The acute myeloid leukemia fusion protein AML1-ETO targets E proteins via a paired amphipathic helix-like TBP-associated factor homology domain

Abstract

Up to 15% of acute myeloid leukemias (AMLs) are characterized by the abnormal expression of the eight-twenty-one (ETO) transcriptional corepressor within an AML1-ETO fusion protein. The t(8;21) chromosomal translocation serves not only to disrupt WT AML1 function but also to introduce ETO activity during hematopoiesis. AML1-ETO was recently shown to inhibit E protein transactivation by physically displacing WT coactivator proteins in an interaction mediated by ETO. Here, we present the 3D solution structure of the human ETO TAFH (eTAFH) domain implicated in AML1-ETO:E protein interactions and report an unexpected fold similarity to paired amphipathic helix domains from the transcriptional corepressor Sin3. We identify and characterize a conserved surface on eTAFH that is essential for ETO:E protein recognition and show that the mutation of key conserved residues at this site alleviates ETObased silencing of E protein transactivation. Our results address uncharacterized aspects of the corepression mechanism of ETO and suggest that eTAFH may serve to recruit ETO (or AML1-ETO) to DNA-bound transcription factors. Together, these findings imply that a cofactor exchange mechanism, analogous to that described for E protein inhibition, may represent a common mode of action for ETO.corepressor ͉ NMR ͉ protein structure ͉ sin3 C ore binding factor (CBF) is an essential hematopoietic transcriptional regulator that is frequently mutated in acute myeloid leukemias (AMLs) (1, 2). In up to 15% of AML cases, a translocation between chromosomes 8 and 21 replaces a portion of the gene encoding the CBF subunit AML1 with a gene specifying an unrelated corepressor protein, eight-twenty-one (ETO) (3, 4). The subsequent fusion protein, AML1-ETO, contains the DNA-binding domain from AML1 fused to most of ETO. Expression of this chimeric protein and the consequent disruption of WT AML1 function are key events in the production of an AML phenotype (5). In WT cells CBF regulates chromatin remodeling of target genes by recruiting coactivators and histone acetyl transferases (6). In t(8;21) cells, the AML1-ETO fusion still binds CBF target DNA sequences through the AML1 DNA-binding domain; however, the ETO fusion partner functionally dominates by recruiting proteins involved in chromatin repression (e.g., corepressors and histone deacetylases) (1, 7). The formation of a corepressor complex involving AML1-ETO serves to shut down gene expression of genes normally activated by CBF.As an alternative or complementary mechanism involving distinct target genes, recent studies have shown that AML1-ETO can silence E protein transactivation (8). E proteins comprise a family of widely expressed transcription factors involved in regulating cell growth, differentiation, and apoptosis (9-11). DNA-bound E proteins interact with the histone acetyl transferase p300͞CREB binding protein, leading to histone modifications that facilitate transcription initiation (8, 12). However, AML1-ETO very strongly interacts with E proteins through an N-terminal activation...

Mtg16/Eto2 Contributes to Murine T-Cell Development

“…The E-protein-deficient Mtg16 point mutant plasmids were generated using a QuikChange II XL site-directed mutagenesis kit (Agilent Technologies). Primers were designed based upon the structure of the Eto-HEB interaction (35) to mimic the following mutations: F210A-F154A and R220A-R164A. The sequence for the F210A forward primer was 5Ј-CACTGAGGCCGTTTGT TATCCCTGCTCTGAAGGCTAATCTT-3Ј, and that for the F210A reverse primer was 5Ј-AAGATTAGCCTTCAGAGCAGGGATAACAAACGGCCTC AGTG-3Ј.…”

“…on April 7, 2019 by guest http://mcb.asm.org/ R164A mutant that retained the ability to bind to HEB AD1 (35). The F210A and R220A mutants were reintroduced into Mtg16-null LSK cells using MSCV-IRES-GFP, and both Mtg16 and the R220A mutant were capable of rescuing T-cell development (Fig.…”

“…The structure of the NHR1 domain of MTG8 in association with the HEB AD1 domain identified residues that mediate this contact (35). While HEB binds to MTG8 through 2 sites, mutations within the NHR1 domain of MTG8 were sufficient to disrupt repression of E-protein-mediated transcriptional activation (20,35).…”

“…The structure of the NHR1 domain of MTG8 in association with the HEB AD1 domain identified residues that mediate this contact (35). While HEB binds to MTG8 through 2 sites, mutations within the NHR1 domain of MTG8 were sufficient to disrupt repression of E-protein-mediated transcriptional activation (20,35). Therefore, to define the contributions of E-protein functions to the role of Mtg16 in T-cell development, we engineered a point mutation in Mtg16 that is homologous to changes that abrogated the binding of the MTG8 NHR1 domain to HEB AD1.…”

“…Likewise, Mtg16 associates with transcriptional activation domain 1 (AD1) in E proteins to impair E-protein-dependent transcription, and E2A instructs lymphoid development while inhibiting myelopoiesis (2,3,7,35,48). E2A-null mice have decreased numbers of thymocytes due to reduced lymphoidprimed multipotent progenitor (LMPP) and early thymocyte progenitor (ETP) production and impaired progression from the earliest CD4 Ϫ CD8 Ϫ CD44 ϩ CD25 Ϫ double-negative 1 (DN1) thymocytes to the subsequent CD4 Ϫ CD8 Ϫ CD44 ϩ CD25 ϩ DN2 thymocytes (2,4,13,24).…”