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.

BC-miR: Monitoring Breast Cancer-Related miRNA Profile in Blood Sera-A Prosperous Approach for Tumor Detection.

Breast cancer is the most frequent cancer with a high fatality rate amongst women worldwide. Diagnosing at an early stage is challenging, and due to the limitations of the currently used techniques, including mammography and imaging diagnostics, it still remains unascertained. Serum biomarkers can be a solution for this as they can be isolated in a less painful, more cost-effective, and minimally invasive manner. In this study, we shed light on the relevant role of multiple microRNAs (miRNAs) as potential biomarkers in breast cancer diagnosis. We monitored the expressional changes of 15 pre-selected miRNAs in a large cohort, including 65 patients with breast cancer and 42 healthy individuals. We performed thorough statistical analyses on the cohort sample set and determined the diagnostic accuracy of individual and multiple miRNAs. Our study reveals a potential improvement in diagnostics by implicating the monitoring of miR-15a+miR-16+miR-221 expression in breast cancer management.

The role of p53 in the DNA damage-related ubiquitylation of S2P RNAPII.

DNA double-strand breaks are one of the most deleterious lesions for the cells, therefore understanding the macromolecular interactions of the DNA repair-related mechanisms is essential. DNA damage triggers transcription silencing at the damage site, leading to the removal of the elongating RNA polymerase II (S2P RNAPII) from this locus, which provides accessibility for the repair factors to the lesion. We previously demonstrated that following transcription block, p53 plays a pivotal role in transcription elongation by interacting with S2P RNAPII. In the current study, we reveal that p53 is involved in the fine-tune regulation of S2P RNAPII ubiquitylation. Furthermore, we emphasize the potential role of p53 in delaying the premature ubiquitylation and the subsequent chromatin removal of S2P RNAPII as a response to transcription block.

The clinical significance of epigenetic and RNAPII variabilities occurring in clear cell renal cell carcinoma as a potential prognostic marker.

Patients diagnosed with clear cell renal cell carcinoma (ccRCC) have poor prognosis for recurrence and approximately 30-40% of them will later develop metastases. For this reason, the appropriate diagnosis and the more detailed molecular characterisation of the primary tumour, including its susceptibility to metastasis, are crucial to select the proper adjuvant therapy by which the most prosperous outcome can be achieved. Nowadays, clinicopathological variables are used for classification of the tumours. Apart from these, molecular biomarkers are also necessary to improve risk classification, which would be the most beneficial amongst modern adjuvant therapies. As a potential molecular biomarker, to follow the transcriptional kinetics in ccRCC patients (n=30), we analysed epigenetic changes (γH2A.X, H3K4me3, and H3K9me3) and the alterations in the level of RNA polymerase II (RNAPII) by immunohistochemical staining on dissected tissue sections. The variabilities between the tumorous and non-tumorous parts of the tissue were detected using quantitative image analysis by monitoring 30 cells from different positions of either the tumorous or the non-tumorous part of the tissue sections. Data obtained from the analyses were used to identify potential prognostic features and to associate them with the progression. These markers might have a value to predict patient outcomes based on their individual cellular background. These results also support that detection of any alteration in the level of H3K4me3, H3K9me3, and γH2A.X can account for valuable information for presuming the progression of ccRCC and the clinical benefits to select the most efficient personalised therapy.

Usp5, Usp34, and Otu1 deubiquitylases mediate DNA repair in Drosophila melanogaster.

Ubiquitylation is critical for preventing aberrant DNA repair and for efficient maintenance of genome stability. As deubiquitylases (DUBs) counteract ubiquitylation, they must have a great influence on many biological processes, including DNA damage response. To elucidate the role of DUBs in DNA repair in Drosophila melanogaster, systematic siRNA screening was applied to identify DUBs with a reduced survival rate following exposure to ultraviolet and X-ray radiations. As a secondary validation, we applied the direct repeat (DR)-white reporter system with which we induced site-specific DSBs and affirmed the importance of the DUBs Ovarian tumor domain-containing deubiquitinating enzyme 1 (Otu1), Ubiquitin carboxyl-terminal hydrolase 5 (Usp5), and Ubiquitin carboxyl-terminal hydrolase 34 (Usp34) in DSB repair pathways using Drosophila. Our results indicate that the loss of Otu1 and Usp5 induces strong position effect variegation in Drosophila eye following I-SceI-induced DSB deployment. Otu1 and Usp5 are essential in DNA damage-induced cellular response, and both DUBs are required for the fine-tuned regulation of the non-homologous end joining pathway. Furthermore, the Drosophila DR-white assay demonstrated that homologous recombination does not occur in the absence of Usp34, indicating an indispensable role of Usp34 in this process.

Systematic overview on the most widespread techniques for inducing and visualizing the DNA double-strand breaks.

DNA double-strand breaks (DSBs) are one of the most frequent causes of initiating cancerous malformations, therefore, to reduce the risk, cells have developed sophisticated DNA repair mechanisms. These pathways ensure proper cellular function and genome integrity. However, any alteration or malfunction during DNA repair can influence cellular homeostasis, as improper recognition of the DNA damage or dysregulation of the repair process can lead to genome instability. Several powerful methods have been established to extend our current knowledge in the field of DNA repair. For this reason, in this review, we focus on the methods used to study DSB repair, and we summarize the advantages and disadvantages of the most commonly used techniques currently available for the site-specific induction of DSBs and the subsequent tracking of the repair processes in human cells. We highlight methods that are suitable for site-specific DSB induction (by restriction endonucleases, CRISPR-mediated DSB induction and laser microirradiation) as well as approaches [e.g., fluorescence-, confocal- and super-resolution microscopy, chromatin immunoprecipitation (ChIP), DSB-labeling and sequencing techniques] to visualize and follow the kinetics of DSB repair.

CDKL5 kinase controls transcription-coupled responses to DNA damage.

Mutations in the gene encoding the CDKL5 kinase are among the most common genetic causes of childhood epilepsy and can also give rise to the severe neurodevelopmental condition CDD (CDKL5 deficiency disorder). Despite its importance for human health, the phosphorylation targets and cellular roles of CDKL5 are poorly understood, especially in the cell nucleus. Here, we report that CDKL5 is recruited to sites of DNA damage in actively transcribed regions of the nucleus. A quantitative phosphoproteomic screen for nuclear CDKL5 substrates reveals a network of transcriptional regulators including Elongin A (ELOA), phosphorylated on a specific CDKL5 consensus motif. Recruitment of CDKL5 and ELOA to damaged DNA, and subsequent phosphorylation of ELOA, requires both active transcription and the synthesis of poly(ADP-ribose) (PAR), to which CDKL5 can bind. Critically, CDKL5 kinase activity is essential for the transcriptional silencing of genes induced by DNA double-strand breaks. Thus, CDKL5 is a DNA damage-sensing, PAR-controlled transcriptional modulator, a finding with implications for understanding the molecular basis of CDKL5-related diseases.

SerpinB10, a Serine Protease Inhibitor, Is Implicated in UV-Induced Cellular Response.

UV-induced DNA damage response and repair are extensively studied processes, as any malfunction in these pathways contributes to the activation of tumorigenesis. Although several proteins involved in these cellular mechanisms have been described, the entire repair cascade has remained unexplored. To identify new players in UV-induced repair, we performed a microarray screen, in which we found SerpinB10 (SPB10, Bomapin) as one of the most dramatically upregulated genes following UV irradiation. Here, we demonstrated that an increased mRNA level of SPB10 is a general cellular response following UV irradiation regardless of the cell type. We showed that although SPB10 is implicated in the UV-induced cellular response, it has no indispensable function in cell survival upon UV irradiation. Nonetheless, we revealed that SPB10 might be involved in delaying the duration of DNA repair in interphase and also in S-phase cells. Additionally, we also highlighted the interaction between SPB10 and H3. Based on our results, it seems that SPB10 protein is implicated in UV-induced stress as a "quality control protein", presumably by slowing down the repair process.

Ubiquitylation-Mediated Fine-Tuning of DNA Double-Strand Break Repair.

The proper function of DNA repair is indispensable for eukaryotic cells since accumulation of DNA damages leads to genome instability and is a major cause of oncogenesis. Ubiquitylation and deubiquitylation play a pivotal role in the precise regulation of DNA repair pathways by coordinating the recruitment and removal of repair proteins at the damaged site. Here, we summarize the most important post-translational modifications (PTMs) involved in DNA double-strand break repair. Although we highlight the most relevant PTMs, we focus principally on ubiquitylation-related processes since these are the most robust regulatory pathways among those of DNA repair.

Emerging Roles of Post-Translational Modifications in Nucleotide Excision Repair.

Nucleotide excision repair (NER) is a versatile DNA repair pathway which can be activated in response to a broad spectrum of UV-induced DNA damage, such as bulky adducts, including cyclobutane-pyrimidine dimers (CPDs) and 6-4 photoproducts (6-4PPs). Based on the genomic position of the lesion, two sub-pathways can be defined: (I) global genomic NER (GG-NER), involved in the ablation of damage throughout the whole genome regardless of the transcription activity of the damaged DNA locus, and (II) transcription-coupled NER (TC-NER), activated at DNA regions where RNAPII-mediated transcription takes place. These processes are tightly regulated by coordinated mechanisms, including post-translational modifications (PTMs). The fine-tuning modulation of the balance between the proteins, responsible for PTMs, is essential to maintain genome integrity and to prevent tumorigenesis. In this review, apart from the other substantial PTMs (SUMOylation, PARylation) related to NER, we principally focus on reversible ubiquitylation, which involves E3 ubiquitin ligase and deubiquitylase (DUB) enzymes responsible for the spatiotemporally precise regulation of NER.

PARylation During Transcription: Insights into the Fine-Tuning Mechanism and Regulation.

Transcription is a multistep, tightly regulated process. During transcription initiation, promoter recognition and pre-initiation complex (PIC) formation take place, in which dynamic recruitment or exchange of transcription activators occur. The precise coordination of the recruitment and removal of transcription factors, as well as chromatin structural changes, are mediated by post-translational modifications (PTMs). Poly(ADP-ribose) polymerases (PARPs) are key players in this process, since they can modulate DNA-binding activities of specific transcription factors through poly-ADP-ribosylation (PARylation). PARylation can regulate the transcription at three different levels: (1) by directly affecting the recruitment of specific transcription factors, (2) by triggering chromatin structural changes during initiation and as a response to cellular stresses, or (3) by post-transcriptionally modulating the stability and degradation of specific mRNAs. In this review, we principally focus on these steps and summarise the recent findings, demonstrating the mechanisms through which PARylation plays a potential regulatory role during transcription and DNA repair.

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.