Abstract
The TRIM37 (also known as MUL) gene is located in the 17q23 chromosomal region, which is amplified in up to ∼40% of breast cancers1. TRIM37 contains a RING finger domain, a hallmark of E3 ubiquitin ligases2, but its protein substrate(s) is unknown. Here we report that TRIM37 mono-ubiquitinates histone H2A, a chromatin modification associated with transcriptional repression3. We find that in human breast cancer cell lines containing amplified 17q23, TRIM37 is upregulated and, reciprocally, the major H2A ubiquitin ligase RNF2 (also known as RING1B)3,4 is downregulated. Genome-wide chromatin immunoprecipitation (ChIP)-chip experiments in 17q23-amplified breast cancer cells identified many genes, including multiple tumour suppressors, whose promoters were bound by TRIM37 and enriched for ubiquitinated H2A. However, unlike RNF2, which is a subunit of polycomb repressive complex 1 (PRC1)3,4,5, we find that TRIM37 associates with polycomb repressive complex 2 (PRC2). TRIM37, PRC2 and PRC1 are co-bound to specific target genes, resulting in their transcriptional silencing. RNA-interference-mediated knockdown of TRIM37 results in loss of ubiquitinated H2A, dissociation of PRC1 and PRC2 from target promoters, and transcriptional reactivation of silenced genes. Knockdown of TRIM37 in human breast cancer cells containing amplified 17q23 substantially decreases tumour growth in mouse xenografts. Conversely, ectopic expression of TRIM37 renders non-transformed cells tumorigenic. Collectively, our results reveal TRIM37 as an oncogenic H2A ubiquitin ligase that is overexpressed in a subset of breast cancers and promotes transformation by facilitating silencing of tumour suppressors and other genes.
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Acknowledgements
We thank C. Peterson for providing Xenopus nucleosomes; R. Weinberg and M. Cristofanilli for providing cell lines; A. Virbasius and the University of Massachusetts Medical School (UMMS) RNAi Core Facility for providing shRNAs; P. Spatrick at the UMMS Genomics Core Facility; the UMMS Proteomics and Mass Spectroscopy Facility for mass spectrometry analysis; and S. Deibler for providing editorial assistance. N.W. is a Sidney Kimmel Scholar for Cancer Research and is supported by young investigator awards from the National Lung Cancer Partnership/Uniting Against Lung Cancer, Melanoma Research Alliance and International Association for the Study of Lung Cancer. This work was also supported by grants from CEA-DSV, ATIGE Genopole and Centre National de la Recherche Scientifique (CNRS) to C.G., and from the National Institutes of Health (R01GM033977) to M.R.G. M.R.G. is an investigator of the Howard Hughes Medical Institute.
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Contributions
C.G. made the initial observation that TRIM37 has an H2A mono-ubiqutination activity. S.B., C.G., J.S.T. and M.R.G. designed the experiments. S.B., L.C., X.Z., L.L. and N.W. performed the research. C.-M.V. provided critical reagents. J.O. and L.J.Z. performed biostatistical analysis for ChIP-chip experiments and database mining. S.B. and M.R.G. analysed and interpreted the data and wrote the paper. All authors reviewed the paper and provided comments.
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Extended data figures and tables
Extended Data Figure 1 Control experiments for Fig. 1.
a, ChIP monitoring H2Aub enrichment on Fas in Kras NIH 3T3 cells expressing a non-silencing (NS) shRNA or a Trim37, Rnf2 or Bmi1 shRNA. Three regions of the Fas promoter were analysed: the core promoter/transcription start site (CP/TSS), and 1 and 2 kb upstream of the TSS. Actb and Gapdh are shown as negative controls. Error bars indicate s.e.m.; n = 9 (three biological replicates with three technical replicates per sample). b, H2Aub ChIP as described in a using a second Trim37, Rnf2 or Bmi1 shRNA unrelated to that used in a. c, qRT–PCR analysis monitoring knockdown efficiencies of Trim37 and Rnf2 shRNAs in NIH 3T3 cells. The results are given relative to expression after treatment with an NS shRNA, which was set to 1. For knockdown efficiencies of the Bmi1 shRNAs see ref. 6. Error bars indicate s.e.m.; n = 3 technical replicates of a representative experiment (out of three experiments). d, In vitro ubiquitination assay. Purified H2B was incubated with E1 (UBE1), E2 (UBCH5B), E3 (TRIM37 or BRCA1), ATP and HA-tagged ubiquitin. Blots were probed with antibodies against H2Bub or H2B. The results show that TRIM37 does not ubiquitinate H2B. BRCA1, which is known to ubiquitinate H2B at K120 (refs 39, 40) was used as a positive control. e, f, qRT–PCR (e) and immunoblots (f) monitoring TRIM37 and RNF2 in various cell lines. Expression of TRIM37 and RNF2 was normalized to that obtained in HMECs, which was set to 1. Error bars indicate s.d.; n = 3 technical replicates of a representative experiment (out of three experiments). g, Immunoblot monitoring TRIM37 levels in MCF7 cells expressing an NS or one of two unrelated TRIM37 shRNAs. α-Tubulin (TUBA) was monitored as a loading control. The results indicate that the TRIM37 antibody is highly specific. h, qRT–PCR analysis monitoring knockdown efficiencies of TRIM37 shRNAs in MCF7, BT474 and MDA-MD-361 cells. Error bars indicate s.e.m.; n = 3 technical replicates of a representative experiment (out of three experiments). i, Top, immunoblots monitoring levels of H2Aub and H2Bub in MCF7 cells expressing an NS or one of two unrelated TRIM37 shRNAs. Bottom, quantification of the H2Aub immunoblots relative to TUBA. The relative level of H2Aub in NS cells was set to 1. In this experiment, histones were acid extracted. j, Top, immunoblots monitoring TRIM37, H2Aub, H2A, H2Bub and H4 in BT474 and MDA-MB-361 cells expressing an NS or one of two unrelated TRIM37 shRNAs. Bottom, H2Aub quantification, as described in g. k, qRT–PCR analysis monitoring knockdown efficiencies of RNF2 shRNAs in MCF7 cells. Error bars indicate s.e.m.; n = 3 technical replicates of a representative experiment (out of three experiments). l, Top, immunoblots monitoring TRIM37, H2Aub, H2A and H4 in MDA-MB-231, Hs578T and T47D cells expressing an NS or one of two unrelated TRIM37 shRNAs. Bottom, H2Aub quantification, as described in g. m, Proliferation of cultured MDA-MB-231, Hs578T and T47D cells expressing an NS or TRIM37 shRNA. Error bars indicate s.d.; n = 3 technical replicates of a representative experiment (out of three experiments). The results show that knockdown of TRIM37 has no effect on proliferation of breast cancer cell lines lacking 17q23 amplification. n, qRT–PCR analysis monitoring TRIM37 expression in an MCF10A cell line ectopically expressing TRIM37 or, as a control, empty vector. The results were normalized to TRIM37 expression in MCF10A cells expressing empty vector, which was set to 1. Error bars indicate s.e.m.; n = 3 technical replicates of a representative experiment (out of three experiments). *P < 0.05, **P < 0.01.
Extended Data Figure 2 Extra details related to ChIP-chip analysis.
a, Histograms showing enrichment of TRIM37, H2Aub (in parental MCF7 cells or after TRIM37 knockdown (KD)), EZH2 and H3K27me3 (in parental MCF7 cells or after TRIM37 knockdown) as a function of distance to the nearest transcription start site (TSS). b, Left, box plot of maximal gene-level log2 fold change of enrichment intensity comparing input for H2Aub in parental MCF7 cells and after TRIM37 knockdown. Right, box plot of number of enriched regions per gene for H2Aub in parental MCF7 cells or after TRIM37 knockdown. The results show that there was a significant difference in H2Aub enrichment at TRIM37 target genes after TRIM37 knockdown with respect to both enrichment intensity (P < 1 × 10−22) and number of enriched regions (P = 2.787008 × 10−222). A region with a fold change ≥2 and a false discovery rate <0.1 was considered a differentially enriched site. c, Venn diagram showing overlap between TRIM37- and EZH2-bound genes and H3K27me3-enriched genes in MCF7 cells. d, Left, box plot of maximal gene-level log2 fold change of enrichment intensity comparing input for H3K27me3 in parental MCF7 cells or after TRIM37 knockdown. Right, box plot of number of enriched regions per gene for H3K27me3 in parental MCF7 cells or after TRIM37 knockdown. The results show that there was a significant difference in H3K27me3 enrichment at TRIM37 target genes after TRIM37 knockdown with respect to both enrichment intensity (P = 5.872777 × 10−109) and number of enriched regions (P = 1.178392 × 10−52). A region with a fold change ≥2 and a false discovery rate <0.1 was considered a differentially enriched site. e, Percentage of promoters (defined as 1 kb upstream of the TSS) bound by EZH2 alone that contain a CpG island (3,332/5,869; 56.77%) or co-bound by EZH2 and TRIM37 that contain a CpG island (1,929/3,384; 57.00%). A two-sample test for equality of proportions with continuity correction showed that there was no statistically significant difference in CpG island content between the EZH2-bound and EZH2, TRIM37 co-bound promoters.
Extended Data Figure 3 Control experiments for Fig. 2.
a, ChIP monitoring binding of TRIM37 at promoters of TRIM37 target genes in MCF7 cells expressing an NS or one of two unrelated TRIM37 shRNAs. As a negative control, TRIM37 binding at three non-TRIM37 target genes, ACTB, EEF1A1 and GAPDH, is shown. Black asterisks indicate significance of TRIM37 enrichment compared with the IgG control (from cells expressing an NS shRNA); blue asterisks indicate significant differences in TRIM37 enrichment in cells expressing a TRIM37 shRNA relative to NS shRNA. Error bars indicate s.d.; n = 3 technical replicates of a representative experiment (out of three experiments). The results confirm that TRIM37 occupancy is reduced upon TRIM37 knockdown, demonstrating the specificity of the TRIM37 antibody in ChIP experiments. b, ChIP monitoring enrichment of H2Aub at promoters of TRIM37 target genes in MCF7 cells expressing a second TRIM37 shRNA unrelated to that used in Fig. 2d. The IgG control and H2Aub signal in cells expressing an NS shRNA are the same as those shown in Fig. 2d. Error bars indicate s.d.; n = 3 technical replicates of a representative experiment (out of three experiments). c, ChIP monitoring enrichment of H2Aub at the promoters of TRIM37 target genes in MCF7 cells expressing an NS or one of two unrelated RNF2 shRNAs. Error bars indicate s.d.; n = 3 technical replicates of a representative experiment (out of three experiments). d, qRT–PCR analysis monitoring expression of TRIM37 target genes in MCF7 cells expressing a second TRIM37 shRNA unrelated to that used in Fig. 2e. Expression of each gene was normalized to that obtained with an NS shRNA, which was set to 1. Error bars indicate s.e.m.; n = 3 technical replicates of a representative experiment (out of three experiments). e, qRT–PCR analysis monitoring expression of TRIM37 target genes after RNF2 knockdown in MCF7 cells. Expression of each gene was normalized to that obtained with an NS shRNA, which was set to 1 (indicated by the dotted red line). Error bars indicate s.e.m.; n = 3 technical replicates of a representative experiment (out of three experiments). f, qRT–PCR analysis monitoring expression of TRIM37 target genes after TRIM37 knockdown in BT474 cells. Error bars indicate s.e.m.; n = 3 technical replicates of a representative experiment (out of three experiments). g, qRT–PCR analysis monitoring expression of p14ARF after knockdown of TRIM37 or EZH2 in BT474 cells. Error bars indicate s.e.m.; n = 3 technical replicates of a representative experiment (out of three experiments). The results indicate that knockdown of EZH2 but not TRIM37 de-represses p14ARF expression in BT474 cells. h, qRT–PCR analysis monitoring knockdown efficiencies of TRIM37 (left) and EZH2 (right) shRNAs in BT474 cells. Error bars indicate s.e.m.; n = 3 technical replicates of a representative experiment (out of three experiments). *P < 0.05, **P < 0.01.
Extended Data Figure 4 Additional experiments showing interaction between TRIM37 and PRC2 subunits.
a, Co-immunoprecipitation analysis. MCF7 nuclear extracts were immunoprecipitated with a TRIM37, EZH2 or SUZ12 antibody, or an IgG control, and the immunoprecipitates were analysed for TRIM37, EZH2 or SUZ12 by immunoblotting. b, Co-immunoprecipitation analysis. MCF7 nuclear extracts were immunoprecipitated with a TRIM37 or EZH2 antibody, or an IgG control, in the presence of ethidium bromide, and the immunoprecipitates were analysed for TRIM37 or EZH2 by immunoblotting. The results show that interaction between TRIM37 and EZH2 occurs in the presence of ethidium bromide and is thus not mediated by DNA. c, Co-immunoprecipitation analysis. Nuclear extracts from MCF7 cells ectopically expressing Flag–TRIM37 were immunoprecipitated with anti-Flag magnetic beads, a EZH2 or SUZ12 antibody, or an IgG control, and the immunoprecipitates were analysed for TRIM37, EZH2 or SUZ12 by immunoblotting. d, Mass spectroscopy. Selected results from the liquid chromatography tandem mass spectroscopy analysis listing TRIM37 or PRC2 subunits, and their total spectra score in samples immunoprecipitated using either IgG or a TRIM37 antibody. The probability of interaction, derived from the Scaffold Viewer software, indicates a ≥95% probability of interaction in all cases. See also Supplementary Table 2. e, Tandem mass spectra of representative peptides of proteins identified in the TRIM37 immunoprecipitate are shown with corresponding spectral counts for each protein shown in the table. f, In vitro interaction pull-down assay. Purified glutathione-S-transferase (GST)–TRIM37 was incubated with an in vitro translated biotinylated PRC2 subunit (indicated on left). GST–TRIM37 was purified using GST-agarose beads, and the presence of the PRC2 subunit was analysed by immunoblotting with an anti-biotin antibody. The results indicate that TRIM37 interacts strongly with EZH2 and weakly with RBBP4, and does not detectably interact with AEBP2, EED or SUZ12. g, GAL4–TRIM37 fusion experiment. Top, schematic diagram of TRIM37 showing the location of the RING, BBOX, coiled coil (CC), MATH and nuclear localization sequence (NLS) motifs. Locations of the motifs were obtained from UniProtKB (http://www.uniprot.org/uniprot/O94972#section_attribute). The ΔBBCC deletion comprises the BBOX and first CC motif (Δaa 89–235). Middle, left, ChIP monitoring binding of GAL4–TRIM37, EZH2 or SUZ12 to the adenovirus E1B promoter containing (GAL4(UAS)-E1B) or lacking (E1B) GAL4-binding sites or to an irrelevant, negative control (NC) DNA region. The indicated GAL4–TRIM37 fusion protein was co-expressed with a plasmid containing or lacking five GAL4-binding sites upstream of the adenovirus E1B gene in 293T cells followed by ChIP analysis. Error bars indicate s.d.; n = 3 technical replicates of a representative experiment (out of three experiments). The results indicate that the GAL4–TRIM37 fusion protein was able to recruit both EZH2 and SUZ212 to the GAL4-binding sites. Middle, right, ChIP monitoring binding of EZH2 and SUZ12 in the presence of GAL4–TRIM37 deletion mutants. The GAL4–TRIM37 wild-type (WT) samples are the same as those shown in the left panel. The results indicate that the ability of GAL4–TRIM37 to recruit EZH2 and SUZ212 requires the TRIM37 RING domain and NLS (presumably for nuclear entry), but not the BBCC or MATH domains. Bottom, immunoblot analysis monitoring expression of GAL4–TRIM37 fusion proteins using a GAL4 antibody. α-Tubulin (TUBA) was monitored as a loading control.
Extended Data Figure 5 Confirmation of the results of Fig. 3f, h using second, unrelated shRNAs.
a, ChIP monitoring binding of BMI1 and EZH2 to the promoters of TRIM37 target genes in MCF7 cells expressing an NS shRNA or a second TRIM37 shRNA unrelated to that used in Fig. 3f. The IgG control and BMI1 and EZH2 signal in cells expressing an NS shRNA are the same as those shown in Fig. 3f. Black asterisks indicate significance of BMI1 or EZH2 enrichment compared with the IgG control (from cells expressing an NS shRNA); blue asterisks indicate significant differences in BMI1 or EZH2 enrichment in cells expressing a TRIM37 shRNA relative to NS shRNA. Error bars indicate s.d.; n = 3 technical replicates of a representative experiment (out of three experiments). b, qRT–PCR (top) and immunoblot (bottom) monitoring BMI1 and EZH2 levels in MCF7 cells expressing an NS or TRIM37 shRNA. Error bars indicate s.e.m.; n = 3 technical replicates of a representative experiment (out of three experiments). The results indicate that BMI1 and EZH2 levels are unaffected by TRIM37 knockdown in MCF7 cells. c, Left, qRT–PCR analysis monitoring expression of two genes that are bound by EZH2 but not TRIM37, ADAM7 (top) and YES1 (bottom), after knockdown of TRIM37 or EZH2 in MCF7 cells. Error bars indicate s.e.m.; n = 3 technical replicates of a representative experiment (out of three experiments). The results indicate that knockdown of EZH2 but not TRIM37 de-represses ADAM7 and YES1 expression. Right, ChIP monitoring EZH2 enrichment at the promoters of ADAM7 and YES1 in MCF7 cells expressing an NS or TRIM37 shRNA. Error bars indicate s.d.; n = 3 technical replicates of a representative experiment (out of three experiments). The results show that knockdown of TRIM37 has no effect on EZH2 binding at ADAM7 and YES1 promoters. Thus, loss of EZH2 binding and de-repression after TRIM37 knockdown is not general to PRC2-bound genes but rather is selective for TRIM37 target genes. d, ChIP monitoring H3K27me3 enrichment at promoters of TRIM37 target genes in MCF7 cells expressing an NS or TRIM37 shRNA. Error bars indicate s.d.; n = 3 technical replicates of a representative experiment (out of three experiments). e, qRT–PCR analysis monitoring BMI1 (left) and EZH2 (right) knockdown efficiency in MCF7 cells after shRNA-mediated knockdown using two unrelated shRNAs against each gene. Expression of each gene was normalized to that obtained with an NS shRNA, which was set to 1. Error bars indicate s.e.m.; n = 3 technical replicates of a representative experiment (out of three experiments). f, qRT–PCR monitoring TRIM37 target gene expression in MCF7 cells after knockdown of BMI1 or EZH2 using a second shRNA unrelated to that used in Fig. 3h. Error bars indicate s.e.m.; n = 3 technical replicates of a representative experiment (out of three experiments). *P < 0.05, **P < 0.01.
Extended Data Figure 6 TRIM37/17q23 copy number and promoter methylation analysis for TRIM37 target genes in human breast cancer samples.
a, Analysis of 17q23 copy number in a panel of 71 human breast cancer samples; the region corresponding to the TRIM37 gene is highlighted in yellow. The breast cancer samples shown here are the same as those shown in Fig. 4a. Red indicates increased copy number and green indicates decreased copy number (see Methods). b, Promoter methylation analysis. Each of the 60 TRIM37 target genes shown in Fig. 4a were analysed for promoter CpG methylation in human breast cancer samples. The level of DNA methylation is shown ranging from low (green) to high (red); white indicates data were not available. The first column is a heat map showing expression of TRIM37. The results indicate no significant correlation between TRIM37 expression and promoter methylation of TRIM37 target genes.
Extended Data Figure 7 Control experiments related to Fig. 4.
a, Proliferation of cultured MCF7 cells expressing an NS shRNA or a second TRIM37 shRNA unrelated to that used in Fig. 4d. Error bars indicate s.d.; n = 3 technical replicates of a representative experiment (out of three experiments). b, c, Tumour formation in mice subcutaneously injected with BT474 (b) or FC-IBC02 (c) cells expressing an NS or TRIM37 shRNA. Error bars indicate s.e.m.; n = 3 mice per group. d, Immunoblot monitoring TRIM37 levels in NIH 3T3 cells expressing vector, wild-type (WT) TRIM37 or TRIM37(C18R). α-Tubulin (TUBA) was monitored as a loading control. e, Immunoblot monitoring TRIM37 levels in MCF10AT cells expressing vector or Flag-tagged TRIM37. f, qRT–PCR analysis monitoring knockdown efficiency of a tumour suppression gene in MCF10AT cells expressing the indicated shRNA. The results are given relative to expression after treatment with an NS shRNA, which was set to 1. Error bars indicate s.d.; n = 3 technical replicates of a representative experiment (out of three experiments). g, Soft agar assay monitoring colony formation of MCF10AT cells expressing an NS or the indicated shRNA. Error bars indicate s.e.m.; n = 3 technical replicates of a representative experiment (out of three experiments). h, Tumour formation in mice subcutaneously injected with MCF10AT cells expressing an NS or the indicated shRNA. Error bars indicate s.e.m.; n = 3 mice per group. i, Soft agar assay monitoring colony formation of MCF10A cells expressing vector or TRIM37. Error bars indicate s.e.m.; n = 3 technical replicates of a representative experiment (out of three experiments). j, Tumour formation in mice subcutaneously injected with MCF10A cells expressing vector or TRIM37. Error bars indicate s.e.m.; n = 3 mice per group. k, Tumour formation in mice subcutaneously injected with BPLER cells expressing vector or TRIM37. Error bars indicate s.e.m.; n = 3 mice per group. *P < 0.05, **P < 0.01.
Extended Data Figure 8 Schematic model of TRIM37-directed transcriptional repression and Kaplan–Meier analysis comparing survival of breast cancer patients with high or low TRIM37 expression.
a, Schematic models for canonical (left) and TRIM37 (right) pathways for target gene silencing. b, Kaplan–Meier analysis of survival of patients with low (black) or high (red) TRIM37 expression. The number of surviving patients at 0-, 5-, 10- and 15-year time points is indicated below the graph. High expression of TRIM37 was significantly correlated with lower survival rate (P value = 0.00046, hazard ratio (HR) = 1.46 with 95% confidence interval 1.18–1.81). The analysis was performed using an online survival analysis tool to analyse the effect of gene expression on breast cancer prognosis using microarray data from 1,809 patients41.
Supplementary information
Supplementary Table 1
List of genes identified from the TRIM37 (Sheet 1) and H2A-ub (Sheet 2) ChIP-chip analyses, and their overlap (Sheet 3). List of genes with differential H2A-ub enrichment upon TRIM37 knockdown in MCF7 cells (Sheet 4). (XLS 9114 kb)
Supplementary Table 2
Liquid chromatography tandem mass spectrometry analysis of proteins that interact with TRIM37. (XLS 56 kb)
Supplementary Table 3
List of genes identified from the EZH2 ChIP-chip analysis (Sheet 1), and the overlap with TRIM37 target genes (Sheet 2). (XLS 3242 kb)
Supplementary Table 4
List of genes identified from the H3K27me3 ChIP-chip analysis (Sheet 1), and the overlap with TRIM37 target genes (Sheet 2). List of genes with differential H3K27me3 enrichment upon TRIM37 knockdown in MCF7 cells (Sheet 3). (XLS 3973 kb)
Supplementary Table 5
List of genes whose decreased expression significantly correlates with increased TRIM37 levels in 466 human breast cancer samples. (XLS 694 kb)
Supplementary Table 6
Overlap of proteins and epigenetic marks, along with the P values for each overlap, for the ChIP-chip analyses. (XLS 18 kb)
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Bhatnagar, S., Gazin, C., Chamberlain, L. et al. TRIM37 is a new histone H2A ubiquitin ligase and breast cancer oncoprotein. Nature 516, 116–120 (2014). https://doi.org/10.1038/nature13955
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DOI: https://doi.org/10.1038/nature13955
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