Structure of the AML1-ETO eTAFH domain–HEB peptide complex and its contribution to AML1-ETO activity

Abstract

AML1-ETO is the chimeric protein product of the t(8;21) in acute myeloid leukemia. The ETO portion of the fusion protein includes the eTAFH domain, which is homologous to several TATA binding protein-associated factors (TAFs) and interacts with E proteins (E2A and HEB). It has been proposed that AML1-ETOmediated silencing of E protein function might be important for t(8;21) leukemogenesis. Here, we determined the solution structure of a complex between the AML1-ETO eTAFH domain and an interacting peptide from HEB. On the basis of the structure, key residues in AML1-ETO for HEB association were mutated. These mutations do not impair the ability of AML1-ETO to enhance the clonogenic capacity of primary mouse bone marrow cells and do not eliminate its ability to repress proliferation or granulocyte differentiation. Therefore, the eTAFH-E protein interaction appears to contribute relatively little to the activity of AML1-ETO. IntroductionAML1-ETO is the fusion protein produced from the t(8;21) identified in acute myeloid leukemia (AML) of the M2 subtype. 1 The translocation fuses the N-terminus of RUNX1 with 575 amino acids of ETO ("eight twenty one," encoded by RUNX1T1). AML1-ETO has 5 conserved domains named for its Drosophila homologues Runt and Nervy: the DNA-and CBF␤-binding Runt domain from RUNX1, and Nervy homology domains 1 to 4 (NHR1-4) in ETO. 2-4 NHR1, also known (and referred to here) as the TATA box-binding protein-associated factor homology domain (eTAFH) is homologous to several TATA-binding protein associated factors (TAFs) and interacts with E proteins. 5 The NHR2 domain, or hydrophobic heptad repeat (HHR) is an ␣-helical tetramer that is essential for the activity of AML1-ETO. [6][7][8][9] NHR3 is a predominantly ␣-helical domain that interacts with the regulatory subunit of type II cyclic AMP-dependent protein kinase (PKA RII␣). 4 The NHR4 domain (also known as myeloid-Nervy-DEAF-1, or MYND) was suggested to contain 2 putative non-DNAbinding zinc-fingers. [10][11][12] We previously solved the structure of the MYND domain and showed it to be a member of the RING finger structural family. 13 The eTAFH domain is homologous to several TAFs, including Drosophila TAF110, human TAF130, and human TAF105. 3,14,15 Previous studies in a t(8;21) leukemic cell line showed that the eTAFH domain interacts with E proteins, 5 which are widely expressed DNA-binding transcription factors involved in regulating differentiation, proliferation, and apoptosis whose transactivation is facilitated by recruiting CBP/p300 histone acetyltransferases (HATs). [16][17][18] The eTAFH domain binds the AD1 transactivation domain of the HeLa cell E-boxbinding protein (HEB), an E protein family member, which prevents CBP/p300 and HAT association. This led to the hypothesis that AML1-ETO dominantly silences E proteins in t(8;21) cells, resulting in dysregulation of E protein target genes, and by that mechanism contributes to leukemogenesis. 5 Consistent with this, a recent chromatin immunoprecipitation (ChIP)-chip study shows eviden...

A novel exon in AML1-ETO negatively influences the clonogenic potential of the t(8;21) in acute myeloid leukemia

“…6 By introducing specific mutations into the regions coding for the NHR1 and NHR2 domains of AML1-ETO transcripts, three recent reports have confirmed a limited role of the NHR1 domain. [6][7][8] Although our results are in keeping with these findings, we further show that the novel 6a-containing transcript may influence the clonogenic potential of the full-length transcript. This observation highlights another potential failsafe mechanism by which cells may be able to regulate uncontrolled leukemic growth.…”

Structural basis of CBP/p300 recruitment in leukemia induction by E2A-PBX1

“…9 Furthermore, because wild-type E proteins appear capable of transcriptional suppression in certain cellular contexts, recruitment of wild-type ETO or other corepressors by PCET could also contribute to physiologic transcriptional regulation by E-proteins. 3,20,44 A comparison of our PCET/ KIX structure with the previously determined PCET/eTAFH complex structure 6 illustrates that, although both eTAFH and KIX bind the PCET motif through hydrophobic interactions with the --x-x--sequence, there are notable differences in the PCET conformation and interactions with residues outside the consensus sequence ( Figure 6). When bound to the eTAFH domain, the helical conformation of HEB-PCET (Lys15 to Leu21) is disrupted by a kink at Asp22, 6 while in complex with KIX the PCET helix extends to Phe27.…”

“…3,20,44 A comparison of our PCET/ KIX structure with the previously determined PCET/eTAFH complex structure 6 illustrates that, although both eTAFH and KIX bind the PCET motif through hydrophobic interactions with the --x-x--sequence, there are notable differences in the PCET conformation and interactions with residues outside the consensus sequence ( Figure 6). When bound to the eTAFH domain, the helical conformation of HEB-PCET (Lys15 to Leu21) is disrupted by a kink at Asp22, 6 while in complex with KIX the PCET helix extends to Phe27. Phe23 within the LDFS sequence of HEB-PCET plays a critical role in KIX recognition as evidenced by our structural, biophysical, biochemical, and mammalian 2-hybrid data.…”

“…16 KIX also binds activation domains of several other mammalian transcription factors 10,13,37,38 and viral proteins [39][40][41] involved in the regulation of lymphopoiesis through their LXXLL sequence or more generic -x-x--sequence described by Lee et al, 10 which is typically preceding by an acidic residue (; Figure 5A). 6,11 Our PCET/KIX complex and associated mutagenesis studies described here provide structural insights into these functions.…”

“…1,[3][4][5] A highly conserved 17-residue region within AD1 at the N-terminus of E-proteins is the target of the eTAFH domain from the transcriptional corepressor ETO and the oncogenic fusion protein AML-ETO. [6][7][8][9] This interaction leads to E-protein silencing by preventing the binding of the transcriptional coactivators p300/CBP to the same site on AD1, which has been termed the "p300/CBP and ETO target in E-proteins" (PCET) motif. 9 The PCET motif is composed of 2 overlapping protein recognition sites: a Leu-x-x-Leu-Leu (LXXLL) sequence and a Leu-Asp-Phe-Ser (LDFS) sequence.…”