ZMYND8 Reads the Dual Histone Mark H3K4me1-H3K14ac to Antagonize the Expression of Metastasis-Linked Genes

Abstract

Histone acetylation, including acetylated H3K14 (H3K14ac), is generally linked to gene activation. Monomethylated histone H3 lysine 4 (H3K4me1), together with other gene-activating marks, denotes active genes. In contrast to usual gene-activating functions of H3K14ac and H3K4me1, we here show that the dual histone modification mark H3K4me1-H3K14ac is recognized by ZMYND8 (also called RACK7) and can function to counteract gene expression. We identified ZMYND8 as a transcriptional corepressor of the H3K4 demethylase JARID1D. ZMYND8 antagonized the expression of metastasis-linked genes, and its knockdown increased the cellular invasiveness in vitro and in vivo. The plant homeodomain (PHD) and Bromodomain cassette in ZMYND8 mediated the combinatorial recognition of H3K4me1-H3K14ac and H3K4me0-H3K14ac by ZMYND8. These findings uncover an unexpected role for the signature H3K4me1-H3K14ac in attenuating gene expression and reveal a metastasis-suppressive epigenetic mechanism in which ZMYND8's PHD-Bromo cassette couples H3K4me1-H3K14ac with downregulation of metastasis-linked genes.

Figure 1. ZMYND8 is a JARID1D-associated protein

(A) Immunoaffinity purification and mass spectrometric analysis of JARID1D-associated proteins. JARID1D-associated proteins from nuclear extracts from FLAG-JARID1D-expressing stable H1299 cells were immunopurified with anti-FLAG (α-FLAG) affinity resins. The proteins bands were analyzed by mass spectrometry. Asterisks indicate breakdowns or non-specific polypeptides. (B) Schematic representation of human JARID1D, ZMYND8, and ZMYND8 deletion mutants. (C) Nuclear localization of JARID1D and ZMYND8 in DU145 cells. Nuclear and cytoplasmic fractions of DU145 cells were blotted with the indicated antibodies. p84 and β-actin were used as a nuclear marker and a cytoplasmic marker, respectively. WCE, whole cell extracts. (D) Co-immunoprecipitation between ectopically expressed FLAG-tagged JARID1D and endogenous ZMYND8 protein. Anti-FLAG-immunoprecipitated samples were blotted with anti-FLAG and anti-ZMYND8 (α-ZMYND8) antibodies. (E) Coimmunoprecipitation between endogenous JARID1D and ZMYND8 in DU145 cells. Anti-JARID1D (α-JARID1D)-immunoprecipitated samples were blotted with anti-JARID1D and anti-ZMYND8 antibodies. (F) Mapping of the ZMYND8 region responsible for the interaction with JARID1D. FLAG-JARID1D and HA-ZMYND8 (or its truncated mutants) were ectopically expressed in 293T cells. Expression levels were examined using anti-HA and anti-FLAG antibodies (Left panel). Following co-immunoprecipitation (IP), the eluates were examined by western blot analysis using anti-HA (α-HA) and anti-FLAG antibodies (Right panel). Open rectangular triangles denote specific bands, and asterisks indicate nonspecific polypeptides. (G and H) Mapping of the JARID1D region responsible for the interaction with ZMYND8. Recombinant full-length JARID1D and its deletion mutants were analyzed using anti-JARID1D antibody (G). A Co-IP assay was performed using recombinant JARID1D, JARID1D mutants, and ZMYND8 that were isolated from Sf9 or Sf21 insect cells (H). See also Figure S1.

Figure 2. The chromatin landscape of ZMYND8 greatly overlaps that of JARID1D.

(A) The genomic distribution of ChIP-seq peaks of JARID1D and ZMYND8. ChIP-Seq was performed using DU145 prostate cancer cells. (B) Average enrichment of JARID1D and ZMYND8 in the genic regions, including TSS and TTS. TSS, transcriptional start site; TTS, transcriptional termination site. (C) Heat maps of genomic co-localization of JARID1D and ZMYND8 in DU145 cells. (D) Venn diagram of the overlap between JARID1D and ZMYND8 peaks. (E) Venn diagram of genes co-occupied by JARID1D and ZMYND8. (F) Gene ontology analysis of genes co-occupied by JARID1D and ZMYND8.

Figure 3. ZMYND8 knockdown increases the invasive abilities of prostate cancer cells in vitro and in vivo

(A) Western blot and quantitative RT-PCR analysis of ZMYND8 levels following the treatment of DU145 cells with shZMYND8s (shZMYND8-95 and shZMYND8-97). (B–D) Effects of ZMYND8 knockdown on the proliferation (B), migration (C) and invasion (D) of DU145 cells. Following the cell migration and invasion assays, cells were stained with crystal violet and counted in at least five fields. (E–G) The effect of ZMYND8 knockdown on the in vivo metastatic abilities of DU145 cells in an intravenous mouse xenograft model. DU145 cells with stably expressing firefly luciferase were infected with lentiviruses containing scramble shRNA (shScramble) or shZMYND8-97. The representative bioluminescent images of mice (shScramble, n=6; shZMYND8, n=6) 8-10 weeks after tail vein injection are shown (E), and their quantified bioluminescent signals were individually plotted (F). Representative images of hematoxylin and eosin–stained lung tissues (G). Data are presented as the mean ± SEM (error bars). See also Figure S1.

Figure 4. ZMYND8 is required for the JARID1D-mediated downregulation of metastasis-linked genes.

(A and C) Venn diagrams (A) and gene ontology analysis (C) of genes upregulated by JARID1D and ZMYND8 knockdown. Whole genome expression levels were measured by the RNA-seq. (B and D) Venn diagrams (B) and gene ontology analysis (D) of genes downregulated by JARID1D and ZMYND8 knockdown. (E) The effect of JARID1D or ZMYND8 knockdown on expression levels of multiple metastasis-linked genes in DU145 cells. A quantitative RT-PCR analysis was performed using samples from at least three independent experiments (i.e., n ≥ 3). (F–I) The effect of ZMYND8 knockdown on JARID1D and H3K4me3 levels at Slug (F), CD44 (G), VEGFA (H), and EGFR (I) genes in DU145 cells. Chromatin levels of ZMYND8, JARID1D, and H3K4me3 were measured by quantitative ChIP that was performed using samples from at least three independent experiments (i.e., three biological replicates). IgG was used as a negative control. Data are presented as the mean ± SEM (error bars). ChIP PCR amplicons are indicated by the small blue lines in Figures 7A-7D. See also Figure S2.

Figure 5. ZMYND8 recognizes H3K4me0-H3K14ac and H3K4me1-H3K14ac via the PHD/Bromo cassette

(A) Schematic representation of ZMYND8-PHD/Bromo/PWWP (ZMYND8-PBP), ZMYND8-PHD/Bromo (ZMYND8-PB), and ZMYND8-PB deletion mutants. (B) Peptide pull-down assays using the indicated biotinylated peptides and the GST-tagged ZMYND8-PB. (C) Peptide pull-down assays using H3 (1-21) peptide and either the recombinant GST-ZMYND8-PB or its deletion mutants. (D and E) Isothermal titration calorimetry (ITC) assays to measure the interaction of the indicated peptides with ZMYND8-PB (D) or ZMYND8-PBP (E). (F) Peptide pull-down assays using the indicated biotinylated peptides and recombinant GST-ZMYND8-PBP. (G) ITC assays to measure the interaction of the indicated peptides with the recombinant ZMYND8-PBP. See also Figure S3.

Figure 6. Molecular basis for the interaction of ZMYND8-PHD/Bromo with H3K4me0-H3K14ac and H3K4me1-H3K14ac

(A) Electrostatic surface view of the ZMYND8-PBP in complex with modeled H3K4me0K14ac peptide. Electrostatic potential is shown as a spectrum ranging from red (negative charge) to blue (positive charge). (B) Structural modelling of ZMYND8-PBP bound to unmodified H3 (1-6aa) peptides. ZMYND8-PBP is in ribbon view. The invisible N-terminal fragment (D96-G105) of ZMYND8-PHD is shown as a dotted line. Close-up view shows recognition of H3 peptide by ZMYND8-PHD (blue). Znf (pink), zinc finger motif between Bromo (green) and PWWP (orange); Magenta dashes, hydrogen bonds. (C) Structural modeling of ZMYND8-PBP bound to H3K4me0K14ac (1-14aa) peptides. Blue spheres, Zinc atoms; Magenta dashes, hydrogen bonds. (D) Close-up view of structural modeling for the recognition of H3K14ac by ZMYND8 Bromo (green). The side chains of two residues, Y247 and N248, are shown in magenta sticks, with blue for nitrogen and red for oxygen. Cyan dashes, hydrogen bonds. (E) Electrostatic surface view of the ZMYND8-PBP in complex with modelled H3K4me1K14ac peptide. Electrostatic potential is shown as a spectrum ranging from red (negative charge) to blue (positive charge). (F) Structural modelling of ZMYND8-PBP bound to H3K4me1 (1-6aa) peptides. ZMYND8-PBP is in ribbon view. The invisible N-terminal fragment (D96-G105) of ZMYND8-PHD is shown as dotted line. Close-up view shows recognition of H3 peptide by ZMYND8-PHD (blue). Znf (pink), zinc finger motif between Bromo (green) and PWWP (orange); Magenta dashes, hydrogen bonds. (G) ITC assays to measure the interaction of H3K4me0 (1-25aa) with ZMYND8-PBP or its PHD mutants. (H) ITC assays to measure the interaction of H3K14ac (1-15aa) with ZMYND8-PBP or its Bromo mutants (N248A or Y247A/N248A). For A–F, the modeled H3 peptide is depicted as sticks with yellow for carbon, blue for nitrogen, and red for oxygen. See also Figure S4.

Figure 7. ZMYND8 downregulates metastasis-linked genes by reading the signature H3K4me1-H3K14ac

(A-D) Genome browser view of ChIP-Seq peaks for input (a negative control), ZMYND8, JARID1D, H3K14ac, H3K4me1, H3K4me3, and H3K27me3 at CD44 (A), Slug (B), VEGFA (C), and EGFR (D) genes in DU145 cells. The blue lines indicate ChIP PCR amplicons for E-H. ChIP-Seq signals of JARID1D, ZMYND8, H3K4me1, H3K14ac, H3K4me3, and H3K27me3 in the tracks represent their remainder signals after input signals were subtracted from their original signals. (E-H) The effect of JARID1D knockdown on ZMYND8 occupancy at CD44 (E), Slug (F), VEGFA (G), and EGFR (H) genes in DU145 cells. Chromatin levels of ZMYND8 and JARID1D were measured by quantitative ChIP (n ≥ 3). PCR values were normalized to input. (I) Western blot analysis of ectopic expression of WT FLAG-ZMYND8, its PHD mutants (D108A, F109D), and its Bromo mutant (YN/AA). DU145 cells infected with shZMYND8-97 virus were transfected with control vector, WT FLAG-ZMYND8 or its point mutants. shLuc-treated DU145 cells that were transfected with control vector were used as a control. (J) The effects of ectopic expression of WT FLAG-ZMYND8, its PHD mutants (D108A, F109D), and its Bromo mutant (YN/AA) on expression levels of CD44, Slug, VEGFA, and EGFR genes in ZMYND8-depleted DU145 cells (n = 5). (K and L) The effects of ectopic expression of WT FLAG-ZMYND8, its PHD mutants (D108A, F109D), and its Bromo mutant (YN/AA) on invasive ability in ZMYND8-depleted DU145 cells. Cells were stained with crystal violet (K) and counted in at least five different fields (L). Data are presented as the mean ± SEM (error bars). See also Figures S5 and S6.