Fig 2: GOF p53 requires an intact TAD to get nucleated on upstream sequences of mutant p53 inducible genes in cells expressing mutant p53.Chromatin immunoprecipitation (ChIP) assays were performed to compare the ability of GOF p53 to interact on the promoter-enhancers of mutant p53-activated genes. a Mutant p53 ChIP was performed with murine cells with p53-R172H and p53-L25Q/W26S/R172H or p53–/– as a control. Mutations at 25/26 in GOF p53 impair the ability of the protein to anchor to the promoter as evident by the loss of ChIP. Mutant p53 ChIP was also performed with b H1299 cells expressing p53-R273H, -R273H L22Q/W23S, and vector control or c H1299 cells expressing p53-R175H, -R175H L22Q/W23S, and vector control. Data are normalized with input DNA values for each respective promoter and are presented as the mean ± SEM (n = 3). A two-sided Student’s t-test was performed; a *p = 0.038, **p = 0.001, ***p = 0.0003; b *p = 0.007, **p = 0.001, ***p = 0.0004; c *p = 0.038, **p = 0.0068. Mutations at 22/23 in human GOF p53 impair the ability of the protein to anchor to the promoter as evident by the loss of ChIP in b and c. d A transient transactivation assay was performed in H1299 using either pCMV Bam neo (Vector) or p53-R273H and siRNA against Ets2, Sp1, and VDR to show reduced transactivation of the Chk1 promoter by p53-R273H when Sp1 is knocked down. Data are presented as the mean ± SEM (n = 3, performed twice). *p-value < 0.05, **p-value < 0.01, and ***p-value < 0.001. NS no statistically significant difference from control.

Fig 3: Proposed model for GOF p53 nucleating on an inducible promoter.Arrow toward the right-hand side depicts transcription direction. For simplicity TAF and other factors are not shown.

Fig 4: TAD mutations affect the state of phosphorylation of mutant p53.Western blot analysis of equal amounts of extracts from a H1299 cells expressing p53-R273H, -L22Q/W23S/R273H, and vector control; b H1299 cells expressing p53-R175H, -L22Q/W23S/R175H, and vector control; and c murine lung cells expressing p53-R172H, -L22Q/W23S/R172H, and p53–/– control were developed with antibodies against different phosphorylation sites on p53. The data presented show that phosphorylation at amino acid position 20 went down significantly. d Transactivation assays of mutant p53 and its derivatives by Ala substitution mutations at positions of phosphorylation using a mutant p53 inducible gene promoter. Transient transcriptional assays were carried out in H1299 cells after transfection of NF-κB2-luc and indicated expression plasmid or empty vector. Cells were harvested after 24 h of transfection. Firefly luciferase readings were plotted as relative light units (RLU). Data are presented as the mean ± SEM (n = 3, performed twice). A two-sided Student’s t-test was performed. Ser20Ala substitution reduced the transactivation ability the greatest. Substitution of S20 to Glu or Asp did not regain transactivation ability either. e Western blot analysis showing level of different p53 mutants after transfection. *p-value < 0.05, **p-value < 0.01, and ***p-value < 0.001. NS no statistically significant difference from control.

Fig 5: Lung tumor formation in the GOF p53-expressing mouse lung cancer model is dependent on expression of mutant p53.a Model shows induced depletion of p53 causes tumor remission. Induction of p53 shRNA causes a reduction of p53-R172H in the mouse lung after dox treatment. Dox was added to the drinking water when mice were 1 month old and continued throughout the life of the mice. b Representative images (Magnification: 20×, Scale bar: 100 µM) of tissue sections from the LSL p53-R172H, shp53, K-Ras, CCSP-Cre mouse line showing apparently normal lung Club cells immunostained for p53 (arrows) in small bronchi lined by respiratory epithelium; +Dox shows a decrease in p53 expression indicated by reduced staining intensity; gross lung image shows formation of lung tumor (shown by a black arrow) in –Dox. Rb indicates respiratory bronchiole. c Representative images (Magnification: 20×, Scale bar: 100 µM) of tissue sections from the LSL p53-R172H, shp53, K-Ras, SPC-Cre mouse line showing apparently normal lung Alveolar Type II cells immunostained for p53 (arrows); gross lung image shows formation of lung tumor (shown by black arrows) in –Dox. AR indicates alveolar region. d Representative images of normal tissue (+Dox) and tumor sections (–Dox) from LSL p53-R172H, shp53, K-Ras, CCSP-Cre and LSL p53-R172H, shp53, K-Ras, SPC-Cre mouse lines stained with hematoxylin and eosin (H&E) (Magnification: 10×, Scale bar: 200 µM) and immunostained with p53 antibody (Magnification: 20×, Scale bar: 100 µM). Rb indicates respiratory bronchiole. AR indicates alveolar region. e Images of tumor sections from LSL p53-R172H, shp53, K-Ras, CCSP-Cre and LSL p53-R172H, shp53, K-Ras, SPC-Cre mouse lines immunostained with SPC (red) and CCSP (green) fluorescent antibodies. Nuclei have been labeled by DAPI (4′,6-diamidino-2-phenylindole) (Magnification: 40×, Scale bar: 50 µM). Arrows indicate SPC-positive cells. CCSP-labeled cells were rare, possibly due to cell type conversion. f Representative images (Magnification: 20×, Scale bar: 100 µM) of tumor sections from CCSP and SPC mouse lines stained for thyroid transcription factor 1 (TTF-1) showing tumors are adenocarcinoma in nature. Arrows indicate positively stained cells.