TRIM24 links a non-canonical histone signature to breast cancer

Abstract

Recognition of modified histone species by distinct structural domains within 'reader' proteins plays a critical role in the regulation of gene expression. Readers that simultaneously recognize histones with multiple marks allow transduction of complex chromatin modification patterns into specific biological outcomes. Here we report that chromatin regulator tripartite motif-containing 24 (TRIM24) functions in humans as a reader of dual histone marks by means of tandem plant homeodomain (PHD) and bromodomain (Bromo) regions. The three-dimensional structure of the PHD-Bromo region of TRIM24 revealed a single functional unit for combinatorial recognition of unmodified H3K4 (that is, histone H3 unmodified at lysine 4, H3K4me0) and acetylated H3K23 (histone H3 acetylated at lysine 23, H3K23ac) within the same histone tail. TRIM24 binds chromatin and oestrogen receptor to activate oestrogen-dependent genes associated with cellular proliferation and tumour development. Aberrant expression of TRIM24 negatively correlates with survival of breast cancer patients. The PHD-Bromo of TRIM24 provides a structural rationale for chromatin activation through a non-canonical histone signature, establishing a new route by which chromatin readers may influence cancer pathogenesis.

Figure 1. TRIM24-PHD finger interacts with unmethylated H3K4

a, Diagram of TRIM24 protein domains. b, Biotinylated peptide pulldowns: recombinant PHD fingers and histone peptides. c, GST-pulldowns: recombinant proteins and native histone proteins. d, ITC titration: binding of TRIM24 PHD-Bromo with histone peptides.

Figure 2. TRIM24 PHD-Bromo simultaneously binds H3K4me0 and acetylated histone lysines

a, Stereo view of the crystal structure of TRIM24 PHD-Bromo in the free state. b, Detailed interactions between PHD of TRIM24 PHD-Bromo and H3(1–10)K4 peptide. c, Detailed interactions between Bromodomain of TRIM24 PHD-Bromo and H3(22–29)K23ac peptide. d, Positioning of H3(1–10)K4 and H3(13–32)K23ac peptides on the surface of TRIM24 PHD-Bromo based on structural information e and f, ITC (as in e) or fluorescence polarization (FP) (as in f) based binding curves of wild-type (WT) or mutant forms of TRIM24 PHD-Bromo with H3(1–33) peptides bearing different combination of modifications. Dissociation constants (K_D) derived from ITC experiments are given as inserts.

Figure 3. TRIM24 is recruited with ERα to ERE sites depleted of H3K4me2

a, ChIP of ERα and TRIM24 at ERE’s of GREB1, 15 min and 6 h estradiol (E₂). Vehicle: EtOH. b, Sequential-ChIP: ERα and TRIM24, 6 h E₂. c, d, ChIP for H3 and histone modifications, 15 min and 6 h E₂, normalized for H3. Each bar represents averaged results, n=3 biological replicates, assayed 3 times each; error bars show standard deviations. e, Genome wide TRIM24 and ERα binding sites in MCF7 cells, −E₂ or +E₂. Two independent experiments analyzed. f, Normalized genome wide H3K4me2 within a window of 800 bp, centered at TRIM24 binding sites (designated as 0), +E₂ (blue line) or −E₂ or (red line).

Figure 4. TRIM24 functions as a co-activator and stabilizes ERα-chromatin interactions

a, Stable shControl and shTRIM24 MCF7 cells +/− E₂. b, TRIM24-WT and TRIM24-C840W expressed in stable shTRIM24 MCF7 cells +/− E₂. c, shControl and shTRIM24 MCF7 cells, E₂ range. TRIM24-WT or EGFP control expressed in shTRIM24 MCF7 cells. (in a, b and c) GREB1 RNA levels normalized to GAPDH; untreated shControl MCF7 set as one. Each bar is an average of 3 biological replicates, 3 independent RT-PCR assays of each; error bars show standard deviation. d, ChIP of ERα and TRIM24, histone H3 and histone modifications, 6 h E₂, shControl and shTRIM24 MCF7 cells. Histone modifications normalized for H3 recovery. Each bar represents averaged results, n=3 and 3 assays of each; error bars show standard deviation.

Figure 5. Aberrant expression of TRIM24 correlates with poor survival of breast cancer patients

a, shControl and shTRIM24 MCF7 cells plus E₂ or E₂ plus 4-hydroxytamoxifen, as indicated. Each bar represents the averaged results for three independent colony formation assays in triplicate plates; error bars show standard deviation. b, Immunohistochemistry: 128 surgical specimens of breast cancer immunostained for TRIM24: subcellular localization (N) and staining intensity (strong, +++; moderate, ++; weak or slightly above background, +; none, −). c, The overall survival rate of 128 patients with nonmetastatic disease, classified by TRIM24 expression (as in b), plotted by the Kaplan-Meier method.