Beyond initiation: Human P53 plays role in transcription elongation

The P53 tumor suppressor regulates the transcrip-tion initiation of selected genes by binding to specif-ic DNA sequences at their promoters. Here we report a novel role of P53 in transcription elongation in human cells. Upon transcription elongation block-age P53 is associated with genes, which have not been reported earlier as its direct targets. P53 could be co-immunoprecipitated with active forms of RPB1, highlighting its association with the elongat-ing RNAPII. During normal transcription cycle, P53 and RPB1 localized at distinct regions of selected non-canonical P53-target genes and this pattern of localization was changed upon transcription elonga-tion block. Additionally, transcription elongation block induced the ubiquitylation and the proteo-somal degradation of RPB1. Finally, we showed that the transcription block induced RPB1 degradation is mediated by P53. Our results reveal a novel role of P53 in human cells during transcription elongation blockage that might serve to facilitate the removal of RNAPII from DNA

Human p53 interacts with the elongating RNAPII complex and is required for the release of actinomycin D induced transcription blockage

The p53 tumour suppressor regulates the transcription initiation of selected genes by binding to specific DNA sequences at their promoters. Here we report a novel role of p53 in transcription elongation in human cells. Our data demonstrate that upon transcription elongation blockage, p53 is associated with genes that have not been reported as its direct targets. p53 could be co-immunoprecipitated with active forms of DNA-directed RNA polymerase II subunit 1 (RPB1), highlighting its association with the elongating RNA polymerase II. During a normal transcription cycle, p53 and RPB1 are localised at distinct regions of selected non-canonical p53 target genes and this pattern of localisation was changed upon blockage of transcription elongation. Additionally, transcription elongation blockage induced the proteasomal degradation of RPB1. Our results reveal a novel role of p53 in human cells during transcription elongation blockage that may facilitate the removal of RNA polymerase II from DNA

Identifying Suitable Reference Gene Candidates for Quantification of DNA Damage-Induced Cellular Responses in Human U2OS Cell Culture System

DNA repair pathways trigger robust downstream responses, making it challenging to select suitable reference genes for comparative studies. In this study, our goal was to identify the most suitable housekeeping genes to perform comparable molecular analyses for DNA damage-related studies. Choosing the most applicable reference genes is important in any kind of target gene expression-related quantitative study, since using the housekeeping genes improperly may result in false data interpretation and inaccurate conclusions. We evaluated the expressional changes of eight well-known housekeeping genes (i.e., 18S rRNA, B2M, eEF1α1, GAPDH, GUSB, HPRT1, PPIA, and TBP) following treatment with the DNA-damaging agents that are most frequently used: ultraviolet B (UVB) non-ionizing irradiation, neocarzinostatin (NCS), and actinomycin D (ActD). To reveal the significant changes in the expression of each gene and to determine which appear to be the most acceptable ones for normalization of real-time quantitative polymerase chain reaction (RT-qPCR) data, comparative and statistical algorithms (such as absolute quantification, Wilcoxon Rank Sum Test, and independent samples T-test) were conducted. Our findings clearly demonstrate that the genes commonly employed as reference candidates exhibit substantial expression variability, and therefore, careful consideration must be taken when designing the experimental setup for an accurate and reproducible normalization of RT-qPCR data. We used the U2OS cell line since it is generally accepted and used in the field of DNA repair to study DNA damage-induced cellular responses. Based on our current data in U2OS cells, we suggest using 18S rRNA, eEF1α1, GAPDH, GUSB, and HPRT1 genes for UVB-induced DNA damage-related studies. B2M, HPRT1, and TBP genes are recommended for NCS treatment, while 18S rRNA, B2M, and PPIA genes can be used as suitable internal controls in RT-qPCR experiments for ActD treatment. In summary, this is the first systematic study using a U2OS cell culture system that offers convincing evidence for housekeeping gene selection following treatment with various DNA-damaging agents. Here, we unravel an indispensable issue for performing and assessing trustworthy DNA damage-related differential gene expressional analyses, and we create a “zero set” of potential reference gene candidates

Drosophila as a new tool to study the chromatin structural changes activated by DNA damages

In eukaryotic cells, any processes which involve DNA have to take place in the context of chromatin structure, which affects the probability of the dam-aging agents to cause DNA breaks and the recruit-ment of the repair proteins. The improper repair or persistence of breaks leads to genome instability, which could result in tumor formation. Our goal is to understand what makes cells able to recognize the appearance of DNA break and how the chromatin structure could change around the break. The an-swers to these questions will provide information on whether specific chromatin structures predispose sites for DNA break and whether memory of previ-ous break is retained in the chromatin structure. We started to setup human cell culture-based and Drosophila experimental systems by which we could study how unique histone post-translation-al modifications (PTMs) could affect the DNA repair. We take advantage of the model system where we delete the endogenous histone cluster and we substitute it with mutant histones which permits or mimics unique histone PTMs. We have already started to mutate histone genes and screen 50 different histone PTMs. We will use these flies to check the DNA repair kinetics in those animals which consist of only the mutated histones. Using the Drosophila and the human cell culture based system we have already identified new H3 and H4 histone PTM candidates that play role in chromo-somal rearrangement which could influence the DNA repair processes. The system developed in our laboratory would help in understanding the mecha-nisms, which give rise to frequent chromosomal break points often detected in tumors. Progress in integrating the chromatin dimension in DNA repair will help to understand how DNA damage may impact on genome stability. These results would also help identifying new key targets in DNA dam-age repair and the final goal of the project is to find potential biomarkers which could be used in anti-cancer therapies. Supported by OTKA-PD [112118] and the János Bolyai Research Scholarship of the Hungarian Academy of Sciences

Small extracellular vesicles convey the stress-induced adaptive responses of melanoma cells

Exosomes are small extracellular vesicles (sEVs), playing a crucial role in the intercellular communication in physiological as well as pathological processes. Here, we aimed to study whether the melanoma-derived sEV-mediated communication could adapt to microenvironmental stresses. We compared B16F1 cell-derived sEVs released under normal and stress conditions, including cytostatic, heat and oxidative stress. The miRNome and proteome showed substantial differences across the sEV groups and bioinformatics analysis of the obtained data by the Ingenuity Pathway Analysis also revealed significant functional differences. The in silico predicted functional alterations of sEVs were validated by in vitro assays. For instance, melanoma-derived sEVs elicited by oxidative stress increased Ki-67 expression of mesenchymal stem cells (MSCs); cytostatic stress-resulted sEVs facilitated melanoma cell migration; all sEV groups supported microtissue generation of MSC-B16F1 co-cultures in a 3D tumour matrix model. Based on this study, we concluded that (i) molecular patterns of tumour-derived sEVs, dictated by the microenvironmental conditions, resulted in specific response patterns in the recipient cells; (ii) in silico analyses could be useful tools to predict different stress responses; (iii) alteration of the sEV-mediated communication of tumour cells might be a therapy-induced host response, with a potential influence on treatment efficacy. © 2019, The Author(s)

Predictive Potential of RNA Polymerase B (II) Subunit 1 (RPB1) Cytoplasmic Aggregation for Neoadjuvant Chemotherapy Failure

We aimed to investigate the contribution of co-translational protein aggregation to the chemotherapy resistance of tumor cells. Increased co-translational protein aggregation reflects altered translation regulation that may have the potential to buffer transcription under genotoxic stress. As an indicator for such an event, we followed the cytoplasmic aggregation of RPB1, the aggregation-prone largest subunit of RNA polymerase II, in biopsy samples taken from patients with invasive carcinoma of no special type. RPB1 frequently aggregates co-translationally in the absence of proper HSP90 chaperone function or in ribosome mutant cells as revealed formerly in yeast. We found that cytoplasmic foci of RPB1 occur in larger sizes in tumors that showed no regression after therapy. Based on these results, we propose that monitoring the cytoplasmic aggregation of RPB1 may be suitable for determining—from biopsy samples taken before treatment—the effectiveness of neoadjuvant chemotherapy