Core Binding Factor (CBF) is required for the development of de®nitive hematopoiesis, and the CBF oncoproteins AML1-ETO, TEL-AML1, and CBFb-SMMHC are commonly expressed in subsets of acute leukemia. CBFb-SMMHC slows the G1 to S cell cycle transition in hematopoietic cells, but the mechanism of this e ect is uncertain. We have sought to determine whether inhibition of CBF-mediated trans-activation is su cient to slow proliferation. We demonstrate that activation of KRAB-AML1-ER, a protein containing the AML1 DNA-binding domain, the KRAB repression domain, and the Estrogen receptor ligand binding domain, also slows G1, if its DNA-binding domain is intact. Also, exogenous AML1 overcame CBFb-SMMHC-induced inhibition of proliferation. Representational di erence analysis (RDA) identi®ed cdk4 RNA expression as an early target of KRAB-AML1 activation. Inhibition of CBF activities by KRAB-AML1-ER or CBFb-SMMHC rapidly reduced endogenous cdk4 mRNA levels, even in cells proliferating at or near control rates as a result of exogenous cdk4 expression. Over-expression of cdk4, especially a variant which cannot bind p16 INK4a , overcame cell cycle inhibition resulting from activation of KRAB-AML1-ER, although cdk4 did not accelerate proliferation when expressed alone. These ®ndings indicate that mutations which alter the expression of G1 regulatory proteins can overcome inhibition of proliferation by CBF oncoproteins.

In CBF␤-SMMHC, core binding factor beta (CBF␤) is fused to the ␣-helical rod domain of smooth muscle myosin heavy chain (SMMHC). We generated Ba/F3 hematopoietic cells expressing a CBF␤-SMMHC variant lacking 28 amino acids homologous to the assembly competence domain (ACD) required for multimerization of skeletal muscle myosin. CBF␤-SMMHC(⌬ACD) multimerized less effectively than either wild-type protein or a variant lacking a different 28-residue segment. In contrast to the control proteins, the ⌬ACD mutant did not inhibit CBF DNA binding, AML1-mediated reporter activation, or G 1 to S cell cycle progression, the last being dependent upon activation of CBF-regulated genes. We also linked the CBF␤ domain to 149 or 83 C-terminal CBF␤-SMMHC residues, retaining 86 or 20 amino acids N-terminal to the ACD. CBF␤-SMMHC(149C) multimerized and slowed Ba/F3 proliferation, whereas CBF␤-SMMHC(83C) did not. The majority of CBF␤-SMMHC and CBF␤-SMMHC(149C) was detected in the nucleus, whereas the ⌬ACD and 83C variants were predominantly cytoplasmic, indicating that multimerization facilitates nuclear retention of CBF␤-SMMHC. When linked to the simian virus 40 nuclear localization signal (NLS), a significant fraction of CBF␤-SMMHC(⌬ACD) entered the nucleus but only mildly inhibited CBF activities. As NLS-CBF␤-SMMHC(83C) remained cytoplasmic, we directed the ACD to CBF target genes by linking it to the AML1 DNA binding domain or to full-length AML1. These AML1-ACD fusion proteins did not affect Ba/F3 proliferation, in contrast to AML1-ETO, which markedly slowed G 1 to S progression dependent upon the integrity of its DNA-binding domain. Thus, the ACD facilitates inhibition of CBF by mediating multimerization of CBF␤-SMMHC in the nucleus. Therapeutics targeting the ACD may be effective in acute myeloid leukemia cases associated with CBF␤-SMMHC expression.The core binding factor (CBF) transcription factors contain one of three CBF␣ subunits, CBF␣1/AML3/RUNX2, CBF␣2/ AML1/RUNX1, or CBF␣3/AML2/RUNX3, and a common CBF␤ subunit (3,14,21,35,50). The CBF␣ subunits contact the consensus DNA binding site, 5Ј-(Pu)ACCPuCA-3Ј, via their 127-amino-acid Runt homology domains (3,30). CBF␤ does not bind DNA but increases the DNA affinity of the CBF␣ subunits via allosteric interaction with the Runt domain (36,47,50). Amino acids 1 to 137 of the 182-residue CBF␤ are sufficient for increasing the DNA affinity of CBF␣ subunits (16,19).CBF␤ is widely expressed, whereas AML1 is largely restricted to hematopoietic cells (43, 50). Mice lacking AML1 or CBF␤ do not develop definitive hematopoiesis (33,37,42,51). A role for AML1 during maturation of pluripotent stem cells along the lymphoid and myeloid lineages has been inferred from its ability to transactivate promoters of lineage-restricted genes (34,41,46,58). AML1 possesses only weak intrinsic transactivating potential but cooperates with additional transcription factors to activate genes in hematopoietic cells (7,40,46). AML1 is found in the cell nucleus, whereas the large majority of CBF␤ is cytopla...

The Azotobacter FeSII protein, also known as the Shethna protein, forms a protective complex with nitrogenase during periods when nitrogenase is exposed to oxygen. One possible mechanism for its action is an oxidation state-dependent conformational interaction with nitrogenase whereby the FeSII protein dissociates from the MoFe and Fe proteins of nitrogenase under reducing conditions. Herein we report the construction and characterization of five site-directed mutants of the FeSII protein (H12Q, H55Q, K14A, K15A, and the double mutant K14A/K15A) which were individually purified after being individually overexpressed in Escherichia coli. These mutant FeSII proteins maintain native-like assembly and orientation of the 2Fe-2S center on the basis of EPR and NMR spectroscopic characterization and their redox midpoint potentials, which are within 25 mV of that of the wild type protein. The abilities of the individual mutant proteins to protect nitrogenase were assessed by determining the remaining nitrogenase activities after adding each pure version back to extracts from an FeSII deletion strain, and then exposing the mixture to oxygen. In these assays, the H12Q mutant functioned as well as the wild type protein. However, mutation of His55, a few residues away from a cluster-liganding cysteine, results in much less efficient protection of nitrogenase. These results are consistent with pH titrations in both oxidation states, which show that His12 is insensitive to 2Fe-2S cluster oxidation state. His55's pK is weakly responsive to oxidation state, and the pK increase of 0. 16 pH unit upon 2Fe-2S cluster oxidation is indicative of ionization of another group between His55 and the 2Fe-2S cluster, which could modulate the FeSII protein's affinity for nitrogenase in a redox state-dependent manner. Both K14A and K15A mutant FeSII proteins partially lost their ability to protect nitrogenase, but the lysine double mutant lost almost all its protective ability. The nitrogenase component proteins in an Azotobacter strain bearing the double lysine mutation (in the chromosome) were degraded much more rapidly in vivo than those in the wild type strain under carbon substrate-limited conditions. These results indicate that the two lysines may have an important role in FeSII function, perhaps in the initial steps of recognizing the nitrogenase component proteins.