Human Genes Affecting Chromosome Metabolism and Stress Response

National Institute Of Environmental Health Sciences

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Abstract

p53 is central to human DNA repair, damage checkpoints and many aspects of human biology. Most cancers are altered for p53 function. IDENTIFYING p53 TARGETS. Identifying the universe of p53 direct transcriptional targets is clinically relevant, especially since many of these may have therapeutic value. There is considerable variation in p53 dependent expression across targeted genes leading to differences in p53-mediated biological consequences, due in part to variation in target response element (RE) sequence. We have focused on RE functionality, i.e., the ability of REs to support transactivation by p53 in human cells in culture and ex vivo. We developed a rigorous method for reanalyzing all the raw data from the individual ChIP-seq studies and associated gene expression using a single analysis workflow, then combining information using a common set of criteria. Our approach revealed a large p53 genome-wide cistrome composed of >900 genes directly targeted by p53. CHARACTERIZATION OF THE P53 HUMAN CISTROME IN RESPONSE TO ENVIRONMENTAL STRESSORS. Using a common analysis pipeline, we analyzed 41 data sets from genome-wide ChIP-seq studies of which 16 have associated gene expression data, including our primary data in normal human lymphocytes. The resulting extensive analysis, accessible at the p53 Binding And Expression Resource (BAER) hub via the UCSC browser, provides a robust platform to characterize p53 binding throughout the human genome including its direct influence on gene expression (i.e., cistrome) and underlying mechanisms. This approach led to our identification of more than 700 novel p53 candidates. Using the p53 BAER hub together with our various gene expression datasets we have 1) expanded the p53 transcriptome in DNA repair and nucleic acid metabolism transactions in different human cancer and primary cells with different p53 functional status and 2) identified new autoregulatory loops in the p53 pathway through the analysis of the p53- interactome withp53-cistrome.THE p53 IMMUNE CISTROME. Through our meta-analysis of the p53 transcriptional network following stresses in many cell types, including lymphocytes, monocytes and blood derived macrophages from healthy participants treated with p53 activating drugs, we have identified a p53 Immune Associated Cistrome. These p53 direct targets, consists of 70 genes directly involved in cellular immune and inflammatory processes, including several interleukins, membrane receptors and various viral/bacterial restriction factors. Additionally, we found that activation of wild type p53 as well as many tumor-associated p53 mutants modulates and changes the expression profile of two major innate immune gene families, the TLR (Toll-like Receptor) family and the APOBEC3 (Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like type 3, A3s) family that are, respectively, pathogen sensors and sentinels against viral infections. We have described this p53 influence on immune responses as the p53/immune axis. THE INFLUENCE OF p53 ON HUMAN IMMUNE RESPONSES DURING VIRAL INFECTIONS. We demonstrated that a SNP (rs3761624) in a p53RE could lead to p53-dependent enhanced expression of TLR8, which is involved in virus detection. Not only did the TLR8 p53RE SNP affect the level of inflammatory responses when lymphocytes from healthy donors were challenged with the cognate ligand for this receptor but this SNP was also positively associated with the severity of respiratory syncytial virus (RSV) disease in infants. Using isogenic p53 proficient and p53 null cell lines we also found that activation of p53 by RSV leads to upregulation of several APOBEC3 gene family members that impact virus infectivity and led us to identify A3G as a novel restriction factor against RSV. We have expanded the p53-TLR8-immune axis concept to human immunodeficiency virus (HIV) infection with support that we obtained from the Office of AIDS Research, using an ex-vivo model of human healthy CD4+ T lymphocytes. In a blind design study, we are using the NIEHS Environmental Polymorphism Registry cohort, to recruit healthy participants on the basis of the TLR8 rs3761624 genotypes. Then isolated CD4+ T lymphocytes for each donor are pretreated with vehicle (DMSO) or p53 activating drug Nutlin and then infected with HIV. Consistent with our previous results, we detected the appearance of two clear groups, non-responsive and responsive for TLR8 expression after Nutlin treatment. For those samples where the induction of TLR8 expression is more than 2-fold, a 30-50% reduction in HIV infectivity was observed when compared to the donors with less than 2-fold or no TLR8 induction. Projecting from this work, we have proposed that the rs rs3761624 SNP, with a worldwide ethnic frequency varying from 25-75%, is a good candidate for a functional biomarker of differences in response to COVID19, including ethnicity and gender, and opens up additional approaches to treatments and targets. Based on our efforts with RSV, there are several directions that should be pursued. One immediate direction has been to determine the relationship between the SNP and severity in COVID-19 infected people. There are several opportunities for mechanistic studies as well as interventions. In addition, using CD4+ T lymphocytes of healthy people treated with Nutlin, we have identified around 30 well-known HIV-1 host restriction factors that are potential p53 transcriptional targets, including A3G, A3H, SAMHD1, MX1, TSG101 PRK and ZAP. In collaboration with Johns Hopkins School of Public Health, we identified 263 HIV-1 seroconverts in the Multicenter AIDS Cohort Study (MACS) previously genotyped for TP53 SNP rs1042522 and TLR8 SNP rs37646880, which is in high linkage disequilibrium with TLR8 rs3761624. A preliminary analysis showed that the TP53 codon 72 variant, may be associated with a lower risk of developing AIDS but the presence of both protective alleles (TP53 rs1042522=GG and TLR8 rs37646880=A) might convey an overall 65% reduction in the risk of AIDS. CANCER-ASSOCIATED p53 MUTANTS. With inclusion of immune response-related TLR and APOBEC3 genes into the p53 network, we also found that tumor-associated p53 mutants have the potential to modulate the transcriptional response of these genes,. suggesting that p53 tumor status might be an important factor in adjuvant therapy employing TLR and APOBEC3 pathways to treat cancer. 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 in cells treated (or untreated) with anticancer agents. We have identified several SL targets for two of the most frequent tumor-associated p53 mutants (R175H and R273H) in the presence of the anticancer drug etoposide as well as for the WT p53 and p53 null conditions. We found that ATR-reduced expression as well as pharmacological inhibition of ATR: 1) enhance etoposide sensitivity in p53 dysfunctional cancer cell lines., and 2) created a unique vulnerability of p53-deficient cell lines to agents targeting Top2. Overall, our findings suggest that a combination of etoposide treatment with functional inactivation of DNA repair in p53-defective cells could be used to enhance the therapeutic efficacy of this Top2 targeting agent. This project involves research on human coronavirus, COVID-19, SARS-coronavirus-2, SARS-cov-2, SARS-cov2, SARS-related coronavirus 2, Severe acute respiratory syndrome coronavirus 2, SARS-Associated Coronavirus, SARS-cov, or SARS-Related Coronavirus.