C/EBP and AP-1 Proteins Form Leucine Zipper Heterodimers and Reduce C/EBP Homodimer Formation During Monopoiesis.

Abstract 2606

C/EBP and AP-1 proteins represent two sub-families of the basic region-leucine zipper (BR-LZ or bZIP) family of transcription factors. C/EBPs, including C/EBPα, C/EBPβ, C/EBPδ, and C/EBPε, homodimerize or heterodimerize via their leucine zipper domains to bind DNA at palindromic DNA element 5'-ATTGCGCAAT or related sequences. The AP-1 family consists of several Jun and Fos proteins which heterodimerize to bind the palindromic DNA element 5'-TGACGTCA. We previously utilized directed dimerization via artificial acidic and basic zippers to show that C/EBPα:AP-1 heterodimers bind novel DNA elements and stimulate monocytic differentiation upon transduction into murine marrow cells (Cai et al 2008). We now demonstrate that intact C/EBPα and c-Jun have the capacity to heterodimerize and preferentially bind a hybrid DNA site, 5'-TGACGCAAT. Using in vitro translated, myc-tagged proteins to control input ratios based on myc Western blotting, a 2:1 ratio of c-Jun:C/EBPα was found to be sufficient for preferential binding to the hybrid element in a gel shift/super-shift assay. As a control, we demonstrate that C/EBPαGZ, containing the GCN4 leucine zipper in place of the C/EBPα zipper, does not heterodimerize with excess c-Jun to bind the hybrid element. We next sought to determine whether a 2:1 AP-1:C/EBP ratio might be achievable by proteins expressed endogenously in myeloid cells. The relative affinities of several C/EBP or AP-1 antisera were determined using myc-tagged proteins whose expression ratios could be normalized using myc antisera. Semi-quantitative Western blot analysis of 32Dcl3 or HF-1 cells undergoing granulocytic differentiation in response to G-CSF or of HL-60 or M1 cells undergoing monocytic differentiation in response to phorbol ester or IL-6, respectively, indicated that the summation of c-Jun, JunB, and c-Fos levels approximately equals the sum of C/EBPα and C/EBPβ, demonstrating the feasibility of endogenous C/EBP:AP-1 heterodimer formation. Myeloid nuclear extracts were then evaluated for C/EBP, C/EBP:AP-1, and AP-1 complexes using biotinylated oligonucleotides. Myeloid cell extracts were incubated with biotinylated DNA probes containing C/EBPα, AP-1, or hybrid cis elements, pulled down using streptavidin beads, washed, eluted, and subjected to Western blotting for bound proteins. Induction of AP-1 proteins during monopoiesis favored binding of C/EBP:AP-1 heterodimers, whereas C/EBPα homodimers were more evident during granulocytic differentiation. Global analysis of genes bound and activated by C/EBP:AP-1 complexes is in progress, and we demonstrate that the C/EBPα:c-Jun heterodimer binds the element 5'-TAGCGCAAG in the PU.1 promoter with increased affinity compared with C/EBPα homodimers. In summary, C/EBP:AP-1 leucine zipper complexes readily form when in vitro translated AP-1 proteins are in modest excess to C/EBPα, endogenous AP-1 proteins are expressed at concentrations similar to endogenous C/EBP proteins making heterodimerization feasible, C/EBP:AP-1 complexes are detected in myeloid nuclear extracts, and increased expression of Jun/Fos proteins in response to M-CSF or other external cues is predicted to reduce C/EBP homodimer and increase C/EBP:AP-1 heterodimer formation to favor monopoiesis over granulopoiesis via activation of genes harboring hybrid C/EBP:AP-1 cis elements. Formation of C/EBP:AP-1 complexes is likely also relevant to additional cell lineages and biologic processes.