Heat Shock Affects Mitotic Segregation of Human Chromosomes Bound to Stress-Induced Satellite III RNAs.

Heat shock activates the transcription of arrays of Satellite III (SatIII) DNA repeats in the pericentromeric heterochromatic domains of specific human chromosomes, the longest of which is on chromosome 9. Long non-coding SatIII RNAs remain associated with transcription sites where they form nuclear stress bodies or nSBs. The biology of SatIII RNAs is still poorly understood. Here, we show that SatIII RNAs and nSBs are detectable up to four days after thermal stress and are linked to defects in chromosome behavior during mitosis. Heat shock perturbs the execution of mitosis. Cells reaching mitosis during the first 3 h of recovery accumulate in pro-metaphase. During the ensuing 48 h, this block is no longer detectable; however, a significant fraction of mitoses shows chromosome segregation defects. Notably, most of lagging chromosomes and chromosomal bridges are bound to nSBs and contain arrays of SatIII DNA. Disappearance of mitotic defects at the end of day 2 coincides with the processing of long non-coding SatIII RNAs into a ladder of small RNAs associated with chromatin and ranging in size from 25 to 75 nt. The production of these molecules does not rely on DICER and Argonaute 2 components of the RNA interference apparatus. Thus, massive transcription of SatIII DNA may contribute to chromosomal instability.

The alternative splicing side of cancer.

Alternative splicing emerges as a potent and pervasive mechanism of gene expression regulation that expands the coding capacity of the genome and forms an intermediate layer of regulation between transcriptional and post-translational networks. Indeed, alternative splicing occupies a pivotal position in developmental programs and in the cell response to external and internal stimuli. Not surprisingly, therefore, its deregulation frequently leads to human disease. In this review we provide an updated overview of the impact of alternative splicing on tumorigenesis. Moreover, we discuss the intricacy of the reciprocal interactions between alternative splicing programs and signal transduction pathways, which appear to be crucially linked to cancer progression in response to the tumor microenvironment. Finally, we focus on the recently described interplay between splicing and chromatin organization which is expected to shed new lights into gene expression regulation in normal and cancer cells.

CorrelaGenes: a new tool for the interpretation of the human transcriptome.

BACKGROUND: The amount of gene expression data available in public repositories has grown exponentially in the last years, now requiring new data mining tools to transform them in information easily accessible to biologists. RESULTS: By exploiting expression data publicly available in the Gene Expression Omnibus (GEO) database, we developed a new bioinformatics tool aimed at the identification of genes whose expression appeared simultaneously altered in different experimental conditions, thus suggesting co-regulation or coordinated action in the same biological process. To accomplish this task, we used the 978 human GEO Curated DataSets and we manually performed the selection of 2,109 pair-wise comparisons based on their biological rationale. The lists of differentially expressed genes, obtained from the selected comparisons, were stored in a PostgreSQL database and used as data source for the CorrelaGenes tool. Our application uses a customized Association Rule Mining (ARM) algorithm to identify sets of genes showing expression profiles correlated with a gene of interest. The significance of the correlation is measured coupling the Lift, a well-known standard ARM index, and the χ(2) p value. The manually curated selection of the comparisons and the developed algorithm constitute a new approach in the field of gene expression profiling studies. Simulation performed on 100 randomly selected target genes allowed us to evaluate the efficiency of the procedure and to obtain preliminary data demonstrating the consistency of the results. CONCLUSIONS: The preliminary results of the simulation showed how CorrelaGenes could contribute to the characterization of molecular pathways and biological processes integrating data obtained from other applications and available in public repositories.

Phosphorylation of SRSF1 is modulated by replicational stress.

DNA ligase I-deficient 46BR.1G1 cells show a delay in the maturation of replicative intermediates resulting in the accumulation of single- and double-stranded DNA breaks. As a consequence the ataxia telangiectasia mutated protein kinase (ATM) is constitutively phosphorylated at a basal level. Here, we use 46BR.1G1 cells as a model system to study the cell response to chronic replication-dependent DNA damage. Starting from a proteomic approach, we demonstrate that the phosphorylation level of factors controlling constitutive and alternative splicing is affected by the damage elicited by DNA ligase I deficiency. In particular, we show that SRSF1 is hyperphosphorylated in 46BR.1G1 cells compared to control fibroblasts. This hyperphosphorylation can be partially prevented by inhibiting ATM activity with caffeine. Notably, hyperphosphorylation of SRSF1 affects the subnuclear distribution of the protein and the alternative splicing pattern of target genes. We also unveil a modulation of SRSF1 phosphorylation after exposure of MRC-5V1 control fibroblasts to different exogenous sources of DNA damage. Altogether, our observations indicate that a relevant aspect of the cell response to DNA damage involves the post-translational regulation of splicing factor SRSF1 which is associated with a shift in the alternative splicing program of target genes to control cell survival or cell death.

An insight into the abundant proteome of 46BR.1G1 fibroblasts deficient of DNA ligase I.

This work presents the proteome profile of cultured human skin fibroblasts established from a patient affected by DNA ligase I (Lig I) deficiency syndrome, a rare disorder characterized by immunodeficiency, growth retardation and sun sensitivity. 2-DE (in the 3-10 and 4-7 pH ranges) was the separation technique used for the production of maps. MALDI-TOF/MS and LC-MS/MS were the mass spectrometry platforms applied for the identification of proteins in gel spots. A total of 154 proteins, including 41 never detected before in skin fibroblasts with this approach, were identified in gel spots analyzed. This newly generated extensive database provides for the first time a global picture of abundant proteins in 46BR.1G1 skin fibroblasts. While being relevant to the particular disorder considered, these results may be regarded as an intriguing starting point on the way to achieve a reference map of the proteins highly expressed in an inherited syndrome with defect in DNA replication and repair pathways.

The role of prelamin A post-translational maturation in stress response and 53BP1 recruitment.

Lamin A is a main constituent of the nuclear lamina and contributes to nuclear shaping, mechano-signaling transduction and gene regulation, thus affecting major cellular processes such as cell cycle progression and entry into senescence, cellular differentiation and stress response. The role of lamin A in stress response is particularly intriguing, yet not fully elucidated, and involves prelamin A post-translational processing. Here, we propose prelamin A as the tool that allows lamin A plasticity during oxidative stress response and permits timely 53BP1 recruitment to DNA damage foci. We show that while PCNA ubiquitination, p21 decrease and H2AX phosphorylation occur soon after stress induction in the absence of prelamin A, accumulation of non-farnesylated prelamin A follows and triggers recruitment of 53BP1 to lamin A/C complexes. Then, the following prelamin A processing steps causing transient accumulation of farnesylated prelamin A and maturation to lamin A reduce lamin A affinity for 53BP1 and favor its release and localization to DNA damage sites. Consistent with these observations, accumulation of prelamin A forms in cells under basal conditions impairs histone H2AX phosphorylation, PCNA ubiquitination and p21 degradation, thus affecting the early stages of stress response. As a whole, our results are consistent with a physiological function of prelamin A modulation during stress response aimed at timely recruitment/release of 53BP1 and other molecules required for DNA damage repair. In this context, it becomes more obvious how farnesylated prelamin A accumulation to toxic levels alters timing of DNA damage signaling and 53BP1 recruitment, thus contributing to cellular senescence and accelerated organismal aging as observed in progeroid laminopathies.

Claspin inhibition leads to fragile site expression.

Fragile sites are hot spots for sister chromatid exchanges, translocations, deletions, complex rearrangements, and gene amplification. It has been hypothesized that rearrangements at fragile sites derive from unreplicated regions resulting from stalled forks that escape the ATR replication checkpoint. In the present study, we investigated the role of the Claspin (CLSPN) gene, which codes for an adaptor protein in the ATR pathway, during DNA replication stress in human cells. We show that the inhibition of the CLSPN gene leads to both genome instability and fragile site expression. Following aphidicolin treatment, we found a transient increase of Claspin synthesis due to its requirement to checkpoint activation. However, Claspin synthesis decreased after a prolonged aphidicolin treatment. We propose that CLSPN modulation, following an extreme replication block, allows rare cells to escape checkpoint mechanisms and enter mitosis with a defect in genome assembly. Our observations provide the basis for a better understanding of cell cycle checkpoints deregulation in cancer.

A pRb-independent mechanism preserves the postmitotic state in terminally differentiated skeletal muscle cells.

In skeletal muscle differentiation, the retinoblastoma protein (pRb) is absolutely necessary to establish definitive mitotic arrest. It is widely assumed that pRb is equally essential to sustain the postmitotic state, but this contention has never been tested. Here, we show that terminal proliferation arrest is maintained in skeletal muscle cells by a pRb-independent mechanism. Acute Rb excision from conditional knockout myotubes caused reexpression of E2F transcriptional activity, cyclin-E and -A kinase activities, PCNA, DNA ligase I, RPA, and MCM2, but did not induce DNA synthesis, showing that pRb is not indispensable to preserve the postmitotic state of these cells. Muscle-specific gene expression was significantly down-regulated, showing that pRb is constantly required for optimal implementation of the muscle differentiation program. Rb-deleted myotubes were efficiently reactivated by forced expression of cyclin D1 and Cdk4, indicating a functionally significant target other than pRb for these molecules. Finally, Rb removal induced no DNA synthesis even in pocket-protein null cells. Thus, the postmitotic state of myotubes is maintained by at least two mechanisms, one of which is pocket-protein independent.

TSPYL2 is a novel regulator of SIRT1 and p300 activity in response to DNA damage.

Protein acetylation and deacetylation events are finely regulated by lysine-acetyl-transferases and lysine-deacetylases and constitute an important tool for the activation or inhibition of specific cellular pathways. One of the most important lysine-acetyl-transferases is p300, which is involved in the regulation of gene expression, cell growth, DNA repair, differentiation, apoptosis, and tumorigenesis. A well-known target of p300 is constituted by the tumor suppressor protein p53, which plays a critical role in the maintenance of genomic stability and whose activity is known to be controlled by post-translational modifications, among which acetylation. p300 activity toward p53 is negatively regulated by the NAD-dependent deacetylase SIRT1, which deacetylates p53 preventing its transcriptional activation and the induction of p53-dependent apoptosis. However, the mechanisms responsible for p53 regulation by p300 and SIRT1 are still poorly understood. Here we identify the nucleosome assembly protein TSPY-Like 2 (TSPYL2, also known as TSPX, DENTT, and CDA1) as a novel regulator of SIRT1 and p300 function. We demonstrate that, upon DNA damage, TSPYL2 inhibits SIRT1, disrupting its association with target proteins, and promotes p300 acetylation and activation, finally stimulating p53 acetylation and p53-dependent cell death. Indeed, in response to DNA damage, cells silenced for TSPYL2 were found to be defective in p53 activation and apoptosis induction and these events were shown to be dependent on SIRT1 and p300 function. Collectively, our results shed new light on the regulation of p53 acetylation and activation and reveal a novel TSPYL2 function with important implications in cancerogenesis.

The dispersal of replication proteins after Etoposide treatment requires the cooperation of Nbs1 with the ataxia telangiectasia Rad3-related/Chk1 pathway.

In mammalian cells, DNA replication takes place in functional subnuclear compartments, called replication factories, where replicative factors accumulate. The distribution pattern of replication factories is diagnostic of the different moments (early, mid, and late) of the S phase. This dynamic organization is affected by different agents that induce cell cycle checkpoint activation via DNA damage or stalling of replication forks. Here, we explore the cell response to etoposide, an anticancer drug belonging to the topoisomerase II poisons. Etoposide does not induce an immediate block of DNA synthesis and progressively affects the distribution of replication proteins in S phase. First, it triggers the formation of large nuclear foci that contain the single-strand DNA binding protein replication protein A (RPA), suggesting that lesions produced by the drug are processed into extended single-stranded regions. These RPA foci colocalize with DNA replicated at the beginning of the treatment. Etoposide also triggers the dispersal of replicative proteins, proliferating cell nuclear antigen and DNA ligase I, from replication factories. This event requires the activity of the ataxia telangiectasia Rad3-related (ATR) checkpoint kinase. By comparing the effect of the drug in cell lines defective in different DNA repair and checkpoint pathways, we show that, along with the downstream kinase Chk1, the Nbs1 protein, mutated in the Nijmegen breakage syndrome, is also relevant for this response and for ATR-dependent phosphorylation. Finally, our analysis evidences a critical role of Nbs1 in the etoposide-induced inhibition of DNA replication in early S phase.

The Krebs Cycle Connection: Reciprocal Influence Between Alternative Splicing Programs and Cell Metabolism.

Alternative splicing is a pervasive mechanism that molds the transcriptome to meet cell and organism needs. However, how this layer of gene expression regulation is coordinated with other aspects of the cell metabolism is still largely undefined. Glucose is the main energy and carbon source of the cell. Not surprisingly, its metabolism is finely tuned to satisfy growth requirements and in response to nutrient availability. A number of studies have begun to unveil the connections between glucose metabolism and splicing programs. Alternative splicing modulates the ratio between M1 and M2 isoforms of pyruvate kinase in this way determining the choice between aerobic glycolysis and complete glucose oxidation in the Krebs cycle. Reciprocally, intermediates in the Krebs cycle may impact splicing programs at different levels by modulating the activity of 2-oxoglutarate-dependent oxidases. In this review we discuss the molecular mechanisms that coordinate alternative splicing programs with glucose metabolism, two aspects with profound implications in human diseases.

Identification of S6K2 as a centrosome-located kinase.

Ribosomal S6 kinase 2 (S6K2) acts downstream of the mammalian target of rapamycin (mTOR). Here, we show that some S6K2 localize at the centrosome throughout the cell cycle. S6K2 is found in the pericentriolar area of the centrosome. S6K2 centrosomal localization is unaffected by serum withdrawal or treatment with rapamycin, wortmannin, U0126, or phorbol-12-myristate-13-acetate (PMA). Unlike S6K2, S6 kinase 1 (S6K1) does not localize at the centrosome, suggesting the two kinases may also have nonoverlapping functions. Our data suggest that centrosomal S6K2 may have a role in the phosphoinositide-3-kinase (PI3K)/Akt/mTOR signaling pathway that has also been detected in the centrosome.

Early effects of topoisomerase I inhibition on RNA polymerase II along transcribed genes in human cells.

We have determined the early effects of camptothecin and alpha-amanitin on genomic DNA-binding sites of RNA polymerase II (RNAPII), TATA-binding protein (TBP), DNA topoisomerase I (Top1), and histone components in human transcribed loci by chromatin-immunoprecipitation (ChIP). The two agents caused notably different alterations in active chromatin. Camptothecin induced a specific reduction of RNAPII density at promoter pause sites and histone modifications suggesting an increased chromatin accessibility. alpha-Amanitin caused an accumulation of RNAPII at transcribed genes, a reduction of TBP bound to chromatin and a less accessible chromatin structure. Interestingly, RNAPII reduction at promoter pause sites occurred within 5-10min of camptothecin treatment, and was not a response to replication-dependent DNA breaks. ChIP analyses of RNAPII along transcribed genes indicated that RNAPII levels were transiently increased at internal exons, and that camptothecin effects could be fully reversed by DRB, a cdk inhibitor. Top1 was found to be enriched in active chromatin, therefore suggesting that Top1 inhibition at the transcribed template and/or adjacent regulating regions immediately affects RNAPII at active genes. The findings are novel in vivo evidence of camptothecin effects on RNAPII bound to transcribing genomic regions, and are consistent with the hypothesis that Top1 activity can be involved in transcription regulation at the level of promoter clearance.

Cellular response to etoposide treatment.

Etoposide is a potent anti-tumor drug that belongs to the class of topoisomerase poisons. Although its molecular target, i.e. DNA topoisomerase II, has been identified more than 20 years ago, the cellular response to etoposide is still poorly understood. The cytotoxicity of the drug stems from its ability to stabilize a covalent complex between DNA topoisomerase II and DNA that results in a high level of DNA damage. Here, we review the present knowledge about the strategy used by the cells to deal with the etoposide-induced DNA damage. New and unanticipated effects of topoisomerase II poisoning on cell metabolism are recently emerging, among which the ability to activate cell cycle checkpoint pathways and to affect gene expression at different levels, including chromatin remodeling and alternative splicing of gene transcripts. The elucidation of the effects of etoposide on cell metabolism will increase our ability to exploit this drug in cancer therapy and will expand our comprehension of the cancerous cell.

A missense MT-ND5 mutation in differentiated Parkinson Disease cytoplasmic hybrid induces ROS-dependent DNA Damage Response amplified by DROSHA.

Genome integrity is continuously threatened by endogenous sources of DNA damage including reactive oxygen species (ROS) produced by cell metabolism. Factors of the RNA interference (RNAi) machinery have been recently involved in the cellular response to DNA damage (DDR) in proliferating cells. To investigate the impact of component of RNAi machinery on DDR activation in terminally differentiated cells, we exploited cytoplasmic hybrid (cybrid) cell lines in which mitochondria of sporadic Parkinson's disease patients repopulate neuroblastoma SH-SY5Y-Rho(0) cells. Upon differentiation into dopaminergic neuron-like cells, PD63 cybrid showed increased intracellular level of ROS and chronic DDR activation, compared to other cybrids with the same nuclear background. Importantly, DDR activation in these cells can be prevented by ROS scavenging treatment suggesting that ROS production is indeed causative of nuclear DNA damage. Sequence analysis of the mitogenomes identified a rare and heteroplasmic missense mutation affecting a highly conserved residue of the ND5-subunit of respiratory complex I, which accounts for ROS increase. We demonstrated that the assembly of nuclear DDR foci elicited by oxidative stress in these cells relies on DROSHA, providing the first evidence that components of RNAi machinery play a crucial role also in the mounting of ROS-induced DDR in non-replicating neuronal cells.

Molecular mechanisms of etoposide.

Etoposide derives from podophyllotoxin, a toxin found in the American Mayapple. It was first synthesized in 1966 and approved for cancer therapy in 1983 by the U.S. Food and Drug Administration (Hande, 1998[25]). Starting from 1980s several studies demonstrated that etoposide targets DNA topoisomerase II activities thus leading to the production of DNA breaks and eliciting a response that affects several aspects of cell metabolisms. In this review we will focus on molecular mechanisms that account for the biological effect of etoposide.

Chronic Replication Problems Impact Cell Morphology and Adhesion of DNA Ligase I Defective Cells.

Moderate DNA damage resulting from metabolic activities or sub-lethal doses of exogenous insults may eventually lead to cancer onset. Human 46BR.1G1 cells bear a mutation in replicative DNA ligase I (LigI) which results in low levels of replication-dependent DNA damage. This replication stress elicits a constitutive phosphorylation of the ataxia telangiectasia mutated (ATM) checkpoint kinase that fails to arrest cell cycle progression or to activate apoptosis or cell senescence. Stable transfection of wild type LigI, as in 7A3 cells, prevents DNA damage and ATM activation. Here we show that parental 46BR.1G1 and 7A3 cells differ in important features such as cell morphology, adhesion and migration. Comparison of gene expression profiles in the two cell lines detects Bio-Functional categories consistent with the morphological and migration properties of LigI deficient cells. Interestingly, ATM inhibition makes 46BR.1G1 more similar to 7A3 cells for what concerns morphology, adhesion and expression of cell-cell adhesion receptors. These observations extend the influence of the DNA damage response checkpoint pathways and unveil a role for ATM kinase activity in modulating cell biology parameters relevant to cancer progression.

Pre-mRNA processing factors meet the DNA damage response.

It is well-known that DNA-damaging agents induce genome instability, but only recently have we begun to appreciate that chromosomes are fragile per se and frequently subject to DNA breakage. DNA replication further magnifies such fragility, because it leads to accumulation of single-stranded DNA. Recent findings suggest that chromosome fragility is similarly increased during transcription. Transcripts produced by RNA polymerase II (RNAPII) are subject to multiple processing steps, including maturation of 5' and 3' ends and splicing, followed by transport to the cytoplasm. RNA maturation starts on nascent transcripts and is mediated by a number of diverse proteins and ribonucleoprotein particles some of which are recruited cotranscriptionally through interactions with the carboxy-terminal domain of RNAPII. This coupling is thought to maximize efficiency of pre-mRNA maturation and directly impacts the choice of alternative splice sites. Mounting evidence suggests that lack of coordination among different RNA maturation steps, by perturbing the interaction of nascent transcripts with the DNA template, has deleterious effects on genome stability. Thus, in the absence of proper surveillance mechanisms, transcription could be a major source of DNA damage in cancer. Recent high-throughput screenings in human cells and budding yeast have identified several factors implicated in RNA metabolism that are targets of DNA damage checkpoint kinases: ATM (ataxia telangiectasia mutated) and ATR (ATM-Rad3 related) (Tel1 and Mec1 in budding yeast, respectively). Moreover, inactivation of various RNA processing factors induces accumulation of γH2AX foci, an early sign of DNA damage. Thus, a complex network is emerging that links DNA repair and RNA metabolism. In this review we provide a comprehensive overview of the role played by pre-mRNA processing factors in the cell response to DNA damage and in the maintenance of genome stability.

A new cell-selective three-dimensional microincubator based on silicon photonic crystals.

In this work, we show that vertical, high aspect-ratio (HAR) photonic crystals (PhCs), consisting of periodic arrays of 5 µm wide gaps with depth of 50 µm separated by 3 µm thick silicon walls, fabricated by electrochemical micromachining, can be used as three-dimensional microincubators, allowing cell lines to be selectively grown into the gaps. Silicon micromachined dice incorporating regions with different surface profiles, namely flat silicon and deeply etched PhC, were used as microincubators for culturing adherent cell lines with different morphology and adhesion properties. We extensively investigated and compared the proliferative behavior on HAR PhCs of eight human cell models, with different origins, such as the epithelial (SW613-B3; HeLa; SW480; HCT116; HT29) and the mesenchymal (MRC-5V1; CF; HT1080). We also verified the contribution of cell sedimentation into the silicon gaps. Fluorescence microscopy analysis highlights that only cell lines that exhibit, in the tested culture condition, the behavior typical of the mesenchymal phenotype are able to penetrate into the gaps of the PhC, extending their body deeply in the narrow gaps between adjacent silicon walls, and to grow adherent to the vertical surfaces of silicon. Results reported in this work, confirmed in various experiments, strongly support our statement that such three-dimensional microstructures have selection capabilities with regard to the cell lines that can actively populate the narrow gaps. Cells with a mesenchymal phenotype could be exploited in the next future as bioreceptors, in combination with HAR PhC optical transducers, e.g., for label-free optical detection of cellular activities involving changes in cell adhesion and/or morphology (e.g., apoptosis) in a three-dimensional microenvironment.