Senataxin plays an essential role with DNA damage response proteins in meiotic recombination and gene silencing.

Senataxin, mutated in the human genetic disorder ataxia with oculomotor apraxia type 2 (AOA2), plays an important role in maintaining genome integrity by coordination of transcription, DNA replication, and the DNA damage response. We demonstrate that senataxin is essential for spermatogenesis and that it functions at two stages in meiosis during crossing-over in homologous recombination and in meiotic sex chromosome inactivation (MSCI). Disruption of the Setx gene caused persistence of DNA double-strand breaks, a defect in disassembly of Rad51 filaments, accumulation of DNA:RNA hybrids (R-loops), and ultimately a failure of crossing-over. Senataxin localised to the XY body in a Brca1-dependent manner, and in its absence there was incomplete localisation of DNA damage response proteins to the XY chromosomes and ATR was retained on the axial elements of these chromosomes, failing to diffuse out into chromatin. Furthermore persistence of RNA polymerase II activity, altered ubH2A distribution, and abnormal XY-linked gene expression in Setx⁻/⁻ revealed an essential role for senataxin in MSCI. These data support key roles for senataxin in coordinating meiotic crossing-over with transcription and in gene silencing to protect the integrity of the genome.

Smg1 haploinsufficiency predisposes to tumor formation and inflammation.

SMG1 is a member of the phosphoinositide kinase-like kinase family of proteins that includes ATM, ATR, and DNA-PK, proteins with known roles in DNA damage and cellular stress responses. SMG1 has a well-characterized role in nonsense-mediated decay as well as suggested roles in the DNA damage response, resistance to oxidative stress, regulation of hypoxic responses, and apoptosis. To understand the roles of SMG1 further, we generated a Genetrap Smg1 mouse model. Smg1 homozygous KO mice were early embryonic lethal, but Smg1 heterozygous mice showed a predisposition to a range of cancers, particularly lung and hematopoietic malignancies, as well as development of chronic inflammation. These mice did not display deficiencies in known roles of SMG1, including nonsense-mediated decay. However, they showed elevated basal tissue and serum cytokine levels, indicating low-level inflammation before the development of tumors. Smg1 heterozygous mice also showed evidence of oxidative damage in tissues. These data suggest that the inflammation observed in Smg1 haploinsufficiency contributes to susceptibility to cancer and that Smg1-deficient animals represent a model of inflammation-enhanced cancer development.

A novel role for hSMG-1 in stress granule formation.

hSMG-1 is a member of the phosphoinositide 3 kinase-like kinase (PIKK) family with established roles in nonsense-mediated decay (NMD) of mRNA containing premature termination codons and in genotoxic stress responses to DNA damage. We report here a novel role for hSMG-1 in cytoplasmic stress granule (SG) formation. Exposure of cells to stress causing agents led to the localization of hSMG-1 to SG, identified by colocalization with TIA-1, G3BP1, and eIF4G. hSMG-1 small interfering RNA and the PIKK inhibitor wortmannin prevented formation of a subset of SG, while specific inhibitors of ATM, DNA-PK(cs), or mTOR had no effect. Exposure of cells to H(2)O(2) and sodium arsenite induced (S/T)Q phosphorylation of proteins. While Upf2 and Upf1, an essential substrate for hSMG-1 in NMD, were present in SG, NMD-specific Upf1 phosphorylation was not detected in SG, indicating hSMG-1's role in SG is separate from classical NMD. Thus, SG formation appears more complex than originally envisaged and hSMG-1 plays a central role in this process.

Low levels of ATM in breast cancer patients with clinical radiosensitivity.

BACKGROUND AND PURPOSE: Adjuvant radiotherapy for cancer can result in severe adverse side effects for normal tissues. In this respect, individuals with anomalies of the ATM (ataxia telangiectasia) protein/gene are of particular interest as they may be at risk of both breast cancer and clinical radiosensitivity. The association of specific ATM gene mutations with these pathologies has been well documented, however, there is uncertainty regarding pathological thresholds for the ATM protein. RESULTS: Semi-quantitative immuno-blotting provided a reliable and reproducible method to compare levels of the ATM protein for a rare cohort of 20 cancer patients selected on the basis of their severe adverse normal tissue reactions to radiotherapy. We found that 4/12 (33%) of the breast cancer patients with severe adverse normal tissue reactions following radiotherapy had ATM protein levels < 55% compared to the mean for non-reactor controls. CONCLUSIONS: ATM mutations are generally considered low risk alleles for breast cancer and clinical radiosensitivity. From results reported here we propose a tentative ATM protein threshold of ~55% for high-risk of clinical radiosensitivity for breast cancer patients.

Functional role for senataxin, defective in ataxia oculomotor apraxia type 2, in transcriptional regulation.

Ataxia oculomotor apraxia type 2 (AOA2) is an autosomal recessive neurodegenerative disorder characterized by cerebellar ataxia and oculomotor apraxia. The gene mutated in AOA2, SETX, encodes senataxin, a putative DNA/RNA helicase which shares high homology to the yeast Sen1p protein and has been shown to play a role in the response to oxidative stress. To investigate further the function of senataxin, we identified novel senataxin-interacting proteins, the majority of which are involved in transcription and RNA processing, including RNA polymerase II. Binding of RNA polymerase II to candidate genes was significantly reduced in senataxin deficient cells and this was accompanied by decreased transcription of these genes, suggesting a role for senataxin in the regulation/modulation of transcription. RNA polymerase II-dependent transcription termination was defective in cells depleted of senataxin in keeping with the observed interaction of senataxin with poly(A) binding proteins 1 and 2. Splicing efficiency of specific mRNAs and alternate splice-site selection of both endogenous genes and artificial minigenes were altered in senataxin depleted cells. These data suggest that senataxin, similar to its yeast homolog Sen1p, plays a role in coordinating transcriptional events, in addition to its role in DNA repair.

Stress-induced gene expression profiling in the black tiger shrimp Penaeus monodon.

Cultured shrimp are continuously exposed to variable environmental conditions that have been associated with stress and subsequent outbreaks of disease. To investigate the effect of environmental stress on Penaeus monodon gene expression, a 3,853 random cDNA microarray chip was generated with clones originating from six stress-enriched hemocyte libraries generated by suppression subtractive hybridization and a normal hemocyte cDNA library. Changes in temporal gene expression were analyzed from shrimp exposed to hypoxic, hyperthermic, and hypoosmotic conditions; 3.1% of the cDNAs were differentially expressed in response to at least one of the environmental stressors, and 72% of the differentially expressed clones had no significant sequence similarity to previously known genes. Among those genes with high identity to known sequences, the most common functional groups were immune-related genes and non-long terminal repeat retrotransposons. Hierarchical clustering revealed a set of cDNAs with temporal and stress-specific gene expression profiles as well as a set of cDNAs indicating a common stress response between stressors. Hypoxic and hyperthermic stressors induced the most severe short-term response in terms of gene regulation, and the osmotic stress had the least variation in expression profiles relative to the control. These expression data agree with observed differences in shrimp physical appearance and behavior following exposure to stress conditions.

Senataxin, defective in ataxia oculomotor apraxia type 2, is involved in the defense against oxidative DNA damage.

A defective response to DNA damage is observed in several human autosomal recessive ataxias with oculomotor apraxia, including ataxia-telangiectasia. We report that senataxin, defective in ataxia oculomotor apraxia (AOA) type 2, is a nuclear protein involved in the DNA damage response. AOA2 cells are sensitive to H2O2, camptothecin, and mitomycin C, but not to ionizing radiation, and sensitivity was rescued with full-length SETX cDNA. AOA2 cells exhibited constitutive oxidative DNA damage and enhanced chromosomal instability in response to H2O2. Rejoining of H2O2-induced DNA double-strand breaks (DSBs) was significantly reduced in AOA2 cells compared to controls, and there was no evidence for a defect in DNA single-strand break repair. This defect in DSB repair was corrected by full-length SETX cDNA. These results provide evidence that an additional member of the autosomal recessive AOA is also characterized by a defective response to DNA damage, which may contribute to the neurodegeneration seen in this syndrome.

The effect of larval age on morphology and gene expression during ascidian metamorphosis.

Metamorphosis is both an ecological and a developmental genetic transition that an organism undergoes as a normal part of ontogeny. Many organisms have the ability to delay metamorphosis when conditions are unsuitable. This strategy carries obvious benefits, but may also result in severe consequences for older larvae that run low on energy. In the marine environment, some lecithotrophic larvae that have prolonged periods in the plankton may begin forming postlarval and juvenile structures that normally do not appear until after settlement and the initiation of metamorphosis. This precocious activation of the postlarval developmental program may reflect an adaptation to increase the survival of older, energy-depleted larvae by allowing them to metamorphose more quickly. In the present study, we investigate morphological and genetic consequences of delay of metamorphosis in larvae of Herdmania momus (a solitary stolidobranch ascidian). We observe significant morphological and genetic changes during prolonged larval life, with older larvae displaying significant changes in RNA levels, precocious migration of mesenchyme cells, and changes in larval shape including shortening of the tail. While these observations suggest that the older H. momus larvae are functionally different from younger larvae and possibly becoming more predisposed to undergo metamorphosis, we did not find any significant differences in gene expression levels between postlarvae arising from larvae that metamorphosed as soon as they were competent and postlarvae developing from larvae that postponed metamorphosis. This recalibration, or convergence, of transcript levels in the early postlarva suggests that changes that occur during prolonged larval life of H. momus are not necessarily associated with early activation of adult organ differentiation. Instead, it suggests that an autonomous developmental program is activated in H. momus upon the induction of metamorphosis regardless of the history of the larva.

Deficiency in the catalytic subunit of DNA-dependent protein kinase causes down-regulation of ATM.

Previous reports have suggested a connection between reduced levels of the catalytic subunit of DNA-dependent protein kinases (DNA-PKcs), a component of the nonhomologous DNA double-strand breaks end-joining system, and a reduction in ATM. We studied this possible connection in other DNA-PKcs-deficient cell types, and following knockdown of DNA-PKcs with small interfering RNA, Chinese hamster ovary V3 cells, lacking DNA-PKcs, had reduced levels of ATM and hSMG-1, but both were restored after transfection with PRKDC. Atm levels were also reduced in murine scid cells. Reduction of ATM in a human glioma cell line lacking DNA-PKcs was accompanied by defective signaling through downstream substrates, post-irradiation. A large reduction of DNA-PKcs was achieved in normal human fibroblasts after transfection with two DNA-PKcs small interfering RNA sequences. This was accompanied by a reduction in ATM. These data were confirmed using immunocytochemical detection of the proteins. Within hours after transfection, a decline in PRKDC mRNA was seen, followed by a more gradual decline in DNA-PKcs protein beginning 1 day after transfection. No change in ATM mRNA was observed for 2 days post-transfection. Only after the DNA-PKcs reduction occurred was a reduction in ATM mRNA observed, beginning 2 days post-transfection. The amount of ATM began to decline, starting about 3 days post-treatment, then it declined to levels comparable to DNA-PKcs. Both proteins returned to normal levels at later times. These data illustrate a potentially important cross-regulation between the nonhomologous end-joining system for rejoining of DNA double-strand breaks and the ATM-dependent damage response network of pathways, both of which operate to maintain the integrity of the genome.

Regulation of the aprX-lipA operon of Pseudomonas fluorescens B52: differential regulation of the proximal and distal genes, encoding protease and lipase, by ompR-envZ.

The production of lipase and protease from psychrotrophic strains of Pseudomonas fluorescens may result in spoilage of dairy products. The lipase (lipA) and alkaline metalloprotease (aprX) genes of P. fluorescens B52 are regulated by temperature and are located at opposite ends of an operon which contains eight genes and spans 14 kb. In this report, we show that lipase activity in the supernatant of cultures of P. fluorescens strain B52 is also regulated by the homologue of the Escherichia coli EnvZ-OmpR two-component regulatory system. Differences in the regulation of lipase and protease may be related to the proximal and distal locations of aprX and lipA within the operon.

Gene expression during early ascidian metamorphosis requires signalling by Hemps, an EGF-like protein.

Hemps, a novel epidermal growth factor (EGF)-like protein, is expressed during larval development and early metamorphosis in the ascidian Herdmania curvata and plays a direct role in triggering metamorphosis. In order to identify downstream genes in the Hemps pathway we used a gene expression profiling approach, in which we compared post-larvae undergoing normal metamorphosis with larval metamorphosis blocked with an anti-Hemps antibody. Molecular profiling revealed that there are dynamic changes in gene expression within the first 30 minutes of normal metamorphosis with a significant portion of the genome (approximately 49%) being activated or repressed. A more detailed analysis of the expression of 15 of these differentially expressed genes through embryogenesis, larval development and metamorphosis revealed that while there is a diversity of temporal expression patterns, a number of genes are transiently expressed during larval development and metamorphosis. These and other differentially expressed genes were localised to a range of specific cell and tissue types in Herdmania larvae and post-larvae. The expression of approximately 24% of the genes that were differentially expressed during early metamorphosis was affected in larvae treated with the anti-Hemps antibody. Knockdown of Hemps activity affected the expression of a range of genes within 30 minutes of induction, suggesting that the Hemps pathway directly regulates early response genes at metamorphosis. In most cases, it appears that the Hemps pathway contributes to the modulation of gene expression, rather than initial gene activation or repression. A total of 151 genes that displayed the greatest alterations in expression in response to anti-Hemps antibody were sequenced. These genes were implicated in a range of developmental and physiological roles, including innate immunity, signal transduction and in the regulation of gene transcription. These results suggest that there is significant gene activity during the very early stages of H. curvata metamorphosis and that the Hemps pathway plays a key role in regulating the expression of many of these genes.

Ecological regulation of development: induction of marine invertebrate metamorphosis.

In the marine environment a wide range of invertebrates have a pelagobenthic lifecycle that includes planktonic larval and benthic adult phases. Transition between these morphologically and ecologically distinct phases typically occurs when the developmentally competent larva comes into contact with a species-specific environmental cue. This cue acts as a morphogenetic signal that induces the completion of the postlarval/juvenile/adult developmental program at metamorphosis. The development of competence often occurs hours to days after the larva is morphologically mature. In the non-feeding--lecithotrophic--larvae of the ascidian Herdmania curvata and the gastropod mollusc Haliotis asinina, gene expression patterns in pre-competent and competent stages are markedly different, reflecting the different developmental states of these larval stages. For example, the expression of Hemps, an EGF-like signalling peptide required for the induction of Herdmania metamorphosis, increases in competent larvae. Induction of settlement and metamorphosis results in further changes in developmental gene expression, which apparently is necessary for the complete transformation of the larval body plan into the adult form.

The aprX-lipA operon of Pseudomonas fluorescens B52: a molecular analysis of metalloprotease and lipase production.

Extracellular protease and lipase production by psychrotrophic strains of Pseudomonas fluorescens is repressed by iron and regulated by temperature. The regulation of protease and lipase has been investigated in P. fluorescens B52. Whereas lipase production is increased below the optimum growth temperature ('low-temperature regulation'), protease production was relatively constant and only decreased above the optimum growth temperature. The genes encoding protease (aprX) and lipase (lipA) are encoded at opposite ends of a contiguous set of genes which also includes protease inhibitor, Type I secretion functions and two autotransporter proteins. Evidence is presented indicating that these genes constitute an operon, with a promoter adjacent to aprX which has been identified by S1 nuclease analysis. The regulation of aprX and lipA has been investigated at the RNA level and using lacZ fusion strains. Whereas the data are consistent with iron regulation at the transcriptional level, a lipA'-'lacZ fusion is not regulated by temperature, suggesting that temperature regulation is post-transcriptional or post-translational. The possibility of regulation at the level of mRNA decay is discussed.

Temperature regulation of protease in Pseudomonas fluorescens LS107d2 by an ECF sigma factor and a transmembrane activator.

The production of extracellular enzymes by Pseudomonas fluorescens is important with respect to phytopathogenesis and, in the case of psychrotrophic strains, food spoilage. The production of extracellular protease has been previously reported to be dependent on temperature in psychrotrophic strains of P. fluorescens; production is decreased above the optimum growth temperature with a relatively small change in growth rate. In this work, a transposon mutant of P. fluorescens LS107d2 has been isolated which, in contrast to the wild-type strain, is completely protease deficient at 29 degrees C, above the optimum growth temperature of 25 degrees C, but which produces protease at 23 degrees C. Further analysis revealed that this mutation is in a gene (prtR) which is part of a dicistronic operon, prtIR, in which the two genes are translationally coupled. Evidence is presented that prtI encodes a sigma factor related to others involved in extracytoplasmic functions (ECF sigma factors) and that prtR encodes a novel transmembrane activator of PrtI. PrtI, like PrtR, is also required for protease production at 29 degrees C but not at 23 degrees C. Analysis of the amino acid sequence of PrtR indicates that it is functionally related to a group of membrane-associated anti-sigma factors and a few transmembrane regulators, but is not significantly sequence related. Complementation analysis indicates that PrtR may also interact with sigma factors other than PrtI. The promoter region of the protease-encoding gene (aprX) in LS107d2 has been identified and has sequence features which could indicate interaction with either an ECF sigma factor or a primary sigma factor.