The MYND Motif Is Required for Repression of Basal Transcription from the Multidrug Resistance 1 Promoter by the t(8;21) Fusion Protein

Abstract

Chromosomal translocations in acute leukemia that affect the AML-1/CBF␤ transcription factor complex create dominant inhibitory proteins. However, the mechanisms by which these proteins act remain obscure. Here we demonstrate that the multidrug resistance 1 (MDR-1) promoter is a target for AML/ETO transcriptional repression. This repression is of basal, not activated, expression from the MDR-1 promoter and thus represents a new mechanism for AML/ETO function. We have defined two domains in AML/ETO that are required for repression of basal transcription from the MDR-1 promoter: a hydrophobic heptad repeat (HHR) motif and a conserved zinc finger (ZnF) domain termed the MYND domain. The HHR mediates formation of AML/ETO homodimers and AML/ETO-ETO heterodimers. Single serine substitutions at conserved cysteine residues within the predicted ZnFs also abrogate transcriptional repression. Finally, we observe that AML/ ETO can also inhibit Ets-1 activation of the MDR-1 promoter, indicating that AML/ETO can disrupt both basal and Ets-1-dependent transcription. The fortuitous inhibition of MDR-1 expression in t(8;21)-containing leukemias may contribute to the favorable response of these patients to chemotherapeutic drugs.AML-1 is the direct or indirect target of multiple chromosomal translocations in acute B-cell and myeloid leukemia. t(8;21) and inv(16) disrupt AML-1 and its heterodimeric partner, CBF␤, respectively, and are the most frequent translocations in acute myeloid leukemia (AML). These translocations are found in the leukemic blasts of up to 30% of patients with AML with discernable translocations (28, 37). t(12;21) also disrupts AML-1 in B-cell acute lymphocytic leukemias of children (43). Thus, AML-1 is one of the most frequently mutated genes in human leukemia. Interestingly, patients containing these translocations uniformly respond better to chemotherapy, with an increased 5-year survival rate (3,9,21,42). t(8;21)(q22;q22) fuses the N-terminal 177 amino acids (aa) of AML-1 to the C-terminal 575 aa of ETO to form the chimeric AML/ETO protein (36). An analysis of the structure of AML/ETO reveals that the DNA binding runt domain of AML-1 is not altered but that the transactivation domain of AML-1 has been replaced by ETO. This led to the hypothesis that the fusion protein acts as a dominant inhibitor of AML-1B function (32, 34). AML/ETO interfered with AML-1B-activated transcription of the T-cell receptor ␤ (TCR␤) enhancer, and the interleukin 3 and granulocyte-macrophage colonystimulating factor (GM-CSF) promoters, but did not affect the basal expression of these promoters (13,32,45). The dominant inhibitory action of the t(8;21) and the inv(16) fusion proteins has been confirmed biologically by expressing these fusion proteins during murine development (4, 48). These mice display the same phenotype as that displayed by AML-1 (and CBF␤-)-deficient mice (39, 46).Multiple mechanisms have been proposed for transcriptional repression, including competition for binding sites and interaction with surrounding facto...

Bop encodes a muscle-restricted protein containing MYND and SET domains and is essential for cardiac differentiation and morphogenesis

“…As m-Bop has repressor activity and contains a MYND domain similar to that shown in the ETO protein to recruit HDAC activity 24 , we used the potent HDAC inhibitor trichostatin A (TSA) 25 to test whether m-Bop2 might repress transcription by recruiting HDACs. Addition of 150 µM TSA significantly abrogated GAL4-m-Bop2 repression of SV40 promoter-driven luciferase other SET domain-containing factors.…”

RUNX1 associates with histone deacetylases and SUV39H1 to repress transcription

“…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.…”

The adenovirus E1A binding protein BS69 is a corepressor of transcription through recruitment of N-CoR

“…Since the MYND domain is a putative interaction molecule for the nuclear co-repressors N-CoR and SMRT (Lutterbach et al, 1998a;Gelmetti et al, 1998;Wang et al, 1998) we tested whether the MYND domain of BS69 also mediates N-CoR binding in a coimmunoprecipitation experiment. We transfected human U2OS osteosarcoma cells with expression vectors for FLAG tagged N-CoR and full-length human BS69 fused to the DNA binding domain of the yeast transcription factor GAL4 at the N-terminus ( Figure 2).…”

“…The C-terminus harbours a region with homology to the MYND domain, a two zinc ®nger motif ®rst identi®ed in DEAF-1 (Gross and McGinnis, 1996) and in MTG8/ETO (Lutterbach et al, 1998a). This domain was shown to be required for both repression of basal transcription by the AML/ETO fusion protein and binding of the transcriptional corepressors N-CoR and SMRT (Lutterbach et al, 1998b;Gelmetti et al, 1998;Wang et al, 1998).…”

“…Both these domains are present in many proteins involved in transcriptional regulation (Aasland et al, 1995;Jeanmougin et al, 1997). The extreme C-terminus of BS69 and its splice variant BRAM1 contain a motif consisting of two zinc ®ngers, ®rst identi®ed in Drosophila DEAF-1 (Gross and McGinnis, 1996) and the mammalian transcription factor MTG8/ETO (Lutterbach et al, 1998a). Figure 1b shows the homology of the human BS69 MYND domain to the domains of human MTG8/ETO and the human DEAF-1 related protein.…”