Genetic ablation of cis-regulatory translational control by the C/EBPβ uORF. (A) Three protein isoforms (LAP* [38 kDa], LAP [35 kDa], and LIP [20 kDa]) are translated from consecutive in-frame initiation codons in the same transcript (Descombes and Schibler 1991). The C/EBPβ mRNA contains a conserved cis-regulatory small uORF (30 base pairs [bp], orange) terminating 4 bp upstream of the LAP initiation site in a different reading frame. (bd) Binding; (pA) poly(A) tail. (B) Translation of the uORF serves to strip ribosomes from their initiating Met-tRNA_i^Met (green to white) and prevents initiation at the proximate LAP initiation codon. Upon reloading of ribosomes with the ternary eIF2–GTP–Met-tRNA_i^Met complex (white to green), translation reinitiation from the downstream AUG codon generates LIP. In C/EBPβ^ΔuORF mice, an A-to-U point mutation was designed to abrogate ribosomal initiation at the uORF start codon without changing the amino acid sequence of the C/EBPβ isoforms. Most ribosomes will thus initiate at the LAP AUG instead. (Display of LAP* translation was omitted for simplicity. For details on alternative start site selection, see Supplemental Fig. S1.) (C) Upon i.p. injection of LPS, LIP is strongly induced in C/EBPβ^WT (WT) but not in C/EBPβ^ΔuORF livers (Δ). (h) Hours of LPS treatment; (α-tub.) α-tubulin; (k.o.) lysate of C/EBPβ knockout mouse. (D) In MEFs, LPS induces LIP expression in C/EBPβ^WT but not in C/EBPβ^ΔuORF cells. (E) Representative luciferase reporter assay (n = 3) demonstrating increased luciferase reporter activity (luc.) in C/EBPβ^ΔuORF (open triangles) as compared with C/EBPβ^WT (black squares) MEFs at indicated times after LPS treatment. Error bars show SEM.

The C/EBPβ^ΔuORF mutation impairs osteoclast differentiation. (A) Tibia sections showing tartrate-resistant acid phosphatase (TRACP)-stained osteoclasts (red staining, light-green counterstain) in C/EBPβ^ΔuORF as compared with C/EBPβ^WT mice. (Arrowheads) Multinucleated osteoclasts; bars, 50 μm. The bar graph displays average osteoclast sizes as determined from six mice per genotype at 8 wk of age. (B) TRACP staining (red) showing osteoclast differentiation of bone marrow-derived precursors of C/EBPβ^ΔuORF and C/EBPβ^WT mice after 6 d in culture with M-CSF and RANK-L (n = 6). (Arrowheads) Multinucleated osteoclasts. The bar graph displays the differential quantification of osteoclasts by the number of nuclei (n) per cell. (C) Immunoblot analysis showing LIP expression in C/EBPβ^WT but not in C/EBPβ^ΔuORF osteoclasts at day 2 of culture. (D) Immunoblot analysis showing increased MafB protein in C/EBPβ^ΔuORF as compared with C/EBPβ^WT osteoclasts. (E) Real-time PCR analysis showing decreased expression of MafB-regulated osteoclast markers in C/EBPβ^ΔuORF (open bars) as compared with C/EBPβ^WT (black bars) osteoclasts. Normalized to Gapdh (glyceraldehyde-3-phosphate-dehydrogenase) and presented relative to C/EBPβ^WT (set to 1, dashed line). Error bars show SEM; (*) P < 0.05; (***) P < 0.001.

The C/EBPβ^ΔuORF mutation causes superinduction of C/EBPβ target genes. (A) Induction of LIP in C/EBPβ^WT livers upon PH is abolished in C/EBPβ^ΔuORF animals. (B) ELISA showing elevated average levels of serum IL-6 at 3 h and 6 h after PH in C/EBPβ^ΔuORF (open triangles) as compared with C/EBPβ^WT (black squares) animals (n = 6; [**] P < 0.01). (C) Real-time PCR analysis demonstrating elevated mRNA contents of acute-phase response genes in C/EBPβ^ΔuORF (open bars) as compared with C/EBPβ^WT (black bars) livers at indicated times after PH (n = 6; [*] P < 0.05; [**] P < 0.01). Error bars show SEM.

Cell proliferation defect in C/EBPβ^ΔuORF mice. (A) In vitro proliferation assay demonstrating reduced expansion of C/EBPβ^ΔuORF (open triangles) as compared with C/EBPβ^WT (black squares) MEF cultures (n = 5 independent embryos per genotype; [**] P < 0.01). (B) Quantification of BrdU-labeled hepatocyte nuclei (2-h pulse-labeling) in liver sections showing a reduced proportion of hepatocytes in S phase in C/EBPβ^ΔuORF (open bars) as compared with C/EBPβ^WT (black bars) and C/EBPβ^LIP (gray bars) livers at 36 and 48 h after PH (n = 8, [***] P < 0.001; n = 7, [*] P < 0.05 vs. wild type, respectively). (C) BrdU immunofluorescence stainings of C/EBPβ^WT, C/EBPβ^ΔuORF, and C/EBPβ^LIP liver sections 36 h after PH. Bars, 100 μm. (D) Real-time PCR analysis showing reduced mRNA contents of CcnA1, CcnA2, CcnB1, CcnE1, CcnE2, and Pcna in C/EBPβ^ΔuORF (open bars) as compared with C/EBPβ^WT (black bars) andC/EBPβ^LIP (gray bars) livers at indicated times after PH. (n = 6, [*] P < 0.05, [**] P < 0.01 vs. wild type). (n.d.) Not determined. Error bars show SEM.

The C/EBPβ^ΔuORF mutation causes repression of E2F target genes. (A) Graphic representation of a genome-wide microarray expression analysis comparing transcript levels in C/EBPβ^WT and C/EBPβ^ΔuORF liver at 36 h after PH. (B) Representative ChIP assay on C/EBPβ^WT liver chromatin showing the association of E2F3, C/EBPα, and C/EBPβ to indicated gene promoters in regenerating liver at 36 h after PH (n = 2). (C) Luciferase reporter assay demonstrating the repressive function of long (black bars), but not of truncated (open bars) C/EBPα and C/EBPβ isoforms on the pGL3TATAbasic-6xE2F reporter construct (n = 3). (luc) Luciferase activity; (p42 and p30) long and truncated C/EBPα isoforms. (D) Luciferase reporter assay with constant, intermediately repressive C/EBPα p42 expression (luciferase activity set to 0.5) showing the corepressive function of LAP* and LAP (black bars) and the derepressive function of LIP (open bars) on the same E2F-responsive reporter construct as used in C (n = 3). Error bars show SEM.