A conserved H3K14ub-driven H3K9me3 for chromatin compartmentalization
Article Date: 15 October 2025
Article URL: https://www.nature.com/articles/s41586-025-09624-5
Article Title: A conserved H3K14ub-driven H3K9me3 for chromatin compartmentalization
Article Image: (not provided)
Summary
This Nature paper identifies G2E3 as an H3K14-specific E3 ubiquitin ligase whose catalysed H3K14ub promotes H3K9 trimethylation (H3K9me3) and is essential for pericentromeric heterochromatin formation and proper chromatin compartmentalisation. The authors use a combination of specific H3K14ub antibodies, in vitro ubiquitination assays, CRISPR knockouts, immunofluorescence, ChIP–seq and RNA‑seq to show that loss of G2E3 reduces pericentromeric H3K9me3, HP1 and SUV39H recruitment, redistributes H3K9me3 to euchromatin and increases repeat transcripts. Mechanistic data indicate SUV39H1/2 N-terminal chromodomain regions bind H3K14ub and that H3K14ub potentiates SUV39H activity; the process is cell-cycle regulated and RNA-dependent. Datasets are available at GEO (GSE202148 and GSE202149) and source data and code references are provided in the paper.
Key Points
- G2E3 is identified as an H3K14 mono‑ubiquitin E3 ligase that specifically modifies histone H3 at lysine 14 (H3K14ub).
- H3K14ub promotes H3K9me3 by recruiting and potentiating SUV39H methyltransferases, aiding pericentromeric heterochromatin assembly.
- Knockout or knockdown of G2E3 leads to reduced pericentromeric H3K9me3, HP1α/β and SUV39H levels, plus mislocalisation of these factors to euchromatin and elevated repeat transcripts.
- Rescue experiments show wildtype G2E3, but not an E3‑inactive mutant, restores H3K14ub and H3K9me3—demonstrating catalytic activity is required.
- SUV39H1/2 N‑terminal chromodomain regions can bind H3K14ub; H3K14ub acts synergistically with existing H3K9me3 to nucleate heterochromatin during mitotic exit.
- G2E3 association with pericentromeric chromatin is RNA‑dependent and G2E3 levels peak in G2/M, linking cell‑cycle dynamics to heterochromatin reassembly.
- Genome‑wide ChIP–seq shows redistribution of H3K9me3 in G2E3 KO cells, indicating a role in global chromatin compartmentalisation.
Content summary
The authors first generated and validated a highly specific H3K14ub antibody and used it to screen for candidate E3 ligases. G2E3 emerged as a robust H3K14 ubiquitin ligase (validated in vitro and in cells). Mutation of G2E3 PHD domains abolished H3K14ub activity while HECT domain alterations did not, pinpointing required structural elements.
Loss‑of‑function (shRNA and CRISPR KO) experiments in multiple cell lines show that G2E3 deficiency slows proliferation, causes mitotic defects and markedly reduces H3K9me3 at pericentromeric regions. Immunofluorescence, fractionation and rescue assays demonstrate that re‑expression of wildtype G2E3 restores H3K14ub and H3K9me3, whereas an E3‑dead mutant does not.
Mechanistically, SUV39H1/2 N‑terminal chromodomain regions mediate binding to H3K14ub; structural predictions and peptide pulldown data support this. The authors propose a model in which pericentromeric transcripts recruit G2E3 in G2/M, G2E3 deposits H3K14ub, which recruits and activates SUV39H to re‑establish H3K9me3 and then HP1 binding, thereby reassembling pericentromeric heterochromatin after S phase. Genome‑wide ChIP–seq and RNA‑seq corroborate redistribution of H3K9me3 and derepression of repeats in G2E3 KO cells.
All sequencing data are publicly available (GSE202148, GSE202149); software used is listed in Methods and publicly accessible. The paper includes extensive extended data supporting antibody specificity, enzyme specificity, cell biology, and genomic analyses.
Context and relevance
This work connects two histone post‑translational marks—H3K14 ubiquitination and H3K9 trimethylation—in a conserved pathway that controls heterochromatin nucleation and maintenance. H3K9me3 is a central heterochromatin mark tied to genome stability, repeat silencing and nuclear organisation; showing that H3K14ub acts upstream to recruit and potentiate SUV39H adds an important layer to our understanding of chromatin compartmentalisation and epigenetic inheritance.
For those following chromatin biology, epigenetic regulation of repeats, cell‑cycle reassembly of chromatin, or histone PTM crosstalk, this paper is highly relevant. It also uses modern tools (specific PTM antibodies, CRISPR KOs, ChIP–seq/RNA‑seq, AlphaFold‑based structural insight) that echo current trends in the field.
Why should I read this?
Short answer: if you care about how heterochromatin gets rebuilt after replication or why repeats stay silent, this paper gives you a neat mechanism — H3K14ub by G2E3 helps set up H3K9me3 and pulls SUV39H/HP1 back to pericentromeres. It’s dense with useful assays and genome‑wide data, so you can nick methods or ideas. We skimmed it and pulled out the essentials so you don’t have to read every extended figure unless you want the nitty‑gritty.
Author style
Punchy: the authors deliver a clear mechanistic advance with solid multi‑level evidence. This is not incremental—identifying a conserved E3 ligase (G2E3) that functionally links H3K14 ubiquitination to H3K9me3 and chromatin compartmentalisation is a significant addition to epigenetics. If your work touches heterochromatin, chromatin dynamics in the cell cycle, or histone code crosstalk, the detailed experiments and datasets here are worth close attention.