. The p53 tumor suppressor and master regulator is central to human DNA repair, damage checkpoints and many aspects of human biology. Importantly, most cancers are altered for p53 function. We discovered an expanded universe of p53 targets and human diversity as well as variations in stress responses. The DNA binding and transactivation of p53 is critical for tumor suppression. There is considerable variation in p53 dependent expression across 100s of targeted genes leading to differences in p53-mediated biological consequences, due in part to variation in target response element (RE) sequence. We found that the target sequence motif differs considerably from the in vitro derived RE consensus target sequence previously described as 2 copies of RRRCA/TT/AGYYY separated by a spacer of up to 13 bases. We have focused on RE functionality, i.e., the ability of REs to support transactivation by p53. To assess transactivation responsiveness of human REs, we developed promoter systems in budding yeast for variable p53 expression. Recently, we identified super-transactivating sequences that provide high transactivation at low p53 levels. The yeast system has has been used to establish the functional evolution of REs across species and was summarized in our piano model that describes functional variability within a transcriptional network. We have translated many of the findings about human p53 in yeast to human cells in culture and ex vivo. Our previous studies showed that a SNP in the Flt1 gene promoter generates a perfect p53-site and enables p53-mediated transactivation of FLT-1, placing the VEGF system directly in the p53 network. We found that p53 can transactivate through noncanonical binding sequences including half-sites. This greatly expands the p53 master regulatory network. We extended these studies genome-wide using ChIP-Seq analysis to identify p53 binding sites and associated gene expression changes following p53 activation by different agents in cancer cells. Interestingly, binding often is not associated with transactivation. A de novo motif search for the in vivo consensus binding sequence revealed the p53 canonical motif is, in fact, 2 tandem RRRCWWGYYY decamers without spacer. Although less efficient, p53 can bind non-canonical p53 motifs composed of either two-decamers with spacers or just a half-site. INTERACTION OF p53 AND ER REGULATORY NETWORKS. We had identified a 1/2-site estrogen receptor RE that greatly increased p53 transactivation at the FLT1 1/2-site p53 RE, establishing a new dimension to the p53 regulatory network. Recently, we addressed the generality of synergistic transactivation by p53 and ER. p53 transactivation was greatly enhanced by ligand-activated ER acting in cis. The increased transactivation extends to several cancer-associated p53 mutants, suggesting ER-dependent mutant p53 activity for at least some REs and possibilities for reactivation of cancer mutants. We propose a general synergistic relationship between the p53 family and ER master regulators in transactivation of p53 target canonical and noncanonical REs which might be poorly responsive to p53 on their own. In collaboration with Alberto Inga (Trento University) and Ken Korach (NIEHS), we are evaluating effects of different ER mutants that affect DNA and accessory protein interactions on p53 mediated transactivation of genes with in cis ER and p53 REs. We developed a functional matrix tool for genome-wide searches for putative p53 target genes. Several new p53 target genes have been identified including the DNA repair-associated RAP80 gene as well as several members of the Toll-like receptors (TLRs) family that determine innate immunity (discussed below). As predicted for RAP80 as well as for several TLRs, including TLRs 2, 5 and 10, p53 and ER can cooperate to mediate its transcription both in cancer cell lines and human lymphocytes. CANCER-ASSOCIATED P53 MUTANTS. Nearly all cancers have mutant or reduced expression of the p53 tumor suppressor gene. Using yeast-based and human cell systems, we found that p53 mutations can lead to considerable diversity in the spectrum of responses from REs including 1) decrease/loss-of-function;2) subtle changes;3) altered specificity;and 4) super-transactivation all of which lead to variation in biological responses. Most of the functional mutants were able to function at 1/2 sites and several can cooperate with ERs to mediate expression of FLT1 revealing a new level of functional interaction between these two master regulators. With the inclusion of immune response-related TLR genes into the p53 network, we evaluated the effect of a panel of 25 tumor-associated p53 mutants on TLR gene family expression after transient transfection in p53 null cancer cell lines. Changes in TLR transactivation patterns, including change-of-spectrum were observed, suggesting that p53 tumor status might be an important factor in adjuvant therapy employing TLR pathways to treat cancer. P53 NETWORK EVOLUTION. We are investigating evolution of REs in terms of responsiveness to p53. Individual REs exhibited marked differences in potential transactivation as well as widespread turnover of functional REs during p53 network evolution. Only 1/3 of the REs found in humans are predicted to be functionally conserved in rodents. Importantly, we found functional conservation of weakly responding REs including 1/2 sites. Among validated p53 REs conserved between rodents and humans, one third were comprised of 1/2- or 3/4-sites, each with a perfect consensus-site suggesting a selective advantage in retaining weak p53 REs. Importantly, the integration of the TLR gene family into the p53 network also appears unique to primates. With our CHIP seq based whole genome p53 binding map generated in primary and cancer cell lines we are addressing evolutionary sequence conservation. REGULATION OF THE IMMUNE RESPONSE BY p53. We found that among the 10 human TLRs, nine had canonical and noncanonical p53 REs. Using primary human cells obtained in a collaboration with the Clinical Research Unit we examined expression of the entire TLR gene family following exposure to anti-cancer p53 inducing agents. Expressions of all TLR genes in blood lymphocytes and alveolar macrophages from healthy volunteers were inducible by DNA metabolic stressors. However, there is considerable inter-individual variability. Furthermore, a polymorphism in the TLR8 promoter provides the first human example of a p53 target RE sequence specifically responsible for endogenous gene induction. Similarly p53 dependent TLR expression is detected in human cancer cell lines. For some TLRs the p53 control seems to enhance the inflammatory responses, mediated by activation of TLRs in the presence of natural ligands. In particular, we found a p53-dependent increase in response to the TLR5 ligand flagellin as measured by both mRNA and protein production of the downstream cytokines IL-6 and IL-8 accompanied by a specific increase in phosphorylation of p38 MAP kinase which is one of the mediators of TLR signaling. We also found that in the absence of TLR ligands, induced p53 can cooperate with Nuclear Factor-kappa beta (NFkappa-beta) to induce pro-inflammatory cytokines in primary human macrophages. YEAST SYSTEM FOR RAPID ASSESSMENT of CHEMICAL AFFECTING p53 and OTHER TRANSCRIPTION FACTORS. We expanded our yeast system to create high-throughput systems for addressing chemical modification of wild type and mutant human p53, factors that interact with p53 as well as adapting the system to address components of the NFkappa regulator. The human proteins are expressed in yeast at varying levels and their ability to function as transcription factors at known target sequences is determined. Chemical modification is assessed as changes in expression of reporters.

Project Start
Project End
Budget Start
Budget End
Support Year
19
Fiscal Year
2012
Total Cost
$1,547,306
Indirect Cost
City
State
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Currier, Jenna M; Cheng, Wan-Yun; Menendez, Daniel et al. (2016) Developing a Gene Biomarker at the Tipping Point of Adaptive and Adverse Responses in Human Bronchial Epithelial Cells. PLoS One 11:e0155875
Menendez, Daniel; Lowe, Julie M; Snipe, Joyce et al. (2016) Ligand dependent restoration of human TLR3 signaling and death in p53 mutant cells. Oncotarget :
Sharma, Vasundhara; Jordan, Jennifer J; Ciribilli, Yari et al. (2015) Quantitative Analysis of NF-κB Transactivation Specificity Using a Yeast-Based Functional Assay. PLoS One 10:e0130170
Shatz, Maria; Shats, Igor; Menendez, Daniel et al. (2015) p53 amplifies Toll-like receptor 5 response in human primary and cancer cells through interaction with multiple signal transduction pathways. Oncotarget 6:16963-80
Lowe, Julie M; Menendez, Daniel; Bushel, Pierre R et al. (2014) p53 and NF-κB coregulate proinflammatory gene responses in human macrophages. Cancer Res 74:2182-92
Menendez, Daniel; Anderson, Carl W (2014) p53 vs. ISG15: Stop, you're killing me. Cell Cycle 13:2160-2161
Lowe, Julie M; Menendez, Daniel; Fessler, Michael B (2014) A new inflammatory role for p53 in human macrophages. Cell Cycle 13:2983-4
Nguyen, Thuy-Ai; Menendez, Daniel; Resnick, Michael A et al. (2014) Mutant TP53 posttranslational modifications: challenges and opportunities. Hum Mutat 35:738-55
Menendez, Daniel; Shatz, Maria; Resnick, Michael A (2013) Interactions between the tumor suppressor p53 and immune responses. Curr Opin Oncol 25:85-92
Ciribilli, Yari; Monti, Paola; Bisio, Alessandra et al. (2013) Transactivation specificity is conserved among p53 family proteins and depends on a response element sequence code. Nucleic Acids Res 41:8637-53

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