Fig 1.
Schematic of CBP and p300 showing identity over various regions.
CBP and p300 have molecular weights of approximately 300kDa and are encoded over 33 and 31 exons and consist of 2441 and 2414 amino acids (aa), respectively. β-catenin, with direct small molecule CBP/catenin (PRI-724/ICG-001) antagonist, competitively binds to CBP’s distal N-terminus, the least conserved region within these two Kat3 coactivators. CBP, cAMP response element binding protein (CREB)-binding protein; p300, E1A-binding protein, 300 kDa; Br, Bromodomain; CH, Cysteine/histidine; KIX, kinase-inducible domain interacting domain.
Fig 2.
Absence of “p300 insertion” sequence sterically inhibits nuclear receptor and β-catenin from simultaneously binding to CBP; however, nuclear receptor may bind concurrently to p300 (due to lack of steric inhibition) and thereby antagonize CBP/β-catenin signaling.
A. Sequence alignment of the distal N-termini of p300 (P1) and CBP (C1), depicting conserved sites for binding of β-catenin (DELI motif) and for binding of nuclear receptor (LXXLL motif), as well as the 9aa “p300 insertion” sequence which is deleted/absent from CBP. (Note: Sequences depicted are for human.) B. Absence of “p300 insertion” sequence prevents nuclear receptor and β-catenin from simultaneously binding to CBP due to steric inhibition (yellow triangle), whereas presence of the sequence allows concurrent binding and synergy of β-catenin and nuclear receptor due to abrogation of steric hindrance (lavender crescent). C. Three-dimensional structural modeling of the N-terminus of CBP/p300 and its putative interactions with β-catenin and nuclear receptor ligand binding domain of RXR alpha are shown in a ribbon representation. (Left) Interaction between the N-terminus of CBP (CBP-NT) (pink color) and β-catenin (white color) is shown. The nuclear receptor (NR) binding motif (LXXLL) is highlighted in red. (Right) Interaction between the N-terminus of p300 (p300-NT) and β-catenin (white color) and nuclear receptor (RXR alpha) (green color). The nuclear receptor binding motif in p300 (LXXLL) is highlighted in yellow color.
Fig 3.
CRISPR/Cas9 editing/deletion of p300 insertion sequence in P19 mouse embryonal carcinoma cells.
A. Schematic outlining the design of CRISPR/Cas9 editing in P19 mouse embryonal carcinoma cells to delete the 27bp insertion in the amino terminus of p300 (as detailed in the Experimental Procedures). Guide RNA (gRNA), CRISPR RNA (crRNA), protospacer adjacent motif (PAM). B. Immunoblotting demonstrated that protein expression levels of both CBP and p300 in the “edited P19 cells” (Edited) are equivalent to those in the wild type P19 cells (WT), confirming that the expression levels of both Kat3 coactivators were not affected by the CRISPR/Cas9 editing. n = 3.
Fig 4.
Edited P19 cells exhibited higher β-catenin/TCF transcription, as well as increased survivin/BIRC5 expression.
A. Edited P19 cells exhibited significantly higher β-catenin/TCF transcription compared with wild type (WT) cells under basal conditions, as assessed by Topflash/Fopflash (TOP/FOP) activity. n = 3, *p < 0.05. B and C. In both wild type and edited P19 cells, Wnt3a significantly activated the Survivin/luciferase reporter construct (compared with empty vehicle). This activation was effectively inhibited by specific, direct small molecule CBP/catenin antagonist ICG-001 in wild type cells (B). However, in the P19 edited cells, although Wnt3a increased Survivin/luciferase activity, ICG-001 did not reduce the activity (C). Representative of n = 3, *p < 0.05, **p < 0.01. D. Expression of survivin/BIRC5 mRNA, which is highly dependent on CBP, was enhanced by Wnt3a in wild type (WT) and to a lesser extent in p300 edited P19 cells. Whereas treatment with ICG-001 in WT cells significantly decreased survivin/BIRC5 message, there was essentially no effect of ICG-001on survivin/BIRC5 expression in edited cells. n = 3, **p < 0.01. Vehicle/control data (set to 1) as indicated with a red, dashed horizontal.
Fig 5.
More β-catenin is associated with p300 in edited P19 cells than in wild type (WT) cells, as demonstrated by immunoblot for β-catenin using samples of nuclear protein subjected to immunoprecipitation with CBP, p300, or (control) rabbit IgG antibody.
n = 3. (5% input/control immunoblotted for β-catenin is depicted on the far left).
Fig 6.
p300 editing affects Wnt and retinoic acid signaling interactions.
A. Wnt3a and all-trans retinoic acid (ATRA), individually, induced Stra6 (stimulated by retinoic acid 6) mRNA expression in both wild type (WT) and p300 edited P19 cells, as assessed by realtime RTPCR. Whereas there was a strong additive effect on the expression of Stra6 with combined Wnt3a and ATRA treatment in WT cells, no additivity was demonstrated in p300 edited cells. n = 3, *p < 0.05. Vehicle/control data (set to 1) as indicated with a red, dashed horizontal. B. The additivity of Wnt3a and ATRA was also reflected at the protein level for Stra6 in wild type but not edited P19 cells, as assessed by densitometry of immunoblot for Stra6. n = 3, *p < 0.05, **p < 0.01. C. Similarly, with Stra8 (stimulated by retinoic acid 8), there is a clear additive effect of Wnt3a and ATRA in wild type (WT) cells, but there is no additive effect in edited P19 cells, as assessed by realtime RTPCR. n = 3, *p < 0.05. Vehicle/control data (set to 1) as indicated with a red, dashed horizontal.
Fig 7.
p300 edited P19 cells do not undergo neuronal differentiation.
A. Expression of ephrin B1, a gene critical for neuronal migration and the maintenance of neuronal progenitors, was induced by Wnt and all-trans retinoic acid (ATRA) in an additive manner in wild type P19 cells, as assessed by realtime RTPCR. Although expression of ephrin B1 was induced by Wnt3a and ATRA, individually, there was antagonism between the two treatments in the edited cells. n = 3, *p < 0.05, **p < 0.01. Vehicle/control data (set to 1) as indicated with a red, dashed horizontal. B and C. Wild type (WT) and edited P19 cells were subjected to an established neuronal differentiation protocol (as detailed in the Experimental Procedures). As demonstrated with light microscopy, WT cells stopped proliferating and neurite outgrowth ensued, whereas edited cells continued to proliferate and neurite outgrowth was not observed (20X magnification) (B). By immuno-fluorescence microscopy, ephrin B1 was clearly expressed after subjected to the differentiation protocol in WT but not edited P19 cells (20X magnification) (C).