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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
Cancer Cell. Author manuscript; available in PMC 2008 June 1.
Published in final edited form as:
PMCID: PMC1978186
NIHMSID: NIHMS25625

Structural basis for recognition of SMRT/N-CoR by the MYND domain and its contribution to AML1/ETO's activity

Summary

AML1/ETO results from the t(8;21) associated with 12-15% of acute myeloid leukemia. The AML1/ETO MYND domain mediates interactions with the co-repressors SMRT and N-CoR and contributes to AML1/ETO's ability to repress proliferation and differentiation of primary bone marrow cells as well as to enhance their self-renewal in vitro. We solved the solution structure of the MYND domain and show it to be structurally homologous to the PHD and RING finger families of proteins. We also determined the solution structure of an MYND-SMRT peptide complex. We demonstrated that a single amino acid substitution that disrupts the interaction between the MYND domain and the SMRT peptide attenuated AML1/ETO's effects on proliferation, differentiation, and gene expression.

Significance

Chromosomal rearrangements often result in the production of chimeric proteins with altered function. In many cases, aberrant recruitment of co-repressor complexes has been shown to contribute substantially to the oncogenic potential of these chimeric proteins. The MYND domain (NHR4) of ETO, responsible for recruitment of the co-repressors SMRT and N-CoR, becomes fused to the AML1 protein as a result of the 8;21 translocation. We have solved the structure of an MYND domain/SMRT complex. Based on the structural information, we have used a point mutation to show that SMRT/N-CoR recruitment by the MYND domain plays a key role in the block in proliferation and differentiation seen with AML1/ETO and alters the expression of genes associated with these functions.

Introduction

AML1/ETO is produced as a result of the t(8;21) found in acute myeloid leukemia (AML) of the M2 subtype (Miyoshi et al., 1993). It consists of the N terminus of AML1, including its DNA-binding Runt domain, and almost the entire ETO protein. AML1/ETO causes leukemia in mice in cooperation with other mutated genes such as TEL/PDGFRβ, ISCBP deficiency, and the FLT3 internal tandem duplication (Grisolano et al., 2003; Schessl et al., 2005; Schwieger et al., 2002). It interacts with many other proteins including CBFβ, SMRT/N-CoR, PLZF, GFI-1, SIN3, and numerous histone deacetylases (Hug and Lazar, 2004). Determining which of these interactions contributes to AML1/ETO's activity and which pathways are affected by these interactions is necessary in order to develop targeted therapy for this leukemia.

Many of AML1/ETO's interactions with other proteins are mediated by the five domains that are conserved with the Drosophila Runt, Lozenge, and Nervy proteins. The most N-terminal domain is the Runt domain from the AML1 fusion partner, which is an s-type immunoglobulin fold that binds both DNA and CBFβ (Bravo et al., 2001; Tahirov et al., 2001). DNA binding by the Runt domain is thought to be essential for AML1/ETO's leukemogenic activity (Grisolano et al., 2003). C-terminal to the Runt domain is the Nervy homology region 1 (NHR1), otherwise known as the ETO TBP-associated factor homology domain (eTAFH) (Zhang et al., 2004). NHR2, or the hydrophobic heptad repeat (HHR) domain is a helical tetramer that mediates oligomerization with AML1/ETO, ETO, or the ETO homologues MTG16 and MTGR1 (Kitabayashi et al., 1998; Liu et al., 2006; Minucci et al., 2000; Zhang et al., 2004). Oligomerization through the HHR domain appears to be essential for AML1/ETO's activity in repressing cell proliferation and differentiation, and in promoting the clonogenic ability of primary hematopoietic progenitors (Hug et al., 2002; Liu et al., 2006; Shimada et al., 2000; Zhang et al., 2001). NHR3 interacts with the regulatory subunit of type II cyclic AMP-dependent protein kinase (PKA RIIα) (Fukuyama et al., 2001). Its in vivo contribution to AML1/ETO function is not well characterized.

NHR4, also known as the myeloid-Nervy-DEAF-1 (MYND) domain was predicted based on its primary sequence to have two putative, non-DNA-binding zinc-fingers (Gross and McGinnis, 1996) and binds the silencing mediator of retinoid and thyroid hormone receptor (SMRT) and nuclear receptor corepressor (N-CoR) complexes (Gelmetti et al., 1998; Lutterbach et al., 1998a; Wang et al., 1998). The MYND domain is defined by a C6HC zinc chelating motif that is found in a number of other nuclear proteins such as Nervy, DEAF-1, BS69, PDCD2, and Bop. MYND domains have frequently been implicated in transcriptional repression (Ansieau and Leutz, 2002; Gottlieb et al., 2002; Ladendorff et al., 2001; Lausen et al., 2004; Scarr and Sharp, 2002; Sims et al., 2002). Recruitment of N-CoR/SMRT and their associated histone deacetylases (HDACs) to AML1 target genes is one mechanism by which AML1/ETO is thought to aberrantly repress transcription and contribute to leukemogenesis (Gelmetti et al., 1998; Lutterbach et al., 1998b).

Here we report the solution structure of the AML1/ETO MYND domain, showing it is structurally homologous to the PHD and RING finger families of proteins. In addition, we solved the structure of a complex between the AML1/ETO MYND domain and a peptide from SMRT. Based on that structure, we introduced single amino acid substitutions into the AML1/ETO MYND domain to assess the role of the SMRT/N-CoR interaction in vivo. We show that mutations that disrupt SMRT/N-CoR binding reverse AML1/ETO's repression of cell proliferation, mildly affect its ability to repress granulocyte differentiation, and attenuate alterations in gene expression caused by AML1/ETO in lineage negative, Sca-1+ c-kit+ (LSK) bone marrow cells.

Results

Solution structure of the MYND domain from AML1/ETO

The 15N-1H HSQC spectrum of the AML1/ETO MYND domain (aa 658-707) clearly indicated a structured entity (Figure 1A), but upon addition of 5mM EDTA it collapsed to a poorly dispersed spectrum characteristic of an unfolded species (data not shown). Refolding was readily achieved by dialysis against 50μM ZnCl2, confirming that Zn2+ is essential for maintaining the MYND structure. The putative zinc chelating residues include cysteines 663, 666, 674, 677, 683, 687, 699, and one of the three consecutive histidines (695, 696 and 697). To unequivocally identify which histidine is chelated to Zn, we ran a long range 15N-1H HSQC (Pelton et al., 1993) at pH 6.0 which clearly indicated that the Nε2 of H695 is resistant to protonation and thus is the zinc-chelating atom (Supplemental Figure 1A). Mutation of H695 to Ala resulted in an HSQC spectrum similar to that obtained in the presence of EDTA (Supplemental Figure 2D and not shown).

Figure 1
Structure of the ETO MYND domain and its interactions with SMRT and N-CoR

We solved a high-resolution structure of the AML1/ETO MYND domain using a combination of NOEs, chemical shifts, and extensive dipolar couplings (Table 1). The overall fold of the AML1/ETO MYND domain adopts an interleaved zinc-chelating topology as observed in the PHD and RING domains. Zinc center I consists of Cys 663, 666, 683, and 687, and zinc center II is formed by Cys 674, 677, 699, and His695 (Figure 1B). The relative positioning of secondary structure elements follows the ββα motif frequently observed in PHD and RING fingers (Pascual et al., 2000; Zheng et al., 2002). These structural features greatly differ from those of the LIM zinc finger domain, which also contains two zinc atoms but adopts a sequential zinc chelating topology and forms two juxtaposed domains with a certain degree of structural similarity (Velyvis et al., 2001).

Table 1
Statistics for NMR data collection and structure calculations

Spadaccini et al. (Spadaccini et al., 2006) recently reported that the DEAF-1 MYND domain (backbone RMSD = 7.8 Å to our ETO MYND domain) has a sequential zinc-chelating topology like that of the LIM domains. This is a completely different fold than we describe here, which is surprising given the very high sequence similarity between MYND domains (Figure 1C). The ETO MYND domain structure has been refined and validated with residual dipolar couplings from two independent alignments. Fitting only the conserved residues in the DEAF-1 MYND domain structure to the 19 corresponding NH dipolar couplings obtained for the ETO MYND domain yields an average Q factor of 84%, i.e. the structure is highly inconsistent with the dipolar couplings measured for the ETO MYND domain. A more extensive discussion of the differences between the two structures and the experimental data that validates the ETO MYND domain structure is provided in the Supplementary Data, including Supplemental Figure 3.

The AML1/ETO MYND domain recognizes a “PPPLI” motif in SMRT and N-CoR

Residues 1031-1273 in repression domain III (RDIII) of SMRT were previously shown to contain the binding site for ETO (Zhang et al., 2001). We confirmed that an aa 1031-1273 SMRT polypeptide bound the MYND domain by HSQC titration (Figure 1A). N-CoR and SMRT are predicted to be mostly unstructured in their repression domains suggesting a peptide recognition motif. We therefore screened a peptide library spanning the SMRT interacting region using fluorescence anisotropy to identify the minimal MYND binding consensus sequence in SMRT. We identified three consecutive peptides that displayed significantly elevated anisotropy in the presence of the MYND domain (Figure 1D) and share the common sequence motif NPPPLI, corresponding to SMRT residues 1104-1109. A sequence similarity search revealed a similar binding site with the sequence PPPLI in N-CoR (aa 1033-1037). Isothermal titration calorimetry (ITC) was employed to determine the stoichiometry and binding affinity between the MYND domain and the SMRT and N-CoR peptides (Figure 1E). A further sequence matching in N-CoR and SMRT using a looser standard yielded one more potential binding site in both proteins: N-CoR 606-615 and SMRT 1664-1673 (Figure 1C). Interaction of the MYND domain with SMRT 1664-1673 was confirmed by an HSQC titration that showed similar chemical shift changes as seen for SMRT 1101-1113, though the affinity appeared weaker (data not shown).

Solution structure of the SMRT-MYND complex

We attempted to solve the structure of a MYND domain/SMRT peptide complex, however half-filtered NOESY spectra of a differentially labeled complex (13C/15N-MYND + unlabeled SMRT) yielded only 12 inter-molecular NOEs, which were insufficient to define the structure of the complex. Complexes with modest affinity often suffer from limited inter-molecular NOE information due to significant off-rates. To decrease the off-rate and thereby facilitate the collection of NOE data we fused the SMRT peptide sequence (SMRT 1101-1113) to the N-terminus of the MYND domain (AML1/ETO 658-707) with a 7 aa linker (ENLYFQG) between them (subsequently referred to as SMRT-MYND). This coupling served to increase the effective local concentration of the SMRT peptide and resulted in longer residence times and increased NOEs. 15N-1H HSQC spectra of SMRT-MYND showed that resonances from the MYND portion of SMRT-MYND overlapped with resonances from the MYND + SMRT non-covalent complex, but not with those of the free MYND domain (Supplemental Figure 1B). Furthermore, the 12 NOEs previously identified in the non-covalent complex were all present in NOESY spectra of SMRT-MYND (data not shown), indicating that the fusion did not change the mode of interaction. Indeed, {1H}15N NOE measurements showed that the linker region is flexible, while the MYND and SMRT portions of the fusion protein display the same dynamic behavior as one another, consistent with a well-formed complex (Figure 2A).

Figure 2
Structure of SMRT-MYND

The structure of MYND-SMRT showed that SMRT binds in an extended conformation to a hydrophobic pocket in MYND (Figure 2B) burying a total solvent-accessible surface area of 476 Å2. L1108, I1109 and S1110 from SMRT form a short anti-parallel β sheet with E672, T673, and C674 of the MYND domain, delineated by 4 inter-strand hydrogen bonds (Figure 2C). The side-chain of L1108 protrudes deep into the hydrophobic pocket of MYND (Figure 2B). This provides a structural rationalization for the importance of Leu at this position, which has been noted by others (Lausen et al., 2004). P1105 packs its side-chain on top of W692 of MYND (Figure 2C). Such a proline-tryptophan packing arrangement has been frequently observed in protein-protein interactions (Glaser et al., 2001). Another residue showing 100% conservation among MYND domains is Q688, which forms a hydrogen bond to the CO of SMRT P1106 through Hε22 (Figure 2C). S675 also has a high degree of conservation among MYND domains (Figure 1C). The S675 γ proton potentially forms a hydrogen bond with L1108 CO of SMRT. Indeed this γ proton undergoes unusually slow exchange with water and displays a downfield shifted chemical shift of 6.47 ppm in MYND-SMRT, indicative of hydrogen bonding.

Mutations in the MYND domain disrupt SMRT peptide binding

We used the structure of the SMRT-MYND complex to rationally design mutations that disrupt SMRT/N-CoR binding, in order to examine the importance of this interaction for AML1/ETO's function. H695 is a zinc-chelating residue, and 15N-1H HSQC spectra showed the MYND domain structure was severely disrupted upon substitution of H695 with Ala (Supplemental Figure 2D), resulting in a protein which cannot bind to the SMRT peptide. Substitution of H689 with Ala, on the other hand, had little or no effect on either the structure or on SMRT binding (Supplemental Figure 2D and Figure 3A). The W692A mutation resulted in perturbations only of spatially proximal residues in the 15N-1H HSQC spectrum (Figure 3B and Supplemental Figure 2A), consistent with this mutation causing no significant change in the protein structure. Substitution of W692 with Ala substantially blocked binding to the SMRT peptide, increasing the Kd more than 12-fold (Figure 3A). Ala substitutions for Q688 and S675 preserved the MYND structure (Supplemental Figures 2B,C) and impaired SMRT peptide binding 12-fold and 2.5-fold, respectively (Figure 3A).

Figure 3
AML1/ETO function is impaired by mutations in the MYND domain that disrupt SMRT binding

Despite the fact that the W692A and H695A mutations decreased binding of the isolated MYND domain to the SMRT peptide, we were unable to detect a difference in full-length AML1/ETO's ability to co-immunoprecipitate full length SMRT or N-CoR when proteins containing either of these two mutations were overexpressed in Cos7 cells (not shown). This was not entirely unexpected, since we previously found that a C-terminal truncation of AML1/ETO that removed both the Nervy and MYND domains entirely did not affect SMRT or N-CoR binding using the same approach (Liu et al., 2006). AML1/ETO can oligomerize with itself, ETO, and the ETO homologs MTGR1 and MTGR2 (MTG16) (Kitabayashi et al., 1998; Liu et al., 2006; Minucci et al., 2000; Zhang et al., 2001). Therefore our inability to detect an effect of the MYND mutations in co-immunoprecipitation assays could be due to AML1/ETO's ability to form mixed tetramers with endogenous ETO homologues that have intact MYND domains. In addition, a second N-CoR binding site was mapped to sequences between eTAFH and HHR (Amann et al., 2001), which could also explain the retention of N-CoR and SMRT binding by the full length, MYND-mutated AML1/ETO proteins.

Contribution of the MYND-SMRT/N-CoR interaction to AML1/ETO function

Previous studies showed that deletion of the MYND domain impaired AML1/ETO's ability to repress the differentiation of established hematopoietic cell lines (Gelmetti et al., 1998; Klampfer et al., 1996; Lutterbach et al., 1998a). However, in primary bone marrow cells, the MYND domain's contribution to AML1/ETO's repression of granulocyte differentiation and proliferation, and to its ability to confer serial replating was only observed when coupled to an HHR domain deletion (Hug et al., 2002). We transduced primary, lineage depleted (CD5, B220, Mac-1, Gr-1, Ter119; Lin) mouse bone marrow cells with retroviruses expressing green fluorescent protein (GFP) alone (MIGR1), AML1/ETO, or an AML1/ETO protein truncated immediately N-terminal to the MYND domain (W664X) (Figure 3C), and assessed granulocyte differentiation after 7 days of culture in the presence of IL-3, IL-6, SCF, and G-CSF in the successfully transduced (GFP+) cells. Consistent with data reported by Hug et al. (Hug et al., 2002), we observed that deletion of the entire MYND domain and sequences C-terminal to it did not impair AML1/ETO's ability to inhibit the differentiation of Gr-1+ Mac-1+ cells from Lin bone marrow cells in vitro (Figure 3D,E). We therefore mutated seven Leu resides in the HHR domain at the tetramer interface that prevent its oligomerization [m7] (Liu et al., 2006), and assessed the effects of the MYND mutants in the context of the m7 HHR mutation. As we showed previously (Liu et al., 2006), the m7 mutation partially ameliorated AML1/ETO's inhibition of granulocyte differentiation, and deletion of the MYND domain in the context of this m7 mutation further weakened AML1/ETO's negative effect on granulocyte differentiation (Figure 3D,E). The W692A and H695A mutations in the context of the m7 mutation also significantly impaired, but did not completely reverse AML1/ETO's repression of granulocyte differentiation (Figure 3D,E). All three m7 + MYND mutant AML1/ETO proteins accumulated to the same level as the m7 mutant in retrovirally transduced NIH3T3 cells (Figure 3F). We could not detect the proteins by Western blot in the transduced primary bone marrow cells (not shown).

AML1/ETO inhibits the short-term proliferation of human and mouse primary bone marrow cells (Hug et al., 2002; Mulloy et al., 2002). It was previously reported that deletion of the MYND domain had no effect on AML1/ETO's anti-proliferative activity (Hug et al., 2002). In our hands, deletion of the MYND domain (W664X) ameliorated this anti-proliferative effect, although the percentage of cells that incorporated BrdU was still significantly lower than for MIGR1 transduced cells (Figure 3G). Both the H695A and W692A mutations alleviated AML1/ETO's repressive effect on proliferation to a similar extent as the W664X truncation (Figure 3G,H).

Deletion of the MYND domain did not affect AML1/ETO's ability to confer increased self-renewal capacity on hematopoietic progenitors in vitro, consistent with data from Hug et al. (Hug et al., 2002) (not shown), so presumably the W692A and H595A mutations would also not affect AML1/ETO's serial replating activity, although we did not examine this directly. Since in our hands, the m7 mutation in the HHR domain destroyed AML1/ETO's ability to confer increased self-renewal (Liu et al., 2006), we could not use the m7 mutant as a sensitized background upon which to further examine the effects of the W692A and H695A mutations.

Loss of the MYND-SMRT/N-CoR interaction attenuates the AML1/ETO signature in LSK cells

To gain insights into the molecular basis by which eliminating SMRT/N-CoR binding through the MYND domain relieves the proliferation block caused by AML1/ETO in Lin bone marrow cells but not its ability to confer serial replating capacity, we analyzed the effect of the W692A mutation on gene expression changes caused by AML1/ETO. We prepared Lin Sca-1+ mouse bone marrow cells, subdivided them into 11 pools, and independently transduced these cell pools with MIGR1, AML1/ETO, or the AML1/ETO W692A mutant (Figure 4A). Two days later, ~25,000 transduced (GFP+) Lin Sca1+ c-kit+ (LSK) cells from each sample were sorted by FACS into RNA lysis buffer for RNA purification, labeling, and hybridization to Affymetrix Mouse Genome 430 2.0 arrays (Figure 4A, B). We drew a wide gate around the Sca-1 population for sorting purposes (Figure 4B) because cell surface Sca-1 levels varied considerably between samples (Figure 4F).

Figure 4
Gene expression profiles of LSK cells expressing AML1/ETO and the AML1/ETO W692 mutant

We uploaded the raw expression data to Iobion Informatic's GeneTraffic v3.2 server (Irizarry et al., 2003) and set expression values from the three MIGR1-transduced samples as the baseline for comparison to AML1/ETO and W692A transduced samples. Unsupervised clustering separately grouped the MIGR1, AML1/ETO, and W692A samples, indicating that the three gene profiles were significantly different (not shown). We screened the data by Significance Analysis of Microarrays (SAM) (Tusher et al., 2001), and found genes corresponding to 2229 probe sets were differentially expressed in AML1/ETO-transduced LSK cells as compared to LSK cells transduced with MIGR1 alone (Figure 4C). These included genes previously reported to be dysregulated in the presence of AML1/ETO in myelomonocytic cell lines (Alcalay et al., 2001; Shimada et al., 2000; Yan et al., 2004) (Figure 4E). One upregulated gene was Ly6a, which encodes the Sca-1 protein, and indeed increased levels of Sca-1 were detected on AML1/ETO-transduced LSK cells (Figure 4F). c-kit, on the other hand, was not differentially expressed either at the mRNA (not shown, inferred from microarray data) or protein level (Figure 4F).

A smaller number of genes (823 probe sets) were differentially expressed in LSK cells transduced with the AML1/ETO W692A mutant as compared to LSK cells transduced with MIGR1 (Figure 4C). We compiled the two probe sets (AML1/ETO versus MIGR1 plus W692A versus MIGR1) into a single “master” list. There was considerable overlap between the two probe sets, thus the master list consisted of 2444 probe sets representing 1727 annotated genes (Figure 4C,D and Supplemental Tables 1,2). We screened the 1727 annotated genes by SAM to identify those that were differentially regulated by AML1/ETO versus W692A, which yielded a list of 1231 genes (Figure 4C, Supplemental Table 2). The expression of 1113 (90%) of these differentially expressed genes was attenuated by the W692A mutation. The remaining 118 differentially expressed genes were either dysregulated in the opposite direction by AML1/ETO and the W692A mutant, or dysregulated to a greater extent by the AML1/ETO W692A protein than by AML1/ETO. Thus the most prominent pattern was that the W692A mutation attenuated AML1/ETO's activity (Supplemental Table 2).

A trivial explanation for attenuated activity would be that the relative levels of the AML1/ETO W692A protein were lower. Unfortunately, we were unable to detect either the AML1/ETO or W692A proteins by Western blot analysis of purified GFP+ cells from retrovirally transduced primary bone marrow cells, and therefore could not directly rule out this possibility. However, the levels of the bicistronic mRNAs encoding the AML1/ETO and W692A proteins were similar, based both on the intensity of GFP fluorescence (not shown), and by qRT-PCR analysis (Supplemental Figure 5). It still remains possible that the W692A protein accumulated at lower levels in LSK cells because it was no longer stabilized by the association with protein complexes normally recruited by the MYND domain.

We identified 496 genes (28.7% of the master gene list) whose expression was dysregulated to the same extent by AML1/ETO and the W692A mutant (Figure 4C and Supplemental Table 2). Therefore if the W692A mutation did affect AML1/ETO protein levels, it impacted the expression of some genes and not others.

We identified 79 cell cycle-related genes whose expression was affected to a relatively small (ranging from 1.51 – 5.4 fold) but significantly different extent by AML1/ETO and the W692A mutant (Table 2). The differential expression of these genes presumably contributes to the major biological difference we observed, namely the repression of proliferation by AML1/ETO and the relief of that repression by the W692A mutation. The more minor effects we observed in granulocyte differentiation correlate with differences in the expression of genes required for that process (Table 3).

Table 2
Cell cycle related genes dysregulated ≥2 fold by AML1/ETO and to a lesser extent or not at all by the AML1/ETO W692A mutant.
Table 3
Genes involved in myeloid cell differentiation and/or function that were differentially expressed by AML1/ETO (≥2 fold) and the AML1/ETO W692A mutant.

Discussion

The structure of the MYND domain/SMRT peptide complex provided insights into how specificity is achieved and structure-based mutations enabled us to assess the contribution of SMRT/N-CoR binding through the MYND domain to AML1/ETO function. As is the case with other domains such as SH3 and WW that bind to Pro-rich sequences, the question arises how specificity can be achieved with a binding motif consisting exclusively of non-polar residues that cannot form specific hydrogen bonds or electrostatic interactions. In the case of the MYND domain/SMRT (N-CoR) binding, specificity is achieved through a combination of proline-tryptophan packing interactions, other van der Waals interactions via the complementary surfaces, and by means of the conformational rigidity of the Pro-rich sequence that minimizes the entropic cost of binding and positions backbone nuclei favorably for hydrogen bond formation. The first Pro in the “PPPLIP” motif packs against W692. The carbonyls of the second and third prolines form hydrogen bonds with Q688 and S675, respectively. Although hydrogen bonds to the backbone generally cannot provide specificity, the relative geometrical positions of W692, Q688, and S675 on the MYND domain favor substrates with a certain backbone conformation for hydrogen bond formation. N-CoR binds the MYND domain with higher affinity than SMRT, and the only difference in the directly interacting region is that P1038 of N-CoR, which is one of the three consecutive residues forming a β strand with the MYND domain, is replaced by S1110 in SMRT. S1110, with its HN pointing away from the β strand, does not seem to participate in any specific interaction, and it is not obvious why a Pro at this position increases binding affinity. It is tempting to speculate that MYND's higher affinity for N-CoR is because the proline reduces the entropic penalty for binding. Indeed, ITC data show that ΔH is quite similar for the MYND/N-CoR and MYND/SMRT interactions (−9883 cal mol−1 and −9836 cal mol−1, respectively) while it is ΔS that dominates the difference in binding affinity (−12.4 cal mol−1 K−1 and −14.5 cal mol−1 K−1, respectively).

Using the structural information, we introduced mutations in the AML1/ETO MYND domain that specifically disrupted SMRT (and presumably N-CoR) binding without significantly affecting the MYND domain structure. Impairing SMRT/N-CoR binding to the MYND domain attenuated AML1/ETO's activity only very modestly in primary bone marrow cells, as had been shown by others using an MYND domain deletion (Hug et al., 2002). AML1/ETO's acute repression of cell proliferation was alleviated but not completely eliminated, and its negative effect on granulocyte differentiation was ameliorated, but only in the context of a mutation that impaired oligomerization. In fact, the presence of an oligomerization domain (HHR) in AML1/ETO complicates efforts to document the importance of other ETO sequences for AML/ETO's activity since the formation of mixed tetramers with endogenous ETO or the ETO homologs MTGR1 and MTGR2 could dampen a mutation's effects. Nevertheless, the effect of a single amino acid substitution that decreased SMRT/N-CoR binding by 12-fold was relatively extensive, at least with respect to the number of genes (1231) whose expression was significantly different by SAM analysis. Presumably included among these are genes whose differential expression underlies the perturbations in proliferation and granulocyte differentiation that were observed by us and others (Gelmetti et al., 1998; Hug et al., 2002; Klampfer et al., 1996).

Cell cycle-related genes that were differentially expressed included those with established roles in cell cycle regulation and checkpoint control, DNA repair, DNA replication, and mitosis. Many genes listed in Table 2 promote these processes, and were down-regulated in the presence of AML1/ETO, with this effect attenuated by the W692A mutation. Conversely, several genes that negatively regulate cell cycle progression or proliferation (Ccng2, G0s2, Tgfb1) were upregulated by AML1/ETO and less so by the AML1/ETO W692A mutant. The largest absolute change in expression among this group was observed for Vegfc which is 75-fold upregulated with AML1-ETO and attenuated to 14-fold with AML1-ETO W692A. Elevated levels of VEGF have been observed for AML patients and correlate with reduced survival as well as reduced complete remission rates in these patients (Aguayo et al., 2000; Aguayo et al., 2002), arguing for the potential applicability of antiangiogenic therapy in t(8;21) leukemia.

AML1/ETO expression also resulted in the downregulation of genes involved in myeloid cell differentiation (Table 3). The most highly differentially expressed gene in this group is Gfi1, which promotes T cell proliferation, granulocyte maturation, and restricts the proliferation and thus maintains the quiescence of hematopoietic stem cells (Hock and Orkin, 2006). Gfi1 expression was upregulated by AML1/ETO (5.3 fold), and that upregulation was attenuated by the W692A mutation. Cebpa, a known suppressor of leukemogenesis and positive regulator of granulopoiesis (Nerlov, 2004) was downregulated 8.3 fold in the presence of AML1/ETO, and that repression was alleviated by the W692A mutation.

Some of the more interesting genes are likely to be those that are highly dysregulated by both AML1/ETO and the W692A mutant. Deletion of the MYND domain did not affect one of AML1/ETO's most important properties, that is its ability to confer serial replating to primary bone marrow cells, which is a readout of self renewal potential (Hug et al., 2002). Furthermore, a truncated form of AML1/ETO or a naturally occurring splice variant that lacks both the Nervy and MYND domains was leukemogenic in mice (Yan et al., 2004). In fact, the truncated AML1/ETO was more highly leukemogenic than the full length protein, and could cooperate with full length AML1/ETO to induce a rapid disease (Yan et al., 2004; Yan et al., 2006). One of the genes whose expression was most highly upregulated by both AML1/ETO (24 fold) and the W692 mutant (23 fold) was high mobility group protein 2 (Hmga2). HMGA proteins are small, architechural transcription factors involved in proliferation, apoptosis, differentiation, and senescence (Narita et al., 2006; Reeves, 2001). Upregulation of HMGA2 expression has been found in a variety of solid tumors in humans, and chromosomal rearrangments of HMGA2 are common in benign mesenchymal tumors (Reeves, 2001). In senescent cells, HMGA was shown to repress MCMs and cyclin A (Narita et al., 2006). As shown in Table 2, cyclin A and MCMs 2, 3, 6, 7, and 8 are all downregulated by AML1/ETO as well as the mutant AML1/ETO, albeit to a lesser extent, suggesting increased HMGA2 may well contribute to the AML1/ETO phenotype.

In summary, our results demonstrate that recruitment of SMRT/N-CoR by the MYND domain contributes to the transcriptional dysregulation caused by AML1/ETO, and to its repression of both proliferation and granulocyte differentiation. The two-hit hypothesis for leukemia development posits the need for both a block in differentiation and a proliferative advantage (Gilliland and Tallman, 2002). It is well-established in mouse models that AML1-ETO by itself does not cause leukemia but requires additional cooperating mutations (Grisolano et al., 2003; Schessl et al., 2005; Schwieger et al., 2002). Full-length AML1-ETO can clearly achieve the block in differentiation posited in the two-hit model but is not able to provide the necessary proliferative advantage. The recent demonstration by Zhang and co-workers (Yan et al., 2004; Yan et al., 2006) that a truncated form of AML1-ETO lacking the Nervy and MYND domains causes leukemia, unlike AML1-ETO itself, suggests that a truncated form of the protein can provide both the differentiation block and the proliferative advantage necessary. This is in good agreement with our results showing that loss of SMRT/N-CoR binding to the MYND domain results in increased proliferation, i.e. abrogation of MYND domain function provides or contributes significantly to the necessary proliferative advantage. The loss of this domain in the truncation in the case of truncated AML1-ETO or altered regulation, perhaps by phosphorylation, of this domain in the case of other cooperating mutations may provide a mechanism to achieve both hits through one protein. In the latter case, the signaling pathways involved in such regulation may therefore prove to be useful targets for therapeutic intervention.

Experimental Procedures

Protein Expression and Purification

All proteins were cloned, expressed, and purified using standard procedures (details in Supplementary Data).

Peptide binding measurements by fluorescence anisotropy

All fluorescence experiments were carried out at 25°C on a FluroMax-3 fluorescence spectrophotometer (Horiba Jobin Yvon, Edison NJ) with an external temperature control unit. The concentration of fluorescein-labeled peptides was kept at 5μM during SMRT binding epitope screening and 1μM during MYND mutant binding experiments. Peptides 12 aa in length with 9 aa overlaps spanning the entire SMRT sequence and with an N-terminal fluorescein were employed for epitope mapping. The excitation and emission wavelengths were set to 494 nm and 525 nm, respectively. Anisotropy values were averaged over 1 second during the epitope screening experiments and over 5 seconds during the MYND mutant binding experiments following a 2-minuate pre-equilibration at 25° C.

Peptide binding measurements by isothermal titration calorimetry

ITC experiments were carried out on a MCS-ITC isothermal titration calorimetric system (MicroCal, Northampton MA) at 26° C. The calorimetric cell containing 170 or 257 μM MYND domain was titrated with 3.3 mM SMRT (1101-1113: YG-TISNPPPLISSAK) or 2 mM N-CoR (N-CoR 1000-1011; YG-TRPPPPLIPSSK) peptides. Both MYND and SMRT/N-CoR were thoroughly dialyzed into the same buffer containing 25 mM Bis-Tris (pH 7.5), 0.1 M NaCl, 1 mM DTT, and 50 μM ZnCl2. Data were analyzed using Origin 7.0 (OriginLab, Northampton MA).

Analytical ultracentrifugation

Sedimentation velocity runs were done at 20 degrees Celsius using an AN-60 titanium rotor (Beckman) and SedVel60 centerpieces with 1.2cm path length and quartz windows. The runs were done at 35,000 rpm and interference scans were acquired using Beckman software. Analyses were done using Sedfit (Schuck, 2000) with (96 resolution, 0.5 to 10 s) radial & time independent noise at 95% confidence intervals.

NMR Spectroscopy

Samples for structure determination were prepared in 25 mM Bis-Tris (pH 6.8), 50 μM ZnCl2, and 1mM DTT. One hundred millimolar NaCl was included in the buffer for charged gel samples to alleviate line broadening due to protein-gel interaction. Data were collected on a 600 MHz Varian Inova Spectrometer (Palo Alto, CA) equipped with a cryogenically cooled probe at 32 °C. Resonance assignment and NOESY data collection was carried out using standard experiments.

For alignment, a negatively charged gel compressed to 4% concentration and consisting of 50% 2-acrylamido-2-methyl-1-propanesulfonic acid and 50% acrylamide (50 - M) and a zwitterionic charged gel compressed to 4% concentration consisting of 50% of a 1:1 mixture of (3-acrylamidopropyl)-trimethylammonium chloride/acrylic acid and 50% acrylamide (50+MA) were employed (Cierpicki and Bushweller, 2004). Three types of dipolar couplings, 1DHN, 1DNC', and 1DC'Cα, were measured using IPAP (Ottiger et al., 1998) and 3D HNCO-based experiments (Yang et al., 1999). All NMR spectra were processed and analyzed using NMRPipe and Sparky (T.D. Goddard and J.M. Kneller, University of California, San Francisco).

Structure Calculations

Structure calculations were carried out using the simulated annealing protocol in CNS (Brunger et al., 1998). Details are provided in the Supplementary Data.

Retroviral transductions

Mutated AML1/ETO proteins were transferred from pBluescript into the MIGR1 vector (Pear et al., 1998), and retroviruses prepared as described previously (Liu et al., 2006). Primary bone marrow mononuclear cells were harvested from 5-6 week old C57BL/6 mice (Jackson Labs, Bar Harbor, ME) and subjected to immunomagnetic negative selection using the Lineage cell depletion Kit (Miltenyi Biotech, Auburn, CA). For the microarray experiments the Lin cells were further enriched for Sca1+ cells by positive selection using the Anti-Sca-1 MicroBead Kit (Miltenyi Biotech). Cells (6×105 in 3ml) were plated in ultra-low adhesion 6-well plates (Costar, Corning, NY) and incubated overnight at 37°C, 5% CO2 in transplant media [RPMI, 20% FCS, penicillin and streptomycin, 10 ng/ml IL-3, 20 ng/ml IL-6 (R&D Systems, Minneapolis, MN), 10 ng/ml SCF (Stem Cell technologies, Vancouver, Canada)]. Twelve hours later, 6×105 cells in 2ml of fresh transplant media were added to 6-well plates (Cellstar, GBO, NC) coated with 100 μg Retronectin (Takara, Madison WI). Retroviral supernatants (2 ml), 4 μl of 40 mg/ml polybrene and 40μl 1M Hepes were added to each well and the cells centrifuged for 90 minutes at 1400 g, 37°C. Cells for microarray experiments were subjected to a second round of transduction 24 hours later as described above.

Granulocyte differentiation

Granulocyte differentiation was assessed as described previously (Liu et al., 2006).

Cell cycle kinetics

BrdU incorporation by Lin bone marrow cells was assessed 48 hours after retroviral transduction according to the manufacturer's instructions (APC-BrdU flow kit, BD PharMingen, San Diego, CA). Cells were incubated with 30 μM BrdU for 1 hour and stained with Allophcocyanin (APC) conjugated anti BrdU antibody and 7-AAD. Data were collected on a FACSCalibur (BD Biosciences, San Jose CA) and analyzed by FlowJo (version 6.1.1, Tree Star, San Carlos, CA).

Microarray Analyses

Lin Sca1+ bone marrow cells independently transduced with MIGR1 (three separate pools), AML1/ETO (three pools) or AML1/ETO W692A (four pools) were stained 48 hours later with PE conjugated anti-CD117 (clone 2B8, BD Bioscience), PerCp-Cy5.5 conjugated Streptavidin (BD Bioscience), APC-conjugated anti-Sca1 (clone D7, Ebiosciences, SanDiego, CA) and biotin conjugated antibody cocktail (Miltenyi Biotech). Lin Sca-1+ c-kit+ GFP+ cells were isolated on a FACSAria (Becton Dickinson). Approximately 25,000 cells were collected for each sample. Cells were sorted into 600μl of RNA lysis buffer (Qiagen buffer RLT w/ β-mercaptoethanol, Valencia CA), incubated at 42°C for 1h, and vortexed for 1min to facilitate cell-lysis. Total RNA was extracted and DnaseI-treated using Rneasy Mini and DnaseI kits (Qiagen) according to the manufacturer's protocol. Total RNA quantity was measured using a ND-1000 spectrophotometer (Nano-Drop, Wilmington, DE) and RNA integrity verified on an Agilent Bioanalyzer using a RNA 6000 Pico chip (Agilent Technologies, Santa Clara CA). To minimize potential sample variability, 50ng of total RNA from each sample was used as template for T7-based linear RNA amplification (RiboAmp kit, Arcturus, Mountain View CA). Poly-inosinic poly-cytidilic acid (200 ng, Sigma, St Louis MO) was added as a carrier during the first round of cDNA synthesis. 1μg of double-stranded cDNA derived from a second round of cDNA synthesis (RiboAmp kit) was used as template for generating biotinylated cRNA (BioArray High Yield Transcription kit, Enzo Life Sciences, Farmingdale NY). Labeled cRNA was then DnaseI-treated and purified on Rneasy mini-columns (Qiagen). Samples were submitted to the Norris Cotton Cancer Center and Dartmouth Medical School Microarray Shared Resource facility for fragmentation and hybridization to Mouse Whole Genome 430 2.0 arrays (Affymetrix, Santa Clara CA).

The coordinates for the MYND and SMRT-MYND structures have been deposited in the PDB with accession codes 2OD1and 2ODD, respectively. The microarray data discussed in this publication have been deposited in NCBIs Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) with accession number GSE7324.

Supplementary Material

01

02

Acknowledgments

We thank Carol Ringelberg and Richard Cowper for bioinformatics support. This work was supported by RO1 CA108056 (JHB and NAS). MDC was supported by T32 GM08704 and JJG by T32 AR07576.

Footnotes

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