A PWWP Domain of Histone-Lysine N-Methyltransferase NSD2 Binds to Dimethylated Lys-36 of Histone H3 and Regulates NSD2 Function at Chromatin

Abstract

The readout of histone modifications plays a critical role in chromatin-regulated processes. Dimethylation at Lys-36 on histone H3 (H3K36me2) is associated with actively transcribed genes, and global up-regulation of this modification is associated with several cancers. However, the molecular mechanism by which H3K36me2 is sensed and transduced to downstream biological outcomes remains unclear. Here we identify a PWWP domain within the histone lysine methyltransferase and oncoprotein NSD2 that preferentially binds to nucleosomes containing H3K36me2. In cells, the NSD2 PWWP domain interaction with H3K36me2 plays a role in stabilizing NSD2 at chromatin. Furthermore, NSD2's ability to induce global increases in H3K36me2 via its enzymatic activity, and consequently promote cellular proliferation, is compromised by mutations within the PWWP domain that specifically abrogate H3K36me2-recognition. Together, our results identify a pivotal role for NSD2 binding to its catalytic product in regulating its cellular functions, and suggest a model for how this interaction may facilitate epigenetic spreading and propagation of H3K36me2.

Keywords: chromatin; chromatin modification; chromatin regulation; histone methylation; histone modification.

FIGURE 1.

Proteome-wide screen for H3K36 methyl binding proteins using modified nucleosome substrates. A, schematic of proteomic approach to screen for candidate H3K36 methyl binders from SILAC nuclear extract. B, enrichment of proteins bound to MLA H3K_C36me2 (top) or H3K_C36me3 (bottom) nucleosomes over unmodified nucleosome substrates. Identified proteins are plotted by SILAC ratio in the forward (x axis) and reverse (y axis) experiments, with candidate H3K36 methyl binders in the top right quadrant. Proteins enriched with ratio >1.8 (log2 ratio > 0.85) in the forward SILAC experiment and <0.55 (log2 ratio < −0.85) in the reverse SILAC experiment are labeled and categorized as PWWP domain proteins (orange), HNRNP family (cyan), RAN-associated (magenta), or Other (gray). C, Western blot analysis of direct pulldown assays testing binding of the indicated GST fusion proteins to H3 tail (21–44 aa) peptide substrates bearing umodified (unmod), monomethyl (me1), dimethyl (me2), or trimethyl (me3) H3K36, or the indicated MLA H3K_C36 nucleosomes.

FIGURE 2.

Candidate screen of human PWWP domains reveals NSD2_PWWP1 and NSD3_PWWP1 as H3K36 methyl readers. A, sequence alignment of selected human PWWP domains. The PWWP motif is highlighted. Arrows indicate residues that are similar between NSD2 and NSD3 but not NSD1. * , conserved residue. : , similar residues. B–D, Western blot analysis of pulldown assays testing direct binding of the indicated PWWP domains as in Fig. 1C. H3K36me3-binding domains are grouped in B, domains with preferential binding to H3K36me2 are shown in C, and the non-binding or nonspecific binding domains are grouped in D.

FIGURE 3.

NSD2_PWWP1 preferentially binds H3K36me2-modified nucleosomes. A, Western blot analysis as in Fig. 2B of GST-NSD2_PWWP1 binding to nucleosomes carrying dimethyl analogs at the indicated H3 lysine residues. B, titration of GST-NSD2_PWWP1 against MLA H3K_C36me2 (top) or H3K_C36me3 (bottom) nucleosomes in EMSA. Bands indicated correspond to free nucleosome core particle (NCP) and nucleosome bound by one (+1) or two (+2) PWWP molecules. C, binding curves quantified from EMSA experiments of GST-NSD2_PWWP1 on MLA H3K_C36me2 or H3K_C36me3 nucleosomes. Error bars represent S.E. from three independent experiments. D, apparent binding affinity (K_dAPP) for H3K_C36me2 and H3K_C36me3 nucleosomes calculated from binding curves for GST-NSD2_PWWP1 in C and for GST-PSIP1_PWWP (data not shown). Error indicates S.E. from three independent experiments.

FIGURE 4.

PWWP-H3K36me2 binding stabilizes NSD2 chromatin association. A, sequence alignment of PSIP1_PWWP and NSD2_PWWP1. Residues involved in the PSIP1_PWWP H3K36me3-binding aromatic cage are highlighted. Boxes indicate residues chosen for mutational analysis. B, mutation of the indicated aromatic cage residues abrogates NSD2_PWWP1 binding to H3K_C36me2 in direct pulldown assays as in Figs. 1C and 2, B–D. C, schematic of modified Stillman fractionation method to biochemically separate chromatin-associated proteins from soluble nuclear proteins in cellular extracts. D, Western blot analysis of biochemical fractions defined in C from HT1080 cells expressing full-length FLAG-myc-NSD2 wild-type or F266A PWWP1 mutant shows altered chromatin association for the PWWP1 mutant derivative. Tubulin and H3 blots serve as controls for intact fractions. WCE, whole cell extract. nuc 150, 150 mm salt soluble nuclear fraction. nuc 300, 300 mm salt soluble nuclear fraction. chromatin, chromatin fraction. E, ChIP analysis of FLAG-myc-NSD2 wild-type or F266A occupancy across CDC42 gene. A schematic of the gene (top panel) indicates the location of primers used for qPCR analysis of FLAG (middle panel) or IgG control (bottom panel) ChIP from each stable cell line. vector, HT1080 cells transduced with empty vector. Error bars indicate S.E. from three experiments. F and G, ChIP analysis as in E at TSS-proximal sites (analogous to CDC42 primer 2) of NSD2 gene targets MYC and TGFA, respectively.

FIGURE 5.

PWWP-H3K36me2 interaction regulates NSD2 cellular functions. A, Western blot analysis with the indicated antibodies on lysates from HT1080 cells expressing NSD2 wild-type or mutant derivatives. vector, HT1080 cells transduced with empty vector. CDM, catalytic dead mutant. B, proliferation assays using the cells described in A counted over 8 days. Error bars indicate S.E. from three experiments. C, cell counts from Day 8 of the proliferation assay in B. p values were calculated using a two-tailed Student's t test. *, p < 0.01. n.s., not significant.

FIGURE 6.

A model for propagation of H3K36me2 by NSD2. A, the N-terminal PWWP domain and SET domain of NSD2 cooperate to recognize nucleosomes carrying H3K36me2 and deposit the same mark on neighboring nucleosomes, thus propagating this modification. B, by this mechanism of propagation, NSD2 mediates the spreading of H3K36me2 across a genomic region (top) or maintenance of H3K36me2 patterns in daughter cells after DNA replication, when parental histones have been distributed between daughter cells and new histones have been incorporated (bottom).