Nucleolar retention of a translational C/EBPalpha isoform stimulates rDNA transcription and cell size

Abstract

The messenger RNA of the intronless CEBPA gene is translated into distinct protein isoforms through the usage of consecutive translation initiation sites. These translational isoforms have distinct functions in the regulation of differentiation and proliferation due to the presence of different N-terminal sequences. Here, we describe the function of an N-terminally extended protein isoform of CCAAT enhancer-binding protein alpha (C/EBPalpha) that is translated from an alternative non-AUG initiation codon. We show that a basic amino-acid motif within its N-terminus is required for nucleolar retention and for interaction with nucleophosmin (NPM). In the nucleoli, extended-C/EBPalpha occupies the ribosomal DNA (rDNA) promoter and associates with the Pol I-specific factors upstream-binding factor 1 (UBF-1) and SL1 to stimulate rRNA synthesis. Furthermore, during differentiation of HL-60 cells, endogenous expression of extended-C/EBPalpha is lost concomitantly with nucleolar C/EBPalpha immunostaining probably reflecting the reduced requirement for ribosome biogenesis in differentiated cells. Finally, overexpression of extended-C/EBPalpha induces an increase in cell size. Altogether, our results suggest that control of rRNA synthesis is a novel function of C/EBPalpha adding to its role as key regulator of cell growth and proliferation.

Figure 1

The extended-C/EBPα isoform is translated from a non-AUG codon. (A) Schematic representation of the C/EBPα messenger RNA with three alternative translation initiation sites, indicated as A (CUG or GUG), B (AUG) and C (AUG), and the initiation site for the cis-regulatory uORF, indicated as D (AUG), respectively. The mRNA can be translated into three separate protein isoforms extended- (Extα), full-length- (Flα) or truncated-C/EBPα (Trα) that contain different sets of the functional domains TAD (Transactivation Domain), BD (Basic Domain) and LZ (Leucine Zipper). (B) Amino-acid sequence comparison of the extended domains of rat, mouse and human C/EBPα; conserved amino-acid residues are shaded in grey. (C) Immunoblots comparing expression from wild-type and mutant rat (rC/EBPα) and human (hC/EBPα) C/EBPα constructs in COS-1 cells: Wild-types (wt), mutants lacking the translation initiation site A (ΔA^1/2), constructs devoid of the 5′leader (Δ5′), and constructs with the CUG or GUG exchanged by an AUG codon (A^opt).

Figure 2

Nucleolar localization of the extended-C/EBPα isoform. (A) Subcellular localization of extended- (Extα), full-length- (Flα) or truncated- (Trα) C/EBPα proteins fused to EGFP after transient transfection in C33A cells. DNA was stained with DAPI to visualize the nucleus and immunostaining with the Fibrillarin-specific antibody served to locate the nucleolus. (B) Alignment of the RRXR motifs within the domains mediating nucleolar localization of extended-C/EBPα and other proteins with known nucleolar localization. (C) Subcellular localization of the isolated extended domain of C/EBPα either harbouring the wt sequence (Extα-Dom) or a mutation of its RRQR motive (Extα-DomΔNoLS) fused to EGFP. Bars, 10 μm.

Figure 3

Pseudo-phosphorylation of serine 299 in extended-C/EBPα stimulates its nucleolar retention. (A) Subcellular distribution of S299D mutants of extended- (Extα^S299D), extended ΔNoLS (Extα^S299DΔNoLS), full-length- (Flα^S299D) or truncated-C/EBPα (Trα^S299D) proteins fused to EGFP. (B) Nucleolar retention of extended-C/EBPα^S299D in HeLa, COS-1, Hek293T, MEF and U2OS cells. C/EBPα^S299D-EGFP constructs were transiently transfected into the different cell lines and subcellular localization was analysed as described in Figure 2. Bars, 10 μm.

Figure 4

C/EBPα isoform expression and subcellular localization on phorbolester (TPA)-induced monocytic differentiation of HL-60 cells. (A) Immunoblot showing endogenous expression of the extended- (Extα), full-length- (Flα) and truncated-C/EBPα (Trα) hC/EBPα isoforms in untreated proliferating HL-60 cells and after treatment with 200 nM phorbolester (TPA) for 12 h. α-tubulin staining in the lower blot serves as loading control. (B) Immunostainings of TPA-treated cells (+TPA) and control cells (Solvent) were performed with an anti-C/EBPα antibody against a C-terminal epitope (14AA). DNA was stained with DAPI to visualize the nucleus and immunostaining with a Fibrillarin-specific antibody served to located the nucleolus. Bars, 10 μm.

Figure 5

Extended-C/EBPα interacts with UBF-1, SL1 and NPM. Immunoprecipitations (IPs) were performed with specific antibodies or with mouse IgG as control from total lysates of HEK293 cells expressing one of the C/EBPα isoforms encoded: wild type (wt) extended-C/EBPα (Extα or Ext), extended-C/EBPα^S299D (SD), extended-C/EBPα^S299DΔNoLS (ΔN), full-length-C/EBPα (Fl), truncated-C/EBPα (Tr). Immunoblots of immunoprecipitates (IP) and total lysates (Input) were stained as indicated. (A) Extended-C/EBPα co-precipitates with UBF-1 using anti-UBF-1 antibodies (B) extended- and full-length-C/EBPα but not truncated-C/EBPα co-precipitate with UBF-1 using anti-UBF-1 antibodies. Immunoglobulin light chain is marked by IgGL. (C) TBP co-precipitates with extended-C/EBPα using anti-C/EBPα antibodies. Immunoglobulin heavy chain is marked by IgGH. (D) Extended-C/EBPα^S299D but not the ΔNoLS mutation co-precipitates with TAF_I48 using anti-TAF_I48 antibodies (upper panel). TAF_I48 co-precipitates more efficiently with TBP using anti-TBP antibodies only in cells expressing extended-C/EBPα^S299D (lower panel). (E) NPM co-precipitates with extended-C/EBPα^S299D but not with the extended-C/EBPα^S299DΔNoLS using anti-C/EBPα antibodies.

Figure 6

Nucleolar retention of extended-C/EBPα depends on ongoing rDNA synthesis. Blocking RNA polymerase I activity with actinomycin D (50 ng/ml) abrogates nucleolar retention of extended-C/EBPα^S299D, whereas blocking RNA polymerase II with α-amanitin (50 μg/ml) or solvent treatment has no effect. C33A cells transiently transfected with extended-C/EBPα^S299D-EGFP were treated for 2 h as indicated and subcellular localization was analysed as described in Figure 2. Bars, 10 μm.

Figure 7

Extended-C/EBPα stimulates RNA Pol I-dependent rDNA transcription. (A) Hek293T cells were transiently transfected with the human rRNA minigene reporter construct pHrP2-BH together with expression constructs for wild type extended-C/EBPα (wt), the extended-C/EBPα^S299D (S²⁹⁹D) mutant, the latter fused to EGFP (S²⁹⁹D EGFP), c-Myc or the empty vector (Vec) as control as indicated. Half of the cells were analysed for transcription from the rRNA minigene reporter by Northern blotting using 10 μg of total RNA and a construct-specific probe. The other half of the cells were used for protein analysis. (B) The levels of the endogenous 45S rRNA precursor in C33A cells stably expressing wild type (wt) extended-, full-length-, truncated-C/EBPα or the S229D mutants thereof (S²⁹⁹D), or the empty vector as control was analysed by Northern blotting from 30 μg of total RNA. (C) The levels 45S rRNA precursor in C33A cells stably expressing wild type (wt) extended-C/EBPα or the S229D or ΔNoLS mutants thereof, or the empty vector as control was analysed by Northern blotting from 30 μg of total RNA. (D) The levels 45S rRNA precursor in C33A cells stably expressing wild type (wt) extended-C/EBPα, the S229D mutant or the empty vector as control untreated or treated with 50 μg/ml α-amanitin (α-am) was analysed by Northern blotting from 30 μg of total RNA. A human β-actin probe or ethidium bromide (EtBr) inverted staining of the gel was used for loading control. Total protein extracts of the cells were blotted for detection of expression of C/EBPα with anti-C/EBP antibodies or expression of c-Myc, with anti-Myc antibodies as indicated.

Figure 8

Selective elimination of extended-C/EBPα impedes rRNA synthesis. (A) Lentiviral shRNA-mediated knock-down of endogenous human C/EBPα (Cα) compared with control scrambled shRNA (Co) in HL-60 cells. (B) Retroviral-mediated expression in C/EBPα knock-down HL-60 (shRNA-Cα) cells from rat C/EBPα (wtα) or the ΔCUGα mutant cDNAs. (C) Retroviral-mediated expression of extended-C/EBPα and the ΔNoLS mutant thereof in C/EBPα knock-down HL-60 (shRNA-Cα) cells. Immunoblots were probed with anti-C/EBPα antibodies (14AA), and with anti-α-tubulin antibodies for loading control. The Xα-labelled band is of indefinite C/EBPα origin; possibly sumoylated truncated-C/EBPα (data not shown). Asterisk (^*) indicates non-specific bands. Levels of 45S pre-rRNA were measured by Northern blotting of 15 μg of total RNA. Ethidium bromide (EtBr) inverted staining of the gel was used for loading control.

Figure 9

Occupation of rDNA sequences by extended-C/EBPα facilitates UBF-1 and TBP recruitment and induces histone acetylation. (A) Schematic representation of part of the rDNA repeat with the transcribed rRNA and C/EBP consensus recognition sequences as indicated. The rDNA sequences analysed in the ChIP assays are designated Cd (C/EBP distal), Cp (C/EBP promoter) and M (c-Myc). (B) Fold enrichment of rDNA obtained with chromatin cross-linking and immunoprecipitation (ChIP) analysis for the regions Cd, M and Cp with anti-C/EBPα or anti-UBF-1 as indicated versus non-specific rabbit IgG. ChIP assays were done with C33A cells expressing empty vector, extended-C/EBPα-wt, -S299D or -S299D-ΔNoLS mutant as indicated. (C) DNA gel of semi-quantitative PCR of region Cp with input, and ChIP assays with the antibodies as indicated for C33A cells expressing empty vector, extended-C/EBPα-wt, -S299D or -S299D-ΔNoLS mutant. (D) Fold enrichment of rDNA obtained with ChIP analysis for the regions M and Cp with anti-TBP versus non-specific rabbit IgG. (E) Fold enrichment of rDNA obtained with ChIP analysis for the regions M and Cp with anti-H3-AC or anti-H4-AC as indicated versus non-specific rabbit IgG. DNA was quantitated by real-time PCR and data are presented as means and standard deviation from two independent ChIP experiments each analyses by two independent PCR reactions.

Figure 10

Overexpression of extended-C/EBPα results in an increase in cell volume. C33A cells expressing empty vector, extended-C/EBPα-wt, -S299D or -S299D-ΔNoLS mutants were analysed for cell volume. The percentage of change in the mean cell volume compared with empty vector was calculated from two independent cultures, each measured in triplicate. The error bars represent the data variation.