AP-1 protein induction during monopoiesis favors C/EBP: AP-1 heterodimers over C/EBP homodimerization and stimulates FosB transcription

Abstract

AP-1 proteins heterodimerize via their LZ domains to bind TGACGTCA or TGACTCA, whereas C/EBPs dimerize to bind ATTGCGCAAT. We demonstrate that intact C/EBPα also heterodimerizes with c-Jun or c-Fos to bind a hybrid DNA element, TGACGCAA, or more weakly to TGATGCAA. A 2:1 ratio of c-Jun:C/EBPα or c-Fos:C/EBPα was sufficient for preferential binding. Semiquantitative Western blot analysis indicates that the summation of c-Jun, JunB, and c-Fos levels in differentiating myeloid cells is similar to or exceeds the entirety of C/EBPα and C/EBPβ, indicating the feasibility of heterodimer formation. Induction of AP-1 proteins during monocytic differentiation favored formation of C/EBP:AP-1 heterodimers, with C/EBPα homodimers more evident during granulopoiesis. Approximately 350 human and 300 murine genes contain the TGACGCAA motif between -2 kb and +1 kb of their transcription start sites. We focused on the murine Fosb promoter, which contains a C/EBP:AP-1 cis element at -56 and -253, with the hFOSB gene containing an identical site at -253 and a 1-bp mismatch at -56. C/EBPα:AP-1 heterodimers bound either site preferentially in a gel-shift assay, C/EBPα:c-Fos ER fusion proteins induced endogenous Fosb mRNA but not in the presence of CHX, C/EBP and AP-1 proteins bound the endogenous Fosb promoter, mutation of the -56 cis element reduced reporter activity fivefold, and endogenous FosB protein was expressed preferentially during monopoiesis versus granulopoiesis. Increased expression of Jun/Fos proteins elevates C/EBP:AP-1 heterodimer formation to potentially activate novel sets of genes during monopoiesis and potentially during other biologic processes.

Figure 1.. C/EBPα zippers with c-Jun or c-Fos when expressed by IVT.

(A) Equal volumes of IVT reaction programmed to encode no protein or Myc-tagged C/EBPα, c-Jun, or c-Fos were subjected to Western blotting (WB) using Myc antibody. (B) Gel-shift assay was carried out using 1 ng of the indicated αα- or αJ-radiolabeled oligonucleotide probes and the indicated C/EBPα, C/EBPα + c-Jun (1:8 mol ratio), or C/EBPα + c-Fos (1:2 mol ratio) protein combinations (upper). retic, Reticulocyte lysate. Specificity was confirmed by competition with 0, 10, or 50 ng unlabeled αα or αJ probe (lower). (C) Gel-shift/super-shift assays were carried out using the αα or αJ probes, C/EBPα + c-Jun (1:8 mol ratio), and 200 ng C/EBPα or c-Jun antiserum. An arrow indicates the super-shift species (upper). Lack of super-shift by normal rabbit Ig was confirmed as a control (lower).

Figure 2.. C/EBPα zippers with c-Jun or c-Fos when expressed in mammalian cells.

(A) Nuclear extracts (12 μg) prepared from 293T cells transiently transfected with CMV vectors encoding no protein (–) or Myc-tagged C/EBPα, c-Jun, or c-Fos were subjected to Western blotting using Myc antibody. (B) Gel-shift/super-shift assay was carried out using 1 ng of the indicated αα- or αJ-radiolabeled oligonucleotide probes labeled to similar specific activity, the indicated C/EBPα, C/EBPα + c-Jun (1:8 mol ratio), C/EBPα + c-Fos (1:8 mol ratio) protein combinations, and 200 ng of the indicated C/EBPα, c-Jun, or c-Fos antiserum or rabbit Ig. An arrow indicates the super-shift species.

Figure 3.. Formation of a DNA-binding C/EBPα:c-Jun complex at different mol ratios.

Gel-shift assay was carried out using the αα or αJ oligonucleotide-radiolabeled probes and IVT C/EBPα + c-Jun at the indicated mol ratios. The specific gel-shift complex is indicated (*).

Figure 4.. Semiquantitative analysis of C/EBP and AP-1 family protein levels in myeloid cells.

(A) Increasing volumes (ratio 1:2:4:8) of IVT Myc-tagged C/EBPα, C/EBPβ, c-Jun, JunB, or c-Fos were subjected to Western blotting with protein-specific antisera (upper panels) or Myc antibody (lower panels). The upper or lower sets of blots were exposed to the same secondary antibody solution for an identical time period and were subjected simultaneously to the chemiluminescence reagents and autoradiography. Relative antibody affinities were estimated by densitometric band analysis, correcting the intensities obtained with the specific antisera using the relative protein expression, as determined using the Myc antibody. (B) Nuclear extracts (2.5, 5.0, or 10.0 μg; ratio 1:2:4) from HF-1 cells cultured with G-CSF for 24 h or HL-60 cells cultured with PMA for 6 h were subjected to Western blotting with the indicated antisera. For each cell line, the series of blots were exposed to the same secondary antibody solution for an identical time period and were subjected simultaneously to the chemiluminescence reagents and autoradiography. Protein expression ratios, setting the C/EBPα (α) level to 1 in each cell line, were estimated from densitometric analysis, correcting intensities using the relative antibody affinities. βL and βS are the long and short C/EBPβ isoforms, LAP and LIP.

Figure 5.. Gel-shift analysis of proteins in myeloid extracts using C/EBP, AP-1, or hybrid sites.

(A) Nuclear extracts (12 μg) from HF-1 cells cultured in G-CSF for 1 day were subject to gel-shift/super-shift assay using the αα- or αJ-radiolabeled probes. Antisera used were normal rabbit Ig (Ig), C/EBPα (α), C/EBPβ (β), C/EBPε (ε), c-Jun (cJ), JunB (JB), or c-Fos (cF). Arrows indicate super-shift complexes. Positions of C/EBPα:C/EBPα and C/EBPβ:C/EBPβ gel-shift complexes are also indicated (α and β). (B) Nuclear extracts (12 μg) from HL-60 cells cultured in PMA for 6 h were subjected similarly to gel-shift/super-shift assay using the αα-, αJ-, and JJ-radiolabeled probes.

Figure 6.. Oligonucleotide pull-down analysis of proteins in myeloid extracts using C/EBP, AP-1, or hybrid sites.

(A) Nuclear extracts prepared from HF-1 cells cultured with G-CSF (G) for 24 h or from noninduced HF-1 cells (NI) were incubated with biotinylated αα, αJ, or JJ oligonucleotides, followed by streptavidin beads, washed, and subjected to Western blotting for the indicated proteins. (B) Similar analysis was conducted using nuclear extracts from HL-60 cells cultured with PMA for 6 h or from noninduced HL-60 cells. Input samples represent 10% of the extract used for oligonucleotide pull-down.

Figure 7.. Regulation of the FosB promoter by C/EBP:AP-1 heterodimers.

(A) Gel-shift assay was conducted with the indicated radiolabeled probes with IVT, myc-tagged proteins C/EBPα (α) and eightfold c-Jun (J) on a per-mol basis, or their combination at a 1:8 mol ratio (αJ). Where indicated, super-shift was conducted by further addition of C/EBPα or c-Jun antisera. Arrows indicate super-shift complexes. (B) HL-60 cells cultured in PMA for 1 day were subjected to ChIP assay using rabbit IgG or C/EBPα, C/EBPβ, c-Jun JunB, and c-Fos antiserum. Shown is the signal obtained, expressed as percent input using primers centered on the FosB –150-bp promoter region or on a more distal –5-kb control region (mean and se). *P < 0.05 versus Ig control. (C) FosB RNA levels were evaluated in the Ba/F3 cell lines expressing C/EBPαLZK-ER (αLZK) together with empty vector (–), C/EBPαLZE-ER (αLZE), c-JunLZE-ER (cJLZE), or c-FosLZE-ER (cFLZE) 24 h after culture in the presence or absence of E2, with triplicate data expressed as an induction ratio (+E2/–E2; left, mean and se). *P < 0.05 versus empty vector cells. Induction of FosB RNA was then assessed similarly at 8 h using cells expressing C/EBPαLZK-ER and c-FosLZE-ER with no addition (–), CHX, E2, or both (E2/CHX). CHX was added 30 min prior to E2 (right, mean and se). *P < 0.05 for E2 versus no addition; #P < 0.05 for E2 versus E2/CHX. (D) FosB(–497/+1)-Luc (5 μg) or the indicated mutant variants were transiently transfected together with CMV-β-galactosidase (0.5 μg) into 5 × 10⁶ 32Dcl3 cells in IL-3, and luciferase and β-galactosidase assays were conducted 48 h later. FosB promoter activity relative to WT (set to 1.00) is shown (mean and se from three experiments). *P < 0.05 versus WT. (E) Total cellular proteins from 32Dcl3 cells cultured in G-CSF for 0–3 days, M1 cells cultured in IL-6 for 0–3 days, HL-60 cells cultured in PMA for 0–2 days, or M1 or HL-60 cells exposed to bzATP for 5 min were subjected to Western blotting for FosB and β-actin. *Locations of FosB and ΔFosB induced in the monocytic lines.