasnferase I (DNMT1) bound directly the H3 ubiquitylation marks and recruited ubiquitin specific protease 7 (Usp7) and Sex comb on midleg-like 2 (SCML2) to the modified chromatin. We showed that Usp7 could remove all three H3 ubiquitylation marks. SCML2 stimulated Usp7's deubiquitylation activity when mononucleosomes and nucleosomal arrays were used as substrates. We showed that SCML2 competed with DNMT1 for Usp7 binding and proposed that SCML2 stimulated Usp7 by stabilising an activating conformation in the enzyme directly on chromatin. We suggest that SCML2 controls DNMT1 recycling from H3 ubiquitylated chromatin to insure faithful maintenance of DNA methylation across the genome. Our work establishes native chemical ligation as an efficient method to prepare site-specifically ubiquitylated histones and affirms chromatin affinity purification coupled with mass spectrometry as a reliable tool for identification of novel interactors of modified chromatin templates.