Abstract
Ikaros family transcription factors play important roles in the control of hematopoiesis. Family members are predicted to contain up to six classic zinc fingers that are arranged into N- and C-terminal domains. The N-terminal domain is responsible for site-specific DNA binding, whereas the C-terminal domain primarily mediates the homo- and hetero-oligomerization between family members. Although the mechanisms of action of these proteins are not completely understood, the zinc finger domains are known to play a central role. In the current study, we have sought to understand the physical and functional properties of these domains, in particular the C-terminal domain. We show that the N-terminal domain from Eos, and not its C-terminal region, is required to recognize GGGA consensus sequences. Surprisingly, in contrast to the behavior exhibited by Ikaros, the C-terminal domain of Eos inhibits the DNA-binding activity of the full-length protein. In addition, we have used a range of biophysical techniques to demonstrate that the C-terminal domain of Eos mediates the formation of complexes that consist of nine or ten molecules. These results constitute the first direct demonstration that Ikaros family proteins can form higher order complexes in solution, and we discuss this unexpected result in the context of what is currently known about the family members and their possible mechanism of action.
Highlights
Most members of the Ikaros family of transcriptional regulators, including Ikaros [1], Aiolos [2], and Helios [3, 4], are expressed primarily in the hematopoietic system and are essential for normal hemolymphopoiesis [1, 5, 6]
These sequences conform to a similar consensus to that obtained for Ikaros, namely GGGA. These results demonstrate that Eos has the highest affinity for DNA sequences with this core motif and is consistent with the fact that the N-terminal zinc finger (ZnF) domain of Eos is highly homologous to that of Ikaros
MBP1⁄7EosC (49.6 kDa) did not elute in the excluded volume, but rather eluted as a single species slightly before a ferritin standard (440 kDa). This result suggests that Eos was chosen. This polypeptide (EosC) mediates the formation of a complex that consists of approximately eight molecules, because maltose-binding protein (MBP) elutes as a monomer in gel filtration chromatography (GFC)
Summary
Subcloning and Production of Proteins Used in DNA Binding Studies—For binding site selection experiments, the DNA-binding domain of Eos (EosN) was subcloned into pGEX-2T (Amersham Biosciences) to. Cells were re-centrifuged, and the nuclei were resuspended in 30 l of Solution C (20 mM HEPES, pH 7.8, 25% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM DTT, 50 ng/ml PMSF, 5 g/ml leupeptin, and 5 g/ml aprotinin). MBP fusion proteins were either further purified by gel filtration chromatography (GFC) using a Superose-12 fast-protein liquid chromatography column (Amersham Biosciences, 0.8 ml/min, 4 °C) run in 50 mM Na2HPO4, 200 mM NaCl, 1 mM dithiothreitol (DTT) or cleaved in solution by the addition of thrombin (4 °C, 2 h). Based on an attempt to fit the sizes of the sample particles to a monomodal (one particle size) distribution, the polydispersity gave an indication of the homogeneity of particle sizes in the sample
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