Abstract
The DNA-binding ETS transcription factor Spi-1/PU.1 is of central importance in determining the myeloid-erythroid developmental switch and is required for monocyte and osteoclast differentiation. Many monocyte genes are dependent upon this factor, including the gene that codes for interleukin-1beta. It has long been known that the conserved ETS DNA-binding domain of Spi-1/PU.1 functionally cooperates via direct association with a diverse collection of DNA-binding proteins, including members of the basic leucine zipper domain (bZIP) family. However, the molecular basis for this interaction has long been elusive. Using a combination of approaches, we have mapped a single residue on the surface of the ETS domain critical for protein tethering by the C/EBPbeta carboxyl-terminal bZIP domain. This residue is also important for nuclear localization and DNA binding. In addition, dependence upon the leucine zipper suggests a novel mode for both protein-DNA interaction and functional cooperativity.
Highlights
The ETS family of transcription factors serves a variety of roles in development and differentiation [1]
Relative binding of these two factors at the upstream site is reversed relative to the downstream site and may play an especially critical role in serving to tether Spi-1 to C/EBP bound at a site in the IL1B upstream induction sequence (UIS) enhancer [19], based upon a greater loss of general enhancer function when the upstream site is deleted from the promoter [7]
These residues are well recognized for their role in DNA binding, our data support other functions relating to nuclear localization and protein tethering
Summary
The ETS family of transcription factors serves a variety of roles in development and differentiation [1]. C/EBP DNA binding at this site appears to be supported by a protein interaction with the adjacent DNA-binding domain of Spi-1 [18] Relative binding of these two factors at the upstream site is reversed relative to the downstream site and may play an especially critical role in serving to tether Spi-1 to C/EBP bound at a site (located between Ϫ2768 and Ϫ2761) in the IL1B upstream induction sequence (UIS) enhancer [19], based upon a greater loss of general enhancer function when the upstream site is deleted from the promoter [7]. While investigating the nature of the Spi-1-C/EBP interaction, we uncovered new functions for the highly conserved arginines of helix 3 (Arg232 and Arg235) within the DNA recognition site These functions expand upon the classic role these residues play in DNA binding and suggest new mechanisms of functional cooperativity and protein-DNA interaction for transcription factors
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