In non Hodgkin lymphoma the

In non-Hodgkin lymphoma the catalytic SET domain of the histone methyltransferase EZH2 is subject to recurrent heterozygous missense mutations . These alterations have been observed in ∼20% of patients with diffuse large B cell lymphoma (DLBCL) and ∼10% of patients with follicular lymphoma (FL). These ‘change-of-function’ mutations alter the substrate preference for EZH2: whereas wild-type EZH2 preferentially methylates unmodified and mono-methylated histone H3 at lysine 27 (H3K27me0 and H3K27me1, respectively), mutant EZH2 prefers dimethylated H3 lysine 27 (H3K27me2) as its substrate (A) . The joint activities of the wild-type and mutant EZH2 enzymes thus coordinately increase H3K27me3 production at the expense of H3K27me2 , . Recent studies have correlated these global changes in H3K27me2 and H3K27me3 with decreased expression of genes required for terminal B cell differentiation . These findings suggest that EZH2 mutant Disuflo Cy3 azide have impaired differentiation potential. These cells, when blocked in an undifferentiated or partially differentiated state, may acquire additional genetic or chromatin alterations, allowing them to progress toward full-blown malignancy. The EZH2 protein is a component of the Polycomb repressive complex 2 (PRC2), together with SUZ12 and EED, and is responsible for all mono-, di-, and trimethylation at H3K27 , . The H3K27me3 modification is found at the promoters of key lineage-defining genes and serves as a binding platform for the canonical PRC1 complex which acts to compact chromatin via its CBX and PHC components , . The role of the H3K27me2 modification is less clear, but it is distributed broadly on chromatin and has been proposed to serve as a ‘repressive blanket’ to prevent aberrant activation of enhancer elements . A key question remains – what are the effects of increased H3K27me3 in lymphoma cells, and in particular how does this correlate with the observed decreases in the expression of genes required for B cell differentiation? In this new study, Donaldson-Collier and colleagues used sophisticated approaches to study the 3D genome, including HiC (chromosome conformation capture, 3C, combined with high-throughput sequencing) and 3C-on-chip (4C)-seq. These data were integrated with chromatin immunoprecipitation and deep sequencing (ChIP-seq) for H3K27me3 and global gene expression measurements. They found that increases in H3K27me3 levels in EZH2 mutant cells do not alter the overall topology of the genome because the boundaries between adjacent topologically associated domains (TADs) are largely unaffected. However, integration of global gene expression analyses with HiC data highlighted that multiple genes within a subset of TADs are coordinately repressed in EZH2 mutant cells, coincidently with increased H3K27me3. Consistent with previous studies, genes that fall into this category are enriched in those that regulate B cell differentiation. Moreover, treatment of lymphoma cells with EZH2 inhibitors, currently under clinical investigation in EZH2 mutant lymphoma patients, led to increased expression of many of the genes within these ‘repressed TADs’ . These findings suggest that H3K27me3 within these TADs is necessary to downregulate their expression. Of note, despite the fact that the vast majority of TADs have higher levels of H3K27me3 in EZH2 mutant cells, only a small subset of TADs behave in the manner highlighted in the present study. Therefore, it remains important to identify and understand what additional factors distinguish the TADs that become repressed in EZH2 mutant cells from the majority that do not. By way of ascribing a mechanism to the observed gene repression within TADs, the authors searched for intra-TAD structural changes. Interestingly, within an individual repressed TAD, the authors observed changes in chromatin looping dynamics leading to alterations in chromatin contacts . These dynamics appeared to be closely linked to the activity of mutant EZH2 because introduction of the mutant enzyme into lymphoma cells was sufficient to establish the altered chromatin configuration, whereas its inhibition reversed these changes (B). Further analyses of this individual TAD suggest that a more condensed chromatin structure was associated with mutant EZH2 activity. However, it is unclear how generalizable these phenomena are. Therefore, it will be important to broaden these analyses to search for chromatin looping dynamics within all TADs throughout the genome.