Two members of the MTG/ETO family of transcriptional corepressors, MTG8 and MTG16, are disrupted by chromosomal translocations in up to 15% of acute myeloid leukemia cases. The third family member, MTGR1, was identified as a factor that associates with the t(8;21) fusion protein RUNX1-MTG8. We demonstrate that Mtgr1 associates with mSin3A, N-CoR, and histone deacetylase 3 and that when tethered to DNA, Mtgr1 represses transcription, suggesting that Mtgr1 also acts as a transcriptional corepressor. To define the biological function of Mtgr1, we created Mtgr1-null mice. These mice are proportionally smaller than their littermates during embryogenesis and throughout their life span but otherwise develop normally. However, these mice display a progressive reduction in the secretory epithelial cell lineage in the small intestine. This is not due to the loss of small intestinal progenitor cells expressing Gfi1, which is required for the formation of goblet and Paneth cells, implying that loss of Mtgr1 impairs the maturation of secretory cells in the small intestine.Chromosomal translocations disrupt master regulatory genes that control cellular proliferation, apoptosis, and the lineage decisions that affect stem cell self-renewal and differentiation of progenitor cells (15,29). The myeloid translocation gene on chromosome 8 (MTG8, also known as eighttwenty-one or ETO) is disrupted by t(8;21) in up to 15% of acute myeloid leukemia cases (7,26,27). MTG8 is the founding member of a gene family that includes the myeloid translocation gene on chromosome 16 (MTG16 or ETO2), which is disrupted by t(16;21), and myeloid translocation gene-related 1 (MTGR1) (5, 6, 12, 18). t(8;21) and t(16;21) fuse MTG8 and MTG16, respectively, to the DNA binding domain of Runtrelated 1 (RUNX1, also known as acute myeloid leukemia 1 or AML1) (7,12,26,27). The resulting fusion proteins repress RUNX1-regulated genes (11,20,25). For RUNX1-MTG8, this repression requires the MTG8 sequences, leading to the hypothesis that MTG8 is a transcriptional corepressor (20). Consistent with this hypothesis, MTG8 associates with multiple corepressors, including N-CoR/SMRT, mSin3, and histone deacetylase 1 (HDAC1), HDAC2, and HDAC3 (1,13,14,23,34).MTG family members display approximately 85% sequence similarity (3) and contain four conserved subdomains with up to 95% identity (5, 8). Based on homology to MTG8, it was anticipated that MTG16 and MTGR1 also act as transcriptional corepressors. MTG16 is 92% homologous to MTG8, and the murine form of MTG16, Eto2, interacts with multiple HDACs and N-CoR (1). In contrast to MTG8, Eto2 failed to interact with mSin3A (1). The MTG family members also heterodimerize, and this property allowed the identification of MTGR1 as a RUNX1-MTG8-associated protein (18). Although it associates with MTG8 and the t(8;21) fusion protein, the molecular function of MTGR1 is unknown.While two of the three MTG family members are disrupted by chromosomal translocations, the MTG family members are widely expressed, suggesting that this gene f...

Canonical Wnt signaling is mediated by a molecular "switch" that regulates the transcriptional properties of the T-cell factor (TCF) family of DNA-binding proteins. Members of the myeloid translocation gene (MTG) family of transcriptional corepressors are frequently disrupted by chromosomal translocations in acute myeloid leukemia, whereas MTG16 may be inactivated in up to 40% of breast cancer and MTG8 is a candidate cancer gene in colorectal carcinoma. Genetic studies imply that this corepressor family may function in stem cells. Given that mice lacking Myeloid Translocation Gene Related-1 (Mtgr1) fail to maintain the secretory lineage in the small intestine, we surveyed transcription factors that might recruit Mtgr1 in intestinal stem cells or progenitor cells and found that MTG family members associate specifically with TCF4. Coexpression of ␤-catenin disrupted the association between these corepressors and TCF4. Furthermore, when expressed in Xenopus embryos, MTG family members inhibited axis formation and impaired the ability of ␤-catenin and XLef-1 to induce axis duplication, indicating that MTG family members act downstream of ␤-catenin. Moreover, we found that c-Myc, a transcriptional target of the Wnt pathway, was overexpressed in the small intestines of mice lacking Mtgr1, thus linking inactivation of Mtgr1 to the activation of a potent oncogene.Canonical Wnt signaling plays a critical regulatory role in development and in stem cell functions and cellular differentiation (41, 43). Wnts initiate a signaling cascade that leads to the nuclear accumulation of ␤-catenin, which associates with T-cell factor 4 (TCF4), releasing the TCF4-associated transcriptional corepressors and recruiting coactivators to stimulate TCF4-dependent transcription. The intestinal epithelium has been a rich source of information about this pathway. For example, Tcf4 is required for small intestinal stem cell selfrenewal; mice lacking this transcription factor exhaust the capacity for continued replenishment of the epithelium in utero (24). Conversely, hyperactive Wnt signaling is closely associated with colorectal carcinoma, most commonly via inactivation of the APC tumor suppressor, which regulates the levels of ␤-catenin (41, 43). At the end point of the Wnt signaling cascade, ␤-catenin opposes the action of transcriptional corepressors for binding to TCFs to regulate genes that affect cell fate decisions.Two members of the myeloid translocation gene (MTG) family of transcriptional corepressors, MTG on chromosome 8 (MTG8; also known as ETO or RUNX1T1) and MTG on chromosome 16 (MTG16; also known as ETO2 or CBFA2T3), are disrupted by chromosomal translocations in acute myeloid leukemia (10, 29), which suggests that these factors are key regulators of cellular proliferation or differentiation. As would be expected of master regulatory factors, these targets of chromosomal translocations in acute leukemia are commonly mutated in other tumor types as well. A genomewide screen to detect mutations of genes in colorectal and breast can...

The localization of the adenovirus E1B-55K-E4orf6 protein complex is critical for its function. Prior studies demonstrated that E4orf6 directs the nuclear localization of E1B-55K in human cells and in rodent cells that contain part of human chromosome 21. We show here that the relevant activity on chromosome 21 maps to RUNX1. RUNX1 proteins are transcription factors that serve as scaffolds for the assembly of proteins that regulate transcription and RNA processing. After transfection, the RUNX1a, RUNX1b, and RUNX1-⌬N variants allowed E4orf6-directed E1B-55K nuclear localization. The failure of RUNX1c to allow nuclear colocalization was relieved by the deletion of amino-terminal residues of this protein. In the adenovirusinfected mouse cell, RUNX1 proteins were localized to discrete structures about the periphery of viral replication centers. These sites are enriched in viral RNA and RNA-processing factors. RUNX1b and RUNX1a proteins displaced E4orf6 from these sites. The association of E1B-55K at viral replication centers was enhanced by the RUNX1a and RUNX1b proteins, but only in the absence of E4orf6. In the presence of E4orf6, E1B-55K occurred in a perinuclear cytoplasmic body resembling the aggresome and was excluded from the nucleus of the infected mouse cell. We interpret these findings to mean that a dynamic relationship exists between the E4orf6, E1B-55K, and RUNX1 proteins. In cooperation with E4orf6, RUNX1 proteins are able to modulate the localization of E1B-55K and even remodel virus-specific structures that form at late times of infection. Subsequent studies will need to determine a functional consequence of the interaction between E4orf6, E1B-55K, and RUNX1.

The function of the NF-E2 transcription factor, a p45/small Maf heterodimer, was analyzed in the erythroleukemia cell lines MEL and CB3. In contrast to MEL cells, CB3 cells are null for p45 and thus express only extremely low levels of adult globin transcripts upon induction by agents promoting erythroid differentiation. We investigated the response of erythroleukemia cells to hemin treatment. Hemin rapidly induces beta-globin gene transcript levels in MEL cells, but not in CB3 cells. Stable expression of the large p45 NF-E2 subunit in CB3 cells restores hemin mediated beta-globin gene transcription, suggesting that the presence of a functional NF-E2 is required for strong induction of beta-globin mRNA levels by hemin in erythroleukemia cells. We performed mutagenesis of two potential heme-regulatory motifs (HRMs) in p45 NF-E2 and found that the mutated versions are expressed and can still recognize a NF-E2 DNA binding element. In addition, we showed that p45 NF-E2 HRM mutants are able to restore beta-globin gene transcription in CB3 cells upon induction by hemin. Our results suggest that globin gene activation by heme appears to be independent of the putative HRMs in the p45 subunit of the NF-E2 transcription factor.

The t(8;21) is one of the most frequent chromosomal translocations associated with acute myelogenous leukemia (AML). This translocation generates a fusion protein, RUNX1-ETO, consisting of the N-terminus of RUNX1 fused to a nearly full-length ETO protein. The RUNX1-ETO fusion protein stimulates the expression of genes that are regulated by Wnt signaling. The Wnt signaling pathway plays a key role in embryonic development and aberrations to this pathway are frequently involved in tumor formation. Therefore, we sought to define the molecular mechanism by which RUNX1-ETO may stimulate Wnt signaling. We have demonstrated that the ETO family member, Mtgr1, functions as a corepressor for TCF4 and that the levels of the TCF-regulated gene, c-Myc, are upregulated in Mtgr1-null mice. Here we show that the Xenopus homolog of Mtgr1, XETOR, can impair Wnt signaling and induce ventralization in a Xenopus axis duplication assay, a classical assay used to define the hierarchy of components in the Wnt pathway. Specifically, microinjection of in vitro transcribed XETOR mRNA was performed in the marginal zone of both dorsal blastomeres at the 2 to 4 cell stage with increasing amounts of XETOR. Embryos were monitored through stage 26. Compared to control embryos, the embryos injected with XETOR mRNA were ventralized and failed to develop head structures. Conversely, although each of the ETO family member proteins associated with TCF4, RUNX1-ETO failed to bind to TCF4 in co-immunoprecipitation experiments. Mtgr1 was originally identified as a RUNX1-ETO-associated protein. Therefore, we tested whether the fusion protein impairs the action of Mtgr1 as a co-repressor for TCF4. RUNX1-ETO associated with Mtgr1, and Mtgr1 failed to associate with TCF4 when RUNX1-ETO was co-expressed. Thus, RUNX1-ETO appears to stimulate TCF-dependent transcription by interfering with the action of the ETO family of transcriptional corepressors.

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