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Emerging roles of the MAGE protein family in stress response pathways

Open AccessPublished:September 13, 2020DOI:https://doi.org/10.1074/jbc.REV120.008029
      The melanoma antigen (MAGE) proteins all contain a MAGE homology domain. MAGE genes are conserved in all eukaryotes and have expanded from a single gene in lower eukaryotes to ∼40 genes in humans and mice. Whereas some MAGEs are ubiquitously expressed in tissues, others are expressed in only germ cells with aberrant reactivation in multiple cancers. Much of the initial research on MAGEs focused on exploiting their antigenicity and restricted expression pattern to target them with cancer immunotherapy. Beyond their potential clinical application and role in tumorigenesis, recent studies have shown that MAGE proteins regulate diverse cellular and developmental pathways, implicating them in many diseases besides cancer, including lung, renal, and neurodevelopmental disorders. At the molecular level, many MAGEs bind to E3 RING ubiquitin ligases and, thus, regulate their substrate specificity, ligase activity, and subcellular localization. On a broader scale, the MAGE genes likely expanded in eutherian mammals to protect the germline from environmental stress and aid in stress adaptation, and this stress tolerance may explain why many cancers aberrantly express MAGEs. Here, we present an updated, comprehensive review on the MAGE family that highlights general characteristics, emphasizes recent comparative studies in mice, and describes the diverse functions exerted by individual MAGEs.

      Introduction: A comparative view of the MAGE gene family

      Discovery of MAGEs

      Classic studies in the 1940s and 1950s provided experimental evidence for the concept that the immune system can recognize and reject tumor cells (
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      The three Es of cancer immunoediting.
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      Cause and consequence of cancer/testis antigen activation in cancer.
      ) and opened the floodgates for identifying and characterizing tumor antigens, which could be targeted for cancer therapy. In addition to mutated, fused, overexpressed, and oncoviral proteins (
      • Whitehurst A.W.
      Cause and consequence of cancer/testis antigen activation in cancer.
      ), male germ cell–specific proteins were added to the inventory in 1991 when melanoma antigen 1 (MAGE-1) was discovered in the melanoma cell line MZ2-MEL (
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      A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma.
      ). MZ2-MEL cells were established from a patient (MZ-2) who had, for 10 years, presented with strong T-cell reactivity against autologous tumor cells in culture (
      • Jager E.
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      The discovery of cancer/testis antigens by autologous typing with t cell clones and the evolution of cancer vaccines.
      ). This patient had stage IV amelanotic melanoma of an unknown primary tumor and never achieved complete remission despite multiple surgical interventions followed by chemotherapy. Remarkably, continued vaccination with autologous melanoma cell clones that had been mutagenized in vitro and lethally irradiated led to the patient surviving for more than 30 years without disease recurrence. To identify the tumor-associated antigens recognized by the cytotoxic T cells in this patient, Boon and his group (
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      • Chomez P.
      • Lurquin C.
      • De Plaen E.
      • Van den Eynde B.
      • Knuth A.
      • Boon T.
      A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma.
      ) applied autologous typing and transfection of a cosmid library into the patient-derived MZ2-E cell line. Their efforts led to the discovery of MAGE-1, the first human tumor antigen, which was later renamed MAGE-A1 upon the identification of additional gene family members (
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      • Knuth A.
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      A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma.
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      ) identified a whole family of MAGE genes, present in all placental mammals. Humans and mice have ∼40 MAGE genes, which include some designated as pseudogenes, that are further subdivided into two major categories based on their sequence homology, tissue expression pattern, and chromosomal location (Figure 1, Figure 2) (
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      An overview of the mage gene family with the identification of all human members of the family.
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      The melanoma antigen genes—any clues to their functions in normal tissues?.
      ). The type I MAGEs include the MAGE-A, -B, primate-specific -C, and mouse-specific Mage-a–like (-al and -k1) subfamily members. Type I MAGEs are also called cancer-testis antigens (CTAs) because they are primarily expressed in the testis but are normally silent in other tissues (Fig. 2A) (
      • Chomez P.
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      • Bertrand M.
      • De Plaen E.
      • Boon T.
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      An overview of the mage gene family with the identification of all human members of the family.
      ,
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      MAGE cancer-testis antigens protect the mammalian germline under environmental stress.
      ); however, they are often aberrantly reactivated during oncogenic transformation (Fig. 2B) and code for antigens recognized by cytolytic T lymphocytes (
      • Chomez P.
      • De Backer O.
      • Bertrand M.
      • De Plaen E.
      • Boon T.
      • Lucas S.
      An overview of the mage gene family with the identification of all human members of the family.
      ). In contrast, the type II MAGEs, consisting of the MAGE-D, -E, -F, -G, -H, -L, and NECDIN genes, are more ubiquitously expressed in humans and mice and not typically associated with human cancer (
      • Chomez P.
      • De Backer O.
      • Bertrand M.
      • De Plaen E.
      • Boon T.
      • Lucas S.
      An overview of the mage gene family with the identification of all human members of the family.
      ,
      • Barker P.A.
      • Salehi A.
      The MAGE proteins: emerging roles in cell cycle progression, apoptosis, and neurogenetic disease.
      ,
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      • Lord T.
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      MAGE cancer-testis antigens protect the mammalian germline under environmental stress.
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      Evolutionary history of the cancer immunity antigen mage gene family.
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      ).
      Figure thumbnail gr2
      Figure 2Expression of MAGEs in normal tissues and cancer. A, human and mouse MAGE expression during different life stages is indicated. Starting with the top part of the outer circle, the expression of MAGEs is depicted during spermatogenesis, in ES cells, in an embryo, and finally in adults. B, the heatmap displays the percentage of various tumors that express each type I MAGE. The results are based upon data generated by the TCGA Research Network (RRID:SCR_003193).
      Figure thumbnail gr1
      Figure 1Overview of the MAGE gene family in humans and mice. A, phylogenetic tree showing the relationship between human and mouse MAGE proteins. The tree was created by the neighbor-joining construction method using the Jukes–Cantor protein distance measurement from the CLC Main Workbench 20. B, chromosomal location of human and mouse MAGE genes. C, locations of MAGE genes on the human and mouse X chromosome based on the recent NCBI's genome assembly HRCh38.p13 and GRCm38.p6. For all figures, the type II MAGEs are represented in green, MAGE-A and -C subfamilies in red, and MAGE-B subfamily in blue. Light colors indicate mouse Mages and dark colors indicate human MAGEs.
      Since the discovery of MAGEs, a major research focus has been developing MAGE-targeted immunotherapies. Despite promising results from initial clinical trials (
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      Selection of immunostimulant AS15 for active immunization with MAGE-A3 protein: results of a randomized phase II study of the European Organisation for Research and Treatment of Cancer Melanoma Group in Metastatic Melanoma.
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      Adjuvant MAGE-A3 immunotherapy in resected non–small-cell lung cancer: phase II randomized study results.
      ), MAGE-A3 vaccines ultimately failed in Phase III due to a lack of efficacy (
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      MAGE-A3 immunotherapeutic as adjuvant therapy for patients with resected, MAGE-A3-positive, stage III melanoma (derma): a double-blind, randomised, placebo-controlled, phase 3 trial.
      ,
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      Efficacy of the MAGE-A3 cancer immunotherapeutic as adjuvant therapy in patients with resected MAGE-A3-positive non-small-cell lung cancer (MAGRIT): a randomised, double-blind, placebo-controlled, phase 3 trial.
      ), which suggested that activation of the T-cell response to MAGE-A3 antigen is not sufficient to inhibit disease progression (
      • Daud A.I.
      Negative but not futile: MAGE-A3 immunotherapeutic for melanoma.
      ). Furthermore, some patients treated with anti-MAGE therapies developed serious off-target effects, like neuro- and cardiotoxicity (
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      • Bennett A.D.
      • et al.
      Cardiovascular toxicity and titin cross-reactivity of affinity-enhanced t cells in myeloma and melanoma.
      ,
      • Morgan R.A.
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      • Miller A.D.
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      Cancer regression and neurological toxicity following anti-MAGE-A3 TCR gene therapy.
      ). The neurotoxicity may have been caused by the anti-MAGE-A3-TCR–engineered T cells recognizing a similar MAGE that is expressed in the brain (i.e. MAGE-A12) (
      • Fon Tacer K.
      • Montoya M.C.
      • Oatley M.J.
      • Lord T.
      • Oatley J.M.
      • Klein J.
      • Ravichandran R.
      • Tillman H.
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      • Pruett-Miller S.M.
      • Bookout A.L.
      • Binshtock E.
      • Kamiński M.M.
      • Potts P.R.
      MAGE cancer-testis antigens protect the mammalian germline under environmental stress.
      ), and the cardiotoxicity was attributed to vaccine recognition of an unrelated peptide (titin) in the heart (
      • Linette G.P.
      • Stadtmauer E.A.
      • Maus M.V.
      • Rapoport A.P.
      • Levine B.L.
      • Emery L.
      • Litzky L.
      • Bagg A.
      • Carreno B.M.
      • Cimino P.J.
      • Binder-Scholl G.K.
      • Smethurst D.P.
      • Gerry A.B.
      • Pumphrey N.J.
      • Bennett A.D.
      • et al.
      Cardiovascular toxicity and titin cross-reactivity of affinity-enhanced t cells in myeloma and melanoma.
      ,
      • Morgan R.A.
      • Chinnasamy N.
      • Abate-Daga D.
      • Gros A.
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      • Zheng Z.
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      • Phan G.Q.
      • Hughes M.S.
      • Kammula U.S.
      • Miller A.D.
      • Hessman C.J.
      • et al.
      Cancer regression and neurological toxicity following anti-MAGE-A3 TCR gene therapy.
      ). Besides inefficacy and unexpected side effects, resistance has been another major roadblock. For example, MAGE-A expression correlates with poor response to the CTLA-4 checkpoint inhibitors in melanoma patients (
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      • Clement K.
      • Cartun Z.J.
      • et al.
      Cancer-germline antigen expression discriminates clinical outcome to CTLA-4 blockade.
      ) and faster development of resistance to the epidermal growth factor receptor tyrosine kinase inhibitors and chemotherapy (
      • Jin J.
      • Liu B.-Z.
      • Wu Z.-M.
      Evaluation of melanoma antigen gene A3 expression in drug resistance of epidermal growth factor receptor-tyrosine kinase inhibitors in advanced nonsmall cell lung cancer treatment.
      ,
      • Chen Y.
      • Zhao H.
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      • Tang H.
      • Qiu C.
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      • Fu B.
      LINC01234/MicroRNA-31-5p/MAGEA3 axis mediates the proliferation and chemoresistance of hepatocellular carcinoma cells.
      ,
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      Melanoma associated antigen (MAGE)-A3 promotes cell proliferation and chemotherapeutic drug resistance in gastric cancer.
      ). Despite these setbacks, research is ongoing to improve clinical outcomes and limit off-target effects of MAGE-based immunotherapies (
      • Sun Q.
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      T-cell receptor gene therapy targeting melanoma-associated antigen-A4 by silencing of endogenous TCR inhibits tumor growth in mice and human.
      ,
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      In vitro generation of cytotoxic T cells with potential for adoptive tumor immunotherapy of multiple myeloma.
      ). Alternative methods to target MAGE-expressing cancers by utilizing combinations of conventional therapy and immunotherapy are also being investigated (
      • Bao L.
      • Dunham K.
      • Lucas K.
      MAGE-A1, MAGE-A3, and NY-ESO-1 can be upregulated on neuroblastoma cells to facilitate cytotoxic t lymphocyte-mediated tumor cell killing.
      ,
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      ,
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      A phase I trial combining decitabine/dendritic cell vaccine targeting MAGE-A1, MAGE-A3 and NY-ESO-1 for children with relapsed or therapy-refractory neuroblastoma and sarcoma.
      ). To successfully and safely target the type I MAGEs, it is important to understand the mechanisms by which these proteins contribute to oncogenesis, how they are regulated, and what they normally do in physiological contexts. In this review, we dive into what is known about the diverse functions of individual MAGEs, as well as their roles in cancer and other diseases. Although MAGE proteins have diverse functions, emerging studies suggest that responding to stress is a unifying theme of MAGEs.

      Genomic organization and structure of human and mouse MAGEs

      Most of the MAGE genes are located in clusters that are preserved in diverse mammalian species; however, each cluster has undergone a different degree of expansion by duplication or retrotransposition, leading to a number of species-specific genes (
      • Zhao Q.
      • Caballero O.L.
      • Simpson A.J.G.
      • Strausberg R.L.
      Differential evolution of mage genes based on expression pattern and selection pressure.
      ). As shown in Fig. 1, human and mouse genomes encompass different numbers of MAGE subfamily members. They also differ in that only humans possess MAGE-C genes, and mice possess additional Mage-a–like genes that form another subfamily (Fig. 1) (
      • Fon Tacer K.
      • Montoya M.C.
      • Oatley M.J.
      • Lord T.
      • Oatley J.M.
      • Klein J.
      • Ravichandran R.
      • Tillman H.
      • Kim M.
      • Connelly J.P.
      • Pruett-Miller S.M.
      • Bookout A.L.
      • Binshtock E.
      • Kamiński M.M.
      • Potts P.R.
      MAGE cancer-testis antigens protect the mammalian germline under environmental stress.
      ). Consistent with their classification as CTAs, the type I MAGE subfamilies reside in syntenic regions on the X chromosome (Fig. 1, B and C) (
      • Whitehurst A.W.
      Cause and consequence of cancer/testis antigen activation in cancer.
      ,
      • Chomez P.
      • De Backer O.
      • Bertrand M.
      • De Plaen E.
      • Boon T.
      • Lucas S.
      An overview of the mage gene family with the identification of all human members of the family.
      ,
      • Fon Tacer K.
      • Montoya M.C.
      • Oatley M.J.
      • Lord T.
      • Oatley J.M.
      • Klein J.
      • Ravichandran R.
      • Tillman H.
      • Kim M.
      • Connelly J.P.
      • Pruett-Miller S.M.
      • Bookout A.L.
      • Binshtock E.
      • Kamiński M.M.
      • Potts P.R.
      MAGE cancer-testis antigens protect the mammalian germline under environmental stress.
      ,
      • Scanlan M.J.
      • Gure A.O.
      • Jungbluth A.A.
      • Old L.J.
      • Chen Y.-T.
      Cancer/testis antigens: an expanding family of targets for cancer immunotherapy.
      ,
      • Caballero O.L.
      • Chen Y.-T.
      Cancer/testis (CT) antigens: potential targets for immunotherapy.
      ), where testis-expressed genes are overrepresented (
      • Warburton P.E.
      • Giordano J.
      • Cheung F.
      • Gelfand Y.
      • Benson G.
      Inverted repeat structure of the human genome: the X-chromosome contains a preponderance of large, highly homologous inverted repeats that contain testes genes.
      ,
      • Skaletsky H.
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      • Chinwalla A.
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      The male-specific region of the human Y chromosome is a mosaic of discrete sequence classes.
      ,
      • Mueller J.L.
      • Skaletsky H.
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      • Rock S.
      • Graves T.
      • Auger K.
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      • Wilson R.K.
      • Page D.C.
      Independent specialization of the human and mouse X chromosomes for the male germ line.
      ). The autosomal murine gene Mage-b3 is an exception, as it resides on chromosome 2. Another distinction between humans and mice is that Mage-a genes map to two different loci on the murine X chromosome, which could be the result of an interchromosomal recombination event during genome evolution in rodents (Fig. 1, B and C) (
      • Ross M.T.
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      The DNA sequence of the human X chromosome.
      ). In contrast to the uniform genomic location of type I MAGEs, type II MAGE genes are located on both autosomes and the X chromosome (Fig. 1, B and C). The type II MAGEs also exhibit species-specific copy number variations. For example, the mouse genome has only three Mage-d genes and an additional Mage-g gene, Mage-g2 (
      • Fon Tacer K.
      • Montoya M.C.
      • Oatley M.J.
      • Lord T.
      • Oatley J.M.
      • Klein J.
      • Ravichandran R.
      • Tillman H.
      • Kim M.
      • Connelly J.P.
      • Pruett-Miller S.M.
      • Bookout A.L.
      • Binshtock E.
      • Kamiński M.M.
      • Potts P.R.
      MAGE cancer-testis antigens protect the mammalian germline under environmental stress.
      ,
      • Jeong J.
      • Jin S.
      • Choi H.
      • Kwon J.
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      • Cho C.
      Characterization of MAGEG2 with testis-specific expression in mice.
      ). Intriguingly, Mage-f1 has a point mutation in rodents and is predicted to either be a pseudogene or code for a truncated protein (
      • Weon J.L.
      • Yang S.W.
      • Potts P.R.
      Cytosolic iron-sulfur assembly is evolutionarily tuned by a cancer-amplified ubiquitin ligase.
      ); thus, Mage-g2 may be a rodent adaptation to this Mage-f1 mutation loss and may serve important functions during germ cell development (
      • Jeong J.
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      • Kim J.
      • Kim J.
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      Characterization of MAGEG2 with testis-specific expression in mice.
      ).
      Although most MAGE proteins (and their murine homologs) are encoded by a single exon, the coding regions of the MAGE-D subfamily span across more than 11 exons (
      • Chomez P.
      • De Backer O.
      • Bertrand M.
      • De Plaen E.
      • Boon T.
      • Lucas S.
      An overview of the mage gene family with the identification of all human members of the family.
      ,
      • Zhao Q.
      • Caballero O.L.
      • Simpson A.J.G.
      • Strausberg R.L.
      Differential evolution of mage genes based on expression pattern and selection pressure.
      ). The MAGE-D subfamily is also the most conserved subfamily between species, with over 90% identity in the coding sequences (
      • Zhao Q.
      • Caballero O.L.
      • Simpson A.J.G.
      • Strausberg R.L.
      Differential evolution of mage genes based on expression pattern and selection pressure.
      ), and the genomic structure of the murine Mage-d genes closely resembles that of humans (
      • Lucas S.
      • Brasseur F.
      • Boon T.
      A new mage gene with ubiquitous expression does not code for known mage antigens recognized by T cells.
      ). The majority of the type I genes acquired several 5′ noncoding exons, potentially allowing for differential regulation of expression (
      • De Plaen E.
      • Traversari C.
      • Gaforio J.J.
      • Szikora J.P.
      • De Smet C.
      • Brasseur F.
      • van der Bruggen P.
      • Lethé B.
      • Lurquin C.
      • Chomez P.
      • De Backer O.
      Structure, chromosomal localization, and expression of 12 genes of the mage family.
      ,
      • Rogner U.C.
      • Wilke K.
      • Steck E.
      • Korn B.
      • Poustka A.
      The melanoma antigen gene (MAGE) family is clustered in the chromosomal band xq28.
      ). Some mouse Mage-b genes that were originally thought to be pseudogenes (Mage-b7, -b8, and -b17) because they have the structure of a processed transcript (
      • Forslund K.Ö.
      • Nordqvist K.
      The melanoma antigen genes—any clues to their functions in normal tissues?.
      ) code for full proteins and are expressed on the transcriptional level in a cell-specific manner in the testis, suggesting a functional role in spermatogenesis (
      • Fon Tacer K.
      • Montoya M.C.
      • Oatley M.J.
      • Lord T.
      • Oatley J.M.
      • Klein J.
      • Ravichandran R.
      • Tillman H.
      • Kim M.
      • Connelly J.P.
      • Pruett-Miller S.M.
      • Bookout A.L.
      • Binshtock E.
      • Kamiński M.M.
      • Potts P.R.
      MAGE cancer-testis antigens protect the mammalian germline under environmental stress.
      ). Furthermore, during primate evolution, human MAGE-A11 acquired three additional 5′ coding exons that are unique among the type I genes (
      • Willett C.S.
      • Wilson E.M.
      Evolution of melanoma antigen-A11 (MAGEA11) during primate phylogeny.
      ). Together, the genomic organization and structure of the MAGE gene family indicate that it has expanded by retrotransposition and local duplication events. After splitting from their phylogenetic ancestor, the MAGE genes independently evolved in each species, with the type I MAGEs evolving most rapidly (
      • Zhao Q.
      • Caballero O.L.
      • Simpson A.J.G.
      • Strausberg R.L.
      Differential evolution of mage genes based on expression pattern and selection pressure.
      ).

      Evolution of the MAGE gene family

      The MAGE gene family is evolutionarily conserved across eukaryotes. Unlike the large multigene family found in placental mammals, earlier eukaryotes, from protozoa to nonplacental mammals like the platypus, possess a single MAGE gene (
      • Barker P.A.
      • Salehi A.
      The MAGE proteins: emerging roles in cell cycle progression, apoptosis, and neurogenetic disease.
      ,
      • Zhao Q.
      • Caballero O.L.
      • Simpson A.J.G.
      • Strausberg R.L.
      Differential evolution of mage genes based on expression pattern and selection pressure.
      ,
      • López-Sánchez N.
      • González-Fernández Z.
      • Niinobe M.
      • Yoshikawa K.
      • Frade J.M.
      Single mage gene in the chicken genome encodes CMage, a protein with functional similarities to mammalian type II mage proteins.
      ,
      • Lee A.K.
      • Potts P.R.
      A comprehensive guide to the mage family of ubiquitin ligases.
      ). The first expansion of the MAGE gene family possibly occurred in marsupials, but with the emergence of the placenta and LINE elements in eutherian mammals, the family rapidly expanded (
      • Zhao Q.
      • Caballero O.L.
      • Simpson A.J.G.
      • Strausberg R.L.
      Differential evolution of mage genes based on expression pattern and selection pressure.
      ). During eutherian radiation, the subfamily ancestors were formed by retrotransposition and expanded by gene duplications (
      • Chomez P.
      • De Backer O.
      • Bertrand M.
      • De Plaen E.
      • Boon T.
      • Lucas S.
      An overview of the mage gene family with the identification of all human members of the family.
      ,
      • Katsura Y.
      • Satta Y.
      Evolutionary history of the cancer immunity antigen mage gene family.
      ).
      Although most of the MAGEs that exist today appear to derive from a single ancestral gene, the identity of the founder family member is still a matter of debate. The unique genomic architecture of the MAGE-D genes suggests that one of them is the founder (
      • Chomez P.
      • De Backer O.
      • Bertrand M.
      • De Plaen E.
      • Boon T.
      • Lucas S.
      An overview of the mage gene family with the identification of all human members of the family.
      ,
      • Katsura Y.
      • Satta Y.
      Evolutionary history of the cancer immunity antigen mage gene family.
      ); however, functional studies of MAGE-G1 imply that it is most closely related to the ancestral MAGE (
      • López-Sánchez N.
      • González-Fernández Z.
      • Niinobe M.
      • Yoshikawa K.
      • Frade J.M.
      Single mage gene in the chicken genome encodes CMage, a protein with functional similarities to mammalian type II mage proteins.
      ,
      • Nishimura I.
      • Shimizu S.
      • Sakoda J-y.
      • Yoshikawa K.
      Expression of Drosophila MAGE gene encoding a necdin homologous protein in postembryonic neurogenesis.
      ). Nevertheless, the type II MAGEs clearly appeared earlier, as evidenced by the high homology shared between the human and mouse orthologs (>80% nucleotide sequence identity) (
      • Zhao Q.
      • Caballero O.L.
      • Simpson A.J.G.
      • Strausberg R.L.
      Differential evolution of mage genes based on expression pattern and selection pressure.
      ,
      • Lee A.K.
      • Potts P.R.
      A comprehensive guide to the mage family of ubiquitin ligases.
      ). In contrast, the type I MAGE paralogs within species are more similar to their subfamily members than to their orthologs between species (Fig. 1A), suggesting that these duplications occurred after the separation of the species. Mice also lack MAGE-C genes, whereas humans lack Mage-a–like genes (
      • Fon Tacer K.
      • Montoya M.C.
      • Oatley M.J.
      • Lord T.
      • Oatley J.M.
      • Klein J.
      • Ravichandran R.
      • Tillman H.
      • Kim M.
      • Connelly J.P.
      • Pruett-Miller S.M.
      • Bookout A.L.
      • Binshtock E.
      • Kamiński M.M.
      • Potts P.R.
      MAGE cancer-testis antigens protect the mammalian germline under environmental stress.
      ), further implying that the type I MAGE subfamilies underwent a more recent and rapid evolution.
      Within type II MAGE proteins, the N- and C-terminal regions that flank the MAGE homology domain (MHD) are completely different in paralogs but are highly conserved between human and mouse orthologs. This pattern further indicates that the type II genes independently evolved before the phylogenetic separation of the two species, which is also evident by the branching of the human and mouse MAGE phylogenetic tree (Fig. 1A) (
      • Chomez P.
      • De Backer O.
      • Bertrand M.
      • De Plaen E.
      • Boon T.
      • Lucas S.
      An overview of the mage gene family with the identification of all human members of the family.
      ). Integrative analysis of genomic structures and codon changes of MAGEs and their distinct evolution patterns indicates that negative or purifying selection maintained the established essential, nonredundant functions of type II MAGEs, whereas positive selection allowed the redundant type I MAGEs to diversify or acquire additional functions (
      • Zhao Q.
      • Caballero O.L.
      • Simpson A.J.G.
      • Strausberg R.L.
      Differential evolution of mage genes based on expression pattern and selection pressure.
      ).
      The MAGE gene family is unique among cancer-testis antigens and the multigenic families of the X chromosome. Although the X chromosome is generally considered to be the most evolutionarily stable chromosome in placental mammals, which is true of the single-copy genes (
      • Mueller J.L.
      • Skaletsky H.
      • Brown L.G.
      • Zaghlul S.
      • Rock S.
      • Graves T.
      • Auger K.
      • Warren W.C.
      • Wilson R.K.
      • Page D.C.
      Independent specialization of the human and mouse X chromosomes for the male germ line.
      ,
      • Ohno S.
      ), its ampliconic regions are rapidly evolving (
      • Wang P.J.
      • McCarrey J.R.
      • Yang F.
      • Page D.C.
      An abundance of X-linked genes expressed in spermatogonia.
      ,
      • Mueller J.L.
      • Mahadevaiah S.K.
      • Park P.J.
      • Warburton P.E.
      • Page D.C.
      • Turner J.M.A.
      The mouse X chromosome is enriched for multicopy testis genes showing postmeiotic expression.
      ,
      • Liu W.-S.
      Mammalian sex chromosome structure, gene content, and function in male fertility.
      ,
      • Vicoso B.
      • Charlesworth B.
      Evolution on the X chromosome: unusual patterns and processes.
      ,
      • Khil P.P.
      • Smirnova N.A.
      • Romanienko P.J.
      • Camerini-Otero R.D.
      The mouse X chromosome is enriched for sex-biased genes not subject to selection by meiotic sex chromosome inactivation.
      ). MAGE genes fall into both categories of genes, as single-copy type II genes are conserved among mammals, whereas several of the type I genes recently expanded (Fig. 1). The rapid expansion of multicopy/ampliconic genes on the X chromosome is thought to be driven by male X chromosome hemizygosity and the benefits these genes offer to male reproductive fitness (
      • Coyne J.A.
      Genetics and speciation.
      ). Due to rapid and selective evolution, these genes often lack murine counterparts, barring traditional in vivo genetic studies (
      • Stevenson B.J.
      • Iseli C.
      • Panji S.
      • Zahn-Zabal M.
      • Hide W.
      • Old L.J.
      • Simpson A.J.
      • Jongeneel C.V.
      Rapid evolution of cancer/testis genes on the X chromosome.
      ,
      • Wyckoff G.J.
      • Wang W.
      • Wu C.-I.
      Rapid evolution of male reproductive genes in the descent of man.
      ,
      • Gibbs Z.A.
      • Whitehurst A.W.
      Emerging contributions of cancer/testis antigens to neoplastic behaviors.
      ). Type I MAGEs are an intriguing exception, as they are present in all mammals, which enables investigation into their physiological function in animal models (
      • Fon Tacer K.
      • Montoya M.C.
      • Oatley M.J.
      • Lord T.
      • Oatley J.M.
      • Klein J.
      • Ravichandran R.
      • Tillman H.
      • Kim M.
      • Connelly J.P.
      • Pruett-Miller S.M.
      • Bookout A.L.
      • Binshtock E.
      • Kamiński M.M.
      • Potts P.R.
      MAGE cancer-testis antigens protect the mammalian germline under environmental stress.
      ). Even though several type I genes have diversified in a species-specific manner, they expanded to the same extent in both mice and humans, resulting in a similar number of genes in both species, which suggests that they convergently evolved to serve similar functions.

      Comparative MAGE expression

      MAGEs in the adult tissues

      Upon the initial discovery and characterization of MAGE genes, their expression was only detectable in tumor samples and could not be identified in the limited set of normal somatic tissues available to the Boon group (
      • van der Bruggen P.
      • Traversari C.
      • Chomez P.
      • Lurquin C.
      • De Plaen E.
      • Van den Eynde B.
      • Knuth A.
      • Boon T.
      A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma.
      ). Later, mRNA of MAGE-A, -B, and -C subfamily members was discovered in the testis and, in some cases, the placenta, hence their classification as CTAs (
      • Chomez P.
      • De Backer O.
      • Bertrand M.
      • De Plaen E.
      • Boon T.
      • Lucas S.
      An overview of the mage gene family with the identification of all human members of the family.
      ,
      • De Plaen E.
      • Traversari C.
      • Gaforio J.J.
      • Szikora J.P.
      • De Smet C.
      • Brasseur F.
      • van der Bruggen P.
      • Lethé B.
      • Lurquin C.
      • Chomez P.
      • De Backer O.
      Structure, chromosomal localization, and expression of 12 genes of the mage family.
      ,
      • Chomez P.
      • Williams R.
      • De Backer O.
      • Boon T.
      • Vennström B.
      The smage gene family is expressed in post-meiotic spermatids during mouse germ cell differentiation.
      ,
      • Rogner U.C.
      • Wilke K.
      • Steck E.
      • Korn B.
      • Poustka A.
      The melanoma antigen gene (MAGE) family is clustered in the chromosomal band xq28.
      ,
      • De Backer O.
      • Verheyden A.M.
      • Martin B.
      • Godelaine D.
      • De Plaen E.
      • Brasseur R.
      • Avner P.
      • Boon T.
      Structure, chromosomal location, and expression pattern of three mouse genes homologous to the human MAGE genes.
      ,
      • Clotman F.
      • De Backer O.
      • De Plaen E.
      • Boon T.
      • Picard J.
      Cell- and stage-specific expression of mage genes during mouse spermatogenesis.
      ,
      • Osterlund C.
      • Töhönen V.
      • Forslund K.O.
      • Nordqvist K.
      Mage-b4, a novel melanoma antigen (MAGE) gene specifically expressed during germ cell differentiation.
      ,
      • Takahashi K.
      • Shichijo S.
      • Noguchi M.
      • Hirohata M.
      • Itoh K.
      Identification of MAGE-1 and MAGE-4 proteins in spermatogonia and primary spermatocytes of testis.
      ,
      • Jurk M.
      • Kremmer E.
      • Schwarz U.
      • Förster R.
      • Winnacker E.L.
      MAGE-11 protein is highly conserved in higher organisms and located predominantly in the nucleus.
      ). Additional studies identified more distant family members that are broadly expressed in normal tissues and are now referred to as type II MAGEs (
      • Chomez P.
      • De Backer O.
      • Bertrand M.
      • De Plaen E.
      • Boon T.
      • Lucas S.
      An overview of the mage gene family with the identification of all human members of the family.
      ,
      • Lucas S.
      • Brasseur F.
      • Boon T.
      A new mage gene with ubiquitous expression does not code for known mage antigens recognized by T cells.
      ,
      • Põld M.
      • Zhou J.
      • Chen G.L.
      • Hall J.M.
      • Vescio R.A.
      • Berenson J.R.
      Identification of a new, unorthodox member of the MAGE gene family.
      ,
      • Boccaccio I.
      • Glatt-Deeley H.
      • Watrin F.
      • Roëckel N.
      • Lalande M.
      • Muscatelli F.
      The human MAGEL2 gene and its mouse homologue are paternally expressed and mapped to the Prader-Willi region.
      ). Comparative anatomical and developmental gene expression profiling of the entire MAGE family revealed five distinct subgroups (Fig. 2A) that may predict the functional categories and tissue-specific activities of MAGE proteins (https://mage.stjude.org/) (
      • Fon Tacer K.
      • Montoya M.C.
      • Oatley M.J.
      • Lord T.
      • Oatley J.M.
      • Klein J.
      • Ravichandran R.
      • Tillman H.
      • Kim M.
      • Connelly J.P.
      • Pruett-Miller S.M.
      • Bookout A.L.
      • Binshtock E.
      • Kamiński M.M.
      • Potts P.R.
      MAGE cancer-testis antigens protect the mammalian germline under environmental stress.
      ).
      Type I MAGEs show expression restricted to either the testis only (referred to as type Ia MAGEs) or to the testis and placenta (type Ib) (Fig. 2A). In mice, several type Ib genes are also expressed in the ovary (
      • Osterlund C.
      • Töhönen V.
      • Forslund K.O.
      • Nordqvist K.
      Mage-b4, a novel melanoma antigen (MAGE) gene specifically expressed during germ cell differentiation.
      ,
      • Nelson P.T.
      • Zhang P.J.
      • Spagnoli G.C.
      • Tomaszewski J.E.
      • Pasha T.L.
      • Frosina D.
      • Caballero O.L.
      • Simpson A.J.G.
      • Old L.J.
      • Jungbluth A.A.
      Cancer/testis (CT) antigens are expressed in fetal ovary.
      ). In contrast to the idea that expression of type I MAGEs is completely restricted to reproductive organs (
      • Chomez P.
      • De Backer O.
      • Bertrand M.
      • De Plaen E.
      • Boon T.
      • Lucas S.
      An overview of the mage gene family with the identification of all human members of the family.
      ,
      • De Plaen E.
      • Traversari C.
      • Gaforio J.J.
      • Szikora J.P.
      • De Smet C.
      • Brasseur F.
      • van der Bruggen P.
      • Lethé B.
      • Lurquin C.
      • Chomez P.
      • De Backer O.
      Structure, chromosomal localization, and expression of 12 genes of the mage family.
      ,
      • De Plaen E.
      • De Backer O.
      • Arnaud D.
      • Bonjean B.
      • Chomez P.
      • Martelange V.
      • Avner P.
      • Baldacci P.
      • Babinet C.
      • Hwang S.Y.
      • Knowles B.
      • Boon T.
      A new family of mouse genes homologous to the human MAGE genes.
      ,
      • De Backer O.
      • Verheyden A.M.
      • Martin B.
      • Godelaine D.
      • De Plaen E.
      • Brasseur R.
      • Avner P.
      • Boon T.
      Structure, chromosomal location, and expression pattern of three mouse genes homologous to the human MAGE genes.
      ,
      • Gaugler B.
      • Van den Eynde B.
      • van der Bruggen P.
      • Romero P.
      • Gaforio J.J.
      • De Plaen E.
      • Lethé B.
      • Brasseur F.
      • Boon T.
      Human gene MAGE-3 codes for an antigen recognized on a melanoma by autologous cytolytic T lymphocytes.
      ), several type I MAGEs (type Ic) are expressed in a variety of organs in both species, including bladder, brain, spleen, small intestine, skeletal muscle, heart, and esophagus (
      • Fon Tacer K.
      • Montoya M.C.
      • Oatley M.J.
      • Lord T.
      • Oatley J.M.
      • Klein J.
      • Ravichandran R.
      • Tillman H.
      • Kim M.
      • Connelly J.P.
      • Pruett-Miller S.M.
      • Bookout A.L.
      • Binshtock E.
      • Kamiński M.M.
      • Potts P.R.
      MAGE cancer-testis antigens protect the mammalian germline under environmental stress.
      ). Besides indicating potential function(s) outside the gonads, this unexpected expression pattern may also explain some cancer immunotherapy side effects, such as the neurological toxicities observed in patients treated with genetically engineered anti-MAGE-A3 T cells (
      • Linette G.P.
      • Stadtmauer E.A.
      • Maus M.V.
      • Rapoport A.P.
      • Levine B.L.
      • Emery L.
      • Litzky L.
      • Bagg A.
      • Carreno B.M.
      • Cimino P.J.
      • Binder-Scholl G.K.
      • Smethurst D.P.
      • Gerry A.B.
      • Pumphrey N.J.
      • Bennett A.D.
      • et al.
      Cardiovascular toxicity and titin cross-reactivity of affinity-enhanced t cells in myeloma and melanoma.
      ,
      • Morgan R.A.
      • Chinnasamy N.
      • Abate-Daga D.
      • Gros A.
      • Robbins P.F.
      • Zheng Z.
      • Dudley M.E.
      • Feldman S.A.
      • Yang J.C.
      • Sherry R.M.
      • Phan G.Q.
      • Hughes M.S.
      • Kammula U.S.
      • Miller A.D.
      • Hessman C.J.
      • et al.
      Cancer regression and neurological toxicity following anti-MAGE-A3 TCR gene therapy.
      ). This finding has important implications in cancer vaccine and immunotherapy development because MAGEs are one of the most frequently targeted proteins, and several clinical trials are under way (
      • Scanlan M.J.
      • Gure A.O.
      • Jungbluth A.A.
      • Old L.J.
      • Chen Y.-T.
      Cancer/testis antigens: an expanding family of targets for cancer immunotherapy.
      ,
      • Goldman B.
      • DeFrancesco L.
      The cancer vaccine roller coaster.
      ,
      • Brichard V.G.
      • Lejeune D.
      GSK's antigen-specific cancer immunotherapy programme: pilot results leading to phase III clinical development.
      ).
      Type II MAGEs display a more ubiquitous pattern of tissue expression and are expressed at higher absolute levels than the type I genes in both species (
      • Barker P.A.
      • Salehi A.
      The MAGE proteins: emerging roles in cell cycle progression, apoptosis, and neurogenetic disease.
      ,
      • Fon Tacer K.
      • Montoya M.C.
      • Oatley M.J.
      • Lord T.
      • Oatley J.M.
      • Klein J.
      • Ravichandran R.
      • Tillman H.
      • Kim M.
      • Connelly J.P.
      • Pruett-Miller S.M.
      • Bookout A.L.
      • Binshtock E.
      • Kamiński M.M.
      • Potts P.R.
      MAGE cancer-testis antigens protect the mammalian germline under environmental stress.
      ,
      • Kuwako K-I.
      • Taniura H.
      • Yoshikawa K.
      Necdin-related mage proteins differentially interact with the E2F1 transcription factor and the p75 neurotrophin receptor.
      ,
      • Lee S.
      • Kozlov S.
      • Hernandez L.
      • Chamberlain S.J.
      • Brannan C.I.
      • Stewart C.L.
      • Wevrick R.
      Expression and imprinting of MAGEL2 suggest a role in Prader-Willi syndrome and the homologous murine imprinting phenotype.
      ,
      • Hao Y.H.
      • Fountain M.D.
      • Fon Tacer K.
      • Xia F.
      • Bi W.
      • Kang S.H.L.
      • Patel A.
      • Rosenfeld J.A.
      • Le Caignec C.D.
      • Isidor B.
      • Krantz I.D.
      • Noon S.E.
      • Pfotenhauer J.P.
      • Morgan T.M.
      • Moran R.
      • et al.
      USP7 acts as a molecular rheostat to promote wash-dependent endosomal protein recycling and is mutated in a human neurodevelopmental disorder.
      ,
      • Bertrand M.
      • Huijbers I.
      • Chomez P.
      • De Backer O.
      Comparative expression analysis of the MAGED genes during embryogenesis and brain development.
      ). The type IIa genes are uniformly and highly expressed in the majority of tissues, and the type IIb MAGEs show enriched expression in the brain (Fig. 2A) (
      • Fon Tacer K.
      • Montoya M.C.
      • Oatley M.J.
      • Lord T.
      • Oatley J.M.
      • Klein J.
      • Ravichandran R.
      • Tillman H.
      • Kim M.
      • Connelly J.P.
      • Pruett-Miller S.M.
      • Bookout A.L.
      • Binshtock E.
      • Kamiński M.M.
      • Potts P.R.
      MAGE cancer-testis antigens protect the mammalian germline under environmental stress.
      ,
      • Kuwako K-I.
      • Taniura H.
      • Yoshikawa K.
      Necdin-related mage proteins differentially interact with the E2F1 transcription factor and the p75 neurotrophin receptor.
      ,
      • Lee S.
      • Kozlov S.
      • Hernandez L.
      • Chamberlain S.J.
      • Brannan C.I.
      • Stewart C.L.
      • Wevrick R.
      Expression and imprinting of MAGEL2 suggest a role in Prader-Willi syndrome and the homologous murine imprinting phenotype.
      ,
      • Hao Y.H.
      • Fountain M.D.
      • Fon Tacer K.
      • Xia F.
      • Bi W.
      • Kang S.H.L.
      • Patel A.
      • Rosenfeld J.A.
      • Le Caignec C.D.
      • Isidor B.
      • Krantz I.D.
      • Noon S.E.
      • Pfotenhauer J.P.
      • Morgan T.M.
      • Moran R.
      • et al.
      USP7 acts as a molecular rheostat to promote wash-dependent endosomal protein recycling and is mutated in a human neurodevelopmental disorder.
      ). Notably, some type IIa genes are also expressed at high levels in the brain, such as MAGE-D in the cerebral cortex, medulla, and hippocampus (
      • Barker P.A.
      • Salehi A.
      The MAGE proteins: emerging roles in cell cycle progression, apoptosis, and neurogenetic disease.
      ,
      • Mouri A.
      • Sasaki A.
      • Watanabe K.
      • Sogawa C.
      • Kitayama S.
      • Mamiya T.
      • Miyamoto Y.
      • Yamada K.
      • Noda Y.
      • Nabeshima T.
      MAGE-D1 regulates expression of depression-like behavior through serotonin transporter ubiquitylation.
      ). As a type IIb MAGE, MAGE-L2 is widely expressed in various human adult tissues and highly enriched in the brain, particularly in the hypothalamus (
      • Fon Tacer K.
      • Montoya M.C.
      • Oatley M.J.
      • Lord T.
      • Oatley J.M.
      • Klein J.
      • Ravichandran R.
      • Tillman H.
      • Kim M.
      • Connelly J.P.
      • Pruett-Miller S.M.
      • Bookout A.L.
      • Binshtock E.
      • Kamiński M.M.
      • Potts P.R.
      MAGE cancer-testis antigens protect the mammalian germline under environmental stress.
      ,
      • Lee S.
      • Kozlov S.
      • Hernandez L.
      • Chamberlain S.J.
      • Brannan C.I.
      • Stewart C.L.
      • Wevrick R.
      Expression and imprinting of MAGEL2 suggest a role in Prader-Willi syndrome and the homologous murine imprinting phenotype.
      ,
      • Hao Y.H.
      • Fountain M.D.
      • Fon Tacer K.
      • Xia F.
      • Bi W.
      • Kang S.H.L.
      • Patel A.
      • Rosenfeld J.A.
      • Le Caignec C.D.
      • Isidor B.
      • Krantz I.D.
      • Noon S.E.
      • Pfotenhauer J.P.
      • Morgan T.M.
      • Moran R.
      • et al.
      USP7 acts as a molecular rheostat to promote wash-dependent endosomal protein recycling and is mutated in a human neurodevelopmental disorder.
      ). In mice, Mage-l2 expression is even more restricted to the brain, and enrichment in the hypothalamus is already detectable in the later embryonic stages (
      • Lee S.
      • Kozlov S.
      • Hernandez L.
      • Chamberlain S.J.
      • Brannan C.I.
      • Stewart C.L.
      • Wevrick R.
      Expression and imprinting of MAGEL2 suggest a role in Prader-Willi syndrome and the homologous murine imprinting phenotype.
      ,
      • Hao Y.H.
      • Fountain M.D.
      • Fon Tacer K.
      • Xia F.
      • Bi W.
      • Kang S.H.L.
      • Patel A.
      • Rosenfeld J.A.
      • Le Caignec C.D.
      • Isidor B.
      • Krantz I.D.
      • Noon S.E.
      • Pfotenhauer J.P.
      • Morgan T.M.
      • Moran R.
      • et al.
      USP7 acts as a molecular rheostat to promote wash-dependent endosomal protein recycling and is mutated in a human neurodevelopmental disorder.
      ), suggesting a role for Mage-l2 during neural development and in the adult brain. Prominent Mage-l2–expressing neurons are located in regions (i.e. the arcuate nuclei, suprachiasmatic nuclei, paraventricular nuclei, and supraoptic nuclei) involved in appetite and feeding behaviors, underscoring the phenotypes seen in Prader–Willi (PWS) and Schaaf–Yang syndrome (SYS) patients, which will be explained in more detail in later sections of this review (
      • Maillard J.
      • Park S.
      • Croizier S.
      • Vanacker C.
      • Cook J.H.
      • Prevot V.
      • Tauber M.
      • Bouret S.G.
      Loss of MAGEL2 impairs the development of hypothalamic anorexigenic circuits.
      ).

      MAGE expression during embryonic development

      The expression of type I and II MAGEs in placenta and several fetal tissues in human and mouse suggest developmental functions (
      • De Plaen E.
      • Traversari C.
      • Gaforio J.J.
      • Szikora J.P.
      • De Smet C.
      • Brasseur F.
      • van der Bruggen P.
      • Lethé B.
      • Lurquin C.
      • Chomez P.
      • De Backer O.
      Structure, chromosomal localization, and expression of 12 genes of the mage family.
      ,
      • Fon Tacer K.
      • Montoya M.C.
      • Oatley M.J.
      • Lord T.
      • Oatley J.M.
      • Klein J.
      • Ravichandran R.
      • Tillman H.
      • Kim M.
      • Connelly J.P.
      • Pruett-Miller S.M.
      • Bookout A.L.
      • Binshtock E.
      • Kamiński M.M.
      • Potts P.R.
      MAGE cancer-testis antigens protect the mammalian germline under environmental stress.
      ,
      • Nelson P.T.
      • Zhang P.J.
      • Spagnoli G.C.
      • Tomaszewski J.E.
      • Pasha T.L.
      • Frosina D.
      • Caballero O.L.
      • Simpson A.J.G.
      • Old L.J.
      • Jungbluth A.A.
      Cancer/testis (CT) antigens are expressed in fetal ovary.
      ,
      • Gjerstorff M.F.
      • Harkness L.
      • Kassem M.
      • Frandsen U.
      • Nielsen O.
      • Lutterodt M.
      • Møllgård K.
      • Ditzel H.J.
      Distinct GAGE and MAGE-A expression during early human development indicate specific roles in lineage differentiation.
      ,
      • Gjerstorff M.F.
      • Kock K.
      • Nielsen O.
      • Ditzel H.J.
      MAGE-A1, GAGE and NY-ESO-1 cancer/testis antigen expression during human gonadal development.
      ). Human placenta expresses several MAGE-A genes (
      • De Plaen E.
      • Traversari C.
      • Gaforio J.J.
      • Szikora J.P.
      • De Smet C.
      • Brasseur F.
      • van der Bruggen P.
      • Lethé B.
      • Lurquin C.
      • Chomez P.
      • De Backer O.
      Structure, chromosomal localization, and expression of 12 genes of the mage family.
      ); in contrast, mouse Mage-a genes are restricted to expression in the testis, whereas the Mage-a–like genes (Mage-al2 and -al3) are highly enriched in the mouse placenta (
      • Fon Tacer K.
      • Montoya M.C.
      • Oatley M.J.
      • Lord T.
      • Oatley J.M.
      • Klein J.
      • Ravichandran R.
      • Tillman H.
      • Kim M.
      • Connelly J.P.
      • Pruett-Miller S.M.
      • Bookout A.L.
      • Binshtock E.
      • Kamiński M.M.
      • Potts P.R.
      MAGE cancer-testis antigens protect the mammalian germline under environmental stress.
      ). This finding suggests that Mage-al genes may be the functional orthologs of human MAGE-A8, -A10, and -A11 in this tissue (
      • Fon Tacer K.
      • Montoya M.C.
      • Oatley M.J.
      • Lord T.
      • Oatley J.M.
      • Klein J.
      • Ravichandran R.
      • Tillman H.
      • Kim M.
      • Connelly J.P.
      • Pruett-Miller S.M.
      • Bookout A.L.
      • Binshtock E.
      • Kamiński M.M.
      • Potts P.R.
      MAGE cancer-testis antigens protect the mammalian germline under environmental stress.
      ).
      Unlike the adult tissues, expression of the type Ia MAGE genes is not restricted to the male gonad during embryonic development. Expression in the developing testis and ovary implicates a role for type I MAGEs in gametogenesis of both sexes (
      • Fon Tacer K.
      • Montoya M.C.
      • Oatley M.J.
      • Lord T.
      • Oatley J.M.
      • Klein J.
      • Ravichandran R.
      • Tillman H.
      • Kim M.
      • Connelly J.P.
      • Pruett-Miller S.M.
      • Bookout A.L.
      • Binshtock E.
      • Kamiński M.M.
      • Potts P.R.
      MAGE cancer-testis antigens protect the mammalian germline under environmental stress.
      ,
      • Takahashi K.
      • Shichijo S.
      • Noguchi M.
      • Hirohata M.
      • Itoh K.
      Identification of MAGE-1 and MAGE-4 proteins in spermatogonia and primary spermatocytes of testis.
      ,
      • Nelson P.T.
      • Zhang P.J.
      • Spagnoli G.C.
      • Tomaszewski J.E.
      • Pasha T.L.
      • Frosina D.
      • Caballero O.L.
      • Simpson A.J.G.
      • Old L.J.
      • Jungbluth A.A.
      Cancer/testis (CT) antigens are expressed in fetal ovary.
      ,
      • Gjerstorff M.F.
      • Kock K.
      • Nielsen O.
      • Ditzel H.J.
      MAGE-A1, GAGE and NY-ESO-1 cancer/testis antigen expression during human gonadal development.
      ). Consistent with mouse expression, human MAGE-A1 and -A4 proteins have been detected in premeiotic germ cells (
      • Takahashi K.
      • Shichijo S.
      • Noguchi M.
      • Hirohata M.
      • Itoh K.
      Identification of MAGE-1 and MAGE-4 proteins in spermatogonia and primary spermatocytes of testis.
      ) and in fetal ovary (
      • Nelson P.T.
      • Zhang P.J.
      • Spagnoli G.C.
      • Tomaszewski J.E.
      • Pasha T.L.
      • Frosina D.
      • Caballero O.L.
      • Simpson A.J.G.
      • Old L.J.
      • Jungbluth A.A.
      Cancer/testis (CT) antigens are expressed in fetal ovary.
      ,
      • Gjerstorff M.F.
      • Kock K.
      • Nielsen O.
      • Ditzel H.J.
      MAGE-A1, GAGE and NY-ESO-1 cancer/testis antigen expression during human gonadal development.
      ), suggesting that human and mouse MAGE-A genes might share similar functions in premeiotic germ cell development of both species.
      Type II MAGEs are broadly expressed during embryonic development in humans (
      • Bertrand M.
      • Huijbers I.
      • Chomez P.
      • De Backer O.
      Comparative expression analysis of the MAGED genes during embryogenesis and brain development.
      ,
      • Langnaese K.
      • Kloos D.U.
      • Wehnert M.
      • Seidel B.
      • Wieacker P.
      Expression pattern and further characterization of human maged2 and identification of rodent orthologues.
      ) and mice (Fig. 2A) (
      • Barker P.A.
      • Salehi A.
      The MAGE proteins: emerging roles in cell cycle progression, apoptosis, and neurogenetic disease.
      ,
      • Fon Tacer K.
      • Montoya M.C.
      • Oatley M.J.
      • Lord T.
      • Oatley J.M.
      • Klein J.
      • Ravichandran R.
      • Tillman H.
      • Kim M.
      • Connelly J.P.
      • Pruett-Miller S.M.
      • Bookout A.L.
      • Binshtock E.
      • Kamiński M.M.
      • Potts P.R.
      MAGE cancer-testis antigens protect the mammalian germline under environmental stress.
      ,
      • Bertrand M.
      • Huijbers I.
      • Chomez P.
      • De Backer O.
      Comparative expression analysis of the MAGED genes during embryogenesis and brain development.
      ,
      • Mouri A.
      • Sasaki A.
      • Watanabe K.
      • Sogawa C.
      • Kitayama S.
      • Mamiya T.
      • Miyamoto Y.
      • Yamada K.
      • Noda Y.
      • Nabeshima T.
      MAGE-D1 regulates expression of depression-like behavior through serotonin transporter ubiquitylation.
      ). The high expression of type II genes in the brain suggests a role in the development and/or function of the central nervous system (
      • Fon Tacer K.
      • Montoya M.C.
      • Oatley M.J.
      • Lord T.
      • Oatley J.M.
      • Klein J.
      • Ravichandran R.
      • Tillman H.
      • Kim M.
      • Connelly J.P.
      • Pruett-Miller S.M.
      • Bookout A.L.
      • Binshtock E.
      • Kamiński M.M.
      • Potts P.R.
      MAGE cancer-testis antigens protect the mammalian germline under environmental stress.
      ,
      • Salehi A.H.
      • Roux P.P.
      • Kubu C.J.
      • Zeindler C.
      • Bhakar A.
      • Tannis L.L.
      • Verdi J.M.
      • Barker P.A.
      NRAGE, a novel mage protein, interacts with the p75 neurotrophin receptor and facilitates nerve growth factor-dependent apoptosis.
      ,
      • Kendall S.E.
      • Goldhawk D.E.
      • Kubu C.
      • Barker P.A.
      • Verdi J.M.
      Expression analysis of a novel p75ntr signaling protein, which regulates cell cycle progression and apoptosis.
      ,
      • Lifantseva N.
      • Koltsova A.
      • Krylova T.
      • Yakovleva T.
      • Poljanskaya G.
      • Gordeeva O.
      Expression patterns of cancer-testis antigens in human embryonic stem cells and their cell derivatives indicate lineage tracks.
      ). For example, MAGE-D1 is highly expressed in the neural tube during early human development and later in the ventricular zone, subplate, and cortical plate (
      • Kendall S.E.
      • Goldhawk D.E.
      • Kubu C.
      • Barker P.A.
      • Verdi J.M.
      Expression analysis of a novel p75ntr signaling protein, which regulates cell cycle progression and apoptosis.
      ,
      • Di Certo M.G.
      • Corbi N.
      • Bruno T.
      • Iezzi S.
      • De Nicola F.
      • Desantis A.
      • Ciotti M.T.
      • Mattei E.
      • Floridi A.
      • Fanciulli M.
      • Passananti C.
      NRAGE associates with the anti-apoptotic factor Che-1 and regulates its degradation to induce cell death.
      ). Interestingly, several type IIb brain-enriched genes, such as Ndn and Mage-l2, are more ubiquitously expressed during embryonic development, which implies involvement in a diverse array of biological functions during embryonic development and in pathogenesis of neurodevelopmental disorders (
      • Lee A.K.
      • Potts P.R.
      A comprehensive guide to the mage family of ubiquitin ligases.
      ). In later sections focusing on MAGE-D1, -D2, -G1, and -L2, we cover these roles in further detail.
      Besides expression during late embryonic development, MAGEs are also expressed in human and mouse embryonic stem (ES) cells (Fig. 2A) (
      • Fon Tacer K.
      • Montoya M.C.
      • Oatley M.J.
      • Lord T.
      • Oatley J.M.
      • Klein J.
      • Ravichandran R.
      • Tillman H.
      • Kim M.
      • Connelly J.P.
      • Pruett-Miller S.M.
      • Bookout A.L.
      • Binshtock E.
      • Kamiński M.M.
      • Potts P.R.
      MAGE cancer-testis antigens protect the mammalian germline under environmental stress.
      ,
      • Gjerstorff M.F.
      • Harkness L.
      • Kassem M.
      • Frandsen U.
      • Nielsen O.
      • Lutterodt M.
      • Møllgård K.
      • Ditzel H.J.
      Distinct GAGE and MAGE-A expression during early human development indicate specific roles in lineage differentiation.
      ,
      • Gaspar J.A.
      • Srinivasan S.P.
      • Sureshkumar P.
      • Doss M.X.
      • Hescheler J.
      • Papadopoulos S.
      • Sachinidis A.
      Depletion of Mageb16 induces differentiation of pluripotent stem cells predominantly into mesodermal derivatives.
      ,
      • Gordeeva O.
      • Gordeev A.
      • Khaydukov S.
      Expression dynamics of Mage family genes during self-renewal and differentiation of mouse pluripotent stem and teratocarcinoma cells.
      ). Like in adult tissues, MAGE-D1 and -D2 are the most highly expressed MAGEs in human (
      • Kendall S.E.
      • Goldhawk D.E.
      • Kubu C.
      • Barker P.A.
      • Verdi J.M.
      Expression analysis of a novel p75ntr signaling protein, which regulates cell cycle progression and apoptosis.
      ,
      • Lifantseva N.
      • Koltsova A.
      • Krylova T.
      • Yakovleva T.
      • Poljanskaya G.
      • Gordeeva O.
      Expression patterns of cancer-testis antigens in human embryonic stem cells and their cell derivatives indicate lineage tracks.
      ) and mouse ES cells (
      • Fon Tacer K.
      • Montoya M.C.
      • Oatley M.J.
      • Lord T.
      • Oatley J.M.
      • Klein J.
      • Ravichandran R.
      • Tillman H.
      • Kim M.
      • Connelly J.P.
      • Pruett-Miller S.M.
      • Bookout A.L.
      • Binshtock E.
      • Kamiński M.M.
      • Potts P.R.
      MAGE cancer-testis antigens protect the mammalian germline under environmental stress.
      ,
      • Gjerstorff M.F.
      • Harkness L.
      • Kassem M.
      • Frandsen U.
      • Nielsen O.
      • Lutterodt M.
      • Møllgård K.
      • Ditzel H.J.
      Distinct GAGE and MAGE-A expression during early human development indicate specific roles in lineage differentiation.
      ,
      • Gaspar J.A.
      • Srinivasan S.P.
      • Sureshkumar P.
      • Doss M.X.
      • Hescheler J.
      • Papadopoulos S.
      • Sachinidis A.
      Depletion of Mageb16 induces differentiation of pluripotent stem cells predominantly into mesodermal derivatives.
      ,
      • Gordeeva O.
      • Gordeev A.
      • Khaydukov S.
      Expression dynamics of Mage family genes during self-renewal and differentiation of mouse pluripotent stem and teratocarcinoma cells.
      ), teratocarcinoma cells, and extraembryonic endoderm cells (
      • Lifantseva N.
      • Koltsova A.
      • Krylova T.
      • Yakovleva T.
      • Poljanskaya G.
      • Gordeeva O.
      Expression patterns of cancer-testis antigens in human embryonic stem cells and their cell derivatives indicate lineage tracks.
      ). Furthermore, expression of several type II MAGEs is increased by retinoic acid–induced differentiation (
      • Gordeeva O.
      • Gordeev A.
      • Khaydukov S.
      Expression dynamics of Mage family genes during self-renewal and differentiation of mouse pluripotent stem and teratocarcinoma cells.
      ,
      • Liu Y.
      • Yang S.
      • Yang J.
      • Que H.
      • Liu S.
      Relative expression of type ii mage genes during retinoic acid-induced neural differentiation of mouse embryonic carcinoma p19 cells: a comparative real-time PCR analysis.
      ). Additional research is warranted to define the contribution of MAGEs in regulation of stemness, differentiation of pluripotent stem cells, and embryonic development.

      MAGE expression during spermatogenesis and folliculogenesis

      From the early mapping of the MAGE gene family (
      • De Plaen E.
      • Traversari C.
      • Gaforio J.J.
      • Szikora J.P.
      • De Smet C.
      • Brasseur F.
      • van der Bruggen P.
      • Lethé B.
      • Lurquin C.
      • Chomez P.
      • De Backer O.
      Structure, chromosomal localization, and expression of 12 genes of the mage family.
      ), it was evident that the majority of type I MAGEs exhibit male germline-restricted expression in both humans and mice (
      • Chomez P.
      • Williams R.
      • De Backer O.
      • Boon T.
      • Vennström B.
      The smage gene family is expressed in post-meiotic spermatids during mouse germ cell differentiation.
      ,
      • Fon Tacer K.
      • Montoya M.C.
      • Oatley M.J.
      • Lord T.
      • Oatley J.M.
      • Klein J.
      • Ravichandran R.
      • Tillman H.
      • Kim M.
      • Connelly J.P.
      • Pruett-Miller S.M.
      • Bookout A.L.
      • Binshtock E.
      • Kamiński M.M.
      • Potts P.R.
      MAGE cancer-testis antigens protect the mammalian germline under environmental stress.
      ,
      • Clotman F.
      • De Backer O.
      • De Plaen E.
      • Boon T.
      • Picard J.
      Cell- and stage-specific expression of mage genes during mouse spermatogenesis.
      ,
      • Osterlund C.
      • Töhönen V.
      • Forslund K.O.
      • Nordqvist K.
      Mage-b4, a novel melanoma antigen (MAGE) gene specifically expressed during germ cell differentiation.
      ,
      • Takahashi K.
      • Shichijo S.
      • Noguchi M.
      • Hirohata M.
      • Itoh K.
      Identification of MAGE-1 and MAGE-4 proteins in spermatogonia and primary spermatocytes of testis.
      ,
      • Gjerstorff M.F.
      • Kock K.
      • Nielsen O.
      • Ditzel H.J.
      MAGE-A1, GAGE and NY-ESO-1 cancer/testis antigen expression during human gonadal development.
      ,
      • Lurquin C.
      • De Smet C.
      • Brasseur F.
      • Muscatelli F.
      • Martelange V.
      • De Plaen E.
      • Brasseur R.
      • Monaco A.P.
      • Boon T.
      Two members of the human MAGEB gene family located in xp21.3 are expressed in tumors of various histological origins.
      ,
      • Jungbluth A.A.
      • Busam K.J.
      • Kolb D.
      • Iversen K.
      • Coplan K.
      • Chen Y.T.
      • Spagnoli G.C.
      • Old L.J.
      Expression of MAGE-antigens in normal tissues and cancer.
      ,
      • Lucas S.
      • De Plaen E.
      • Boon T.
      MAGE-B5, MAGE-B6, MAGE-C2, and MAGE-C3: Four new members of the mage family with tumor-specific expression.
      ,
      • Lee A.K.
      • Klein J.
      • Fon Tacer K.
      • Lord T.
      • Oatley M.J.
      • Oatley J.M.
      • Porter S.N.
      • Pruett-Miller S.M.
      • Tikhonova E.B.
      • Karamyshev A.L.
      • Wang Y.D.
      • Yang P.
      • Korff A.
      • Kim H.J.
      • Taylor J.P.
      • et al.
      Translational repression of G3BP in cancer and germ cells suppresses stress granules and enhances stress tolerance.
      ), which implicated that the potential physiological function of these proteins is related to spermatogenesis. Mammalian spermatogenesis is a highly coordinated and cyclic process of male germ cell generation entailing cell divisions and differentiation to ultimately yield a large number of haploid spermatozoa. Spermatogenesis takes place in the seminiferous tubules of the testis, where somatic Sertoli cells develop an epithelium to support male germ cell proliferation and differentiation (
      • Lord T.
      • Oatley J.M.
      Spermatogonial response to somatic cell interactions.
      ). In the basal compartment (i.e. the gap between the basement membrane and the Sertoli cell tight junction), spermatogonial stem cells (SSCs) give rise to progenitors, also referred to as undifferentiated spermatogonia, which undergo a series of rapid transit-amplifying mitotic divisions. A surge in retinoic acid signals for progenitors to differentiate and go through a few more rounds of division to ultimately give rise to spermatocytes (
      • Tagelenbosch R.A.J.
      • de Rooij D.G.
      A quantitative study of spermatogonial multiplication and stem cell renewal in the C3H/101 F1 hybrid mouse.
      ). Spermatocytes then enter meiotic division and cross the blood-testis barrier (BTB) to become pachytene spermatocytes. In the apical compartment of seminiferous tubules, spermatocytes then undergo two meiotic divisions to generate haploid round spermatids that undergo morphological changes to eventually mature into spermatozoa that are released into the lumen. This process takes ∼35 days in mice and ∼75 days in humans. Cyclic retinoic acid pulsation, which occurs every 8.6 days in the mouse testis, ensures continuity in spermatogenesis and a permanent supply of sperm throughout the life of a male (
      • de Rooij D.G.
      The nature and dynamics of spermatogonial stem cells.
      ,
      • Griswold M.D.
      Spermatogenesis: the commitment to meiosis.
      ,
      • Oatley J.M.
      • Brinster R.L.
      Regulation of spermatogonial stem cell self-renewal in mammals.
      ,
      • Hogarth C.A.
      • Arnold S.
      • Kent T.
      • Mitchell D.
      • Isoherranen N.
      • Griswold M.D.
      Processive pulses of retinoic acid propel asynchronous and continuous murine sperm production.
      ,
      • Schlatt S.
      • Ehmcke J.
      Regulation of spermatogenesis: an evolutionary biologist's perspective.
      ).
      The first round of mouse spermatogenesis is a distinctive program that provides a good model system to study gene expression during sperm development, as specific germ cell types (i.e. spermatogonia, spermatocytes, and spermatids) appear postnatally in a well-defined order (
      • Bellvé A.R.
      • Cavicchia J.C.
      • Millette C.F.
      • O'Brien D.A.
      • Bhatnagar Y.M.
      • Dym M.
      Spermatogenic cells of the prepuberal mouse: isolation and morphological characterization.
      ,
      • Schultz N.
      • Hamra F.K.
      • Garbers D.L.
      A multitude of genes expressed solely in meiotic or postmeiotic spermatogenic cells offers a myriad of contraceptive targets.
      ). Analysis of age-dependent MAGE expression patterns following initiation of spermatogenesis revealed that the majority of type I MAGEs are expressed at distinct stages in premeiotic, meiotic, and postmeiotic cells during sexual maturation (Fig. 2A) (
      • Fon Tacer K.
      • Montoya M.C.
      • Oatley M.J.
      • Lord T.
      • Oatley J.M.
      • Klein J.
      • Ravichandran R.
      • Tillman H.
      • Kim M.
      • Connelly J.P.
      • Pruett-Miller S.M.
      • Bookout A.L.
      • Binshtock E.
      • Kamiński M.M.
      • Potts P.R.
      MAGE cancer-testis antigens protect the mammalian germline under environmental stress.
      ). Specifically, Mage-b4 and -b16 are expressed in spermatogonia, including SSCs (
      • Fon Tacer K.
      • Montoya M.C.
      • Oatley M.J.
      • Lord T.
      • Oatley J.M.
      • Klein J.
      • Ravichandran R.
      • Tillman H.
      • Kim M.
      • Connelly J.P.
      • Pruett-Miller S.M.
      • Bookout A.L.
      • Binshtock E.
      • Kamiński M.M.
      • Potts P.R.
      MAGE cancer-testis antigens protect the mammalian germline under environmental stress.
      ,
      • Osterlund C.
      • Töhönen V.
      • Forslund K.O.
      • Nordqvist K.
      Mage-b4, a novel melanoma antigen (MAGE) gene specifically expressed during germ cell differentiation.
      ). Prepachytene spermatocytes exhibit peak expression of all Mage-a subfamily members, whose expression starts in spermatogonia and hits the highest point just before entry into meiosis and the BTB transition (
      • Fon Tacer K.
      • Montoya M.C.
      • Oatley M.J.
      • Lord T.
      • Oatley J.M.
      • Klein J.
      • Ravichandran R.
      • Tillman H.
      • Kim M.
      • Connelly J.P.
      • Pruett-Miller S.M.
      • Bookout A.L.
      • Binshtock E.
      • Kamiński M.M.
      • Potts P.R.
      MAGE cancer-testis antigens protect the mammalian germline under environmental stress.
      ,
      • Clotman F.
      • De Backer O.
      • De Plaen E.
      • Boon T.
      • Picard J.
      Cell- and stage-specific expression of mage genes during mouse spermatogenesis.
      ). Interestingly, the non-X-chromosome–residing MAGE genes, Mage-g1, -g2, and -b3, are expressed in pachytene spermatocytes during meiosis (Fig. 2A) (
      • Fon Tacer K.
      • Montoya M.C.
      • Oatley M.J.
      • Lord T.
      • Oatley J.M.
      • Klein J.
      • Ravichandran R.
      • Tillman H.
      • Kim M.
      • Connelly J.P.
      • Pruett-Miller S.M.
      • Bookout A.L.
      • Binshtock E.
      • Kamiński M.M.
      • Potts P.R.
      MAGE cancer-testis antigens protect the mammalian germline under environmental stress.
      ). The majority of MAGE genes expressed in haploid spermatids are the testis-restricted type Ia Mage genes, including Mage-b1, -b2, and -b5 (Fig. 2A) (
      • Chomez P.
      • Williams R.
      • De Backer O.
      • Boon T.
      • Vennström B.
      The smage gene family is expressed in post-meiotic spermatids during mouse germ cell differentiation.
      ,
      • Fon Tacer K.
      • Montoya M.C.
      • Oatley M.J.
      • Lord T.
      • Oatley J.M.
      • Klein J.
      • Ravichandran R.
      • Tillman H.
      • Kim M.
      • Connelly J.P.
      • Pruett-Miller S.M.
      • Bookout A.L.
      • Binshtock E.
      • Kamiński M.M.
      • Potts P.R.
      MAGE cancer-testis antigens protect the mammalian germline under environmental stress.
      ,
      • Clotman F.
      • De Backer O.
      • De Plaen E.
      • Boon T.
      • Picard J.
      Cell- and stage-specific expression of mage genes during mouse spermatogenesis.
      ). Consistent with the broad expression of type II MAGEs in many tissues and somatic cell types, most type II Mages are expressed predominantly in the Sertoli cells (
      • Fon Tacer K.
      • Montoya M.C.
      • Oatley M.J.
      • Lord T.
      • Oatley J.M.
      • Klein J.
      • Ravichandran R.
      • Tillman H.
      • Kim M.
      • Connelly J.P.
      • Pruett-Miller S.M.
      • Bookout A.L.
      • Binshtock E.
      • Kamiński M.M.
      • Potts P.R.
      MAGE cancer-testis antigens protect the mammalian germline under environmental stress.
      ,
      • Hennuy B.
      • Reiter E.
      • Cornet A.
      • Bruyninx M.
      • Daukandt M.
      • Houssa P.
      • N'Guyen V.H.
      • Closset J.
      • Hennen G.
      A novel messenger ribonucleic acid homologous to human MAGE-D is strongly expressed in rat Sertoli cells and weakly in Leydig cells and is regulated by follitropin, lutropin, and prolactin.
      ).
      Besides the testis, several type I Mages are also expressed in the mouse ovary during follicle growth and maturation (Fig. 2A) (
      • Fon Tacer K.
      • Montoya M.C.
      • Oatley M.J.
      • Lord T.
      • Oatley J.M.
      • Klein J.
      • Ravichandran R.
      • Tillman H.
      • Kim M.
      • Connelly J.P.
      • Pruett-Miller S.M.
      • Bookout A.L.
      • Binshtock E.
      • Kamiński M.M.
      • Potts P.R.
      MAGE cancer-testis antigens protect the mammalian germline under environmental stress.
      ). For example, Mage-b4 is expressed in the first 2 weeks after birth (
      • Fon Tacer K.
      • Montoya M.C.
      • Oatley M.J.
      • Lord T.
      • Oatley J.M.
      • Klein J.
      • Ravichandran R.
      • Tillman H.
      • Kim M.
      • Connelly J.P.
      • Pruett-Miller S.M.
      • Bookout A.L.
      • Binshtock E.
      • Kamiński M.M.
      • Potts P.R.
      MAGE cancer-testis antigens protect the mammalian germline under environmental stress.
      ), when the rate of primordial follicle recruitment into the growth phase is the greatest, which is in line with immunohistochemistry analysis showing that female germ cells express Mage-b4 throughout meiosis and in dormant primary oocytes (
      • Osterlund C.
      • Töhönen V.
      • Forslund K.O.
      • Nordqvist K.
      Mage-b4, a novel melanoma antigen (MAGE) gene specifically expressed during germ cell differentiation.
      ). Mage-a10, -b3, and -b7 are enriched later, during follicle maturation (Fig. 2A) (
      • Fon Tacer K.
      • Montoya M.C.
      • Oatley M.J.
      • Lord T.
      • Oatley J.M.
      • Klein J.
      • Ravichandran R.
      • Tillman H.
      • Kim M.
      • Connelly J.P.
      • Pruett-Miller S.M.
      • Bookout A.L.
      • Binshtock E.
      • Kamiński M.M.
      • Potts P.R.
      MAGE cancer-testis antigens protect the mammalian germline under environmental stress.
      ). Intriguingly, the pseudogene Mage-a9ps, which is not expressed in any other tissue, is expressed during early ovary development, implying a potential regulatory function of this gene in oogenesis (
      • Fon Tacer K.
      • Montoya M.C.
      • Oatley M.J.
      • Lord T.
      • Oatley J.M.
      • Klein J.
      • Ravichandran R.
      • Tillman H.
      • Kim M.
      • Connelly J.P.
      • Pruett-Miller S.M.
      • Bookout A.L.
      • Binshtock E.
      • Kamiński M.M.
      • Potts P.R.
      MAGE cancer-testis antigens protect the mammalian germline under environmental stress.
      ). All type II genes are expressed in the ovary, but only a few are regulated during ovary development, such as Mage-l2, which is enriched during early follicle growth (Fig. 2A). Taken together, these results indicate that MAGE genes are expressed in specific cell types and stages during spermatogenesis or folliculogenesis to perform unique and nonoverlapping functions during germ cell differentiation.

      Epigenetic and transcriptional regulation of MAGE gene expression

      Since first being identified in melanoma, MAGEs have been described in a myriad of tumors of various histological types and stages of progression (Fig. 2B) (
      • van der Bruggen P.
      • Traversari C.
      • Chomez P.
      • Lurquin C.
      • De Plaen E.
      • Van den Eynde B.
      • Knuth A.
      • Boon T.
      A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma.
      ,
      • De Plaen E.
      • Traversari C.
      • Gaforio J.J.
      • Szikora J.P.
      • De Smet C.
      • Brasseur F.
      • van der Bruggen P.
      • Lethé B.
      • Lurquin C.
      • Chomez P.
      • De Backer O.
      Structure, chromosomal localization, and expression of 12 genes of the mage family.
      ,
      • Lurquin C.
      • De Smet C.
      • Brasseur F.
      • Muscatelli F.
      • Martelange V.
      • De Plaen E.
      • Brasseur R.
      • Monaco A.P.
      • Boon T.
      Two members of the human MAGEB gene family located in xp21.3 are expressed in tumors of various histological origins.
      ,
      • Jungbluth A.A.
      • Busam K.J.
      • Kolb D.
      • Iversen K.
      • Coplan K.
      • Chen Y.T.
      • Spagnoli G.C.
      • Old L.J.
      Expression of MAGE-antigens in normal tissues and cancer.
      ,
      • Lucas S.
      • De Plaen E.
      • Boon T.
      MAGE-B5, MAGE-B6, MAGE-C2, and MAGE-C3: Four new members of the mage family with tumor-specific expression.
      ,
      • Weber J.
      • Salgaller M.
      • Samid D.
      • Johnson B.
      • Herlyn M.
      • Lassam N.
      • Treisman J.
      • Rosenberg S.A.
      Expression of the MAGE-1 tumor antigen is up-regulated by the demethylating agent 5-aza-2‘-deoxycytidine.
      ,
      • Sigalotti L.
      • Coral S.
      • Nardi G.
      • Spessotto A.
      • Cortini E.
      • Cattarossi I.
      • Colizzi F.
      • Altomonte M.
      • Maio M.
      Promoter methylation controls the expression of MAGE2, 3 and 4 genes in human cutaneous melanoma.
      ,
      • Suyama T.
      • Ohashi H.
      • Nagai H.
      • Hatano S.
      • Asano H.
      • Murate T.
      • Saito H.
      • Kinoshita T.
      The MAGE-A1 gene expression is not determined solely by methylation status of the promoter region in hematological malignancies.
      ,
      • Li B.
      • Qian X.P.
      • Pang X.W.
      • Zou W.Z.
      • Wang Y.P.
      • Wu H.Y.
      • Chen W.F.
      Hca587 antigen expression in normal tissues and cancers: correlation with tumor differentiation in hepatocellular carcinoma.
      ,
      • Honda T.
      • Tamura G.
      • Waki T.
      • Kawata S.
      • Terashima M.
      • Nishizuka S.
      • Motoyama T.
      Demethylation of MAGE promoters during gastric cancer progression.
      ,
      • Weon J.L.
      • Potts P.R.
      The mage protein family and cancer.
      ). Given this widespread expression in different cancers, many studies have sought to identify and understand the underlying mechanisms that lead to the ectopic expression of MAGEs in cancer. Both the distinct stage-specific expression of MAGE CTAs in the male germline (Fig. 2A) (
      • Fon Tacer K.
      • Montoya M.C.
      • Oatley M.J.
      • Lord T.
      • Oatley J.M.
      • Klein J.
      • Ravichandran R.
      • Tillman H.
      • Kim M.
      • Connelly J.P.
      • Pruett-Miller S.M.
      • Bookout A.L.
      • Binshtock E.
      • Kamiński M.M.
      • Potts P.R.
      MAGE cancer-testis antigens protect the mammalian germline under environmental stress.
      ) and the diverse pattern of activation in specific tumor types (Fig. 2B) (
      • Lee A.K.
      • Potts P.R.
      A comprehensive guide to the mage family of ubiquitin ligases.
      ,
      • Weon J.L.
      • Potts P.R.
      The mage protein family and cancer.
      ) suggest that a combination of epigenetic alterations with tissue-specific transcription factors is required to permit stable transcriptional activation of MAGE expression, although the precise regulatory mechanisms are still not fully understood.

      DNA methylation

      The discovery that a methyltransferase inhibitor, 5′-aza-2′-deoxycytidine (DAC) was capable of inducing MAGE-A1 expression indicated that DNA methylation status contributes to MAGE silencing in normal tissues and aberrant expression in cancer (
      • Weber J.
      • Salgaller M.
      • Samid D.
      • Johnson B.
      • Herlyn M.
      • Lassam N.
      • Treisman J.
      • Rosenberg S.A.
      Expression of the MAGE-1 tumor antigen is up-regulated by the demethylating agent 5-aza-2‘-deoxycytidine.
      ). Accordingly, the level of promoter methylation of various MAGEs inversely correlates with their expression in cancers (
      • Sigalotti L.
      • Coral S.
      • Nardi G.
      • Spessotto A.
      • Cortini E.
      • Cattarossi I.
      • Colizzi F.
      • Altomonte M.
      • Maio M.
      Promoter methylation controls the expression of MAGE2, 3 and 4 genes in human cutaneous melanoma.
      ,
      • Honda T.
      • Tamura G.
      • Waki T.
      • Kawata S.
      • Terashima M.
      • Nishizuka S.
      • Motoyama T.
      Demethylation of MAGE promoters during gastric cancer progression.
      ,
      • De Smet C.
      • De Backer O.
      • Faraoni I.
      • Lurquin C.
      • Brasseur F.
      • Boon T.
      The activation of human gene MAGE-1 in tumor cells is correlated with genome-wide demethylation.
      ,
      • De Smet C.
      • Loriot A.
      • Boon T.
      Promoter-dependent mechanism leading to selective hypomethylation within the 5′ region of gene MAGE-A1 in tumor cells.
      ,
      • De Smet C.
      • Lurquin C.
      • Lethé B.
      • Martelange V.
      • Boon T.
      DNA methylation is the primary silencing mechanism for a set of germ line- and tumor-specific genes with a CpG-rich promoter.
      ,
      • Karpf A.R.
      • Bai S.
      • James S.R.
      • Mohler J.L.
      • Wilson E.M.
      Increased expression of androgen receptor coregulator MAGE-11 in prostate cancer by DNA hypomethylation and cyclic amp.
      ,
      • Pattani K.M.
      • Soudry E.
      • Glazer C.A.
      • Ochs M.F.
      • Wang H.
      • Schussel J.
      • Sun W.
      • Hennessey P.
      • Mydlarz W.
      • Loyo M.
      • Demokan S.
      • Smith I.M.
      • Califano J.A.
      MAGEB2 is activated by promoter demethylation in head and neck squamous cell carcinoma.
      ,
      • Furuta J.
      • Umebayashi Y.
      • Miyamoto K.
      • Kikuchi K.
      • Otsuka F.
      • Sugimura T.
      • Ushijima T.
      Promoter methylation profiling of 30 genes in human malignant melanoma.
      ,
      • Jones P.A.
      • Baylin S.B.
      The fundamental role of epigenetic events in cancer.
      ). The predominant methyltransferase involved in the maintenance of CpG (5′-C-phosphate-G-3′) methylation of MAGE promoters is DNMT1 (
      • Loriot A.
      • De Plaen E.
      • Boon T.
      • De Smet C.
      Transient down-regulation of DNMT1 methyltransferase leads to activation and stable hypomethylation of MAGE-A1 in melanoma cells.
      ). In addition, methyl-CpG–binding domain proteins contribute to the silencing of MAGE-A genes (
      • Wischnewski F.
      • Friese O.
      • Pantel K.
      • Schwarzenbach H.
      Methyl-CpG binding domain proteins and their involvement in the regulation of the MAGE-A1, MAGE-A2, MAGE-A3, and MAGE-A12 gene promoters.
      ,
      • Liu S.
      • Liu F.
      • Huang W.
      • Gu L.
      • Meng L.
      • Ju Y.
      • Wu Y.
      • Li J.
      • Liu L.
      • Sang M.
      MAGE-A11 is activated through TFCP2/ZEB1 binding sites de-methylation as well as histone modification and facilitates escc tumor growth.
      ), further implying the important role of DNA methylation in transcriptional regulation of MAGE genes. Although the role of DNA methylation in physiological regulation of MAGEs is mostly unknown, the methylation reprograming pattern during gametogenesis (
      • Trasler J.M.
      Epigenetics in spermatogenesis.
      ) suggests that it could contribute to the cell type–specific MAGE expression pattern during spermatogenesis.
      In line with this idea, BORIS (brother of the regulator of imprinted sites), a demethylation factor involved in regulation of the site specificity and timing of epigenetic reprogramming in germ cells, was recently found to promote aberrant activation of MAGEs in human tumors (
      • Vatolin S.
      • Abdullaev Z.
      • Pack S.D.
      • Flanagan P.T.
      • Custer M.
      • Loukinov D.I.
      • Pugacheva E.
      • Hong J.A.
      • Morse 3rd, H.
      • Schrump D.S.
      • Risinger J.I.
      • Barrett J.C.
      • Lobanenkov V.V.
      Conditional expression of the CTCF-paralogous transcriptional factor BORIS in normal cells results in demethylation and derepression of MAGE-A1 and reactivation of other cancer-testis genes.
      ,
      • Schwarzenbach H.
      • Eichelser C.
      • Steinbach B.
      • Tadewaldt J.
      • Pantel K.
      • Lobanenkov V.
      • Loukinov D.
      Differential regulation of MAGE-A1 promoter activity by BORIS and Sp1, both interacting with the TATA binding protein.
      ). Thus, BORIS—itself a cancer-testis gene—highlights a possible overlap between the regulatory system for induction of MAGE genes in both normal male germ cells and cancer cells with respect to CpG methylation. The involvement of BORIS also suggests that aberrant activation of MAGEs might not just be a random consequence of genome-wide demethylation in cancer, as previously thought, but rather a process of targeted epigenetic modifications (
      • Vatolin S.
      • Abdullaev Z.
      • Pack S.D.
      • Flanagan P.T.
      • Custer M.
      • Loukinov D.I.
      • Pugacheva E.
      • Hong J.A.
      • Morse 3rd, H.
      • Schrump D.S.
      • Risinger J.I.
      • Barrett J.C.
      • Lobanenkov V.V.
      Conditional expression of the CTCF-paralogous transcriptional factor BORIS in normal cells results in demethylation and derepression of MAGE-A1 and reactivation of other cancer-testis genes.
      ). Expression of BORIS in male germ cells overlaps with several MAGEs that are expressed from spermatogonia to spermatocytes and also coincides with erasure of the global methylation pattern (
      • Fon Tacer K.
      • Montoya M.C.
      • Oatley M.J.
      • Lord T.
      • Oatley J.M.
      • Klein J.
      • Ravichandran R.
      • Tillman H.
      • Kim M.
      • Connelly J.P.
      • Pruett-Miller S.M.
      • Bookout A.L.
      • Binshtock E.
      • Kamiński M.M.
      • Potts P.R.
      MAGE cancer-testis antigens protect the mammalian germline under environmental stress.
      ,
      • Vatolin S.
      • Abdullaev Z.
      • Pack S.D.
      • Flanagan P.T.
      • Custer M.
      • Loukinov D.I.
      • Pugacheva E.
      • Hong J.A.
      • Morse 3rd, H.
      • Schrump D.S.
      • Risinger J.I.
      • Barrett J.C.
      • Lobanenkov V.V.
      Conditional expression of the CTCF-paralogous transcriptional factor BORIS in normal cells results in demethylation and derepression of MAGE-A1 and reactivation of other cancer-testis genes.
      ,
      • Martin-Kleiner I.
      Boris in human cancers—a review.
      ). Furthermore, the illegitimate activation of BORIS also correlates with the up-regulation of several MAGEs in cancer (
      • Martin-Kleiner I.
      Boris in human cancers—a review.
      ). However, MAGE-A1 and other CTA genes are expressed in melanoma in the absence of BORIS activation, suggesting more complex activation of these genes (
      • Kholmanskikh O.
      • Loriot A.
      • Brasseur F.
      • De Plaen E.
      • De Smet C.
      Expression of BORIS in melanoma: lack of association with MAGE-A1 activation.
      ).
      Altogether, the importance of DNMTs and BORIS in spermatogenesis, their stage-specific expression during male germline development and their implication in cancer suggest that DNA methylation impacts MAGE expression in germ cells and cancer. Furthermore, the differential acquisition of methylation marks between male and female gametes (
      • Trasler J.M.
      Epigenetics in spermatogenesis.
      ) may also underlie differential expression of MAGE genes in male and female gonads, but further studies are required to provide experimental evidence and molecular details of such regulation.

      Histone modifications

      DNA methylation of MAGE promoters is intertwined with post-translational modification of histones, and both work together to enhance MAGE gene expression in cancer cells (
      • Weber J.
      • Salgaller M.
      • Samid D.
      • Johnson B.
      • Herlyn M.
      • Lassam N.
      • Treisman J.
      • Rosenberg S.A.
      Expression of the MAGE-1 tumor antigen is up-regulated by the demethylating agent 5-aza-2‘-deoxycytidine.
      ,
      • Suyama T.
      • Ohashi H.
      • Nagai H.
      • Hatano S.
      • Asano H.
      • Murate T.
      • Saito H.
      • Kinoshita T.
      The MAGE-A1 gene expression is not determined solely by methylation status of the promoter region in hematological malignancies.
      ,
      • De Smet C.
      • Lurquin C.
      • Lethé B.
      • Martelange V.
      • Boon T.
      DNA methylation is the primary silencing mechanism for a set of germ line- and tumor-specific genes with a CpG-rich promoter.
      ,
      • Karpf A.R.
      • Lasek A.W.
      • Ririe T.O.
      • Hanks A.N.
      • Grossman D.
      • Jones D.A.
      Limited gene activation in tumor and normal epithelial cells treated with the DNA methyltransferase inhibitor 5-aza-2‘-deoxycytidine.
      ). Tumor cells with high expression of MAGE-A1 and -A3 exhibit an enrichment in activation marks with a concomitant decrease in the repressive mark (
      • Rao M.
      • Chinnasamy N.
      • Hong J.A.
      • Zhang Y.
      • Zhang M.
      • Xi S.
      • Liu F.
      • Marquez V.E.
      • Morgan R.A.
      • Schrump D.S.
      Inhibition of histone lysine methylation enhances cancer–testis antigen expression in lung cancer cells: implications for adoptive immunotherapy of cancer.
      ). Inhibition of DNA methyltransferases and histone deacetylases (HDACs) leads to MAGE-A11 expression, supporting the idea that DNA methylation and histone modifications play a synergistic role in regulating MAGE expression (
      • James S.R.
      • Cedeno C.D.
      • Sharma A.
      • Zhang W.
      • Mohler J.L.
      • Odunsi K.
      • Wilson E.M.
      • Karpf A.R.
      DNA methylation and nucleosome occupancy regulate the cancer germline antigen gene magea11.
      ). In thyroid cancer and pituitary tumors, reactivation of the fibroblast growth factor receptor 2-IIIb (FGFR2-IIIb) led to repression of MAGE-A3 and -A6 by increasing histone deacetylation and histone methylation (
      • Kondo T.
      • Zhu X.
      • Asa S.L.
      • Ezzat S.
      The cancer/testis antigen melanoma-associated antigen-A3/A6 is a novel target of fibroblast growth factor receptor 2-IIIB through histone H3 modifications in thyroid cancer.
      ,
      • Zhu X.
      • Asa S.L.
      • Ezzat S.
      Fibroblast growth factor 2 and estrogen control the balance of histone 3 modifications targeting MAGE-A3 in pituitary neoplasia.
      ). Histone deacetylation was also shown to be responsible for the silencing of MAGE-A1, -A2, -A3, and -A12 expression (
      • Wischnewski F.
      • Pantel K.
      • Schwarzenbach H.
      Promoter demethylation and histone acetylation mediate gene expression of MAGE-A1, -A2, -A3, and -A12 in human cancer cells.
      ), whereas in female pituitary tumors, estradiol promoted H3 acetylation and MAGE-A3 expression (
      • Zhu X.
      • Asa S.L.
      • Ezzat S.
      Fibroblast growth factor 2 and estrogen control the balance of histone 3 modifications targeting MAGE-A3 in pituitary neoplasia.
      ). In addition to histone lysine acetylation, histone lysine methylation was also shown to affect MAGE gene expression in cancer cells (
      • Rao M.
      • Chinnasamy N.
      • Hong J.A.
      • Zhang Y.
      • Zhang M.
      • Xi S.
      • Liu F.
      • Marquez V.E.
      • Morgan R.A.
      • Schrump D.S.
      Inhibition of histone lysine methylation enhances cancer–testis antigen expression in lung cancer cells: implications for adoptive immunotherapy of cancer.
      ). G9A, also known as euchromatic histone lysine N-methyltransferase 2 (EHMT2), methylates histone H3 Lys-9 in the MAGE-A2, -A6, and -A8 promoter regions, leading to the maintenance of their heterochromatic and silent state (
      • Tachibana M.
      • Sugimoto K.
      • Nozaki M.
      • Ueda J.
      • Ohta T.
      • Ohki M.
      • Fukuda M.
      • Takeda N.
      • Niida H.
      • Kato H.
      • Shinkai Y.
      G9a histone methyltransferase plays a dominant role in euchromatic histone H3 lysine 9 methylation and is essential for early embryogenesis.
      ). Together, a growing body of evidence indicates that diverse epigenetic mechanisms regulate the expression and silencing of MAGE genes in cancer cells; however, how these epigenetic mechanisms contribute to their expression in the germline is still mostly unknown. Further studies into epigenetic regulation of MAGE gene expression are warranted, in particular, as epigenetic drugs are used in combination with immunotherapy to improve the response of cancer patients (
      • Rao M.
      • Chinnasamy N.
      • Hong J.A.
      • Zhang Y.
      • Zhang M.
      • Xi S.
      • Liu F.
      • Marquez V.E.
      • Morgan R.A.
      • Schrump D.S.
      Inhibition of histone lysine methylation enhances cancer–testis antigen expression in lung cancer cells: implications for adoptive immunotherapy of cancer.
      ,
      • Chiappinelli K.B.
      • Zahnow C.A.
      • Ahuja N.
      • Baylin S.B.
      Combining epigenetic and immunotherapy to combat cancer.
      ).

      Transcription factors and signal transduction pathways

      Both the cell-specific MAGE expression during spermatogenesis and their distinct expression in diverse cancers raise questions about the specificity of the regulation of these genes and potential transcription factors involved. In contrast to epigenetic regulation of MAGE genes, the transcription factors and upstream activating pathways are still mostly undetermined. Mapping of type I MAGE promoter regions using deletional analysis and transcription factor–binding site analysis identified ETS- and SP1-binding elements, which were able to activate MAGE-A1 expression upon binding ETS transcription factors (
      • De Smet C.
      • Courtois S.J.
      • Faraoni I.
      • Lurquin C.
      • Szikora J.P.
      • De Backer O.
      • Boon T.
      Involvement of two Ets binding sites in the transcriptional activation of the MAGE1 gene.
      ). Methylation of ETS- and SP1-binding sites in several MAGE-A promoters was subsequently shown to silence MAGE-A expression by preventing transcription factor binding and recruiting methyl-CpG–binding domain proteins (
      • Wischnewski F.
      • Friese O.
      • Pantel K.
      • Schwarzenbach H.
      Methyl-CpG binding domain proteins and their involvement in the regulation of the MAGE-A1, MAGE-A2, MAGE-A3, and MAGE-A12 gene promoters.