C/EBP alpha:AP-1 leucine zipper heterodimers bind novel DNA elements, activate the PU.1 promoter and direct monocyte lineage commitment more potently than C/EBP alpha homodimers or AP-1

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/EBPalpha 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/EBPalpha zippers with c-Jun, JunB or c-Fos, but not with c-Maf or MafB. To evaluate activities of specific homodimers or heterodimers we utilized LZs with acid (LZE) or basic (LZK) residues in their salt bridge positions. C/EBPalphaLZE:C/EBPalphaLZK preferentially binds a C/EBP site, c-JunLZE:c-FosLZK an AP-1 site and C/EBPalphaLZE:c-JunLZK a hybrid element identified as TTGCGTCAT by oligonucleotide selection. In murine myeloid progenitors, C/EBPalpha:c-Jun or C/EBPalpha:c-Fos LZE:LZK heterodimers induce monocyte lineage commitment with markedly increased potency compared with C/EBPalpha or c-Jun homodimers or c-Jun:c-Fos heterodimers, demonstrating a positive functional consequence of C/EBP:AP-1 bZIP subfamily interaction. C/EBPalpha:cJun binds and activates the endogenous PU.1 promoter, providing one mechanism for induction of monopoiesis by this complex.

Figure 1

The C/EBP and AP-1 LZs interact to enable DNA-binding. (a) Diagram of the zipper swap:gel shift assay. (b–f) The indicated proteins were co-expressed in 293T cells, and nuclear extracts were prepared and subjected to gel shift assay using the NE-C/EBP probe. * marks bZIP proteins, ** marks full-length proteins, and arrows mark heterodimers. Abbreviations: α-C/EBPα; αbZIP, βbZIP, δbZIP - bZIP domains of C/EBPα, C/EBPβ, or C/EBPδ; αJZ, αFZ,αJB, αMBZ, αCMZ - C/EBPα with c-Jun, c-Fos, JunB, MafB, or c-Maf LZs, L12V C/EBPα mutant L317V/L324V; αGZbZIP the C/EBPα BR linked to the GCN4 LZ.

Figure 2

Interactions between C/EBPα and AP-1 proteins. (a) C/EBPα (α) or its L12V variant were co-expressed with C/EBPβ (β), c-Jun, JunB, or c-Fos in 293T cells. Nuclear extracts were subjected to immunoprecipitation (IP) with the indicated antisera followed by Western blotting with C/EBPα antibody. I – 1% of IP input extract. (b) Nuclear extracts corresponding to 4 × 10⁷ WEHI-3B D+ myeloid cells were subjected to IP with rabbit IgG (Ig) or the indicated antisera, followed by Western blotting for C/EBPα.

Figure 3

Directed heterodimerization using acid and basic LZs. (a) Two α-helices dimerizing via hydrophobic a and d residues, with e and g residues forming salt bridges. (b) Diagram of C/EBPα dimerizing via acidic LZE and basic LZK. (c) Gel shift assay using 293T nuclear expressing C/EBPαLZE (αLZE), C/EBPαLZK (αLKZ), or both (E+K) with the NE-C/EBP probe. (d) Gel shift assay using extracts expressing the indicated combinations of LZE/LZK proteins with four DNA probes, NE-αα(ATTGCGCAAT), NE-αJ(tgacGCAAT), NE-JJ(TGACTCA), and NE-α-(GCAAT). The AP-1 half-site in the hybrid αJ site is shown in lower case. (e) Gel shift assay using extracts expressing the indicated LZE+LZK protein combinations, or 293T extract as a control, with the MS-JJ AP-1 site probe.

Figure 4

C/EBPα:c-Jun or C/EBPα:c-Fos induce monocytic differentiation more potently than C/EBPα or c-Jun homodimers or c-Fos:c-Jun heterodimers. (a) 293T cells were transiently transfected with retroviral plasmids expressing the indicated non-ER or ER fusion LZE or LZK proteins. Total cellular proteins harvested at 48 hrs were subjected to Western blotting with C/EBPα (lanes 1–2), c-Jun (lanes 3–4), JunB (lanes 5–6), c-Fos (lanes 7–8), or ER (lanes 9–14) antisera. Lanes were loaded with equal cell numbers except that JunB-LZE and C/EBPα-LZK were loaded 20-fold less due to their higher expression. (b) Marrow cells isolated from mice exposed to 5-FU were transduced with pBabePuro-C/EBPαLZK-ER (αLZK) and MIGR1 vectors expressing C/EBPαLZE-ER (αLZE), c-JunLZE-ER (cJLZE), or c-FosLZE-ER (FLZE). After three days of transduction and one day of puromycin selection, viable cells were isolated and subjected to lineage-depletion. The cells were then cultured +/− E2 for 2 days in IL-3/IL-6/SCF and subjected to FACS for Mac1 and Gr-1, gating on GFP⁺ cells. Representative data is shown. Cells in the upper left, Mac1⁺Gr-1⁻ quadrant are monocytes, and cells in the upper right, Mac1⁺Gr-1⁺ quadrant are granulocytes. Percent monocytes (%M) = Mac1⁺Gr-1⁻/(Mac1⁺Gr-1⁻ + Mac1⁺Gr-1⁺) cells × 100%. (c) The increase in percent monocytes due to E2 addition upon FACS analysis of marrow cells transduced with the indicated combinations of LZE-ER and LZK-ER proteins or empty vectors. (d) Increase in percent monocytes in marrow cells transduced with pBabePuro-C/EBPα-ER and MIGR1 (α), pBabePuro and MIGR1-cJun-ER (cJ), both pBabePuro-C/EBPα-ER and MIGR1-cJun-ER (α:cJ), or MIGR1-cFos-ER (cF). Data are mean and standard error from three experiments, with p values comparing +/− E2 (left). Expression of ER fusions was verified by Western analysis of transfected 293T cells (right).

Figure 5

C/EBPα:c-Jun or C/EBPα:c-Fos induce monocyte lineage specification more potently than C/EBPα homodimers or c-Fos:c-Jun. (a, b) The increase in percent CFU-M due to E2 exposure for 24 hrs just prior to culture of doubly transduced, GFP⁺ and puromycin-resistant cells in methylcellulose with IL-3/IL-6/SCF or with G-CSF/SCF is shown. %CFU-M = CFU-M/(CFU-M + CFU-G) × 100%. Data are mean and standard error from three experiments.

Figure 6

C/EBPα:cJun binds a hybrid DNA element and the PU.1 promoter. (a) Sequences of individual DNAs selected, and their consensus, after immunoprecipitation of complexes bound to either C/EBPαLZK:cJunLZE, c-Jun, or C/EBPα. Bold nucleotide symbols match the consensus. Lower case letters indicate the second most frequent base. (b) Gel shift assay using 293T extracts expressing no exogenous protein (−), C/EBPαLZK:C/EBPαLZK (α:α), C/EBPαLZK:cJunLZE (α:cJ), or C/EBPαLZK:cFosLZE (α:cF) and either a radiolabeled probe containing the PU.1 promoter −68 bp C/EBP site with 6 bp of 5′ and 3′ flanking sequences (PU.1–68) or the same probe with the flanking sequences replaced with guanines (G flank). (c) Gel shift using these proteins and C/EBPαLZK:JunBLZE (α:JB) and probes containing either the C2 or C5 C/EBPα-binding sites in the PU.1 distal enhancer. (d) ChIP assay using 32Dcl3 cells cultured in IL-3 (top panel) or in G-CSF for one day (panels 2–6) and either rabbit Ig, C/EBPα, C/EBPβ, or c-Jun antisera. I input. PCR was done using oligonucleotides derived from the PU.1 promoter (−215 to +25), the PU.1 distal enhancer, the NE promoter (−157 to +92), the βactin gene, or −7 kb of the PU.1 gene. Results shown are representative of two independent experiments.

Figure 7

C/EBPα:cJun binds and activates the endogenous PU.1 promoter. (a) Western blot analysis of parental Ba/F3 cells or subclones expressing the indicated LZK:LZE ER fusion protein combinations, using C/EBPα, c-Jun, c-Fos, or actin antisera (top to bottom). (b) ChIP assay using the Ba/F3 lines and Ig or ER antisera. PCR was done for the PU.1 promoter, the β-actin gene, or −7 kb of the PU.1 gene. Results +E2 (bottom three panels) are representative of two independent experiments, and results −E2 (top panel) are from one experiment. (c) RNA prepared from the Ba/F3 lines cultured with or without E2 for 6 hrs was subjected to real time PCR for PU.1 mRNA, with GAPDH mRNA PCR used for normalization. Mean and standard deviation from three determinations is shown. P value for PU.1 expression with versus without E2 is from the paired student’s t test.