Ezh2 Requires PHF1 To Efficiently Catalyze H3 Lysine 27 Trimethylation In Vivo

Abstract

The mammalian Polycomblike protein PHF1 was previously shown to interact with the Polycomb group (PcG) protein Ezh2, a histone methyltransferase whose activity is pivotal in sustaining gene repression during development and in adulthood. As Ezh2 is active only when part of the Polycomb Repressive Complexes (PRC2-PRC4), we examined the functional role of its interaction with PHF1. Chromatin immunoprecipitation experiments revealed that PHF1 resides along with Ezh2 at Ezh2-regulated genes such as the HoxA loci and the non-Hox MYT1 and WNT1 genes. Knockdown of PHF1 or of Ezh2 led to up-regulated HoxA gene expression. Interestingly, depletion of PHF1 did correlate with reduced occupancy of Bmi-1, a PRC1 component. As expected, knockdown of Ezh2 led to reduced levels of its catalytic products H3K27me2/H3K27me3. However, reduced levels of PHF1 also led to decreased global levels of H3K27me3. Notably, the levels of H3K27me3 decreased while those of H3K27me2 increased at the up-regulated HoxA loci tested. Consistent with this, the addition of PHF1 specifically stimulated the ability of Ezh2 to catalyze H3K27me3 but not H3K27me1/ H3K27me2 in vitro. We conclude that PHF1 modulates the activity of Ezh2 in favor of the repressive H3K27me3 mark. Thus, we propose that PHF1 is a determinant in PcG-mediated gene repression.

Corepressor Protein CDYL Functions as a Molecular Bridge between Polycomb Repressor Complex 2 and Repressive Chromatin Mark Trimethylated Histone Lysine 27

“…Histone Methyltransferase (HMT) Assay-HMT assays were performed as described previously (37). For ChIP Sequencing-MCF-7 cells were maintained in DMEM supplemented with 10% fetal bovine serum.…”

“…4E, top panel). It has been proposed that H3K27me2 is an intermediate H3K27 methylation state that marks genes as being potentially repressible by PRC2 (37). Through efficient binding to both H3K27me2 and H3K27me3, CDYL serves as a more competent bridging factor than EED to connect PRC2 and histone modifications in chromatin.…”

Polycomb-like proteins link the PRC2 complex to CpG islands

“…Polycomb-like proteins (PCLs), including PHF1, MTF2 and PHF19, are PRC2 associated factors that form sub-complexes with PRC2 core components 3 , and have been proposed to modulate PRC2’s enzymatic activity or its recruitment to specific genomic loci 4–13 . Mammalian PRC2 binding sites are enriched in CG content, which correlate with CpG islands that display a low level of DNA methylation 14 .…”

Coordinated activities of wild-type plus mutant EZH2 drive tumor-associated hypertrimethylation of lysine 27 on histone H3 (H3K27) in human B-cell lymphomas

“…A presumptive reduction in the rate of H3K27 methylation due to enzyme heterozygosity seemed to us to be difficult to rationalize as the basis for a malignant phenotype (1, 2), especially in light of previous data indicating that overexpression of EZH2 (3-6), loss-of-function mutations in the corresponding H3K27 demethylase UTX (7), or overexpression of components of the PRC2, such as PHF19/PCL3 involved in increased H3K27 trimethylation (8)(9)(10), all result in malignant phenotypes in specific human cancers. Several recent reports indicate that an important component of early lymphomagenesis is the acquisition of stem cell-like characteristics (11,12).…”