In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

Louis Masclef; Uriarte Maxime; Oumaima Ahmed; Nadine Sen Nkwe; Haithem Barbour; Nicholas  V.G. Iannantuono; Amina Boubekeur; Salima Daou; El Bachir Affar

doi:10.3791/59385

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Method Article

In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones

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DOI:

10.3791/59385

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July 25th, 2019

Louis Masclef^*¹ , Uriarte Maxime^*¹ , Oumaima Ahmed¹ , Nadine Sen Nkwe¹ , Haithem Barbour¹ , Nicholas V.G. Iannantuono¹ , Amina Boubekeur¹ , Salima Daou¹^,² , El Bachir Affar¹^,²

¹Maisonneuve-Rosemont Hospital Research Center and Department of Medicine, University of Montréal, ²Lunenfeld-Tanenbaum Research Institute, Sinai Health System

^* These authors contributed equally

Summary

Ubiquitination is a post-translational modification that plays important roles in cellular processes and is tightly coordinated by deubiquitination. Defects in both reactions underlie human pathologies. We provide protocols for conducting ubiquitination and deubiquitination reaction in vitro using purified components.

Abstract

Ubiquitination is a post-translational modification that plays important roles in various signaling pathways and is notably involved in the coordination of chromatin function and DNA-associated processes. This modification involves a sequential action of several enzymes including E1 ubiquitin-activating, E2 ubiquitin-conjugating and E3 ubiquitin-ligase and is reversed by deubiquitinases (DUBs). Ubiquitination induces degradation of proteins or alteration of protein function including modulation of enzymatic activity, protein-protein interaction and subcellular localization. A critical step in demonstrating protein ubiquitination or deubiquitination is to perform in vitro reactions with purified components. Effective ubiquitination and deubiquitination reactions could be greatly impacted by the different components used, enzyme co-factors, buffer conditions, and the nature of the substrate. Here, we provide step-by-step protocols for conducting ubiquitination and deubiquitination reactions. We illustrate these reactions using minimal components of the mouse Polycomb Repressive Complex 1 (PRC1), BMI1, and RING1B, an E3 ubiquitin ligase that monoubiquitinates histone H2A on lysine 119. Deubiquitination of nucleosomal H2A is performed using a minimal Polycomb Repressive Deubiquitinase (PR-DUB) complex formed by the human deubiquitinase BAP1 and the DEUBiquitinase ADaptor (DEUBAD) domain of its co-factor ASXL2. These ubiquitination/deubiquitination assays can be conducted in the context of either recombinant nucleosomes reconstituted with bacteria-purified proteins or native nucleosomes purified from mammalian cells. We highlight the intricacies that can have a significant impact on these reactions and we propose that the general principles of these protocols can be swiftly adapted to other E3 ubiquitin ligases and deubiquitinases.

Introduction

Ubiquitination is one of the most conserved post-translational modifications and is critical for a wide variety of organisms including yeast, plants and vertebrates. Ubiquitination consists of the covalent attachment of ubiquitin, a highly conserved 76 amino acid polypeptide, to target proteins and occurs in three sequential steps involving three enzymes, i.e., E1-activating, E2-conjugating and E3 ligase¹^,²^,³. This post-translational modification plays central roles in a wide spectrum of biological processes. Indeed, the E3 ligases, which provide the specificity of the reacti....