Structural and functional studies on AML1 and the oncoprotein AML1-ETO

The AML1-CBFbeta complex regulates the transcription of genes essential during hematopoiesis. Both AML1 and CBFbeta are frequent targets of chromosomal translocations involved in human leukemias. Of these, the t(8;21) is associated with 12∼15% of acute myeloid leukemia cases. The resultant AML1-ETO protein contains the Runt domain of AML1 and 4 domains from ETO, namely TAF110, HHR, nervy and MYND. The crystal structure of the HHR domain was solved to a 2.0A resolution and it shows that the HHR domain forms a homo-tetramer. Its tetramerization was found to be critical for AML1-ETO's activities, such as the inhibition of granulocyte differentiation, the immortalization of primary bone marrow cells, and the regulation of endogenous target genes. The ETO MYND domain interacts with co-repressors such as N-CoR and SMRT. The binding consensus sequence of the co-repressors was identified through fluorescence anisotropy screening on a peptide library. The solution structure of the MYND domain was solved as a complexed form with a SMRT peptide. The complex structure reveals conserved features for MYND-substrate interactions. Point mutations were designed that specifically disrupt MYND-corepressor association without disrupting the overall fold of the MYND domain, which provides the biophysical basis for further investigations of the biological significance of the MYND-corepressor interaction. The Runt domain binds both DNA and CBFbeta. CBFbeta allosterically enhances Runt's affinity for DNA without making direct contacts to DNA. Through NMR dynamics, a network of correlated residues in the Runt domain were identified that respond to the allosteric regulation from CBFbeta binding. This work shows how NMR studies can elucidate dynamic behaviors associated with protein allosteric regulation.