A mechanism for oxidative damage repair at gene regulatory elements.

Oxidative genome damage is an unavoidable consequence of cellular metabolism. It arises at gene regulatory elements by epigenetic demethylation during transcriptional activation1,2. Here we show that promoters are protected from oxidative damage via a process mediated by the nuclear mitotic apparatus protein NuMA (also known as NUMA1). NuMA exhibits genomic occupancy approximately 100 bp around transcription start sites. It binds the initiating form of RNA polymerase II, pause-release factors and single-strand break repair (SSBR) components such as TDP1. The binding is increased on chromatin following oxidative damage, and TDP1 enrichment at damaged chromatin is facilitated by NuMA. Depletion of NuMA increases oxidative damage at promoters. NuMA promotes transcription by limiting the polyADP-ribosylation of RNA polymerase II, increasing its availability and release from pausing at promoters. Metabolic labelling of nascent RNA identifies genes that depend on NuMA for transcription including immediate-early response genes. Complementation of NuMA-deficient cells with a mutant that mediates binding to SSBR, or a mitotic separation-of-function mutant, restores SSBR defects. These findings underscore the importance of oxidative DNA damage repair at gene regulatory elements and describe a process that fulfils this function.

Post-transcriptional regulation of gene expression by Unr.

Unr (upstream of N-ras) is a eukaryotic RNA-binding protein that has a number of roles in the post-transcriptional regulation of gene expression. Originally identified as an activator of internal initiation of picornavirus translation, it has since been shown to act as an activator and inhibitor of cellular translation and as a positive and negative regulator of mRNA stability, regulating cellular processes such as mitosis and apoptosis. The different post-transcriptional functions of Unr depend on the identity of its mRNA and protein partners and can vary with cell type and changing cellular conditions. Recent high-throughput analyses of RNA-protein interactions indicate that Unr binds to a large subset of cellular mRNAs, suggesting that Unr may play a wider role in translational responses to cellular signals than previously thought.

The Metabotropic Glutamate 5 Receptor in Sleep and Wakefulness: Focus on the Cortico-Thalamo-Cortical Oscillations.

Sleep is an essential innate but complex behaviour which is ubiquitous in the animal kingdom. Our knowledge of the distinct neural circuit mechanisms that regulate sleep and wake states in the brain are, however, still limited. It is therefore important to understand how these circuits operate during health and disease. This review will highlight the function of mGlu5 receptors within the thalamocortical circuitry in physiological and pathological sleep states. We will also evaluate the potential of targeting mGlu5 receptors as a therapeutic strategy for sleep disorders that often co-occur with epileptic seizures.

The impact of assessment and feedback practice on the student learning experiences in higher education.

Sound knowledge of the learning process and connectedness of assessment in higher education is imperative. Assessment is essential for both learners and teachers to ensure, and to provide evidence, that learning outcomes/objectives against set competency levels have been achieved for the chosen programme of the study. However, many coming into higher education are often unaware of the rules of the game. In this perspective, we will highlight the reasons why assessment is important, how assessment and feedback affect students' learning process and why the process of assessment can often affect students' mental well-being. We will appraise the different methods of assessment with a specific example (Objective Structured Clinical Examination, OSCE) and highlight why it is important that we adopt a holistic approach towards fostering assessment know-how and student well-being.

Mitochondrial DNA replication and repair defects: Clinical phenotypes and therapeutic interventions.

Mitochondria is a unique cellular organelle involved in multiple cellular processes and is critical for maintaining cellular homeostasis. This semi-autonomous organelle contains its circular genome - mtDNA (mitochondrial DNA), that undergoes continuous cycles of replication and repair to maintain the mitochondrial genome integrity. The majority of the mitochondrial genes, including mitochondrial replisome and repair genes, are nuclear-encoded. Although the repair machinery of mitochondria is quite efficient, the mitochondrial genome is highly susceptible to oxidative damage and other types of exogenous and endogenous agent-induced DNA damage, due to the absence of protective histones and their proximity to the main ROS production sites. Mutations in replication and repair genes of mitochondria can result in mtDNA depletion and deletions subsequently leading to mitochondrial genome instability. The combined action of mutations and deletions can result in compromised mitochondrial genome maintenance and lead to various mitochondrial disorders. Here, we review the mechanism of mitochondrial DNA replication and repair process, key proteins involved, and their altered function in mitochondrial disorders. The focus of this review will be on the key genes of mitochondrial DNA replication and repair machinery and the clinical phenotypes associated with mutations in these genes.

SMN-deficient cells exhibit increased ribosomal DNA damage.

Spinal muscular atrophy, the leading genetic cause of infant mortality, is a motor neuron disease caused by low levels of survival motor neuron (SMN) protein. SMN is a multifunctional protein that is implicated in numerous cytoplasmic and nuclear processes. Recently, increasing attention is being paid to the role of SMN in the maintenance of DNA integrity. DNA damage and genome instability have been linked to a range of neurodegenerative diseases. The ribosomal DNA (rDNA) represents a particularly unstable locus undergoing frequent breakage. Instability in rDNA has been associated with cancer, premature ageing syndromes, and a number of neurodegenerative disorders. Here, we report that SMN-deficient cells exhibit increased rDNA damage leading to impaired ribosomal RNA synthesis and translation. We also unravel an interaction between SMN and RNA polymerase I. Moreover, we uncover an spinal muscular atrophy motor neuron-specific deficiency of DDX21 protein, which is required for resolving R-loops in the nucleolus. Taken together, our findings suggest a new role of SMN in rDNA integrity.

DNA Intercalators Inhibit Eukaryotic Ribosomal RNA Synthesis by Impairing the Initiation of Transcription.

In eukaryotes, ribosome biogenesis is driven by the synthesis of the ribosomal RNA (rRNA) by RNA polymerase I (Pol-I) and is tightly linked to cell growth and proliferation. The 3D-structure of the rDNA promoter plays an important, yet not fully understood role in regulating rRNA synthesis. We hypothesized that DNA intercalators/groove binders could affect this structure and disrupt rRNA transcription. To test this hypothesis, we investigated the effect of a number of compounds on Pol-I transcription in vitro and in cells. We find that intercalators/groove binders are potent inhibitors of Pol-I specific transcription both in vitro and in cells, regardless of their specificity and the strength of its interaction with DNA. Importantly, the synthetic ability of Pol-I is unaffected, suggesting that these compounds are not targeting post-initiating events. Notably, the tested compounds have limited effect on transcription by Pol-II and III, demonstrating the hypersensitivity of Pol-I transcription. We propose that stability of pre-initiation complex and initiation are affected as result of altered 3D architecture of the rDNA promoter, which is well in line with the recently reported importance of biophysical rDNA promoter properties on initiation complex formation in the yeast system.

DNA repair and neurological disease: From molecular understanding to the development of diagnostics and model organisms.

In both replicating and non-replicating cells, the maintenance of genomic stability is of utmost importance. Dividing cells can repair DNA damage during cell division, tolerate the damage by employing potentially mutagenic DNA polymerases or die via apoptosis. However, the options for accurate DNA repair are more limited in non-replicating neuronal cells. If DNA damage is left unresolved, neuronal cells die causing neurodegenerative disorders. A number of pathogenic variants of DNA repair proteins have been linked to multiple neurological diseases. The current challenge is to harness our knowledge of fundamental properties of DNA repair to improve diagnosis, prognosis and treatment of such debilitating disorders. In this perspective, we will focus on recent efforts in identifying novel DNA repair biomarkers for the diagnosis of neurological disorders and their use in monitoring the patient response to therapy. These efforts are greatly facilitated by the development of model organisms such as zebrafish, which will also be summarised.

Antioxidants and lipid peroxidation in gestational diabetes--a preliminary study.

Increased free radical activity in gestational diabetes (GDM) can lead to a host of damaging and degenerative maternal and fetal complications. Hence antioxidant levels in blood of GDM mothers and cord blood were estimated. Erythrocyte glutathione (GSH), superoxide dismutase (SOD) and thiobarbituric acid reactive substances (TBARS), plasma vitamins C and E and serum total glutathione-S-transferase (GST), protein thiols and ceruloplasmin (Cp) were estimated spectrophotometrically in maternal blood of age matched controls and mothers with GDM and also in cord blood samples of the above. There was a significant increase in the erythrocytic GSH, serum total GST and protein thiols in GDM maternal blood when compared to controls whereas erythrocytic SOD exhibited a marked decrease in GDM cases. The changes in plasma vitamins C and E, Cp and erythrocytic TBARS in GDM were not significantly different from controls. Cord blood levels of protein thiols were also significantly increased in GDM. No significant changes were observed in the serum Cp and GST levels of the same. Hence, elevated glucose levels can induce oxidative stress in GDM mothers.

UCHL3 Regulates Topoisomerase-Induced Chromosomal Break Repair by Controlling TDP1 Proteostasis.

Genomic damage can feature DNA-protein crosslinks whereby their acute accumulation is utilized to treat cancer and progressive accumulation causes neurodegeneration. This is typified by tyrosyl DNA phosphodiesterase 1 (TDP1), which repairs topoisomerase-mediated chromosomal breaks. Although TDP1 levels vary in multiple clinical settings, the mechanism underpinning this variation is unknown. We reveal that TDP1 is controlled by ubiquitylation and identify UCHL3 as the deubiquitylase that controls TDP1 proteostasis. Depletion of UCHL3 increases TDP1 ubiquitylation and turnover rate and sensitizes cells to TOP1 poisons. Overexpression of UCHL3, but not a catalytically inactive mutant, suppresses TDP1 ubiquitylation and turnover rate. TDP1 overexpression in the topoisomerase therapy-resistant rhabdomyosarcoma is driven by UCHL3 overexpression. In contrast, UCHL3 is downregulated in spinocerebellar ataxia with axonal neuropathy (SCAN1), causing elevated levels of TDP1 ubiquitylation and faster turnover rate. These data establish UCHL3 as a regulator of TDP1 proteostasis and, consequently, a fine-tuner of protein-linked DNA break repair.

C9orf72 expansion disrupts ATM-mediated chromosomal break repair.

Hexanucleotide repeat expansions represent the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia, though the mechanisms by which such expansions cause neurodegeneration are poorly understood. We report elevated levels of DNA-RNA hybrids (R-loops) and double strand breaks in rat neurons, human cells and C9orf72 ALS patient spinal cord tissues. Accumulation of endogenous DNA damage is concomitant with defective ATM-mediated DNA repair signaling and accumulation of protein-linked DNA breaks. We reveal that defective ATM-mediated DNA repair is a consequence of P62 accumulation, which impairs H2A ubiquitylation and perturbs ATM signaling. Virus-mediated expression of C9orf72-related RNA and dipeptide repeats in the mouse central nervous system increases double strand breaks and ATM defects and triggers neurodegeneration. These findings identify R-loops, double strand breaks and defective ATM-mediated repair as pathological consequences of C9orf72 expansions and suggest that C9orf72-linked neurodegeneration is driven at least partly by genomic instability.

Stimulation of translation by human Unr requires cold shock domains 2 and 4, and correlates with poly(A) binding protein interaction.

The RNA binding protein Unr, which contains five cold shock domains, has several specific roles in post-transcriptional control of gene expression. It can act as an activator or inhibitor of translation initiation, promote mRNA turnover, or stabilise mRNA. Its role depends on the mRNA and other proteins to which it binds, which includes cytoplasmic poly(A) binding protein 1 (PABP1). Since PABP1 binds to all polyadenylated mRNAs, and is involved in translation initiation by interaction with eukaryotic translation initiation factor 4G (eIF4G), we investigated whether Unr has a general role in translational control. We found that Unr strongly stimulates translation in vitro, and mutation of cold shock domains 2 or 4 inhibited its translation activity. The ability of Unr and its mutants to stimulate translation correlated with its ability to bind RNA, and to interact with PABP1. We found that Unr stimulated the binding of PABP1 to mRNA, and that Unr was required for the stable interaction of PABP1 and eIF4G in cells. siRNA-mediated knockdown of Unr reduced the overall level of cellular translation in cells, as well as that of cap-dependent and IRES-dependent reporters. These data describe a novel role for Unr in regulating cellular gene expression.

The histone demethylase JMJD2A/KDM4A links ribosomal RNA transcription to nutrients and growth factors availability.

The interplay between methylation and demethylation of histone lysine residues is an essential component of gene expression regulation and there is considerable interest in elucidating the roles of proteins involved. Here we report that histone demethylase KDM4A/JMJD2A, which is involved in the regulation of cell proliferation and is overexpressed in some cancers, interacts with RNA Polymerase I, associates with active ribosomal RNA genes and is required for serum-induced activation of rDNA transcription. We propose that KDM4A controls the initial stages of transition from 'poised', non-transcribed rDNA chromatin into its active form. We show that PI3K, a major signalling transducer central for cell proliferation and survival, controls cellular localization of KDM4A and consequently its association with ribosomal DNA through the SGK1 downstream kinase. We propose that the interplay between PI3K/SGK1 signalling cascade and KDM4A constitutes a mechanism by which cells adapt ribosome biogenesis level to the availability of growth factors and nutrients.

The identification of a novel role for BRCA1 in regulating RNA polymerase I transcription.

The unrestrained proliferation of cancer cells requires a high level of ribosome biogenesis. The first stage of ribosome biogenesis is the transcription of the large ribosomal RNAs (rRNAs); the structural and functional components of the ribosome. Transcription of rRNA is carried out by RNA polymerase I (Pol-I) and its associated holoenzyme complex.Here we report that BRCA1, a nuclear phosphoprotein, and a known tumour suppressor involved in variety of cellular processes such as DNA damage response, transcriptional regulation, cell cycle control and ubiquitylation, is associated with rDNA repeats, in particular with the regulatory regions of the rRNA gene.We demonstrate that BRCA1 interacts directly with the basal Pol-I transcription factors; upstream binding factor (UBF), selectivity factor-1 (SL1) as well as interacting with RNA Pol-I itself. We show that in response to DNA damage, BRCA1 occupancy at the rDNA repeat is decreased and the observed BRCA1 interactions with the Pol-I transcription machinery are weakened.We propose, therefore, that there is a rDNA associated fraction of BRCA1 involved in DNA damage dependent regulation of Pol-I transcription, regulating the stability and formation of the Pol-I holoenzyme during initiation and/or elongation in response to DNA damage.

Androgens and estrogens stimulate ribosome biogenesis in prostate and breast cancer cells in receptor dependent manner.

Ribosome biogenesis is a fundamental cellular process intimately linked to cell growth and proliferation, which is upregulated in most of cancers especially in aggressive cancers. In breast and prostate cancers steroid hormone receptor signalling is the principal stimulus for cancer growth and progression. Here we investigated the link between estrogen and androgen receptor signalling and the initial stage of ribosome biogenesis - transcription of rRNA genes. We have discovered that oestrogen or androgen treatment can positively regulate rRNA synthesis in breast and prostate cancer cells respectively and that this effect is receptor dependent. This novel and interesting finding suggests a previously unidentified link between steroid hormone receptor signalling pathways and the regulation of ribosome biogenesis.

Topoisomerase IIα promotes activation of RNA polymerase I transcription by facilitating pre-initiation complex formation.

Type II DNA topoisomerases catalyse DNA double-strand cleavage, passage and re-ligation to effect topological changes. There is considerable interest in elucidating topoisomerase II roles, particularly as these proteins are targets for anti-cancer drugs. Here we uncover a role for topoisomerase IIα in RNA polymerase I-directed ribosomal RNA gene transcription, which drives cell growth and proliferation and is upregulated in cancer cells. Our data suggest that topoisomerase IIα is a component of the initiation-competent RNA polymerase Iß complex and interacts directly with RNA polymerase I-associated transcription factor RRN3, which targets the polymerase to promoter-bound SL1 in pre-initiation complex formation. In cells, activation of rDNA transcription is reduced by inhibition or depletion of topoisomerase II, and this is accompanied by reduced transient double-strand DNA cleavage in the rDNA-promoter region and reduced pre-initiation complex formation. We propose that topoisomerase IIα functions in RNA polymerase I transcription to produce topological changes at the rDNA promoter that facilitate efficient de novo pre-initiation complex formation.

Crosstalk with insulin and dependence on PI3K/Akt/mTOR rather than MAPK pathways in upregulation of basal growth following long-term oestrogen deprivation in three human breast cancer cell lines.

BACKGROUND: MCF-7, T-47-D, ZR-75-1 human breast cancer cell lines are dependent on oestrogen for growth but can adapt to grow during long-term oestrogen deprivation. This serves as a model for identification of therapeutic targets in endocrine-resistant breast cancer. METHODS: An overlooked complication of this model is that it involves more than non-addition of oestrogen, and inadequate attention has been given to separating molecular events associated with each of the culture manipulations. RESULTS: Insulin and oestradiol were shown to protect MCF-7 cells against upregulation of basal growth, demonstrating a crosstalk in the growth adaptation process. Increased phosphorylation of p44/42MAPK and c-Raf reflected removal of insulin from the medium and proliferation of all three cell lines was inhibited to a lesser extent by PD98059 and U0126 following long-term oestrogen/insulin withdrawal, demonstrating a reduced dependence on the MAPK pathway. By contrast, long-term oestrogen/insulin deprivation did not alter levels of phosphorylated Akt and did not alter the dose-response of growth inhibition with LY294002 in any of the three cell lines. The IGF1R inhibitor picropodophyllin inhibited growth of all MCF-7 cells but only in the long-term oestrogen/insulin-deprived cells was this paralleled by reduction in phosphorylated p70S6K, a downstream target of mTOR. Long-term oestrogen/insulin-deprived MCF-7 cells had higher levels of phosphorylated p70S6K and developed increased sensitivity to growth inhibition by rapamycin. CONCLUSIONS: The greater sensitivity to growth inhibition by rapamycin in all three cell lines following long-term oestrogen/insulin deprivation suggests rapamycin-based therapies might be more effective in breast cancers with acquired oestrogen resistance.

Enhanced sensitivity to rapamycin following long-term oestrogen deprivation in MCF-7, T-47-D and ZR-75-1 human breast cancer cells.

Human breast cancer cells (MCF-7, T-47-D and ZR-75-1) can adapt to circumvent any reduced growth rate during long-term oestrogen deprivation, and this provides three model systems to investigate mechanisms of endocrine resistance in breast cancer. In this paper we report consistent differences in the effects of three growth inhibitors following long-term oestrogen deprivation in all three cell models. Long-term oestrogen deprivation of MCF-7, T-47-D and ZR-75-1 cells resulted in reduced growth inhibition by PD98059 (2-10 µg/ml), implying a loss of dependence on mitogen-activated protein kinase pathways for growth. The growth inhibitor LY294002 (2-10 µM) inhibited growth of both oestrogen-maintained and oestrogen-deprived cells with similar dose-responses, implying continued similar dependence on phosphoinositide 3-kinase (PI3K) pathways with no alteration after adaptation to oestrogen independent growth. However, by contrast, long-term oestrogen deprivation resulted in an increased sensitivity to growth inhibition by rapamycin, which was not reduced by readdition of oestradiol. The enhanced inhibition of long-term oestrogen-deprived MCF-7-ED, T-47-D-ED and ZR-75-1-ED cell growth by combining rapamycin with LY294002 at concentrations where each alone had little effect, offers preclinical support to the development of therapeutic combinations of rapamycin analogues with other PI3K inhibitors in endocrine-resistant breast cancer.