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

Abstract

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 corepressors 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.

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

A. Overlay of ¹⁵N-¹H HSQC spectra of the free MYND domain (red) and MYND + SMRT1031-1273 (blue) collected at pH 6.0 and 37 °C at 500 MHz. B. Overlay of the backbone of 31 conformers representing the solution structure of the MYND domain with α helical residues (S685-H695) in red, β sheet residues (E672-T673 and R681-Y682) in cyan, zinc chelating side-chains in blue, zinc chelating atoms in yellow, and zinc atoms in green. C. Sequence alignment of MYND domains (Zn chelating residues in red, additional conserved residues in green and brown) and interacting regions from SMRT and N-CoR (identical residues in red, conserved residues in green). D. Fluorescence anisotropy measurements on 70 fluorescein-labeled peptides spanning residues 1031-1273 of SMRT. The Y axis corresponds to the difference between the anisotropy in the presence and absence of the MYND domain. E. Isothermal titration calorimetry (ITC) measurements MYND domain binding to peptides derived from SMRT and N-CoR.

Figure 2. Structure of SMRT-MYND

A. Left: Overlay of the backbone of 10 conformers representing the solution structure of SMRT-MYND. Residues with {¹H}¹⁵N NOE > 0.6 in blue, residues with {¹H}¹⁵N NOE < 0.6 in red, and residues with no {¹H}¹⁵N NOE information in green. Right: Overlay of the backbone of 25 conformers of SMRT-MYND, showing only the structured portion of the protein. The MYND domain is black with helical residues in red and β-sheet residues in cyan. SMRT is gold with β–sheet residues in cyan. B. Surface representation of the MYND domain with SMRT peptide bound. Electrostatic potential mapped onto surface (blue – positive electrostatic potential, red – negative electrostatic potential). C. Left: Sequence-specific interactions between the MYND domain and SMRT illustrating the stacking of MYND W692 and SMRT P1105, the hydrogen bond between MYND Q688 and SMRT P1106, and the hydrogen bond between MYND S675 and SMRT-P1107. Hydrogen bonds are displayed as blue dotted lines. Colors are the same as in the right hand figure in panel A. 25 conformers are shown. Right: Schematic illustration of the observed hydrogen bonds between MYND (red) and SMRT (blue). Backbone-backbone hydrogen bonds are black and sidechain-backbone hydrogen bonds are green.

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

A. Fluorescence anisotropy measurements of the SMRT1031-1273 fluorescein-labeled peptide binding with MYND mutants. Plot shows normalized change in occupancy (or normalized change in anisotropy) using the equation [FA(mutant)-FA(H695A))/(FA(WT)-FA(H695A)], where FA(mutant), FA(H695A), and FA(WT) stand for the fluorescence anisotropy values of the peptide in the presence of the mutant MYND, of H695A MYND, and of wild-type MYND, respectively. Since H695A is unfolded, the fluorescence anisotropy of this protein was used as the zero point to correct for the effects of increased viscosity with increasing protein concentration, rather than the value for the peptide alone, on the fluorescence polarization measurement. SMRT peptide concentration was kept at 1 μM and WT and mutant MYND domains at 161 μM. Binding constants of the mutants relative to that of WT were calculated as (([mutant]_total+K_D(mutant))/[mutant]_total) / (([WT]_total+K_D(WT))/[WT]_total) = (FA(WT)-FA(H695A))/(FA(mutant)-FA(H695A)), where [mutant]_total=[WT]_total=161 μM, and K_D(WT) = 109.4 μM based on ITC. Error bars represent the mean ± SD for 3 independent measurements. B. Structure of the MYND domain illustrating the localized chemical shift perturbations caused by the W692A mutation. NH atoms are colored as follows: NH chemical shift change > 0.1 ppm, red; NH chemical shift change between 0.04 and 0.1 ppm, orange; NH chemical shift change < 0.04 ppm, blue; unassigned, black). The side chain of W692 is shown in purple. C. Schematic diagram of AML1/ETO and the truncated W664X mutant. Sequences derived from AML1 are brown, and those from ETO are blue. D. Representative flow of Lin⁻ BM cells infected with MIGR1 retroviruses expressing AML1/ETO and mutated derivatives, following seven days of culture in the presence of IL3, IL6, SCF, and G-CSF. Cells within the forward and side scatter gates were further gated for GFP expression, and GFP positive cells examined for Mac-1 and Gr-1 expression. The experiments were performed twice with triplicate samples. E. Average percentages of Gr-1⁺Mac-1⁺ cells (± SD) from a representative experiment with triplicate samples. F. Western blot probed with an antibody to the Runt domain, demonstrating expression of the m7 AML1/ETO HHR mutant and mutated MYND derivatives thereof in retrovirally-transduced NIH3T3 cells. G. BrdU incorporation 48 hours after transduction of Lin⁻ bone marrow cells with MIGR1 expressing GFP, AML1/ETO, or the AML1/ETO MYND mutants. GFP⁺ cells were analyzed for BrdU and 7-AAD incorporation following a 1 hour BrdU pulse. No differences in sub G1 (apoptotic) cells were observed. Shown is a representative of three experiments. H. Average percentage of gated BrdU⁺ cells from scatter plots in panel G (±SD) (n=3). All samples were significantly different (P ≤ 0.01) from either MIGR1 or AML1/ETO transduced cells.

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

A. Experimental scheme. B. Isolation of the retrovirally transduced (GFP⁺) LSK cells used for microarray analyses. C. Microarray data analysis. Expression values from the three MIGR1-transduced samples were set as the baseline for comparison to AML1/ETO- and AML1/ETO W692A-transduced samples. The AML1/ETO and AML1/ETO W692A data were filtered to remove all genes without at least 3 observations of log2 ratio = 0.58 (1.5 fold) from MIGR1 samples, yielding 6034 probe sets. SAM performed on AML1/ETO versus MIGR1 with a false discovery rate (FDR) of 0.01% identified 2229 significant probe set changes. SAM on AML1/ETO W692A versus MIGR1 with an FDR of 0.03% identified 823 significant changes. These were combined to yield a master list of 2444 probe sets, which are provided in Supplemental Table 1. The 2444 probe sets were manually filtered, removing those corresponding to non-annotated sequences, “hypothetical proteins”, “cDNA sequences”, and “expressed sequences”. In addition, for genes whose expression was reported as significant by multiple probe sets, only one probe set was included in the subsequent data analyses. The filtered list contained 1727 annotated genes (Supplemental Table 2). SAM was performed on these 1727 genes to compare expression changes caused by AML1/ETO and AML1/ETO W692A (relative to MIGR1) with FDR = 1.16%, from which 1231 genes that were significantly differently expressed, and 496 genes that were not significantly differently expressed were identified (Supplemental Table 2). D. Unsupervised hierarchical clustering of the master list of 1727 genes using the average clustering of Pearson correlation coefficient, depicted as a heat map. Yellow represents genes overexpressed relative to the average level in the three MIGR1-transduced samples, and blue represents under-expressed genes. E. Examples of previously identified genes (Shimada et al., 2000; Yan et al., 2004) differentially regulated by AML1/ETO in LSK cells. F. Histograms documenting cell surface expression of Sca-1 and c-kit on the transduced LSK cells.