The GATA-2 transcription factor is essential for early haematopoietic development, and is highly expressed in the early progenitor cells of various blood-cell lineages. It binds to DNA at GATA sites using primarily the second of two adjacent zinc-finger domains (the C-finger; CF). Recently, mutations in this CF region have been linked to a hereditary form of myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML)¹ . Preliminary evidence suggested that the mutations are able to disrupt the DNA-binding activity of the GATA2 protein.

We have cloned, expressed and purified wild-type and mutant human GATA2-CF constructs, and determined through circular dichroism spectroscopy and 1D H¹ NMR that they are all well folded. We have examined their DNA-binding activity for two 29-mer sequences derived from known GATA2-responsive promoters, containing a single or a double GATA site. Gel-shift assays revealed some small differences in affinity between the wild-type and two of the mutant constructs, but did not indicate any differences in binding between the two oligos examined, suggesting that only one of the GATA sites was being utilized in the double-site oligo. Isothermal titration calorimetry (ITC) conducted using the single-site oligo demonstrated a tight binding event with one-to-one stoichiometry, but fitting of the data was difficult due to the presence of a second, weak binding event that is probably the result of non-specific interaction between the DNA and the GATA2-CF protein. However, we were able to confirm that one of the mutants did indeed have a lower DNA-binding affinity. ITC conducted using the wildtype protein and the double-site oligo displayed multiple binding events, suggesting that in solution both GATA sites were being bound. These results confirm that the mutations do have some affect on the DNA-binding activity of GATA2 which could easily disrupt normal GATA2 functions.