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Supplementary Figure 7. Targeting of TRIM37 suppresses TNBC metastasis.

The majority of clinical deaths in patients with triple-negative breast cancer (TNBC) are due to chemoresistance and aggressive metastases, with high prevalence in younger women of African ethnicity. Although tumorigenic drivers are numerous and varied, the drivers of metastatic transition remain largely unknown. Here, we uncovered a molecular dependence of TNBC tumors on the TRIM37 network, which enables tumor cells to resist chemotherapeutic as well as metastatic stress. TRIM37-directed histone H2A monoubiquitination enforces changes in DNA repair that rendered TP53-mutant TNBC cells resistant to chemotherapy. Chemotherapeutic drugs triggered a positive feedback loop via ATM/E2F1/STAT signaling, amplifying the TRIM37 network in chemoresistant cancer cells. High expression of TRIM37 induced transcriptomic changes characteristic of a metastatic phenotype, and inhibition of TRIM37 substantially reduced the in vivo propensity of TNBC cells. Selective delivery of TRIM37-specific antisense oligonucleotides using antifolate receptor 1–conjugated nanoparticles in combination with chemotherapy suppressed lung metastasis in spontaneous metastatic murine models. Collectively, these findings establish TRIM37 as a clinically relevant target with opportunities for therapeutic intervention.Significance:TRIM37 drives aggressive TNBC biology by promoting resistance to chemotherapy and inducing a prometastatic transcriptional program; inhibition of TRIM37 increases chemotherapy efficacy and reduces metastasis risk in patients with TNBC.

Table I: List of TRIM37-associated DSB repair and cell proliferation genes expressed in non-TNBC, TNBC and normal tissue obtained from METABRIC patient cohort.

Supplementary Figure 4. Chemotherapy activates ATM/E2F1/STAT axis to upregulate TRIM37 in TNBC.

Supplementary Methods and Material

The majority of clinical deaths in patients with triple-negative breast cancer (TNBC) are due to chemoresistance and aggressive metastases, with high prevalence in younger women of African ethnicity. Although tumorigenic drivers are numerous and varied, the drivers of metastatic transition remain largely unknown. Here, we uncovered a molecular dependence of TNBC tumors on the TRIM37 network, which enables tumor cells to resist chemotherapeutic as well as metastatic stress. TRIM37-directed histone H2A monoubiquitination enforces changes in DNA repair that rendered TP53-mutant TNBC cells resistant to chemotherapy. Chemotherapeutic drugs triggered a positive feedback loop via ATM/E2F1/STAT signaling, amplifying the TRIM37 network in chemoresistant cancer cells. High expression of TRIM37 induced transcriptomic changes characteristic of a metastatic phenotype, and inhibition of TRIM37 substantially reduced the in vivo propensity of TNBC cells. Selective delivery of TRIM37-specific antisense oligonucleotides using antifolate receptor 1–conjugated nanoparticles in combination with chemotherapy suppressed lung metastasis in spontaneous metastatic murine models. Collectively, these findings establish TRIM37 as a clinically relevant target with opportunities for therapeutic intervention.Significance:TRIM37 drives aggressive TNBC biology by promoting resistance to chemotherapy and inducing a prometastatic transcriptional program; inhibition of TRIM37 increases chemotherapy efficacy and reduces metastasis risk in patients with TNBC.

Lack of effective immune infiltration represents a significant barrier to immunotherapy in solid tumors. Thus, solid tumor‐enriched death receptor‐5 (DR5) activating antibodies, which generates tumor debulking by extrinsic apoptotic cytotoxicity, remains a crucial alternate therapeutic strategy. Over past few decades, many DR5 antibodies moved to clinical trials after successfully controlling tumors in immunodeficient tumor xenografts. However, DR5 antibodies failed to significantly improve survival in phase‐II trials, leading in efforts to generate second generation of DR5 agonists to supersize apoptotic cytotoxicity in tumors. Here we have discovered that clinical DR5 antibodies activate an unexpected immunosuppressive PD‐L1 stabilization pathway, which potentially had contributed to their limited success in clinics. The DR5 agonist stimulated caspase‐8 signaling not only activates ROCK1 but also undermines proteasome function, both of which contributes to increased PD‐L1 stability on tumor cell surface. Targeting DR5‐ROCK1‐PD‐L1 axis markedly increases immune effector T‐cell function, promotes tumor regression, and improves overall survival in animal models. These insights have identified a potential clinically viable combinatorial strategy to revive solid cancer immunotherapy using death receptor agonism., DR5 agonists moved to clinical trials after successfully controlling tumors in immunodeficient xenograft models. By stabling introducing human DR5 extracellular domain into murine tumor cells, this study identifies PD‐L1 pathway as a contributing factor for potential immune evasion in solid tumors.

Background Rett syndrome (RTT) is a neurodevelopmental disorder caused by mutations in the X-linked methyl-CpG binding protein 2 (MeCP2) gene. While MeCP2 mutations are lethal in most males, females survive birth but show severe neurological defects. Because X-chromosome inactivation (XCI) is a random process, approximately 50% of the cells silence the wild-type (WT) copy of the MeCP2 gene. Thus, reactivating the silent WT copy of MeCP2 could provide therapeutic intervention for RTT. Methods Toward this goal, we screened ~ 28,000 small-molecule compounds from several libraries using a MeCP2-luciferase reporter cell line and cortical neurons from a MeCP2-EGFP mouse model. We used gain/increase of luminescence or fluorescence as a readout of MeCP2 reactivation and tested the efficacy of these drugs under different drug regimens, conditions, and cellular contexts. Results We identified inhibitors of the JAK/STAT pathway as XCI-reactivating agents, both by in vitro and ex vivo assays. In particular, we show that AG-490, a Janus Kinase 2 (JAK2) kinase inhibitor, and Jaki, a pan JAK/STAT inhibitor, are capable of reactivating MeCP2 from the inactive X chromosome, in different cellular contexts. Conclusions Our results suggest that inhibition of the JAK/STAT pathway is a new potential pathway to reinstate MeCP2 gene expression as an efficient RTT treatment.

Additional file 1: Supplementary figure 1. Compound validation under different treatment regimens. A) AG490 validation in 6-well plate format. Relative Luminescence Units (RLU) is shown in function of the drug treatment for the clone Xi8. B) qRT-PCR of MeCP2, luciferase, Xist and Rnf12. Left: single treatment for the 72 hours regimen for clone Xi8. Right: double treatment regimen for 72 hr each for clone Xi8. Bars indicate standard error of the mean (SEM). * indicates statistical significance, p ≤ 0.05, n = 3 per genes, average of two independent assays is shown) (C) Top: Representative fluorescent immunostaining images of cortical neurons in presence or absence of the compounds with double treatment (i.e. twice for 72 hr). Bottom: Quantitative analysis of number of GFP positive cells. The % of GFP positive cells increased in the presence of AG490 and Jaki with a double treatment regimen. Bars indicate standard error of the mean (SEM), samples are color-coded, concentrations are shown. * indicates statistical significance, p ≤ 0.05, n = 4 per compound and dose, average of two independent assays is shown).

Additional file 3: Supplementary Table 1. This table shows the libraries used in this screen.

Rett syndrome (RTT) is a genetic disorder resulting from a loss-of-function mutation in one copy of the X-linked gene methyl-CpG–binding protein 2 (MECP2). Typical RTT patients are females and, due to random X chromosome inactivation (XCI), ∼50% of cells express mutant MECP2 and the other ∼50% express wild-type MECP2. Cells expressing mutant MECP2 retain a wild-type copy of MECP2 on the inactive X chromosome (Xi), the reactivation of which represents a potential therapeutic approach for RTT. Previous studies have demonstrated reactivation of Xi-linked MECP2 in cultured cells by biological or pharmacological inhibition of factors that promote XCI (called “XCI factors” or “XCIFs”). Whether XCIF inhibitors in living animals can reactivate Xi-linked MECP2 in cerebral cortical neurons, the cell type most therapeutically relevant to RTT, remains to be determined. Here, we show that pharmacological inhibitors targeting XCIFs in the PI3K/AKT and bone morphogenetic protein signaling pathways reactivate Xi-linked MECP2 in cultured mouse fibroblasts and human induced pluripotent stem cell-derived postmitotic RTT neurons. Notably, reactivation of Xi-linked MECP2 corrects characteristic defects of human RTT neurons including reduced soma size and branch points. Most importantly, we show that intracerebroventricular injection of the XCIF inhibitors reactivates Xi-linked Mecp2 in cerebral cortical neurons of adult living mice. In support of these pharmacological results, we also demonstrate genetic reactivation of Xi-linked Mecp2 in cerebral cortical neurons of living mice bearing a homozygous XCIF deletion. Collectively, our results further establish the feasibility of pharmacological reactivation of Xi-linked MECP2 as a therapeutic approach for RTT.

SUMMARY Receptor clustering is the first and critical step to activate apoptosis by death receptor-5 (DR5). The recent discovery of the autoinhibitory DR5 ectodomain has challenged the long-standing view of its mechanistic activation by the natural ligand Apo2L. Because the autoinhibitory residues have remained unknown, here we characterize a crucial patch of positively charged residues (PPCR) in the highly variable domain of DR5. The PPCR electrostatically separates DR5 receptors to autoinhibit their clustering in the absence of ligand and antibody binding. Mutational substitution and antibody-mediated PPCR interference resulted in increased apoptotic cytotoxic function. A dually specific antibody that enables sustained tampering with PPCR function exceptionally enhanced DR5 clustering and apoptotic activation and distinctively improved the survival of animals bearing aggressive metastatic and recurrent tumors, whereas clinically tested DR5 antibodies without PPCR blockade function were largely ineffective. Our study provides mechanistic insights into DR5 activation and a therapeutic analytical design for potential clinical success., In brief Shivange et al. describe a negative regulatory epitope in the death receptor-5 ectodomain that limits apoptotic cytotoxicity. The negative regulatory domain co-targeting with dual-specificity antibody designs elicits sustained DR5 clustering and a superior anti-tumor response, explaining past clinical failures and revisiting future clinical strategies., Graphical Abstract

Oncogenomic studies indicate that copy number variation (CNV) alters genes involved in tumor progression; however, identification of specific driver genes affected by CNV has been difficult, as these rearrangements are often contained in large chromosomal intervals among several bystander genes. Here, we addressed this problem and identified a CNV-targeted oncogene by performing comparative oncogenomics of human and zebrafish melanomas. We determined that the gene encoding growth differentiation factor 6 (GDF6), which is the ligand for the BMP family, is recurrently amplified and transcriptionally upregulated in melanoma. GDF6-induced BMP signaling maintained a trunk neural crest gene signature in melanomas. Additionally, GDF6 repressed the melanocyte differentiation gene MITF and the proapoptotic factor SOX9, thereby preventing differentiation, inhibiting cell death, and promoting tumor growth. GDF6 was specifically expressed in melanomas but not melanocytes. Moreover, GDF6 expression levels in melanomas were inversely correlated with patient survival. Our study has identified a fundamental role for GDF6 and BMP signaling in governing an embryonic cell gene signature to promote melanoma progression, thus providing potential opportunities for targeted therapy to treat GDF6-positive cancers.

miR-206, miR-1a-1 and miR-1a-2 are induced during differentiation of skeletal myoblasts and promote myogenesis in vitro. miR-206 is required for skeletal muscle regeneration in vivo. Although this microRNA family is hypothesized to play an essential role in differentiation, a triple knockout of the three genes has not been done to test this hypothesis. We report that triple KO C2C12 myoblasts generated using CRISPR/Cas9 method differentiate despite the expected de-repression of the microRNA targets. Surprisingly, their mitochondrial function is diminished. Triple KO mice demonstrate partial embryonic lethality, most likely due to the role of miR-1a in cardiac muscle differentiation. Two triple KO mice survive and grow normally to adulthood with smaller myofiber diameter and diminished physical performance. Thus, unlike other microRNAs important in other differentiation pathways, the miR-206 family is not absolutely essential for myogenesis and is instead a modulator of optimal differentiation of skeletal myoblasts.

X chromosome inactivation (XCI) is the random silencing of one X chromosome in females to achieve gene dosage balance between the sexes. As a result, all females are heterozygous for X-linked gene expression. One of the key regulators of XCI is Xist, which is essential for the initiation and maintenance of XCI. Previous studies have identified 13 trans acting X chromosome inactivation factors (XCIFs) using a large-scale, loss-of-function genetic screen. Inhibition of XCIFs, such as ACVR1 and PDPK1, using short-hairpin RNA or small molecule inhibitors, reactivates X chromosome-linked genes in cultured cells. But the feasibility and tolerability of reactivating the inactive X chromosome in vivo remains to be determined. Towards this goal, a XistΔ:Mecp2/Xist:Mecp2-Gfp mouse model has been generated with non-random XCI due to deletion of Xist on one X chromosome. Using this model, the extent of inactive X reactivation was quantitated in the mouse brain following treatment with XCIF inhibitors. Recently published results show, for the first time, that pharmacological inhibition of XCIFs reactivates Mecp2 from the inactive X chromosome in cortical neurons of the living mouse brain.

X-inactive specific transcript (Xist) is a long noncoding RNA that is essential for initiating and maintaining epigenetic silencing of one copy of the X chromosome in mammalian females. But the mechanism by which Xist localizes and spreads on the X chromosome and facilitates transcriptional silencing remains largely unknown. This limited understanding, at least in part, is due to the technical difficulties in the visualization and functional characterization of Xist. Development of a successful method for Xist tracking is a key to better understanding of the X chromosome silencing, as well as to gain insight into the regulatory role of other long noncoding RNAs. Here, we describe an alternative method for visualization of Xist lncRNA in cells using a CRISPR/Cas9-based approach. This strategy is relatively simple approach to track Xist at different stages of cell differentiation, providing mechanistic insights into the initiation, maintenance, and establishment of X inactivation.

X-inactive specific transcript (Xist) is a long noncoding RNA that is essential for initiating and maintaining epigenetic silencing of one copy of the X chromosome in mammalian females. But the mechanism by which Xist localizes and spreads on the X chromosome and facilitates transcriptional silencing remains largely unknown. This limited understanding, at least in part, is due to the technical difficulties in the visualization and functional characterization of Xist. Development of a successful method for Xist tracking is a key to better understanding of the X chromosome silencing, as well as to gain insight into the regulatory role of other long noncoding RNAs. Here, we describe an alternative method for visualization of Xist lncRNA in cells using a CRISPR/Cas9-based approach. This strategy is relatively simple approach to track Xist at different stages of cell differentiation, providing mechanistic insights into the initiation, maintenance, and establishment of X inactivation.

Therapeutic antibodies targeting ovarian cancer (OvCa)-enriched receptors have largely been disappointing due to limited tumor specific antibody-dependent cellular cytotoxicity (ADCC). Here we report a symbiotic approach that is highly selective and superior compared to investigational clinical antibodies. This Bispecific-Anchored Cytotoxicity-Activator (BaCa) antibody is rationally designed to instigate “cis” and “trans” cytotoxicity by combining specificities against folate receptor alpha-1 (FOLR1) and death receptor 5 (DR5). Whereas the in vivo agonist DR5 signaling requires FcγRIIB interaction, the FOLR1 anchor functions as a primary clustering point to retain and maintain a high-level of tumor-specific apoptosis. The presented proof of concept study strategically makes use of a tumor-cell enriched anchor receptor for agonist death-receptor targeting to potentially generate a clinically viable strategy for OvCa.

Rett syndrome (RTT) is a devastating neurodevelopmental disorder with no cure. RTT typically affects females and is caused by mutations in one of the two copies of the X-linked gene MECP2. Due to X chromosome inactivation, the random silencing of one X chromosome, females with RTT have a mixture of cells that express either normal or mutant MECP2. Importantly, cells expressing mutant MECP2 retain a normal copy of MECP2 on the inactive X chromosome (Xi), the reactivation of which represents a potential therapeutic approach for RTT. Here we use small-molecule inhibitors to reactivate the Xi-linked Mecp2 gene in brain neurons of living mice. Our results further establish the feasibility of Xi-linked MECP2 reactivation-based therapeutics for treatment of RTT.

X-chromosome inactivation (XCI), the random transcriptional silencing of one X chromosome in somatic cells of female mammals, is a mechanism that ensures equal expression of X-linked genes in both sexes. XCI is initiated in cis by the noncoding Xist RNA, which coats the inactive X chromosome (Xi) from which it is produced. However, trans-acting factors that mediate XCI remain largely unknown. Here, we perform a large-scale RNA interference screen to identify trans-acting XCI factors (XCIFs) that comprise regulators of cell signaling and transcription, including the DNA methyltransferase, DNMT1. The expression pattern of the XCIFs explains the selective onset of XCI following differentiation. The XCIFs function, at least in part, by promoting expression and/or localization of Xist to the Xi. Surprisingly, we find that DNMT1, which is generally a transcriptional repressor, is an activator of Xist transcription. Small-molecule inhibitors of two of the XCIFs can reversibly reactivate the Xi, which has implications for treatment of Rett syndrome and other dominant X-linked diseases. A homozygous mouse knockout of one of the XCIFs, stanniocalcin 1 (STC1), has an expected XCI defect but surprisingly is phenotypically normal. Remarkably, X-linked genes are not overexpressed in female Stc1(-/-) mice, revealing the existence of a mechanism(s) that can compensate for a persistent XCI deficiency to regulate X-linked gene expression.

In mammals, gene expression is largely controlled at the transcriptional level. In response to environmental or intrinsic signaling, gene expression is often fine-tuned by epigenetic modifications, including DNA methylation and histone modifications. One such histone modification is ubiquitination that predominately occurs in mono-ubiquitinated forms on histone H2A and H2B. We recently identified and characterized a novel E3 ligase called TRIM37 that ubiquitinates H2A. This study highlights the consequence of aberrant histone ubiquitination at the promoters of tumor suppressor genes in breast cancer. Regulatory mechanism by which TRIM37 and other auxiliary proteins are involved in the initiation and progression of breast cancer is of utmost importance toward generating effective therapeutics. Here, we describe a detailed step-by-step process of carrying out in vitro ubiquitination assay using purified histone proteins or reconstituted nucleosomes and affinity-purified recombinant E3 ligase like TRIM37. These experimental procedures are largely based on our studies in mammalian cells and will be a useful tool to identify substrate for E3 ubiquitin ligase as well as characterizing new E3 ligases.

In mammals, gene expression is largely controlled at the transcriptional level. In response to environmental or intrinsic signaling, gene expression is often fine-tuned by epigenetic modifications, including DNA methylation and histone modifications. One such histone modification is ubiquitination that predominately occurs in mono-ubiquitinated forms on histone H2A and H2B. We recently identified and characterized a novel E3 ligase called TRIM37 that ubiquitinates H2A. This study highlights the consequence of aberrant histone ubiquitination at the promoters of tumor suppressor genes in breast cancer. Regulatory mechanism by which TRIM37 and other auxiliary proteins are involved in the initiation and progression of breast cancer is of utmost importance toward generating effective therapeutics. Here, we describe a detailed step-by-step process of carrying out in vitro ubiquitination assay using purified histone proteins or reconstituted nucleosomes and affinity-purified recombinant E3 ligase like TRIM37. These experimental procedures are largely based on our studies in mammalian cells and will be a useful tool to identify substrate for E3 ubiquitin ligase as well as characterizing new E3 ligases.

Intracellular pathogens and the molecules they express have limited contact with the immune system. Here, we show that macrophages infected with intracellular pathogens Mycobacterium tuberculosis, M bovis BCG, Salmonella typhimurium, or Toxoplasma gondii release from cells small vesicles known as exosomes which contain pathogen-associated molecular patterns (PAMPs). These exosomes, when exposed to uninfected macrophages, stimulate a proinflammatory response in a Toll-like receptor– and myeloid differentiation factor 88–dependent manner. Further, exosomes isolated from the bronchoalveolar lavage fluid (BALF) of M bovis BCG–infected mice contain the mycobacteria components lipoarabinomannan and the 19-kDa lipoprotein and can stimulate TNF-α production in naive macrophages. Moreover, exosomes isolated from M bovis BCG– and M tuberculosis–infected macrophages, when injected intranasally into mice, stimulate TNF-α and IL-12 production as well as neutrophil and macrophage recruitment in the lung. These studies identify a previously unknown function for exosomes in promoting intercellular communication during an immune response to intracellular pathogens, and we hypothesize that extracellular release of exosomes containing PAMPs is an important mechanism of immune surveillance.

Mycobacterium avium is a major opportunistic pathogen in HIV-positive individuals and is responsible for increased morbidity and mortality in AIDS patients. M. avium express glycopeptidolipids (GPLs) as a major cell wall constituent, and recent studies suggest that GPLs play an important role in M. avium pathogenesis. In the present study we show that M. avium-infected macrophages release GPLs, which are trafficked from the phagosome through the endocytic network to multivesicular bodies. Prior studies have shown that multivesicular bodies can fuse with the plasma membrane releasing small 50 to 100 nm vesicles known as exosomes. We found that M. avium-infected macrophages release exosomes containing GPLs leading to the transfer of GPLs from infected to uninfected macrophages. Interestingly, exosomes isolated from M. avium-infected but not from uninfected macrophages can stimulate a proinflammatory response in resting macrophages. This proinflammatory response is dependent on Toll like receptor (TLR) 2, TLR4, and MyD88 suggesting that released exosomes contain M. avium-expressed TLR ligands. Our studies are the first to demonstrate that exosomes isolated from mycobacteria-infected macro-phages can induce a proinflammatory response, and we hypothesize that exosomes play an important role in immune surveillance during intracellular bacteria infections.

Mycobacterium avium is a significant cause of morbidity and mortality in AIDS patients. M. avium can be isolated as three major morphotypes: smooth-transparent (SmT ), smooth-opaque (SmO) and rough (Rg). Studies indicate that many Rg isolates lack or have modified glycopeptidolipids (GPLs). GPLs are major surface constituents of the M. avium cell wall and heterogeneity in their carbohydrate moieties has been used to classify M. avium into different serotypes, with serotypes 1, 4 and 8 being isolated with high frequency from AIDS patients. However, it is unclear what role GPLs play in M. avium pathogenicity. To begin to address how the absence of GPLs affects M. avium-macrophage interaction, we used the well-characterized M. avium 2151 SmT and Rg isolates which differ in GPL expression. We found macrophages infected with the Rg compared with SmT M. avium 2151 showed prolonged activation of the mitogen-activated protein kinases (MAPKs) p38 and ERK1/2. Macrophages infected with the Rg 2151 also showed increased tumour necrosis factor-alpha (TNF-alpha) production. Interestingly, TNF-alpha secretion by macrophages infected with SmO or SmT 2151 was dependent on p38, ERK1/2 and NF-kappaB while TNF-alpha secretion by Rg 2151-infected macrophages was dependent on NF-kappaB but not the MAPKs. Rg 2151-infected macrophages also produced increased levels of IL-6, IL-12, MCP-1 and RANTES relative to macrophages infected with SmT 2151. These results indicate that M. avium 2151 deficient in GPLs promote increased macrophage activation. This disparity in cellular activation stems from a quantitative and qualitative difference in the macrophage signalling response to the Rg and SmT M. avium 2151.

Mycobacterium avium is a major opportunistic pathogen of AIDS patients in the United States. The understanding of M. avium pathogenesis has been hampered by the inability to create gene knockouts by homologous recombination, an important mechanism for defining and characterizing virulence factors. In this study a functional methyltransferase D (mtfD) gene was deleted by allelic replacement in the M. avium strain 104. Methyltransferase D is involved in the methylation of glycopeptidolipids (GPLs); highly antigenic glycolipids found in copious amounts on the M. avium cell surface. Interestingly, the loss of mtfD resulted in M. avium 104 containing only the non-serotype specific GPL. Results also suggest that the mtfD encodes for a 3-O-methyltransferase. The absence of significant amounts of any serotype-specific GPLs as a consequence of mtfD deletion indicates that the synthesis of the core 3,4-di-O-methyl rhamnose is a prerequisite for synthesis of the serotype-specific GPLs. Macrophages infected with the mtfD mutant show elevated production of tumour necrosis factor-alpha (TNF-alpha) and RANTES compared to control infections. In addition, the M. avium 104 mtfD mutant exhibits decreased ability to survive/proliferate in mouse liver and lung compared to wild-type 104, as assessed by bacterial counts. Importantly, the mtfD mutant complemented with a wild-type mtfD gene maintained an infection level similar to wild-type. These experiments demonstrate that the loss of mtfD results in a M. avium 104 strain, which preferentially activates macrophages in vitro and shows attenuated virulence in mice. Together our data support a role for GPLs in M. avium pathogenesis.

The RING finger protein TRIM37 is encoded by a gene that is amplified in certain breast cancers, but its function is unknown; here, it is shown to mono-ubiquitinate histone H2A and repress gene expression, and to function as a breast cancer oncoprotein. The RING finger protein TRIM37 is encoded by a gene amplified in certain breast cancers, but its function was unknown. Michael Green and colleagues now report that TRIM37 can mono-ubiquitinate histone H2A and functions in gene regulation by associating with the polycomb complex PRC2. TRIM37 is proposed to promote transformation by altering the specificity of PRC2 and facilitating silencing of tumour suppressors and other genes. 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.

In mammals, the transcriptional activity of genes is often attenuated by epigenetic silencing mechanisms involving DNA hypermethylation and/or inhibitory histone modifications. Alterations in epigenetic regulation contribute to a number of developmental anomalies, genetic disorders and diseases, including cancer. Therefore, a detailed understanding of the mechanisms involved in epigenetic silencing is expected to shed light on these human disease states and suggest new therapeutic approaches. Inhibitory histone modifications are promoted by a variety of epigenetic regulators including the Polycomb repressive complexes PRC1 and PRC2, which function to silence specific sets of genes.1 In the canonical PRC1/2-mediated silencing pathway, PRC2 first interacts with the promoter and catalyzes histone H3 lysine 27 (H3K27) trimethylation. This repressive mark is then recognized by PRC1, which consists of a histone H2A E3 ubiquitin ligase that mono-ubiquitinates H2A on lysine 119 (Fig. 1, left). In mammalian cells, the PRC1 component RNF2 is the major H2A ubiquitin ligase involved in gene regulation. Figure 1. TRIM37 represses gene expression through a non-canonical PRC1/2-mediated silencing pathway. (Left) The canonical model for PRC1/2-mediated gene silencing. PRC2 first interacts with the promoter and catalyzes H3K27 trimethylation using the PRC2 subunit ... We recently characterized a new epigenetic regulator, called TRIM37, that is involved in PRC1/2-mediated epigenetic repression.2 TRIM37 contains an RING finger domain that is a hallmark of E3 ubiquitin ligases, but its substrate(s) and function(s) remained unidentified. We demonstrated that TRIM37 functions as an H2A E3 ubiquitin ligase that, like RNF2, mono-ubiquitinates histone H2A at lysine 119. Notably, the TRIM37 gene is located in 17q23, a chromosomal region that is amplified in almost 40% of breast cancers. Interestingly, in breast cancer cell lines harboring 17q23 amplification, RNF2 levels are greatly reduced, suggesting TRIM37 might functionally replace RNF2 in 17q23-amplified breast cancer cells. Unexpectedly, however, a variety of biochemical experiments demonstrated that unlike RNF2, TRIM37 is associated with PRC2, not PRC1. To identify TRIM37 target genes, we performed genome-wide chromatin immunoprecipitation (ChIP)-chip and identified 9412 gene promoters that are bound by TRIM37 and enriched for ubiquitinated H2A; importantly, this list of TRIM37 target genes includes many putative tumor suppressors. Moreover, we found that TRIM37 was co-bound with PRC2 and PRC1 to specific target genes, resulting in their transcriptional silencing. Analysis of a published comprehensive dataset of human breast cancer samples revealed a statistically significant correlation between increased TRIM37 levels and decreased expression of TRIM37 target genes, emphasizing the clinical relevance of TRIM37. Our results raised the possibility that in 17q23-amplified breast cancers, the elevated levels of TRIM37 contribute to oncogenesis by promoting silencing of tumor suppressor genes. In support of this model, we showed that RNA interference (RNAi)-mediated knockdown of TRIM37 in breast cancer cells containing 17q23 amplification reduced tumor growth in xenograft mouse model studies. Conversely, ectopic expression of TRIM37 in non-malignant and immortalized pre-malignant breast epithelial lines not only transformed the cells but also induced tumor formation in xenograft mice. Collectively, our findings provide crucial insight into how TRIM37 functions and uncover a non-canonical PRC1/2-mediated silencing pathway (Fig. 1, right). Upon overexpression, TRIM37 associates with and alters the specificity of PRC2, thereby promoting extensive changes in gene expression that include the silencing of multiple tumor suppressors. The promoter-bound TRIM37–PRC2 complex carries out both H3K27 trimethylation and H2A mono-ubiquitination in a mechanism that is distinct from the canonical PRC1/2-mediated silencing pathway. How PRC2 and PRC1 are selectively recruited to specific genomic loci in mammalian cells represents a major unanswered question in the field. Unlike in Drosophila, where so-called Polycomb response elements (PREs) direct PRC1/2 recruitment, such elements appear to be lacking in mammalian genomes.1 Some models propose that mammalian PRCs are directly recruited either by transcription factors or long non-coding RNAs, whereas other models hypothesize that PRCs are indirectly recruited to genes that are already transcriptionally inactive.3,4 The TRIM37-mediated epigenetic silencing model that we have derived and characterized in breast cancers harboring 17q23 amplification provides a powerful tool to investigate the mechanisms of PRC1/2 recruitment. Our study identified TRIM37 as an oncogenic counterpart of RNF2, the main H2A E3 ubiquitin ligase functioning in normal mammalian cells. Inhibition of TRIM37 activity is expected to selectively prevent the growth of 17q23 amplicon-positive tumors without affecting normal cells. Therefore, TRIM37 represents an attractive potential target for the development of novel therapies for breast cancers harboring 17q23 amplification. The characterization of TRIM37 as an oncogene may also provide insight into an autosomal recessive growth disorder called mulibrey nanism. A number of truncating frame-shift mutations in TRIM37 have been identified in individuals with the disease.5,6 In most cases, the truncated TRIM37 protein is predicted to retain an intact and likely functional RING domain, raising the possibility that the oncogenic activity of TRIM37 might contribute to the high frequency of tumors associated with mulibrey nanism.7 However, whether aberrant TRIM37 E3 ubiquitin ligase activity is an underlying cause of the growth defects observed in mulibrey nanism is an interesting question that merits further study.

Mycobacterium avium is a major opportunistic pathogen of AIDS patients in the United States. The understanding of M. avium pathogenesis has been hampered by the inability to create gene knockouts by homologous recombination, an important mechanism for defining and characterizing virulence factors. In this study a functional methyltransferase D (mtfD) gene was deleted by allelic replacement in the M. avium strain 104. Methyltransferase D is involved in the methylation of glycopeptidolipids (GPLs); highly antigenic glycolipids found in copious amounts on the M. avium cell surface. Interestingly, the loss of mtfD resulted in M. avium 104 containing only the non-serotype specific GPL. Results also suggest that the mtfD encodes for a 3-O-methyltransferase. The absence of significant amounts of any serotype-specific GPLs as a consequence of mtfD deletion indicates that the synthesis of the core 3,4-di-O-methyl rhamnose is a prerequisite for synthesis of the serotype-specific GPLs. Macrophages infected with the mtfD mutant show elevated production of tumour necrosis factor-alpha (TNF-alpha) and RANTES compared to control infections. In addition, the M. avium 104 mtfD mutant exhibits decreased ability to survive/proliferate in mouse liver and lung compared to wild-type 104, as assessed by bacterial counts. Importantly, the mtfD mutant complemented with a wild-type mtfD gene maintained an infection level similar to wild-type. These experiments demonstrate that the loss of mtfD results in a M. avium 104 strain, which preferentially activates macrophages in vitro and shows attenuated virulence in mice. Together our data support a role for GPLs in M. avium pathogenesis.