Model of crosstalk between phosphorylation and R3 methylation of C/EBPβ.

LAP^*/C/EBPβ1 R3 mutations alter C/EBPβ target gene activation. LAP^*/C/EBPβ1 constructs were transfected into QT6 fibroblasts as indicated. RNA was extracted 18 h post-transfection and subjected to serial northern hybridization to probes directed to the mim-1 gene, #325 gene, and GAPDH as a control.

Physical and functional interaction between C/EBPβ and PRMT4/CARM1 in eukaryotic cells. (A) K562 cell lysates were incubated with antibodies (IgG, negative control) or anti-C/EBPβ and antigen–antibody complexes were immunoprecipitated. Proteins were analysed by immunoblotting with anti-PRMT4/CARM1 or anti-C/EBPβ as indicated. IB: immunoblot. (B) LAP^*/C/EBPβ1 WT or R3A constructs, as indicated, were transfected in QT6 fibroblasts together with WT or PRMT4/CARM1^mut constructs. RNA blots were subjected to serial hybridization to the mim-1, #325 and GAPDH gene probes.

PRMT4 methylates the N-terminus of C/EBPβ. (A) Scheme of C/EBPβ N-terminal peptides and C/EBPβ N-terminal GST-protein. (B) In vitro methylation of C/EBPβ N-terminal peptides by PRMT3, PRMT4/CARM1, and PRMT5. HA-tagged PRMT3, 4, and 5 were expressed in HEK-293 cells, purified from cell lysates by immunoprecipitation with anti-HA and incubated with 1 μM of the corresponding peptides as substrates in the presence of L-[methyl-³H]methionine as a ³H-methyl-donor. Bars represent relative incorporation of labelled S-adenosyl-L-[methyl-³H]methionine. (C) In vitro methylation of recombinant C/EBPβ N-terminal GST proteins by PRMT4/CARM1. Reactions were carried out as described in (B) with 3 μg GST protein as a substrate. Bars represent relative incorporation of labelled S-adenosyl-L-[methyl-³H]methionine. Histone H3 was used as a positive and the GST moiety as a negative control for PRMT4/CARM1-specific L-[methyl-³H]methionine labelling.

Differential interaction of SWI/SNF and Mediator complexes with R3 methylated and unmethylated LAP^*/C/EBPβ1 N-terminal peptides. (A) The N-terminal sequence of LAP^*/C/EBPβ1 was synthesized with R3 in unmodified form (1–41) or in asymmetrically dimethylated form (1–41 Rme2a). Peptides with covalently attached C-terminal biotin moieties (grey pentagons) were applied to UNIPEX proteomic libraries and interactions were revealed by streptavidin-HRP (SA-HRP) and ECL. Positive clones were identified as characteristic duplicate pattern per square. Libraries contained 48x48 squares with four duplicate clones on two PVDF membranes. A single square containing the brama-related gene 1 (Brg1) expression clone interacts with unmethylated, but not with methylated peptide (arrow heads). (B) Unmethylated and methylated C/EBPβ peptides (1 μM), as shown in (A) were incubated with cell lysates and bound proteins separated by streptavidin Dynabeads. Cell lysates were prepared from Raji cells (top) or HEK-293 MED23-HA-transfected cell lysates (bottom). Western blots were incubated with anti-hBrm, anti-BAF155, anti-BAF47/Ini1, anti-HA, and anti-MED26 as indicated.

Effect of ras/MAPkinase signalling or a phospho-mimetic mutation on the interaction between C/EBPβ and PRMT4/CARM1. (A) FLAG-tagged LAP^*/C/EBPβ1 was co-expressed with HA-tagged PRMT4/CARM1 in QT6 fibroblasts in the absence and presence of ras^V12. Immunoprecipitation (IP) from cell lysates with anti-Flag or anti-HA antibody as indicated (top) and protein expression control (below, expr. control). The anti-P-Thr235 antibody specifically reveals C/EBPβ that is phosphorylated at the MAPkinase site. IP: immunoprecipitation. IB: immunoblot. (B) Lysates from K562 cells (with/without PMA treatment) were immunoprecipitated with anti-C/EBPβ or negative control IgG. Proteins were analysed by immunoblotting with anti-PRMT4/CARM1, anti-C/EBPβ, or anti-P-Thr235, as indicated. Protein expression control below (expr. contr.). IP: immunoprecipitation. IB: immunoblot. (C) Relative quantification of phosphorylation and R3me2 on LAP^*/C/EBPβ1 by multiple-reaction monitoring (MRM). Left: three different tryptic peptides were used for quantification of C/EBPβ in total cell lysates from untreated and PMA-treated K562 cells, as indicated. Middle: quantification of the C/EBPβ Thr-235 phosphorylation MAPkinase site containing tryptic C/EBPβ peptide. Right: quantification of the R3-LAP^*/C/EBPβ1 methylated tryptic peptide in total cell lysates by MRM. (D) FLAG-tagged LAP^*/C/EBPβ1 or LAP^*/C/EBPβ1 T220D constructs were co-expressed with HA-tagged PRMT4/CARM1 in QT6 fibroblasts. Cell lysates were immunoprecipitated with anti-HA and immunoblots (IP) were developed with anti-Flag or anti-HA, respectively (indicated on the right). IP: immunoprecipition. IB: immunoblot. Below: protein expression control (anti-Flag or anti-HA) as indicated. Vertical lines indicate assembly of two separated lanes on the same immunoblot.

C/EBPβ is post-translationally methylated on arginine residues. (A) Scheme of C/EBPβ isoforms that arise by alternative translation initiation from in-frame start codons termed as LAP^*/C/EBPβ1, LAP/C/EBPβ2 and LIP/C/EBPβ3. (B) LAP^*/C/EBPβ1 is precipitated by the ASYM24 anti-Rme2a antibody in the absence of AdOx. LAP^*/C/EBPβ1-transfected fibroblasts were treated with adenosine dialdehyde (AdOx) for 12 h (as indicated) and immunoprecipitated with the ASYM24 antibody. Precipitated proteins were analysed by immunoblotting using an anti-C/EBPβ antibody. Right side shows expression controls in the absence and presence of AdOx. (C) Purification scheme of C/EBPβ. Briefly, His-tagged C/EBPβ was purified under denaturing conditions by affinity chromatography on a nickel chelating resin. Blot shows purified fractions of C/EBPβ. (D) Tandem mass spectrum of an R3me2 and oxidized N-terminal C/EBPβ peptide. b- and y-series of the LysC-generated peptides are indicated. Mass shifts indicate a 28 Da modification, corresponding to dimethylation at position R3. Underneath: alignment of conserved region 1 (CR1) of LAP^*/C/EBPβ1 from various species shows conserved R3 (magenta box) and start sites (arrows) of the two long isoforms.

Myeloid and adipogenic gene regulation. (A) Expression of neutrophile elastase (hELA2) transcripts by C/EBPβ isoforms in NIH 3T3 cells before and after PMA treatment. NIH 3T3 cells were transfected with constructs (as indicated) and after 24 h stimulated with phorbol ester (PMA) for 1.5 h. Total mRNA and cDNA were prepared and hELA2 gene expression was analysed by RT–PCR and normalized to GAPDH expression. (B) Expression control of C/EBPβ isoforms detected by immunoblotting. (C) ChIP assay from either unstimulated or PMA-stimulated U937 cells. Antibodies were used as indicated. Quantitative PCR results are shown as fold binding compared with the IgG control. (D) Activation of PPARγ, aP2, and adipsin expression by LAP^*/C/EBPβ1, LAP/C/EBPβ2 isoforms, and the LAP^*/C/EBPβ1 R3A or R3L mutants in NIH 3T3 L1 cells in the absence of adipogenic differentiation hormone cocktail. NIH 3T3 L1 cells were transfected with vector, LAP^*/C/EBPβ1, LAP/C/EBPβ2, LAP^*/C/EBPβ1 R3A, and LAP^*/C/EBPβ1 R3L and stable transfectants selected by puromycin. Cells were grown to confluency and total mRNA and cDNAs were prepared 10 days after confluency. The results were normalized to GAPDH expression. (E) Oil red O staining of stably transfected cells, ten days post-confluency, as shown in (D). (F) Protein expression control of cells as shown in (D).