Figure 5.
PTM-Specific C/EBPβ Interactions
(A) PTM-dependent protein binding to FPFALRAYLGYQAT peptides of C/EBPβ CR7. Normalized binding intensity relative to the unmodified peptide. Four binding categories are considered: proteins with reduced binding by any PTM (repressed, blue, far left), proteins not responding to modifications on C/EBPβ (independent, yellow, middle), proteins with PTM-specific binding (regulated, yellow-red, small), and enhanced binding by any PTM (enhanced, red, far right).
(B) Immunoprecipitation (IP) from transfected HEK293T cell lysates: FLAG-tagged C/EBPβ wild-type (WT) or mutants, as indicated, abrogating (C/EBPβ R60A; R193A) or mimicking (C/EBPβ R60L, R193L) methylation. Co-immunoprecipitation (coIP) of endogenous YEATS4 and DMAP1 detected by immunoblotting. YEATS4 and DMAP1 bind to C/EBPβR60L and failed to bind to the R60A C/EBPβ mutant. RelA favors WT or C/EBPβR193A binding over R193L. TLE3 preferentially binds to C/EBPβR193L. Underneath: Expression controls of endogenous YEATS4, DMAP1, RelA, and TLE3.
(C) coIP of WT and mutant C/EBPβ, as indicated, with hemagglutinin (HA)-tagged TLE3. C/EBPβR193L, but not WT or R193A, co-IPs with TLE3.
(D) In vitro methylation of C/EBPβ peptides by CARM1. Scheme of C/EBPβ with approximate positions of peptides and R-residues indicated by red bars and dots; R residue sequence positions underneath. Bar graphs show peptide-specific incorporation of methyl group from donor S-adenosyl-L-[methyl-3H] methionine.
(E) coIP of C/EBPβ with TLE3 depends on CARM1. Co-expression of WT C/EBPβ (R) or mutants (R193A, R193L), TLE3, and CARM1 are indicated on the top. WT C/EBPβ, but not R193A, co-immunoprecipitates with TLE3 in the presence of CARM1, whereas the C/EBPβR193L mutation supersedes CARM1 requirement.
(F) RT-PCR of ADIPOQ, CFD, and FABP4 expression (arbitrary units: target gene to internal 36B4 expression control) induced by C/EBPβ WT, R193A, R193L, or vector control in the absence or presence of TLE3 in NIH 3T3 L1 cells without adipogenic differentiation cocktail (upper panel; n = 3, ANOVA; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001; ns: not significant). Oil red O staining of stably transfected NIH 3T3 L1 cells with C/EBPβ constructs, vector control and TLE3, as indicated, 3 weeks post-confluency (middle panel, representative images) and protein expression controls (lower panel).