C/EBPbetaDeltauORF mice--a genetic model for uORF-mediated translational control in mammals

Abstract

Upstream ORFs (uORFs) are translational control elements found predominantly in transcripts of key regulatory genes. No mammalian genetic model exists to experimentally validate the physiological relevance of uORF-regulated translation initiation. We report that mice deficient for the CCAAT/enhancer-binding protein beta (C/EBPbeta) uORF initiation codon fail to initiate translation of the autoantagonistic LIP (liver inhibitory protein) C/EBPbeta isoform. C/EBPbeta(DeltauORF) mice show hyperactivation of acute-phase response genes, persistent repression of E2F-regulated genes, delayed and blunted S-phase entry of hepatocytes after partial hepatectomy, and impaired osteoclast differentiation. These data and the widespread prevalence of uORFs in mammalian transcriptomes suggest a comprehensive role of uORF-regulated translation in (patho)physiology.

Figure 1.

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.

Figure 2.

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.

Figure 3.

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.

Figure 4.

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.

Figure 5.

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.