C/EBPα:AP-1 Leucine Zipper Heterodimers Bind Novel DNA Elements, Activate the PU.1 Promoter, and Direct Monocyte Lineage Commitment More Potently Than C/EBPα Homodimers or AP-1.
Authors: Friedman Alan D. , Cai Dong Hong , Wang Dehua , Keefer Jeffrey , Yeamans Christine , Hensley Kortney 
Published: October 2019

Abstract
The basic-region leucine zipper (BR-LZ or bZIP) transcription factors dimerize via their LZ domains to position the adjacent BRs for DNA-binding. Members of the C/EBP, AP-1, and CREB/ATF bZIP subfamilies form homodimeric or heterodimeric complexes with other members of the same subset and bind specific DNA motifs. Here we demonstrate that C/EBPα also zippers with AP-1 proteins and that this interaction allows contact with novel DNA elements and induction of monocyte lineage commitment in myeloid progenitors. A leucine zipper swap:gel shift assay demonstrates that C/EBPα zippers with c-Jun, JunB, or c-Fos, but not with c-Maf or MafB. In addition, C/EBPα co-ips c-Jun, JunB, or c-Fos, either expressed in 293T cells or as endogenous proteins in a myeloid cell line. Affinity of C/EBPα for AP-1 proteins appears weaker in this assay than affinity of C/EBPα for the related C/EBPβ, suggesting that interaction of C/EBP and AP-1 proteins is not primarily cross-inhibitory but instead generates heterodimers capable of binding novel DNA elements and inducing unique biological effects. To evaluate activities of specific homodimers or heterodimers we utilized LZs with acid (LZE) or basic (LZK) residues in their salt bridge positions. This approach is advantageous over efforts to fuse bZIP proteins together as zippering of tethered proteins may be sterically hindered and disruptive to the remainder of the protein. We find that C/EBPαLZE:C/EBPαLZK preferentially binds a C/EBP site, c-JunLZE:c-FosLZK an AP-1 site, and C/EBPαLZE:c-JunLZK a hybrid element identified as 5′-TTGCGTCAT by oligonucleotide selection. In contrast, c-Jun homodimers selected the consensus site 5′-TGACGTCAT, and C/EBPα homodimers selected 5′-NNGNNGCAAC. Note that 5′-TTGC in the selected hybrid site corresponds to 5′-GCAA in the C/EBPα site. In lineage-negative murine myeloid progenitors, either C/EBPα-ER or c-Jun-ER potently induce monocyte as compared to granulocyte development. Using LZE:LZK proteins linked to the ER ligand-binding domain, we find that C/EBPα:c-Jun or C/EBPα:c-Fos LZE:LZK heterodimers induce monocyte lineage commitment with markedly increased potency compared with C/EBPα or c-Jun homodimers or c-Jun:c-Fos heterodimers, demonstrating a positive functional consequence of C/EBP:AP-1 bZIP subfamily interaction. We propose that C/EBPα induction of monopoiesis requires zippering with endogenous c-Jun and that conversely induction of monopoiesis by c-Jun requires zippering with endogenous C/EBPα. Finally, using Ba/F3 lines stably expressing LZE:LZK ER fusion proteins, we find that C/EBPα:c-Jun binds the endogenous PU.1 promoter as assessed by chromatin immunoprecipitation and that exposure of cells expressing C/EBPαLZK-ER and c-JunLZE-ER to estradiol induces PU.1 mRNA expression as assessed by quantitative RT-PCR, whereas C/EBPαLZK-ER in combination with C/EBPαLZE-ER did not induce PU.1 mRNA in Ba/F3 cells. In addition, both endogenous C/EBPα and c-Jun were detected by the ChIP assay in the vicinity of the PU.1 promoter in 32Dcl3 myeloid cells. Thus, induction of PU.1 may contribute to stimulation of monopoiesis by C/EBPα:c-Jun heterodimers. In addition, as C/EBP and AP-1 proteins are widely expressed, zippering between these bZIP subfamilies may have broad biologic relevance. To gain insight into the spectrum of genes activated specifically by this complex, we have initiated microarray studies using our Ba/F3 cell lines.