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.

Histone H3 trimethylation of lysine 9 (H3K9me3) and proteins of the heterochromatin protein 1 (HP1) family are hallmarks of heterochromatin, a state of compacted DNA essential for genome stability and long-term transcriptional silencing. The mechanisms by which H3K9me3 and HP1 contribute to chromatin condensation have been speculative and controversial. Here we demonstrate that human HP1β is a prototypic HP1 protein exemplifying most basal chromatin binding and effects. These are caused by dimeric and dynamic interaction with highly enriched H3K9me3 and are modulated by various electrostatic interfaces. HP1β bridges condensed chromatin, which we postulate stabilizes the compacted state. In agreement, HP1β genome-wide localization follows H3K9me3-enrichment and artificial bridging of chromatin fibres is sufficient for maintaining cellular heterochromatic conformation. Overall, our findings define a fundamental mechanism for chromatin higher order structural changes caused by HP1 proteins, which might contribute to the plastic nature of condensed chromatin.

Background: Chromatin-HP1 (heterochromatin protein 1) interaction is crucial for heterochromatin assembly. Results: hHP1␤ uses alternative interfaces to bind nucleosomes depending on histone 3 methylation within a highly dynamic complex. Conclusion: hHP1␤ explores chromatin for sites of methyl-mark enrichment where it can bind histone 3 tails from adjacent nucleosomes. Significance: We provide a conceptual framework to understand the molecular basis of dynamic interactions regulated by histone modification.

Despite insights on the cellular level, the molecular details of chromatin reorganization in sperm development, which involves replacement of histone proteins by specialized factors to allow ultra most condensation of the genome, are not well understood. Protamines are dispensable for DNA condensation during Drosophila post-meiotic spermatogenesis. Therefore, we analyzed the interaction of Mst77F, another very basic testis-specific protein with chromatin and DNA as well as studied the molecular consequences of such binding. We show that Mst77F on its own causes severe chromatin and DNA aggregation. An intrinsically unstructured domain in the C-terminus of Mst77F binds DNA via electrostatic interaction. This binding results in structural reorganization of the domain, which induces interaction with an N-terminal region of the protein. Via putative cooperative effects Mst77F is induced to multimerize in this state causing DNA aggregation. In agreement, overexpression of Mst77F results in chromatin aggregation in fly sperm. Based on these findings we postulate that Mst77F is crucial for sperm development by giving rise to a unique condensed chromatin structure.

Transcription factors are frequently altered in leukemia through chromosomal translocation, mutation or aberrant expression. AML1-ETO, a fusion protein generated by the t(8;21) translocation in acute myeloid leukemia (AML), is a transcription factor implicated in both gene repression and activation. AML1-ETO exists as dimer/oligomer through its NHR2 dimerization domain. Given that the AML1-ETO dimerization has been shown to be critical for its leukemogenic activity, it is important to identify coregulatory factors that “read” the NHR2 dimerization and contribute to leukemogenesis. We now show that, in leukemic cells, AML1-ETO resides in and functions through a stable protein complex (AETFC) that contains several hematopoietic transcription factors and cofactors. 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 leukemogenesis. Within the AETFC complex, the AML1-ETO dimerization is required for a specific interaction between the dimerized NHR2 domain and a novel NHR2-binding (N2B) motif in E proteins. Crystallographic study 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 transcription factors create a new protein-binding interface through dimerization. Intriguingly, disruption of this interaction by point mutations abrogates AML1-ETO-induced hematopoietic stem cell self-renewal and leukemogenesis. These results reveal new mechanisms of action of AML1-ETO in leukemogenesis and provide potential therapeutic possibilities for targeting t(8;21)+ AML. Citation Format: Xiao-Jian Sun, Zhanxin Wang, Lan Wang, Yanwen Jiang, T. David Soong, Nils Kost, Wei-Yi Chen, Olivier Elemento, Wolfgang Fischle, Ari Melnick, Dinshaw J. Patel, Stephen D. Nimer, Robert G. Roeder. A stable transcription factor complex nucleated by dimeric AML1-ETO controls leukemogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-111. doi:10.1158/1538-7445.AM2013-LB-111