How can a single genome generate specialized cells at the correct time and place in multicellular organisms? Gene-expression programs that are specific to different cell types are regulated by the addition and removal of chemical groups, known as epigenetic marks, to and from chromatin — the compact protein–DNA complex in which the genome is organized. Two papers in Nature Communications (one by Ragazzini et al.1 and the other by Jain et al.2), together with recent studies3–5, show how a protein called EZHIP can regulate and disrupt the addition of a major epigenetic mark by the Polycomb repressive complex 2 (PRC2) during certain biological events and in a type of cancer.

The Polycomb protein complexes are key epigenetic modulators that act to repress gene expression6. Mammalian PRC2 contains a core complex consisting of several subunits, including either EZH1 or EZH2 as an enzyme subunit7. The core complex can interact with various accessory subunits that influence its enzymatic activity and direct it to specific regions of the genome.

The major substrate of PRC2 is a nucleosome: a complex of eight histone proteins, around which DNA is wrapped; nucleosomes are the basic building blocks of chromatin. PRC2 adds a trimethyl group (me3) to a specific amino-acid residue of the H3 histone (lysine 27; abbreviated as K27), generating an epigenetic mark termed H3K27me3 (Fig. 1a). This modification occurs initially at certain sites and promotes the passage of PRC2 across adjacent regions of the chromatin8.

Figure 1 | The protein EZHIP inhibits the enzymatic activity of the Polycomb repressive complex 2 (PRC2). a, A nucleosome comprises DNA wrapped around a histone protein complex. PRC2 is a group of associated proteins that adds a trimethyl (me3) group to a lysine amino-acid residue (K27) in the histone protein H3, generating the H3K27me3 mark (green). H3K27me3 represses the expression of nearby genes, and further activates PRC2, leading to an increase in H3K27me3 throughout the genomic region. b, A mutant form of H3 known as H3K27M, in which K27 is substituted by a methionine residue, inhibits EZH2, the enzyme subunit of PRC2, leading to reductions in H3K27me3. H3K27M drives a form of brain cancer called diffuse intrinsic pontine glioma (DIPG). c, The protein EZHIP contains an amino-acid sequence that is highly similar to that containing the substituted methionine in H3K27M (the K27M-like peptide; KLP), and Ragazzini et al.1 and Jain et al.2 show that EZHIP’s KLP inhibits EZH2. EZHIP expression is upregulated in a brain cancer called posterior fossa ependymoma group A (PFA)2, but is also needed for germ-cell maturation and fertility in female mice1.

The distribution of the H3K27me3 mark on nucleosomes throughout the genome changes drastically during certain biological events, including in sperm and egg cells when they mature, and in fertilized egg cells9. However, the molecular mechanisms that regulate this redistribution, and its functional consequences, are unknown. EZHIP is expressed only in the testes, ovaries and embryo-surrounding tissues of placental mammals10, and was previously shown to interact with PRC2 in cultured mouse stem cells11,12. Ragazzini and colleagues demonstrate that, in the testes and ovaries of mice, EZHIP binds to and reduces the enzymatic activity of PRC2.

Read the paper: EZHIP constrains Polycomb Repressive Complex 2 activity in germ cells

The authors also show that mice lacking EZHIP can live and develop normally, and are apparently fertile, but that female mice that lack EZHIP show progressive infertility with age. This finding calls for a re-evaluation of genomic studies in women with infertility, to clarify whether certain EZHIP gene variants could increase the risk of this condition. EZHIP protein is found exclusively in placental mammals, so one might speculate that its appearance could have contributed to the evolution of the placenta.

Other reports2,4,5 have found that, when EZHIP is expressed, it associates with PRC2 and inhibits its function — putting a ‘lid’ on the PRC2 marker. EZHIP expression is inversely correlated with the level of the H3K27me3 mark. Indeed, Jain et al. show that reintroducing EZHIP into cultured human cells that normally do not express the protein inhibits PRC2 activity and markedly reduces H3K27me3 levels. Conversely, removing the EZHIP gene from cultured cells that normally express it increases levels of H3K27me31,2,4,5. These results indicate that there is a causal relationship, rather than just a correlation, between EZHIP expression and decreases in H3K27me3 and changes to the overall epigenetic landscape.

Dysregulation of PRC2 function can be highly detrimental. For example, a mutation in one of the H3-encoding genes that leads to lysine 27 being replaced by a methionine residue greatly reduces the effectiveness of the complex (Fig. 1b). The mutant histone, which is termed H3K27M, has been implicated in aggressive childhood brain cancer13, including one such cancer called diffuse intrinsic pontine glioma (DIPG)14.

Comparative analyses2,3 have shown that the EZHIP-encoding gene has changed rapidly over evolution and, as a result, the amino-acid sequence of the protein shows little similarity between different species. However, a stretch of 12 amino acids at one end of the protein is identical in many mammalian species. Notably, this sequence is highly similar to the amino-acid sequence that contains the methionine 27 residue in the DIPG-associated histone variant H3K27M2,3,5. It is therefore known as the K27M-like peptide (KLP) and might mimic the effect of H3K27M on PRC2 function.

Read the paper: PFA ependymoma-associated protein EZHIP inhibits PRC2 activity through a H3 K27M-like mechanism

Jain et al. determine the functionality of EZHIP and, particularly, the function of its KLP. They propose a model in which EZHIP forms a complex with PRC2 that is bound to H3K27me3, and, in a similar way to H3K27M, prevents PRC2 from depositing further H3K27me3 marks across the chromatin (Fig. 1c). Using a computerized model of the co-crystal structure of PRC2 bound to H3K27M, Jain et al. predict that the KLP of EZHIP makes stable contacts with the PRC2 enzyme subunit EZH2 similarly to how H3K27M binds to EZH2.

Moreover, Jain and colleagues show that EZHIP inhibition of PRC2 activity relies on the methionine 406 residue in KLP, and that the ability of EZHIP to inhibit PRC2 is enhanced when PRC2 is activated by H3K27me3 at a different site on the complex from where EZHIP binds. Mouse embryo fibroblast cells that were engineered to express H3K27M or EZHIP show similar reductions in chromatin H3K27me3 and similar changes in gene expression relative to genetically normal mouse embryo fibroblasts. However, Jain et al. also find that some ‘resilient’ parts of the chromatin in the EZHIP-expressing cells retain their H3K27me3 marks; the mechanisms that enable the retention of PRC2 function selectively at these sites are not clear.

H3K27M is a major driving force in the progression of DIPG. However, the PRC2 function that remains in DIPG with H3K27M promotes growth of the tumour, and thus further inhibition of this residual PRC2 activity has been proposed as a promising therapeutic approach14–16. It remains to be seen whether reducing PRC2 function — using small-molecule EZH2 inhibitors or by mimicking the KLP in EZHIP — might be a viable strategy.

Disrupted PRC2 function is a feature of another type of childhood brain cancer called posterior fossa ependymoma group A (PFA)4. However, unlike in DIPG (in which PRC2 function is reduced by H3K27M), PRC2 function in PFA is reduced by an aberrant upregulation of EZHIP2,3. Whether other proteins that have KLP activity might inhibit PRC2 in other human tumours, and whether other chromatin complexes besides PRC2 can similarly be inhibited by other proteins, are questions that should be explored.

Together, the results of the two papers increase our understanding not only of how PRC2 affects gene expression and cell function in different settings, but also of mammalian physiology and human disease. By advancing our understanding of EZHIP functionality, the studies lay the foundation for exploring the role of this protein in driving cancer, and indicate that targeting PRC2 or EZHIP could have therapeutic potential for children with PFA and DIPG.