Part I: Structural and functional insight into the type A CBFbeta-SMMHC protein and acute myeloid leukemia (AML). Part II: Solution structure of the HDGF PWWP domain and implications of its function as a DNA binding domain

Part I. Core-binding factors are heterodimeric transcriptional factors consisting of a DNA-binding Runx1 (CBFalpha) subunit and a CBFbeta subunit. In humans, chromosomal rearrangements that disrupt the Runx1 and CBFB genes are associated with a significant percentage of leukemias. CBFbeta is disrupted in acute myeloid leukemia by inv(16)(p13;q22), t(16;16), and del(16)(q22), resulting in the production of novel fusion proteins containing most of the CBFbeta protein fused to the C-terminal coiled-coil domain from smooth muscle myosin heavy chain (SMMHC). In Part I, we demonstrate that CBFbeta-SMMHC binds to the DNA binding Runt domain from Runx1 with both higher affinity and altered stoichiometry relatives to native CBFbeta. In addition, we demonstrate that CBFbeta-SMMHC inhibits high-affinity DNA binding of the Runx1 Runt domain. We propose that the inhibition of DNA-binding and increased affinity combine to mediate the dysregulation of Runx-regulated genes caused by CBFbeta-SMMHC. These results also clearly suggest that targeting of the CBFbeta-SMMHC protein for drug development may well be a viable approach for the treatment of the associated leukemia.; Part II. Hepatoma Derived Growth Factor (HDGF) is an endogenous nuclear-targeted mitogen that is linked with human disease. HDGF is a member of the weakly conserved PWWP domain family. In Part II, we have determined the NMR structure of the HDGF PWWP domain to high resolution using a combination of NOEs, J-couplings, and dipolar couplings. Comparison of this structure to a previously determined structure of the HDGF PWWP domain shows a significant difference in the C-terminal region. Comparison to structures of other PWWP domains shows a high degree of similarity to the PWWP domain structures from Dnmt3b and mHRP. The results of selected and amplified binding assay and NMR titrations with DNA suggest that the HDGF PWWP domain may function as a non-specific DNA binding domain. Based on the NMR titrations, we propose a model of the interaction of the PWWP domain with DNA.