ETO, a Target of t(8;21) in Acute Leukemia, Makes Distinct Contacts with Multiple Histone Deacetylases and Binds mSin3A through Its Oligomerization Domain

Abstract

t(8;21) and t(16;21) create two fusion proteins, AML-1-ETO and AML-1-MTG16, respectively, which fuse the AML-1 DNA binding domain to putative transcriptional corepressors, ETO and MTG16. Here, we show that distinct domains of ETO contact the mSin3A and N-CoR corepressors and define two binding sites within ETO for each of these corepressors. In addition, of eight histone deacetylases (HDACs) tested, only the class I HDACs HDAC-1, HDAC-2, and HDAC-3 bind ETO. However, these HDACs bind ETO through different domains. We also show that the murine homologue of MTG16, ETO-2, is also a transcriptional corepressor that works through a similar but distinct mechanism. Like ETO, ETO-2 interacts with N-CoR, but ETO-2 fails to bind mSin3A. Furthermore, ETO-2 binds HDAC-1, HDAC-2, and HDAC-3 but also interacts with HDAC-6 and HDAC-8. In addition, we show that expression of AML-1-ETO causes disruption of the cell cycle in the G 1 phase. Disruption of the cell cycle required the ability of AML-1-ETO to repress transcription because a mutant of AML-1-ETO, ⌬469, which removes the majority of the corepressor binding sites, had no phenotype. Moreover, treatment of AML-1-ETO-expressing cells with trichostatin A, an HDAC inhibitor, restored cell cycle control. Thus, AML-1-ETO makes distinct contacts with multiple HDACs and an HDAC inhibitor biologically inactivates this fusion protein.The acute myeloid leukemia 1 (AML-1) gene is one of the most frequently mutated genes in human leukemia and is disrupted by multiple chromosomal translocations in AML, including t(8;21) and t(16;21) (9, 35, 38). t(8;21) is the most frequent of these translocations, and it contains the AML-1 DNA binding domain fused to a transcriptional corepressor, ETO (also known as MTG8) (4, 5, 34). t(16;21), although rarer, fuses the AML-1 DNA binding domain to an ETOrelated protein, MTG16 (9). AML-1 is also indirectly affected by inv(16), which fuses CBF␤, an allosteric regulator of AML-1, to a smooth muscle myosin heavy chain (25).ETO is highly related to MTG16 and a third family member, MTGR1, in mammalian cells and Nervy in Drosophila (6). The mammalian family members are highly conserved throughout the proteins, with four domains conserved in Nervy. These regions are an N-terminal domain that is also homologous to the transcriptional coactivator TAF110 (17), a hydrophobic heptad repeat (HHR) that mediates dimerization (3, 21), a domain of unknown function termed the Nervy domain, and a domain containing two zinc finger motifs that are required for contacting the central domain of N-CoR (29). The murine homologue of MTG16 was identified by low-stringency screening of a cDNA library by using an ETO cDNA as a probe (3). It shares 77% overall identity with human ETO, but within three of four conserved domains, these proteins are 92 to 96% identical, implying that they function similarly. The Nervy domain is the least conserved domain among family members and is 86% identical between these two proteins.ETO is a component of a high-molecular-weight complex containing ...

RUNX1 associates with histone deacetylases and SUV39H1 to repress transcription

“…For example, ETO/MTG8 contains multiple contact sites for mSin3, nuclear hormone co-repressors and histone deacetylases (Lutterbach et al, 1998b;Amann et al, 2001;Hildebrand et al, 2001), but mutation of only one site can significantly impair RUNX1-ETOmediated repression (Lutterbach et al, 1998b;Amann et al, 2001). In fact, a completely inactive form of RUNX1-ETO retains the ability to bind to the nuclear hormone co-repressor N-CoR and histone deacetylase-1 (Lenny et al, 1995;Amann et al, 2001). Our mutagenesis study indicates that elimination of residues 379-430 or 347-387 impaired RUNX1-mediated repression.…”

“…Cells were extracted with phosphate-buffered saline supplemented with 1 mM ethylenediamine tetraacetate (EDTA), 1.5 mg/ml iodoacetamide, 0.2 mM polymethyl sulfonyl fluoride (PMSF) and 0.1 T.I.U/ml aprotinin, and containing 0.5% Triton X-100 unless otherwise noted as described (Amann et al, 2001). Immunoblot analysis was performed as described (Lutterbach et al, 1998a(Lutterbach et al, , 2000Amann et al, 2001). Antibodies to CBFb, RUNX1, RUNX2 and RUNX3 are available from EMD Biosciences, San Diego, CA.…”

AML-1, PU.1, and Sp3 regulate expression of human bactericidal/permeability-increasing protein

“…Given that mutation analysis and EMSA indicated an important role of AML1, this finding was unexpected and might indicate that AML1 requires additional factors, such as CBFb [32][33][34][35] or MOZ [36], to be active. The fusion protein AML1-ETO, present in some cases of acute myeloid leukemia, functions as a strong repressor of AML1-responsive promoters in a dominant negative fashion [37][38][39][40]. To gain further support for participation of AML1 in BPI-expression, we therefore overexpressed AML1-ETO and determined its effect on promoter activity.…”

Aberrant expression of CD19 in AML with t(8;21) involves a poised chromatin structure and PAX5

“…The translocation results in the production of a fusion protein that consists of 177 amino acids of RUNX1 containing the Runt domain fused to 575 amino acids of RUNX1T1 (also known as ETO or MTG8). The Runt domain is involved in DNA binding and heterodimerization with CBFb (Kagoshima et al, 1993) whereas RUNX1T1 can interact with several transcriptional co-repressors (Amann et al, 2001). As a result, the RUNX1-RUNX1T1 fusion protein can recruit transcriptional co-repressor complexes to RUNX1 binding sites and thus represses RUNX1 target genes (Linggi et al, 2002;Follows et al, 2003).…”