Human Genes Affecting Chromosome Metabolism and Stress Response

National Institute Of Environmental Health Sciences

Investigators

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Abstract

The p53 tumor suppressor is central to human DNA repair, damage checkpoints and many aspects of human biology. Importantly, most cancers are altered for p53 function. There is considerable variation in p53 dependent expression across >200 targeted genes leading to differences in p53-mediated biological consequences, due in part to variation in target response element (RE) sequence. We found that RE motifs differ 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 directly assess functionality of human REs, i.e., transactivation responsiveness, we developed promoter systems in budding yeast for variable human p53 expression and have translated many of the findings to human cells in culture and ex vivo. That led us to the identification of super-transactivating sequences (STARES) that provide high transactivation at low p53 levels. Remarkably, we observed that transactivation specificity is conserved among p53 family proteins. We found that p53 can transactivate through noncanonical binding sequences including half-sites, greatly expanding the p53 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 in cancer cells as well as in primary T-lymphocytes after treatment with anti-cancer drugs. Similar to cancer cells, we found that most p53 bound sites contained a canonical p53 response element (2 tandem RRRCWWGYYY decamers without spacer). Importantly, we showed that p53 can also engage transcription using half-sites across the genome and we went on to define the minimal binding unit for p53-mediated transcription as a 1/2-site. Among differentially expressed genes associated with p53 binding several are relevant to immunity and inflammation. Moreover, we are developing mass spectrometry methods to determine posttranslational modifications on p53 from human cancer cell lines and primary cells. INTERACTION OF p53 AND ER 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 network. p53 transactivation was greatly enhanced by ligand-activated ER acting in cis. The increase 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 found nearly 200 synergistically expressed genes following combined treatment with doxorubicin and estradiol in MCF7 cells. We propose a general synergistic relationship between p53 and ER master regulators in transactivation of p53 target canonical and noncanonical REs which might be poorly responsive to p53 on their own. CANCER-ASSOCIATED p53 MUTANTS. With inclusion of immune response-related TLR genes into the p53 network, we evaluated the effect of 25 tumor-associated p53 mutants on TLR gene family expression after transient transfection in p53-null cancer cell. 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. Furthermore, we demonstrated that tumor-associated p53 mutants that induced expression of TLR3, enhanced cytokine and chemokine responses mediated by this receptor after exposing cells to TLR3 ligand poly-I:C alone or in presence of Doxorubicin. We also found that functional rescue of loss-of-function p53 mutants by the p53 reactivating drug RITA, restored TLR gene expression in a mutant p53 cell line and also enhanced DNA damage induced-apoptosis via TLR3 signaling. We propose that chemotherapeutic manipulation of normal or mutant p53 responses along with immune challenges that include TLRs could enhance inflammatory/immune type responses to environmental factors. Furthermore, several p53 mutants also altered the expression of the innate immune related APOBEC3 gene family. In particular, overexpression of a group of tumor-associated p53 mutants in p53 null cancer cells actually promoted expression of APOBEC3B, which normally is repressed by WT p53, suggesting a clear gain of function phenotype for these p53 mutants. Since TP53 gene mutations occur in many human cancers, it is important to identify anticancer drugs that specifically target p53 mutant tumor cells. We are pursuing the identification of synthetic lethal (SL) genes with genome-wide siRNA-based screens, that when reduced in expression in p53 mutant cells cause death or reduced growth. This expected to lead to potential targets and provide opportunities for anticancer drug development. 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. 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. Similar to those observations, we identified in our recent ChIPseq cancer cell study that 60% of the p53 REs were conserved at the sequence level in rodents. Using Gene Ontology (GO) for gene function classification to identify those potential p53 target genes associated with DNA metabolism and repair functions, we found that only a few of the p53 RE sequences were conserved in rodents in agreement with our earlier findings. Surprisingly, the p53-mediated expression of TLRs appears to be also unique to primates, based on phylogenetic analyses of p53-REs in the TLR promoters. Consistent with a lack of p53-REs, most TLR expression in mice is not influenced by DNAdamage. This finding suggests that humans and close relatives evolved enhanced innate immune inflammatory responses to DNA damage.These results may indicate evolutionary selection on p53 dependent chromosomal stress responses. THE INFLUENCE OF P53 ON HUMAN IMMUNE RESPONSES. Recently, our lab established a broad role for WT and mutant p53 in the responses of most Toll-like receptor (TLR) genes of the innate immune system in humans. We have discovered that all the human TLR genes are responsive to chromosomal stress. Most are subject to activation of p53 by common anti-tumor agents leading to modulation in human primary and cancer cell lines and changing responses to cognate ligands. Global gene expression analyses revealed a group of 200 genes that exhibited a p53/ligand synergistic response to chemotherapeutic agents. Included were genes related to immune/inflammation processes. Furthermore, p53 ChIPseq studies, revealed that several of the human cytidine deaminase APOBEC3 genes in the innate immune that deal with retrotransposons and RNA virus infection are also subject to p53 transcriptional regulation. These observations together with discovery of other immune related p53 target genes provide new insights into the relationship between p53, environmental stress and immunity, and they suggest approaches for cancer. We have found that p53 can collaborate with NFkappaB to strongly alter several immune/inflammatory responses in primary lymphocytes and macrophages from healthy volunteers, tumor-conditioned macrophages (TAM-like) and tumor cells. Transcriptome analysis identified global cooperation with p53/NF-kappaB on expression of immune response genes, including chemokines and interleukins. Thus, the human p53 master master regulatory network extends to many stress responding components of the immune system.