Ezh2 requires PHF1 to efficiently catalyze H3 lysine 27 tri-methylation in vivo

Howard Hughes Medical Institute; Department of Biochemistry, NYU-Medical School, 522 First Av., New York, NY 10016, USA; Department of Biochemistry, Division of Nucleic Acids Enzymology, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, 683 Hoes Lane, Piscataway, New Jersey 08854, USA; Department of Molecular Biology and Biochemistry, Rutgers University, Nelson Laboratories, 604 Allison Road, Piscataway, New Jersey 08854, USA; The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104, USA

^* To whom correspondence should be addressed. Email: reinbd01{at}med.nyu.edu.

	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 only active when part of the Polycomb Repressive Complexes (PRC2-4), we examined the functional role of its interaction with PHF1. ChIP 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. Knock-down of either 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, knock-down of Ezh2 led to reduced levels of its catalytic products H3K27me(2/3). 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 H3K27me1/2 in vitro. We conclude that PHF1 modulates the activity of Ezh2 in favour of the repressive H3K27me3 mark. Thus, we propose that PHF1 is a determinant in PcG-mediated gene repression.