Chromatin-modifying Complex Component Nurf55/p55 Associates with Histones H3 and H4 and Polycomb Repressive Complex 2 Subunit Su(z)12 through Partially Overlapping Binding Sites

Florence Baudin,Agnieszka J. Nowak,Christoph W. Müller,Vladimir Rybin,Christian U. Stirnimann,Nga Ly-Hartig,Claudio Alfieri,Doris Lindner

doi:10.1074/jbc.m110.207407

Abstract

Drosophila Nurf55 is a component of different chromatin-modifying complexes, including the PRC2 (Polycomb repressive complex 2). Based on the 1.75-Å crystal structure of Nurf55 bound to histone H4 helix 1, we analyzed interactions of Nurf55 (Nurf55 or p55 in fly and RbAp48/46 in human) with the N-terminal tail of histone H3, the first helix of histone H4, and an N-terminal fragment of the PRC2 subunit Su(z)12 using isothermal calorimetry and pulldown experiments. Site-directed mutagenesis identified the binding site of histone H3 at the top of the Nurf55 WD40 propeller. Unmodified or K9me3- or K27me3-containing H3 peptides were bound with similar affinities, whereas the affinity for K4me3-containing H3 peptides was reduced. Helix 1 of histone H4 and Su(z)12 bound to the edge of the β-propeller using overlapping binding sites. Our results show similarities in the recognition of histone H4 and Su(z)12 and identify Nurf55 as a versatile interactor that simultaneously contacts multiple partners.

Highlights

Polycomb group proteins play important roles in maintaining the silenced state of homeotic genes, as well as in other biological processes, including X chromosome inactivation, germ line development, stem cell pluripotency, and cancer metastasis
Three different major Polycomb repressive complexes have been identified in Drosophila melanogaster: PRC1, PRC2, and PhoRC (Pleiohomeotic-repressive complex)
Nurf55 is a non-catalytic subunit of PRC2 that is necessary in vitro to ensure high-affinity binding of the complex to nucleosomes [7, 9]

Summary

EXPERIMENTAL PROCEDURES

Protein Expression and Purification—Wild-type and mutant Drosophila Nurf (p55) proteins were expressed in Sf21 cells as N-terminal, tobacco etch virus-cleavable, His-tagged fusion proteins and purified using standard procedures as described under supplemental “Experimental Procedures.” The CD spectra of wild-type and mutant Nurf proteins confirmed that the overall structure was preserved in all mutants (supplemental Fig. S1). Protein Expression and Purification—Wild-type and mutant Drosophila Nurf (p55) proteins were expressed in Sf21 cells as N-terminal, tobacco etch virus-cleavable, His-tagged fusion proteins and purified using standard procedures as described under supplemental “Experimental Procedures.”. FLAG-tagged Su(z) and E(z) proteins were expressed separately or coexpressed with wild-type and mutant Nurf proteins in Sf21 insect cells and purified using antiFLAG affinity resin as described [9]. Su(z) fragments were expressed as tobacco etch virus-cleavable GST fusion proteins in Escherichia coli and purified using standard procedures (see supplemental “Experimental Procedures”). Crystallographic Structure Determination—For crystallization, purified Nurf was mixed with the H426–45 peptide at a ratio of 1:3. For competition ITC experiments, we used 8 –12 ␮M Nurf preincubated in the cell with 30 –100 ␮M competitor peptide (see Table 2). To fit an apparent dissociation constant (KD(app)), we used standard procedures [20] implemented in the MicroCal Origin 7.0 software package

RESULTS AND DISCUSSION

Peptide with fixed concentration

Peptide with variable concentration