Transcription Factors TP53 Research Articles

Heterozygous knockout of Synaptotagmin13 phenocopies ALS features and TP53 activation in human motor neurons

Spinal motor neurons (MNs) represent a highly vulnerable cellular population, which is affected in fatal neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA). In this study, we show that the heterozygous loss of SYT13 is sufficient to trigger a neurodegenerative phenotype resembling those observed in ALS and SMA. SYT13+/− hiPSC-derived MNs displayed a progressive manifestation of typical neurodegenerative hallmarks such as loss of synaptic contacts and accumulation of aberrant aggregates. Moreover, analysis of the SYT13+/− transcriptome revealed a significant impairment in biological mechanisms involved in motoneuron specification and spinal cord differentiation. This transcriptional portrait also strikingly correlated with ALS signatures, displaying a significant convergence toward the expression of pro-apoptotic and pro-inflammatory genes, which are controlled by the transcription factor TP53. Our data show for the first time that the heterozygous loss of a single member of the synaptotagmin family, SYT13, is sufficient to trigger a series of abnormal alterations leading to MN sufferance, thus revealing novel insights into the selective vulnerability of this cell population.

The Role and Efficacy of JNK Inhibition in Inducing Lung Cancer Cell Death Depend on the Concentration of Cisplatin.

Toxicity and the emergence of resistance are the main challenges in cancer treatment. The optimal dose of cisplatin, one of the most widely used chemotherapeutic anticancer drugs, is currently being widely debated. Furthermore, the dose-dependent molecular mechanisms of its action are poorly understood. To assess the role of protein kinase JNK (cJun N-terminal kinase) signaling in lung cancer treatment, we combined small-molecule JNK inhibitors and cisplatin. Wild-type p53 (tumor suppressor transcription factor TP53) and mutated RAS-bearing lung adenocarcinoma cell line A549 was used as a model in our studies. Here, we demonstrate cisplatin concentration-dependent opposing roles of JNK in killing cancer cells: a cell-protective role at low cisplatin concentrations and an apoptosis-promoting (or neutral) role at high concentrations. Time- and dose-dependent activation of pro-survival protein kinase AKT and TP53 was shown, with similar activation dynamics in cells exposed to different (low and high) cisplatin concentrations. Selective inhibition of AKT and activation of TP53 (expression and phosphorylation) led to a decrease in cell survival, indicating their involvement in cisplatin-induced cell death regulation. The activation levels of TP53 and AKT in cisplatin-treated A549 cells after cotreatment with the JNK inhibitor SP600125 correlated with their role in regulating cell death. TP53 and AKT were proposed as signaling proteins mediating the outcome of JNK inhibition in A549 cells exposed to different concentrations of cisplatin. Our findings suggest that a combination of stress kinase JNK inhibition and low-dose cisplatin, together with manipulation of drug-induced signaling, could be considered as a promising treatment strategy for certain lung cancers.

Screening of drug targets for tuberculosis on the basis of transcription factor regulatory network and mRNA sequencing technology.

Tuberculosis is a worldwide epidemic disease, posing a serious threat to human health. To find effective drug action targets for Mycobacterium tuberculosis, differentially expressed genes in tuberculosis patients and healthy people were screened by mRNA sequencing in this study. A total of 556 differentially expressed genes in tuberculosis patients and healthy people were screened out by mRNA sequencing technology. 26 transcription factors and 66 corresponding target genes were screened out in the AnimalTFDB 3.0 database, and a transcription factor regulatory network was constructed. Three key transcription factors (TP53, KLF5 and GATA2) and one key gene (AKT1) were screened as new potential drug targets and diagnostic targets for tuberculosis by MCODE cluster analysis, and the key genes and key transcription factors were verified by RT-PCR. Finally, we constructed the and a key factor and KEGG signaling pathway regulatory network to clarify the possible molecular pathogenesis of tuberculosis. This study suggested M. tuberculosis may activate the AKT1 gene expression by regulating transcription factors TP53, KLF5, and GATA2, thus activating the B cell receptor signaling pathway to induce the infection and invasion of M. tuberculosis. AKT1, TP53, KLF5, and GATA2 can be used as new potential drug targets for tuberculosis.

Molecular mechanism of Pulmonary diseases caused by exposure to urban PM2.5 in Chengdu-Chongqing Economic Circle, China.

Chengdu-Chongqing Economic Circle (CD-CQ Economic Circle) is one of China's four major economic circles and five major urban agglomerations located in Southwest China's Sichuan Basin. The CD-CQ Economic Circle, with its strong economic development and dense population, suffers from severe PM2.5 pollution, which is known to cause chronic and acute respiratory ailments. This study examined the lung disease-related hub genes, functions, and pathways that are affected by PM2.5 in summer and winter in the two central megacities of Chengdu and Chongqing. PM2.5 frequently activates lung disease-associated hub genes, most notably the transcription factor TP53. TP53 interacts with the majority of lung disease-related genes and regulates important and commonly occurring biological functions and pathways, including gland development, aging, reactive oxygen species metabolic process, the response to oxygen levels, and fluid shear stress, among others. Thus, PM2.5 has been shown to target TP53 for regulating lung disease genes/functions/pathways, thereby influencing the occurrence and progression of lung illnesses. Notably, PM2.5 may be associated with small cell carcinoma of the lung due to the high number of lung disease genes, hub genes, critical functions, and pathways enriched in this kind of cancer. These findings shed fresh light on the molecular pathophysiology of PM2.5 pollution on the respiratory system in the CD-CQ Economic Circle and aid in the development of novel techniques for mitigating PM2.5 pollution-associated respiratory illness.

DNA hypermethylation modification promotes the development of hepatocellular carcinoma by depressing the tumor suppressor gene ZNF334

DNA methylation plays a pivotal role in the development and progression of tumors. However, studies focused on the dynamic changes of DNA methylation in the development of hepatocellular carcinoma (HCC) are rare. To systematically illustrate the dynamic DNA methylation alternation from premalignant to early-stage liver cancer with the same genetic background, this study enrolled 5 HBV-related patients preceded with liver cirrhosis, pathologically identified as early-stage HCC with dysplastic nodules. Liver fibrosis tissues, dysplastic nodules and early HCC tissues from these patients were used to measure DNA methylation. Here, we report significant differences in the DNA methylation spectrum among the three types of tissues. In the early stage of HCC, DNA hypermethylation of tumor suppressor genes is predominant. Additionally, DNA hypermethylation in the early stage of HCC changes the binding ability of transcription factor TP53 to the promoter of tumor suppressor gene ZNF334, and inhibits the expression of ZNF334 at the transcription level. Furthermore, through a series of in vivo and in vitro experiments, we have clarified the exacerbation effect of tumor suppressor gene ZNF334 deletion in the occurrence of HCC. Combined with clinical data, we found that the overall survival and relapse-free survival of patients with high ZNF334 expression are significantly longer. Thus, we partly elucidated a sequential alternation of DNA methylation modification during the occurrence of HCC, and clarified the biological function and regulatory mechanism of the tumor suppressor gene ZNF334, which is regulated by related DNA methylation sites. Our study provides a new target and clinical evidence for the early diagnosis and sheds light on the precise treatment of liver cancer.

Predicting the Effect of Variants of Unknown Significance in Molecular Tumor Boards with the VUS-Predict Pipeline.

Precision oncology utilizing molecular biomarkers for targeted therapies is one of the hopes to treat cancer. The availability of patient specific molecular profiling through next-generation sequencing, though, increases the amount of available data per patient to an extent that computational support is required to identify potential driver alterations for targeted therapies and rational decision-making in molecular tumor boards (MTBs). For some genetic variants evidence-based drug recommendations are available in public databases, but for the majority, the variants of unknown significance (VUS), this clinical information is missing. Additionally, for most of these variants no information about the functional impact on the protein is accessible. To acquire maximal functional evidence for VUS, the VUS-Predict pipeline collects estimations about the effect of a VUS by integrating multiple pre-existing tools. Pre-existing tools implement different approaches for their predictions, which are summarized by our newly developed tool with a common score and classification in neutral or deleterious variants. The primary tools are chosen based on their sensitivity and specificity on well-known variants of the transcription factor TP53. Resulting negative and positive predictive values are used to calibrate the VUS-Predict pipeline. Further, the pipeline is evaluated using data from public cancer databases and cases of the MTB in Göttingen, both also in comparison with the ensemble method REVEL. The results show that VUS-Predict has clear advantages in a clinical setting due to clear and traceable predictions. In particular, VUS outperforms REVEL in the real-life setting of a MTB. Likewise, an evaluation on variants of public cancer databases confirms the good results of VUS-Predict and shows the need for a reliable gold standard and unambiguous results of the tools under test.

CBIO-22. p53 GAIN-OF-FUNCTION MUTATIONS DRIVE SEX SPECIFIC EFFECTS ON GLIOMA TUMORIGENESIS

Abstract Sex differences in malignant brain tumors are well-established: Males exhibit greater incidence and poorer survival. Understanding the biology behind these sex differences requires investigation of the pathways known to drive gliomagenesis. The transcription factor TP53 (p53) is one of the most commonly mutated genes in glioblastoma. Most p53 mutations are missense mutations in the DNA-binding domain that lead to the expression of a full length mutant p53 protein. These mutations can endow p53 with oncogenic gains-of-function through aberrant DNA binding and regulation of noncanonical cancer-promoting target genes. Previously, we analyzed patient mutation data and identified six p53 mutations with sex differences in prevalence. In this study, we developed an in vitro mutant p53 glioma model to investigate the sex specific effects of three p53 point mutations: R175H, Y205C, and Y220C (Mm R172H, Y202C, and Y217C respectively). Male and female astrocytes isolated from p53flox/- mouse pups were transduced with a retrovirus expressing mutant p53, followed by a lentivirus expressing CRE recombinase to remove the endogenous WTp53. We then assessed cell proliferation, clonogenicity, and in vivo tumorigenesis in these cells. All three mutations assayed displayed sex differences in proliferation, with male cells overexpressing p53:Y202C and p53:Y217C growing faster than female cells, and female cells overexpressing p53:R172H growing faster than male cells. Male Y202C and Y217C expressing astrocytes also exhibited a trend toward greater clonogenicity compared to female astrocytes. This observation is supported by higher expression of the stem cell markers SOX2 and NESTIN in the male cells. We performed parallel flank injections of male and female astrocytes expressing each mutation or p53 KO. Only male astrocytes expressing p53:Y202C or p53:Y217C and female astrocytes expressing p53:R172H mutation were able to form tumors in vivo. Together, these data support a sex specific gain-of-function phenotype for three different p53 mutations observed in glioma.

Tp53 Suppression Promotes Cardiomyocyte Proliferation during Zebrafish Heart Regeneration

SUMMARYZebrafish regenerate heart muscle through division of pre-existing cardiomyocytes. To discover underlying regulation, we assess transcriptome datasets for dynamic gene networks during heart regeneration and identify suppression of genes associated with the transcription factor Tp53. Cardiac damage leads to fluctuation of Tp53 protein levels, concomitant with induced expression of its central negative regulator, mdm2, in regenerating cardiomyocytes. Zebrafish lacking functional Tp53 display increased indicators of cardiomyocyte proliferation during regeneration, whereas transgenic Mdm2 blockade inhibits injury-induced cardiomyocyte proliferation. Induced myocardial overexpression of the mitogenic factors Nrg1 or Vegfaa in the absence of injury also upregulates mdm2 and suppresses Tp53 levels, and tp53 mutations augment the mitogenic effects of Nrg1. mdm2 induction is spatiotemporally associated with markers of de-differentiation in injury and growth contexts, suggesting a broad role in cardiogenesis. Our findings reveal myocardial Tp53 suppression by mitogen-induced Mdm2 as a regulatory component of innate cardiac regeneration.

Molecular mechanisms of resistance to bovine mastitis

Identification of disease-causing genes that underlie complex traits, such as susceptibility to disease, is the primary goal of genetic and biomedical researches. Bovine mastitis is the most prevalent disease with substantial economic losses and animal welfare issues in dairy industry. Quantitative trait loci (QTLs) and single-nucleotide polymorphism or gene association data as biomarkers were used in the selection programs in this industry. In the current microarray-based study, the gene expression profiles of cows selected for high or low mastitis susceptibility affected by a specific QTL on Bos taurus autosome 18 (BTA18) with induced E. coli mastitis in acute phase (time point 24h) were used to perform functional genomics. The results of a functional enrichment analysis showed that the Q genotype (favorable allele) helps to correct suppression of the metabolic process during infection and help to moderate signaling pathways related to immune response and inflammation. The most important pathways suppressed by Q genotype are related to triggering the inflammatory and immune responses which cause moderate responses to infection. Furthermore, the pathways related to growth, cell proliferation and reproductive process enriched by down-regulated expressed genes. The results of network analysis showed that transcription factors TP53, SP1 ligands such as INS, IL1B, IFNG, TGFB1, EGF and protein kinase MAPK1 are the common regulatory factors in the constructed networks.These findings regardless of revealing the new biological process of gene regulatory network analysis of differentially expressed genes (DE)s in bovine mastitis, could also introduce the important key regulatory elements in resistance to mastitis.

TP53 mediated miR-3647-5p prevents progression of cervical carcinoma by targeting AGR2.

Previous studies have shown that miRNAs involved in a number of biological processes, such as cell growth, development, differentiation, and apoptosis. The dysregulation of miRNA expression is associated with various diseases, including cervical cancer. However, the involvement of miR‐3647‐5p in the progression of tumors is unclear. In this study, we confirmed that miR‐3647‐5p was down‐regulated during cervical carcinogenesis and development, which was positively correlated with the prognosis of patients with cervical cancer. In addition, our study showed that miR‐3647‐5p can inhibit the proliferation of cervical cancer cells and promote apoptosis, suggesting that miR‐3647‐5p is involved in the development of cervical cancer as a tumor suppressor gene. Furthermore, we found that transcription factor TP53 could promote the expression of miR‐3647‐5p, suggesting that the dysfunction of miR‐3647‐5p in cervical cancer may be related to TP53. In addition, we also found that miR‐3647‐5p can inhibit the proliferation of cervical cancer cells and promote apoptosis by targeting AGR2. In summary, our research reveals that transcription factor TP53 promotes the expression of miR‐3647‐5p, while up‐regulated miR‐3647‐5p targets AGR2, inhibiting cervical cancer cell proliferation and promoting apoptosis. Our study reveals the mechanism of TP53/miR‐3647‐5p/AGR2 axis in cervical cancer, which may be useful for targeted therapy of cervical cancer.

Experimental Data-Mining Analyses Reveal New Roles of Low-Intensity Ultrasound in Differentiating Cell Death Regulatome in Cancer and Non-cancer Cells via Potential Modulation of Chromatin Long-Range Interactions.

Background: The mechanisms underlying low intensity ultrasound (LIUS) mediated suppression of inflammation and tumorigenesis remain poorly determined.Methods: We used microarray datasets from NCBI GEO Dataset databases and conducted a comprehensive data mining analyses, where we studied the gene expression of 299 cell death regulators that regulate 13 different cell death types (cell death regulatome) in cells treated with LIUS.Results: We made the following findings: (1) LIUS exerts a profound effect on the expression of cell death regulatome in cancer cells and non-cancer cells. Of note, LIUS has the tendency to downregulate the gene expression of cell death regulators in non-cancer cells. Most of the cell death regulator genes downregulated by LIUS in non-cancer cells are responsible for mediating inflammatory signaling pathways; (2) LIUS activates different cell death transcription factors in cancer and non-cancer cells. Transcription factors TP-53 and SRF- were induced by LIUS exposure in cancer cells and non-cancer cells, respectively; (3) As two well-accepted mechanisms of LIUS, mild hyperthermia and oscillatory shear stress induce changes in the expression of cell death regulators, therefore, may be responsible for inducing LIUS mediated changes in gene expression patterns of cell death regulators in cells; (4) LIUS exposure may change the redox status of the cells. LIUS may induce more of antioxidant effects in non-cancer cells compared to cancer cells; and (5) The genes modulated by LIUS in cancer cells have distinct chromatin long range interaction (CLRI) patterns to that of non-cancer cells.Conclusions: Our analysis suggests novel molecular mechanisms that may be utilized by LIUS to induce tumor suppression and inflammation inhibition. Our findings may lead to development of new treatment protocols for cancers and chronic inflammation.

Disturbance of gene expression in primary human hepatocytes by hepatotoxic pyrrolizidine alkaloids: A whole genome transcriptome analysis

1,2-unsaturated pyrrolizidine alkaloids (PA) are plant metabolites predominantly occurring in the plant families Asteraceae and Boraginaceae. Acute and chronic PA poisoning causes severe hepatotoxicity. So far, the molecular mechanisms of PA toxicity are not well understood. To analyze its mode of action, primary human hepatocytes were exposed to a non-cytotoxic dose of 100μM of four structurally different PA: echimidine, heliotrine, senecionine, senkirkine. Changes in mRNA expression were analyzed by a whole genome microarray. Employing cut-off values with a |fold change| of 2 and a q-value of 0.01, data analysis revealed numerous changes in gene expression. In total, 4556, 1806, 3406 and 8623 genes were regulated by echimidine, heliotrine, senecione and senkirkine, respectively. 1304 genes were identified as commonly regulated. PA affected pathways related to cell cycle regulation, cell death and cancer development. The transcription factors TP53, MYC, NFκB and NUPR1 were predicted to be activated upon PA treatment. Furthermore, gene expression data showed a considerable interference with lipid metabolism and bile acid flow. The associated transcription factors FXR, LXR, SREBF1/2, and PPARα/γ/δ were predicted to be inhibited.In conclusion, though structurally different, all four PA significantly regulated a great number of genes in common. This proposes similar molecular mechanisms, although the extent seems to differ between the analyzed PA as reflected by the potential hepatotoxicity and individual PA structure.

Transcription factors TP53 and SP1 and the osteogenic differentiation of dental stem cells

Dental follicle is a loose connective tissue that surrounds the developing tooth. Dental follicle cells (DFCs) have a promising potential for tissue engineering applications including periodontal and bone regeneration. However, little is known about the molecular mechanisms underlying osteogenic differentiation. In a previous study we detected that more than 35% of genes that are regulated during osteogenic differentiation of DFCs have promoter binding sites for the transcription factors TP53 and SP1. However, the role of these transcription factors in dental stem cells is still unknown. We hypothesize that both factors influence the processes of cell proliferation and differentiation in dental stem cells. Therefore, we transiently transfected DFCs and dental pulp stem cells (SHED; Stem cells from human exfoliated decidiuous teeth) with expression vectors for these transcription factors. After overexpression of SP1 and TP53, SP1 influenced cell proliferation and TP53 osteogenic differentiation in both dental cell types. The effects on cell proliferation and differentiation were less pronounced after siRNA mediated silencing of TP53 and SP1. This indicates that the effects we observed after TP53 and SP1 overexpression are indirect and subject of complex regulation. Interestingly, upregulated biological processes in DFCs after TP53-overexpression resemble the downregulated biological processes in SHED after SP1-overexpression. Here, regulated processes are involved in cell motility, wound healing and programmed cell death. In conclusion, our study demonstrates that SP1 and TP53 influence cell proliferation and differentiation and similar biological processes in both SHED and DFCs.

Dental Cell Biology, Biochips and Open Access

Dental cell biology investigates the development and regeneration of dental tissues and biochips are therefore important tools in an advanced molecular research. Here, transcriptomics and proteomics are used for the evaluation of genome wide gene expression profiles of cultivated dental (stem) cells after various treatments or for a comparison of different dental stem cell types or developmental stages of dental tissues. At the beginning of a new project, biochips can be very useful and the most important empirical tool to get first hints for ad hoc hypotheses or to set up new hypotheses for experiments in the lab. On the other hand, however, it is very unlikely that biochip data answer directly a specific question; the data received from biochips require careful analyses by cell biologists to estimate their biological importance. For example after a specific treatment of dental stem cells someone obtain all expressed genes at a given time point without an analysis of the meaning for the biology of the cell. To get for example interesting candidate genes for being key regulators in the process of interest a lot of theoretical work with and without bioinformatics programs is required and after that, the lab work begins to prove the biological significance of these genes. One example makes this obvious: In a study of the dental follicle cell proteome, we found that the transcription factors TP53 and SP1 have promoter binding sites for 35% of all regulated during the osteogenic differentiation [1]. In a microarray study, we found that ZBTB16 is an up-regulated transcription factor during osteogenic differentiation [2]. We postulate that these transcription factors are involved in the process of differentiation. Although we could produce these new hypotheses only by the use of biochips and bioinformatics, new experiments were required to prove their biological significance. In the lab we showed that TP53 and ZBTB16, but not SP1 play roles in the molecular process of osteogenic differentiation in dental stem cells.

P53 binds preferentially to genomic regions with high DNA-encoded nucleosome occupancy

The human transcription factor TP53 is a pivotal roadblock against cancer. A key unresolved question is how the p53 protein selects its genomic binding sites in vivo out of a large pool of potential consensus sites. We hypothesized that chromatin may play a significant role in this site-selection process. To test this, we used a custom DNA microarray to measure p53 binding at approximately 2000 sites predicted to possess high-sequence specificity, and identified both strongly bound and weakly bound sites. When placed within a plasmid, weakly bound sites become p53 responsive and regain p53 binding when stably integrated into random genomic locations. Notably, strongly bound sites reside preferentially within genomic regions whose DNA sequence is predicted to encode relatively high intrinsic nucleosome occupancy. Using in vivo nucleosome occupancy measurements under conditions where p53 is inactive, we experimentally confirmed this prediction. Furthermore, upon p53 activation, nucleosomes are partially displaced from a relatively broad region surrounding the bound p53 sites, and this displacement is rapidly reversed upon inactivation of p53. Thus, in contrast to the general assumption that transcription-factor binding is preferred in sites that have low nucleosome occupancy prior to factor activation, we find that p53 binding occurs preferentially within a chromatin context of high intrinsic nucleosome occupancy.

Proteomic analysis of osteogenic differentiation of dental follicle precursor cells

Recently, there has been an increased interest in unravelling the molecular mechanisms and cellular pathways controlling the differentiation and proliferation of human stem cell lines. Proteome analysis has proven to be an effective approach to comprehensive analysis of the regulatory network of differentiation. In the present study we applied 2-DE combined with capillary-LC-MS/MS analysis to profile differentially regulated proteins upon differentiation of dental follicle precursor cells (DFPCs). Out of 115 differentially regulated proteins, glutamine synthetase, lysosomal proteinase cathepsin B proteins, plastin 3 T-isoform, beta-actin, superoxide dismutases, and transgelin were found to be highly up-regulated, whereas cofilin-1, pro-alpha 1 collagen, destrin, prolyl 4-hydrolase and dihydrolipoamide dehydrogenase were found to be highly down-regulated. The group of up-regulated proteins is associated with actin-bundling and defence against oxidative cellular stress, whereas down-regulated proteins were associated with collagen biosynthesis. Bioinformatic analyses of the entire data set confirmed these findings that represent significant steps towards the understanding of DFPC differentiation. The bioinformatic analyses suggest that proteins associated with cell cycle progression and protein metabolism were down-regulated and proteins involved in catabolism, cell motility and biological quality were up-regulated. These results display the general physiological state of DFPCs before and after osteogenic differentiation. We also identified regulatory proteins, such as the transcription factors TP53 and Sp-1, associated with the differentiation process. Further studies will investigate the impact of identified regulatory proteins for cell proliferation and osteogenic differentiation in DFPCs.

Induction of gene expression as a monitor of exposure to ionizing radiation.

The complex molecular responses to genotoxic stress are mediated by a variety of regulatory pathways. The transcription factor TP53 plays a central role in the cellular response to DNA-damaging agents such as ionizing radiation, but other pathways also play important roles. In addition, differences in radiation quality, such as the exposure to high-LET radiation that occurs during space travel, may influence the pattern of responses. The premise is developed that stress gene responses can be employed as molecular markers for radiation exposure using a combination of informatics and functional genomics approaches. Published studies from our laboratory have already demonstrated such transcriptional responses with doses of gamma rays as low as 2 cGy, and in peripheral blood lymphocytes (PBLs) irradiated ex vivo with doses as low as 20 cGy. We have also found several genes elevated in vivo 24 h after whole-body irradiation of mice with 20 cGy. Such studies should provide insight into the molecular responses to physiologically relevant doses, which cannot necessarily be extrapolated from high-dose studies. In addition, ongoing experiments are identifying large numbers of potential biomarkers using microarray hybridization and various irradiation protocols including expression at different times after exposure to low- and high-LET radiation. Computation-intensive informatics analysis methods are also being developed for management of the complex gene expression profiles resulting from these experiments. With further development of these approaches, it may be feasible to monitor changes in gene expression after low-dose radiation exposure and other physiological stresses that may be encountered during manned space flight, such as the planned mission to Mars.