p53 Functions through Stress- and Promoter-Specific Recruitment of Transcription Initiation Components before and after DNA Damage  Joaquı́n M Espinosa, Ramiro E Verdun, Beverly M Emerson  Molecular Cell  Volume 12, Issue 4, Pages 1015-1027 (October 2003) DOI: 10.1016/S1097-2765(03)00359-9

Figure 1 Activation of the p53-Pathway in Response to UVC and Doxorubicin (A) p53 posttranslational modifications precede stabilization and accumulation. Western blot using total protein extracts from U2OS cells harvested at indicated times after UVC (25 J/m2) or doxorubicin (0.5 μM) treatment. Identical blots were probed with antibodies as indicated. (B) p53 target genes are activated with stress-specific kinetics. RT-PCR showing induction of p53 target genes, p21, and Fas/APO1, after DNA damage. Representative induction profiles are shown at right. Standard deviation is below 20%. (C and D) p21 and Fas/APO1 activation depends on p53 activity. Western blot assays confirm the lack of p53 in SAOS2 and HCT116 p53−/− cells before and 8 hr after damage (C). RT-PCR showing lack of p21 and Fas/APO1 mRNA accumulation in p53 null cells (D). Molecular Cell 2003 12, 1015-1027DOI: (10.1016/S1097-2765(03)00359-9)

Figure 2 Dynamics of Transcription Factor Occupancy at the p21 Promoter after UVC-Mediated Activation Protein extracts from untreated (−) or UVC-treated (+, 8 hr posttreatment) U2OS cells were subjected to ChIP assays using antibodies against the indicated proteins. Control immunoprecipitations with normal IgGs were also performed (Mock lanes). Indicated amounts of total input DNA were also subjected to PCR (Input lanes). PCR products were quantified by densitometry; percentage of immunoprecipitated DNA was calculated by comparison to input signals and plotted over an up-to-scale representation of the p21 locus. Quantification represents the average of three independent experiments. All eight immunoprecipitations were performed using the same two extracts. All five PCR reactions were performed using the same DNA samples as template. For inset in (C), Ac-H4 immunoprecipitations were done using extracts from HCT116 cells treated under identical conditions. PP, proximal promoter. Molecular Cell 2003 12, 1015-1027DOI: (10.1016/S1097-2765(03)00359-9)

Figure 3 Kinetics of Transcription Factor Occupancy during p21 Promoter Activation after UVC Irradiation Protein extracts were prepared from U2OS cells at indicated times after UVC treatment and subjected to ChIP assays using the indicated antibodies. PCR was performed to test for enrichment of the indicated p21 promoter regions. Input and mock controls for the 0 hr time point are shown inside each experiment, and input controls from all seven time points are shown in the bottom right panel. PCR products were quantified by densitometry, and representative kinetics of occupancy (referring to reactions in the top row) are shown at the right of each panel. Standard deviation is below 20%. Conversion of RNA pol II from Ser5- to Ser2-phospho-CTD at the proximal promoter (dotted line in [I]) correlates with CDK9 recruitment. PP, proximal promoter. Molecular Cell 2003 12, 1015-1027DOI: (10.1016/S1097-2765(03)00359-9)

Figure 4 Fast and Transient Activation of the p21 Gene Occurs during Early Stages of the p53 Response (A) Simultaneous visualization of p21 mRNA and p21 protein by RNA-ImmunoFISH. Normal human fibroblasts were fixed at indicated times after UVC treatment and subjected to indirect immunofluorescence using antibodies against p21 protein (green), followed by FISH to detect p21 mRNA employing a specific cDNA probe (red). Dotted ovals designate the nuclear periphery. (B) Discrete nuclear foci of S15P-p53 accumulation appear shortly after DNA damage. RNA-ImmunoFISH assays were performed as in (A) but using S15P-p53 antibodies instead (green). (C) Colocalization of early S15P-p53 nuclear foci with the p21 gene transcription site. Close-up images of nuclei after 1 hr postdamage reveal clear colocalization of a p21 mRNA transcription center with a major S15P-p53 accumulation site. Molecular Cell 2003 12, 1015-1027DOI: (10.1016/S1097-2765(03)00359-9)

Figure 5 Stress-Specific Mechanisms of p21 Promoter Activation Are Revealed by Differential Kinetics of TAFII250 and TFIIB Occupancy during Doxorubicin Treatment (A) Dynamics of transcription factor occupancy before and after doxorubicin treatment. ChIP assays were performed using U2OS protein extracts prepared before (−) or 8 hr after (+) addition of doxorubicin to the culture. (B) Reloading of RNA pol II during doxorubicin treatment correlates with an increase in TFIIB levels. Kinetic assays were performed as in Figure 3 using antibodies indicated at the left of each panel. Representative quantifications are shown at right. Standard deviation is below 20%. (C) Comparative ChIP analyses of p53 wild-type and p53 null cells were performed using extracts prepared 6 hr after UVC or doxorubicin treatment. (D) Western blot assays show no significant change in overall levels of TAFII250 or TFIIB by 6 hr after stress. Molecular Cell 2003 12, 1015-1027DOI: (10.1016/S1097-2765(03)00359-9)

Figure 6 Abundance of RNA Pol II at p53 Target Promoters Varies Greatly before DNA Damage (A and B) Extracts from unstressed U2OS cells were used in ChIP assays with total RNA pol II (A) or S5P-CTD (B) antibodies. Equal fractions of immunopurified DNA were subjected to PCR under conditions of linear amplification using primers against core promoter regions of different p53 target genes (indicated at left). Identical amounts of input DNA were included as controls. Densitometric quantification from three independent immunoprecipitations is shown at right. (C) Comparative analysis of unstressed cells with different p53 status shows requirement of p53 for PIC assembly at the p21 promoter before DNA damage. (D–G) p21 loci localize to bodies of S5P-RNA pol II accumulation as seen by DNA-ImmunoFISH. Unstressed normal human fibroblasts were subjected to indirect immunofluorescence assays using S5P-CTD (D and G), PML (E), or SC35 (F) antibodies (red signals), followed by DNA FISH using p21 or Fas/APO1 probes ([D–F] and [G], respectively, green signals). Colocalization of p21 loci with S5P-CTD signals appears in yellow after image merging (right panels). Molecular Cell 2003 12, 1015-1027DOI: (10.1016/S1097-2765(03)00359-9)

Figure 7 Fas/APO1 Promoter Is Activated by Stress-Specific Mechanisms without Loss of Poised RNA Pol II (A and B) TAFII250 is recruited to the Fas/APO1 promoter only after UVC treatment, and activation occurs without changes in TFIIB levels. ChIP assays using extracts from U2OS cells treated with UVC (A) or doxorubicin (B) were performed with antibodies indicated at the left of each panel. DNA samples were tested for enrichment of three different regions of the Fas/APO1 locus: the intronic p53 binding site (+780), the DPE-containing promoter region (PP), and a control region 4 kb upstream of the transcription start site (−4 kb). Input and mock controls were performed as in Figures 3 and 5. Representative quantifications are shown at right. (C) Model of differential p53 target gene activation. The p21 locus contains two distal p53 binding sites, and the core promoter region harbors initiator and noncanonical TATA box elements to which a PIC is recruited through p53 action before DNA damage. Increase in modified p53 and histone acetylation occurs after both UVC and doxorubicin treatment. However, p53-dependent recruitment of TAFII250 is exclusive to UVC-induced activation. High levels of poised RNA pol II at the p21 promoter are rapidly converted into an elongating form during activation, as reflected by conversion of S5P-CTD to S2P-CTD and recruitment of pTEFb. Efficient elongation exceeds reinitiation and total levels of RNA pol II decrease drastically. Reinitiation is abrogated after UVC by loss of TFIIB, whereas a constant increase of TFIIB during doxorubicin treatment allows reloading of RNA pol II and a second wave of transcription. The Fas/APO1 locus contains low amounts of poised RNA pol II at the core promoter before DNA damage. After an increase in modified p53 and histone acetylation, the appearance of elongating RNA pol II does not correspond with a decline in poised polymerase at the promoter, suggesting that efficient reinitiation is taking place. TAFII250 recruitment also occurs only after UVC damage but, in contrast to p21, low initial levels of TFIIB remain constant during activation. Molecular Cell 2003 12, 1015-1027DOI: (10.1016/S1097-2765(03)00359-9)