Dominant negative effects of Weaver syndrome-associated EZH2 variants

Heterozygous missense mutations in EZH2 cause Weaver syndrome (WS), a developmental disorder characterized by intellectual disability and overgrowth. EZH2encodes the enzymatic subunit of Polycomb Repressive Complex 2 (PRC2), which mediates mono-, di-, and tri-methylation of histone H3 lysine 27 (H3K27me1/2/3). Most WS-associated EZH2 variants lack functional characterization but are presumed loss of function. However, the lack of early truncating mutations in EZH2 led us to hypothesise a dominant-negative mechanism for WS, which was supported by our structural analysis of all known WS-associated EZH2 variants. We isogenically modelled 10 representative variants in embryonic stem cells and showed they reduce global H3K27me2/3 with concomitant increases in H3K27ac and chromatin decompaction. Notably, the pattern of H3K27me2/3 reductions indicated dominant-negative interference on PRC2 activity, even when WS-variants were expressed atlow levels. RNA-seq identified weakly Polycomb-bound genes that lose canonical PRC1 (cPRC1) occupancy and become derepressed, including several phenotypically relevant growth control genes. Comparative analysis of a gain-of-function EZH2 variant causing growth restriction revealed reciprocal chromatin and transcriptional changes compared to WS-associated variants. Taken together, our findings support a model where EZH2 variants associated with opposing developmental growth syndromes affect not only H3K27me3, but also intergenic H3K27me2, chromatin architecture, and cPRC1 recruitment. Overall design: We generated Ezh2 heterozygous knockout mESCs and then exogenously expressed FLAG/HA-tagged human wild-type EZH2 (EZH2-WT) or 5 Weaver syndrome associated EZH2 variants. For comparison, we also expressed growth restriction-associated EZH2-A733T variant. We performed exogenous reference genome-normalised ChIP-seq (ChIP-Rx) for H3K27me2, H3K27me3, H3K27ac, H3K36me2, SUZ12, CBX7, and FLAG in these cell lines.