FUNCTIONAL-CHARACTERIZATION OF ETV6 AND ETV6 CBFA2 IN THE REGULATION OF THE MCSFR PROXIMAL PROMOTER/

The ETV6/CBFA2 (TEL/AML1) fusion gene occurs as a result of the chromo some translocation t(12;21)(p13;q22) in up to 30% of children diagnose d with B cell precursor (cd10(+), cd19(+)) acute lymphoblastic leukemi a. Leukemic cells that have acquired the t(12;21) usually demonstrate loss of the remaining normal ETV6 (TEL) allele, Using reporter gene as says we have functionally characterized both the normal ETV6 and ETV6/ CBFA2 fusion proteins in the regulation of the MCSFR proximal promoter , Neither ETV6 or ETV6/CBFA2 has any significant, detectable effect on the promoter by itself, However, both ETV6 and ETV6/ CBFA2 inhibit th e activation of the promoter by CBFA2B(AML1B) and C/EEPa. We have show n that a 29-bp region of the MCSFR promoter containing the binding sit es for CBFA2B and C/EBPa is sufficient for the inhibition by ETV6 and ETV6/CBFA2. Mutational analysis of the MCSFR promoter revealed that bi nding of both CBFA2B and C/EBPa to their respective sites is necessary for the inhibition by ETV6 and ETV6/CBFA2. Deletion of the helix-loop -helix (HLH) region from the cDNAs of ETV6 and ETV6/CBFA2 decreased bu t did not completely abrogate the ability of either construct to inhib it promoter activation, We also found that the ETS DNA binding region of ETV6 is necessary for inhibition of the promoter, Addition of ETS1 and FLI1, two ETS family members that have homology in the 5' HLH regi on, but not Spi1, an ETS family member without the 5' HLH region, also inhibited reporter gene expression, Our data show that the inhibition mediated by ETV6 and ETV6/CBFA2, in the context of the MCSFR promoter , depend on interactions with other proteins, not just CBFA2B. Our res ults also indicate that the transactivation characteristics of ETV6/CB FA2 are a combination of positive and negative regulatory properties.