A stable transcription factor complex nucleated by oligomeric AML1–ETO controls leukaemogenesis

Abstract

Transcription factors are frequently altered in leukaemia through chromosomal translocation, mutation or aberrant expression1. AML1-ETO, a fusion protein generated by the t(8;21) translocation in acute myeloid leukaemia (AML), is a transcription factor implicated in both gene repression and activation2. AML1-ETO oligomerization, mediated by the NHR2 domain, is critical for leukaemogenesis3–6, making it important to identify coregulatory factors that “read” the NHR2 oligomerization and contribute to leukaemogenesis4. We now show that, in leukaemic cells, AML1-ETO resides in and functions through a stable protein complex (AETFC) that contains several haematopoietic transcription (co)factors. These AETFC components stabilize the complex through multivalent interactions, provide multiple DNA-binding domains for diverse target genes, colocalize genome-wide, cooperatively regulate gene expression, and contribute to leukaemogenesis. Within the AETFC complex, AML1-ETO oligomerization is required for a specific interaction between the oligomerized NHR2 domain and a novel NHR2-binding (N2B) motif in E proteins. Crystallographic analysis of the NHR2-N2B complex reveals a unique interaction pattern in which an N2B peptide makes direct contact with side chains of two NHR2 domains as a dimer, providing a novel model of how dimeric/oligomeric transcription factors create a new protein-binding interface through dimerization/oligomerization. Intriguingly, disruption of this interaction by point mutations abrogates AML1-ETO–induced haematopoietic stem/progenitor cell self-renewal and leukaemogenesis. These results reveal new mechanisms of action of AML1-ETO and a potential therapeutic target in t(8;21)+ AML.

Genome-wide studies identify a novel interplay between AML1 and AML1/ETO in t(8;21) acute myeloid leukemia

“…Furthermore, re-ChIP experiments in Kasumi-1 cells revealed that the interaction of AML1/ETO and AML1 indeed occurred on chromatin ( Figure 4C). In addition, because recent studies have shown that AML1/ETO exists as a stable complex on chromatin with multiple transcription factors, 14 we then assessed whether AML1 was present in this stable complex. As shown in supplemental Figures 5 and 6, the genomic occupancy of AML1 was correlated with those reported in the AML1/ETO complex, ie, E2A, HEB, and LMO2 (r 5 0.492, 0.439, and 0.475, respectively; P , 2.2e216), indicating that AML1 colocalized with the AML1/ETO stable complex on chromatin.…”

“…11 On the other hand, AML1/ETO can also recruit coactivators (such as p300 and PRMT1) to activate genes, especially those involved in stem cell self-renewal, such as ID1, CDKN1A, and EGR1. 12,13 In addition to dynamic interactions with various regulatory (co)factors, a recent study has reported that AML1/ETO resides in and functions through a stable multiprotein complex that at least contains HEB, LYL1, LMO2, and CBFb, 14 providing another layer of regulatory complexity for AML1/ETO in regulating its target genes.…”

“…Libraries were prepared according to the Illumina genomic prep kit (Illumina, San Diego, CA) and then sequenced by Illumina Genome Analyzer II platform (Illumina) to generate 35-bp single-end reads. The detailed analysis of ChIP sequencing (ChIP-seq) data, including our data and published ChIP-seq data, 14,16,20,[23][24][25] is available in supplemental Methods available on the Blood Web site.DNA pull-down, coimmunoprecipitation, GST pull-down, and immunofluorescence microscopy DNA pull-down assays were performed as described previously with several modifications.26 Coimmunoprecipitation (Co-IP) assays in Kasumi-1 and AML1/ETO-induced U937-A/E/9/14/18 cells were performed strictly according to the manufacturer's protocol (Active Motif). Glutathione S-transferase (GST) fusion proteins were generated according to the GST Gene Fusion System Handbook (GE Healthcare, Piscataway, NJ).…”

“…AML1/ETO-AML1 complex-targeted genes exhibit different AML1/ETO and AML1 binding patterns, which correlate with the expression of genes regulated by AML1/ETO To investigate the biological significance of the AML1/ETO-AML1 complex, we then asked how this complex influences the expression of genes regulated by AML1/ETO. We first retrieved genes that were differentially regulated on AML1/ETO knockdown on Kasumi-1 and SKNO-1 cells 14,20 and that were differentially expressed between AML1/ETO-positive and -negative AML-M2 patient samples 29 (details in Methods and supplemental Methods). Based on the gene set analysis, 30 we found that AML1/ETO-downregulated genes were as expected to be enriched in the genes targeted by the AML1/ETO-AML1 complex (Table 1), supporting the notion that AML1/ETO represses the AML1-regulated gene.…”

The RUNX1–PU.1 axis in the control of hematopoiesis

“…RUNX1-ETO retains the ability to interact with the enhancer core DNA sequence and has been shown to interfere with RUNX1-dependent transactivation [11,[121][122][123] and to alter the transcriptional regulation of normal RUNX1 target genes [27,122]. The fusion protein forms a tetrameric complex that interacts with other important hematopoietic regulators, namely the bridging factors LMO2 and LDB1, the E-Box binding factor HEB and the ETS family members FLI1 and ERG [123][124][125][126]. Genome-wide studies demonstrated that the majority of RUNX1-ETO binding sites overlap with binding sites for RUNX1 [122,125] and it was shown later that RUNX1 and RUNX1-ETO form complexes with the same transcriptional regulators and compete for the same binding sites.…”