Related Experiment Video
Updated: Jun 7, 2025
In Vitro Ubiquitination and Deubiquitination Assays of Nucleosomal Histones
Published on: July 25, 2019
An E2 ubiquitin-conjugating enzyme links diubiquitinated H2B to H3K27M oncohistone function
Alan L Jiao1,2, Erdem Sendinc2, Barry M Zee2
1Ludwig Institute for Cancer Research, Nuffield Department of Medicine, University of Oxford, Oxford OX3 7DQ, United Kingdom.
Abstract:
The H3K27M oncogenic histone (oncohistone) mutation drives ~80% of incurable childhood brain tumors known as diffuse midline gliomas (DMGs). The major molecular feature of H3K27M mutant DMGs is a global loss of H3K27 trimethylation (H3K27me3), a phenotype conserved in Caenorhabditis elegans (C. elegans). Here, we perform unbiased genome-wide suppressor screens in C. elegans expressing H3K27M and isolate 20 suppressors, all of which at least partially restore H3K27me3. 19/20 suppressor mutations map to the same histone H3.3 gene in which the K27M mutation was originally introduced. Most of these create single amino acid substitutions between residues R26-Y54, which do not disrupt oncohistone expression. Rather, they are predicted to impair interactions with the Polycomb Repressive Complex 2 (PRC2) and are functionally conserved in human cells. Further, we mapped a single extragenic H3K27M suppressor to ubc-20, an E2 ubiquitin-conjugating enzyme, whose loss rescued H3K27me3 to nearly 50% wild-type levels despite continued oncohistone expression and chromatin incorporation. We demonstrate that ubc-20 is the major enzyme responsible for generating diubiquitinated histone H2B. Our study provides in vivo support for existing models of PRC2 inhibition via direct oncohistone contact and suggests that the effects of H3K27M may be modulated by H2B ubiquitination.
Insights
Researchers identified genetic suppressors of the H3K27M oncohistone mutation in C. elegans, revealing mechanisms to restore H3K27me3 levels in diffuse midline gliomas (DMGs). This work offers new therapeutic targets for childhood brain tumors.
Area of Science:
- Epigenetics and Cancer Biology
- Developmental Biology and Genetics
Background:
- The H3K27M oncohistone mutation drives diffuse midline gliomas (DMGs), characterized by a global loss of H3K27 trimethylation (H3K27me3).
- This H3K27M phenotype is conserved in model organisms like *Caenorhabditis elegans* (*C. elegans*), providing a platform for genetic screening.
Purpose of the Study:
- To identify genetic suppressors of the H3K27M oncohistone mutation in *C. elegans*.
- To elucidate the molecular mechanisms underlying the restoration of H3K27me3 levels in the presence of the H3K27M mutation.
Main Methods:
- Genome-wide suppressor screens were performed in *C. elegans* expressing the H3K27M oncohistone.
- Suppressor mutations were mapped to specific genes, and their effects on H3K27me3 levels and protein interactions were analyzed.
- Functional conservation of identified suppressors in human cells was assessed.
Main Results:
- Twenty suppressors were isolated, all partially restoring H3K27me3 levels.
- Nineteen suppressors mapped to the histone H3.3 gene, causing amino acid substitutions predicted to impair Polycomb Repressive Complex 2 (PRC2) interactions.
- One extragenic suppressor, *ubc-20* (an E2 ubiquitin-conjugating enzyme), was identified, which rescued H3K27me3 by inhibiting H2B ubiquitination.
Conclusions:
- In vivo findings support models of PRC2 inhibition by direct oncohistone contact.
- Modulation of H2B ubiquitination by enzymes like UBC-20 presents a potential therapeutic strategy for H3K27M-driven diffuse midline gliomas.
Related Concept Videos
02:04Covalently Linked Protein Regulators
These groups modify specific amino acids in a protein....
02:32Histone Modification
Acetylation
The enzyme histone acetyltransferase adds acetyl group to the histones. Another enzyme, histone...
02:58Regulated Protein Degradation
Protein degradation plays two important roles in the cells. It helps to protect cells from misfolded or damaged proteins before they lead to a...
00:50Anaphase Promoting Complex
02:25Spreading of Chromatin Modifications
Writers
The writer...
01:13The Proteasome
In this pathway, the target proteins are first tagged with small proteins called ubiquitin. This involves participation of a series of enzymes including— E1 (ubiquitin-activating enzyme), E2 (ubiquitin-conjugating enzyme), and E3...