The developmental basis for scaling of mammalian tooth size.

When evolution leads to differences in body size, organs generally scale along. A well-known example of the tight relationship between organ and body size is the scaling of mammalian molar teeth. To investigate how teeth scale during development and evolution, we compared molar development from initiation through final size in the mouse and the rat. Whereas the linear dimensions of the rat molars are twice that of the mouse molars, their shapes are largely the same. Here, we focus on the first lower molars that are considered the most reliable dental proxy for size-related patterns due to their low within-species variability. We found that scaling of the molars starts early, and that the rat molar is patterned equally as fast but in a larger size than the mouse molar. Using transcriptomics, we discovered that a known regulator of body size, insulin-like growth factor 1 (Igf1), is more highly expressed in the rat molars compared to the mouse molars. Ex vivo and in vivo mouse models demonstrated that modulation of the IGF pathway reproduces several aspects of the observed scaling process. Furthermore, analysis of IGF1-treated mouse molars and computational modeling indicate that IGF signaling scales teeth by simultaneously enhancing growth and by inhibiting the cusp-patterning program, thereby providing a relatively simple mechanism for scaling teeth during development and evolution. Finally, comparative data from shrews to elephants suggest that this scaling mechanism regulates the minimum tooth size possible, as well as the patterning potential of large teeth.

Disrupting IGF Signaling in Adult Mice Conditions Leanness, Resilient Energy Metabolism, and High Growth Hormone Pulses.

Growth hormone (GH) and insulinlike growth factor (IGF) promote aging and age-related pathologies. Inhibiting this pathway by targeting IGF receptor (IGF-1R) is a promising strategy to extend life span, alleviate age-related diseases, and reduce tumor growth. Although anti-IGF-1R agents are being developed, long-term effects of IGF-1R blockade remain unknown. In this study, we used ubiquitous inducible IGF-1R knockout (UBIKOR) to suppress signaling in all adult tissues and screened health extensively. Surprisingly, UBIKOR mice showed no overt defects and presented with rather inconspicuous health, including normal cognition. Endocrine GH and IGF-1 were strongly upregulated without causing acromegaly. UBIKOR mice were strikingly lean with coordinate changes in body composition and organ size. They were insulin resistant but preserved physiological energy expenditure and displayed enhanced fasting metabolic flexibility. Thus, long-term IGF-1R blockade generated beneficial effects on aging-relevant metabolism, but exposed to high GH. This needs to be considered when targeting IGF-1R to protect from neurodegeneration, retard aging, or fight cancer.

Insulin-like growth factor 1 receptor regulates hypothermia during calorie restriction.

When food resources are scarce, endothermic animals can lower core body temperature (Tb). This phenomenon is believed to be part of an adaptive mechanism that may have evolved to conserve energy until more food becomes available. Here, we found in the mouse that the insulin-like growth factor 1 receptor (IGF-1R) controls this response in the central nervous system. Pharmacological or genetic inhibition of IGF-1R enhanced the reduction of temperature and of energy expenditure during calorie restriction. Full blockade of IGF-1R affected female and male mice similarly. In contrast, genetic IGF-1R dosage was effective only in females, where it also induced transient and estrus-specific hypothermia in animals fed ad libitum. These effects were regulated in the brain, as only central, not peripheral, pharmacological activation of IGF-1R prevented hypothermia during calorie restriction. Targeted IGF-1R knockout selectively in forebrain neurons revealed that IGF signaling also modulates calorie restriction-dependent Tb regulation in regions rostral of the canonical hypothalamic nuclei involved in controlling body temperature. In aggregate, these data identify central IGF-1R as a mediator of the integration of nutrient and temperature homeostasis. They also show that calorie restriction, IGF-1R signaling, and body temperature, three of the main regulators of metabolism, aging, and longevity, are components of the same pathway.

The Alzheimer's disease transcriptome mimics the neuroprotective signature of IGF-1 receptor-deficient neurons.

Seminal studies using post-mortem brains of patients with Alzheimer's disease evidenced aberrant insulin-like growth factor 1 receptor (IGF1R) signalling. Addressing causality, work in animal models recently demonstrated that long-term suppression of IGF1R signalling alleviates Alzheimer's disease progression and promotes neuroprotection. However, the underlying mechanisms remain largely elusive. Here, we showed that genetically ablating IGF1R in neurons of the ageing brain efficiently protects from neuroinflammation, anxiety and memory impairments induced by intracerebroventricular injection of amyloid-ß oligomers. In our mutant mice, the suppression of IGF1R signalling also invariably led to small neuronal soma size, indicative of profound changes in cellular homeodynamics. To gain insight into transcriptional signatures leading to Alzheimer's disease-relevant neuronal defence, we performed genome-wide microarray analysis on laser-dissected hippocampal CA1 after neuronal IGF1R knockout, in the presence or absence of APP/PS1 transgenes. Functional analysis comparing neurons in early-stage Alzheimer's disease with IGF1R knockout neurons revealed strongly convergent transcriptomic signatures, notably involving neurite growth, cytoskeleton organization, cellular stress response and neurotransmission. Moreover, in Alzheimer's disease neurons, a high proportion of genes responding to Alzheimer's disease showed a reversed differential expression when IGF1R was deleted. One of the genes consistently highlighted in genome-wide comparison was the neurofilament medium polypeptide Nefm. We found that NEFM accumulated in hippocampus in the presence of amyloid pathology, and decreased to control levels under IGF1R deletion, suggesting that reorganized cytoskeleton likely plays a role in neuroprotection. These findings demonstrated that significant resistance of the brain to amyloid-ß can be achieved lifelong by suppressing neuronal IGF1R and identified IGF-dependent molecular pathways that coordinate an intrinsic program for neuroprotection against proteotoxicity. Our data also indicate that neuronal defences against Alzheimer's disease rely on an endogenous gene expression profile similar to the neuroprotective response activated by genetic disruption of IGF1R signalling. This study highlights neuronal IGF1R signalling as a relevant target for developing Alzheimer's disease prevention strategies.

The human DNA ends proteome uncovers an unexpected entanglement of functional pathways.

DNA ends get exposed in cells upon either normal or dysfunctional cellular processes or molecular events. Telomeres need to be protected by the shelterin complex to avoid junctions occurring between chromosomes while failing topoisomerases or clustered DNA damage processing may produce double-strand breaks, thus requiring swift repair to avoid cell death. The rigorous study of the great many proteins involved in the maintenance of DNA integrity is a challenging task because of the innumerous unspecific electrostatic and/or hydrophobic DNA-protein interactions that arise due to the chemical nature of DNA. We devised a technique that discriminates the proteins recruited specifically at DNA ends from those that bind to DNA because of a generic affinity for the double helix. Our study shows that the DNA ends proteome comprises proteins of an unexpectedly wide functional spectrum, ranging from DNA repair to ribosome biogenesis and cytoskeleton, including novel proteins of undocumented function. A global mapping of the identified proteome on published DNA repair protein networks demonstrated the excellent specificity and functional coverage of our purification technique. Finally, the native nucleoproteic complexes that assembled specifically onto DNA ends were shown to be endowed with a highly efficient DNA repair activity.

DNA enzymes as potential therapeutics: towards clinical application of 10-23 DNAzymes.

INTRODUCTION: Ongoing studies on the inhibition of gene expression at the mRNA level have identified several types of specific inhibitors such as antisense oligonucleotides, small interfering RNA, ribozymes and DNAzymes (Dz). After its discovery in 1997, the 10-23 Dz (which can cleave RNA efficiently and site-specifically, has flexible design, is independent from cell mechanisms, does not require expensive chemical modifications for effective use in vivo) has been employed to downregulate a range of therapeutically important genes. Recently, 10-23 Dzs have taken their first steps into clinical trials. AREAS COVERED: This review focuses predominantly on Dz applications as potential antiviral, antibacterial, anti-cancer and anti-inflammatory agents as well as for the treatment of cardiovascular disease and diseases of CNS, summarizing results of their clinical trials up to the present day. EXPERT OPINION: In comparison with antisense oligonucleotides and small interfering RNAs, Dzs do not usually show off-target effects due to their high specificity and lack of immunogenicity in vivo. As more results of clinical trials carried out so far are gradually becoming available, Dzs may turn out to be safe and well-tolerated therapeutics in humans. Therefore, there is a good chance that we may witness a deoxyribozyme drug reaching the clinic in the near future.

Monitoring DNA triplex formation using multicolor fluorescence and application to insulin-like growth factor I promoter downregulation.

Inhibition of insulin-like growth factor I (IGF-I) signaling is a promising antitumor strategy and nucleic acid-based approaches have been investigated to target genes in the pathway. Here, we sought to modulate IGF-I transcriptional activity using triple helix formation. The IGF-I P1 promoter contains a purine/pyrimidine (R/Y) sequence that is pivotal for transcription as determined by deletion analysis and can be targeted with a triplex-forming oligonucleotide (TFO). We designed modified purine- and pyrimidine-rich TFOs to bind to the R/Y sequence. To monitor TFO binding, we developed a fluorescence-based gel-retardation assay that allowed independent detection of each strand in three-stranded complexes using end-labeling with Alexa 488, cyanine (Cy)3 and Cy5 fluorochromes. We characterized TFOs for their ability to inhibit restriction enzyme activity, compete with DNA-binding proteins and inhibit IGF-I transcription in reporter assays. TFOs containing modified nucleobases, 5-methyl-2'-deoxycytidine and 5-propynyl-2'-deoxyuridine, specifically inhibited restriction enzyme cleavage and formed triplexes on the P1 promoter fragment. In cells, deletion of the R/Y-rich sequence led to 48% transcriptional inhibition of a reporter gene. Transfection with TFOs inhibited reporter gene activity to a similar extent, whereas transcription from a mutant construct with an interrupted R/Y region was unaffected, strongly suggesting the involvement of triplex formation in the inhibitory mechanisms. Our results indicate that nuclease-resistant TFOs will likely inhibit endogenous IGF-I gene function in cells.

Targeting insulin-like growth factor I with 10-23 DNAzymes: 2'-O-methyl modifications in the catalytic core enhance mRNA cleavage.

Insulin-like growth factor I (IGF-I) and its cognate receptor (IGF-1R) contribute to normal cell function and to tumorigenesis. The role of IGF-I signaling in tumor growth has been demonstrated in vivo using nucleic acid-based strategies. Here, we designed the first 10-23 DNAzymes directed against IGF-I mRNA. Unlike antisense approaches and RNA interference that require protein catalysis, DNAzymes catalyze protein-free RNA cleavage. We identified target sequences and measured catalytic properties of differently designed DNAzymes on short synthetic RNA targets and on in vitro transcribed IGF-I mRNA. The most efficient cleavers were then transfected into cells, and their inhibitory effect was analyzed using reporter gene assays. We found that increasing the size of DNAzyme flanking sequences and modifications of the termini with 2'-O-methyl residues improved cleavage rates of target RNAs. Modification of the catalytic loop with six 2'-O-methyl ribonucleotides at nonessential positions increased or decreased catalytic efficiency depending on the mRNA target site. In cells, DNAzymes with 2'-O-methyl-modified catalytic cores and flanking sequences were able to inhibit reporter gene activity because of specific recognition and cleavage of IGF-I mRNA sequences. Mutant DNAzymes with inactive catalytic cores were unable to block reporter gene expression, demonstrating that the RNA cleaving ability of 10-23 DNAzymes contributed to inhibitory mechanisms. Our results show that nuclease-resistant 2'-O-methyl-modified DNAzymes with high catalytic efficiencies are useful for inhibiting IGF-I gene function in cells.

Small interfering RNA targeted to IGF-IR delays tumor growth and induces proinflammatory cytokines in a mouse breast cancer model.

Insulin-like growth factor I (IGF-I) and its type I receptor (IGF-IR) play significant roles in tumorigenesis and in immune response. Here, we wanted to know whether an RNA interference approach targeted to IGF-IR could be used for specific antitumor immunostimulation in a breast cancer model. For that, we evaluated short interfering RNA (siRNAs) for inhibition of in vivo tumor growth and immunological stimulation in immunocompetent mice. We designed 2'-O-methyl-modified siRNAs to inhibit expression of IGF-IR in two murine breast cancer cell lines (EMT6, C4HD). Cell transfection of IGF-IR siRNAs decreased proliferation, diminished phosphorylation of downstream signaling pathway proteins, AKT and ERK, and caused a G0/G1 cell cycle block. The IGF-IR silencing also induced secretion of two proinflammatory cytokines, TNF- α and IFN-γ. When we transfected C4HD cells with siRNAs targeting IGF-IR, mammary tumor growth was strongly delayed in syngenic mice. Histology of developing tumors in mice grafted with IGF-IR siRNA treated C4HD cells revealed a low mitotic index, and infiltration of lymphocytes and polymorphonuclear neutrophils, suggesting activation of an antitumor immune response. When we used C4HD cells treated with siRNA as an immunogen, we observed an increase in delayed-type hypersensitivity and the presence of cytotoxic splenocytes against wild-type C4HD cells, indicative of evolving immune response. Our findings show that silencing IGF-IR using synthetic siRNA bearing 2'-O-methyl nucleotides may offer a new clinical approach for treatment of mammary tumors expressing IGF-IR. Interestingly, our work also suggests that crosstalk between IGF-I axis and antitumor immune response can mobilize proinflammatory cytokines.

Chronodisruption in lung cancer and possible therapeutic approaches.

A customary temporal organization of physiological functions and biological processes is necessary to maintain body homeostasis and an altered body time structure may favour carcinogenesis. There is growing evidence that GH stimulates cancer growth, IGF1 may have a role in carcinogenesis and cancer promotion, GH-IGF1 axis, TRH, TSH, thyroxine, melatonin and cortisol modulate immune cell function and the immune system is often dysfunctional in patients with malignancies. The aim of our study was to evaluate GH-IGF1 axis, hypothalamus-pituitary-thyroid axis, melatonin, cortisol, lymphocyte subsets and IL2 in lung cancer patients. Peripheral blood samples were collected at 4-hour intervals in a 24-hour period from eleven healthy male subjects (age range 35-53 years) and nine male patients suffering from non-small cell lung cancer (age range 43-63 years). In each blood sample, lymphocyte subpopulations (CD3+, CD4+, CD8+, CD16+, CD20+, CD25+, HLA-DR+, γδTcR bearing cells) were analyzed and GH, IGF1, TRH, TSH, FT4, melatonin, cortisol and IL2 were measured. Circadian rhythmicity was evaluated and MESOR, amplitude and acrophase values were compared. In healthy subjects a significant circadian rhythm could be demonstrated with midday peaks for CD8+, CD16+, γδTCR expressing cells and cortisol, and peaks during the night for CD3+, CD4+, GH, TSH and melatonin. A borderline significant rhythm was also observed for CD20+, with a peak late in the evening. IGF1, TRH, FT4 and IL2 values did not show rhythmic variation. In cancer patients a significant circadian rhythm could be demonstrated with diurnal peak for CD16+ and peaks during the night for CD4+ and melatonin. GH, IGF1, TRH, TSH, FT4, cortisol and IL2 values did not show rhythmic variation. MESOR of CD8+ (P<0.0001), CD20+ (P=0.05), γδTCR expressing cells (P=0.01), IGF1 (P<0.001) and TSH (P=0.032) was higher in healthy subjects, whereas MESOR of CD16+ (P<0.0001), CD25+ (P=0.001), GH (P<0.001), TRH (P=0.002), FT4 (P=0.030), cortisol (P=0.01) and IL2 (P=0.02) was higher in cancer patients. Amplitude of circadian variation of γδTCR expressing cells (P=0.01), TSH (P<0.001) and cortisol (P=0.01) was higher in healthy subjects, whereas amplitude of circadian variation of CD4+ was higher in cancer patients (P=0.02). In conclusion, non-small cell lung cancer patients show severe alterations of periodic and quantitative characteristics of neuroendocrine and immune parameters with loss of circadian rhythmicity and internal desynchronization, leading to chronodisruption.

A steric blocker of translation elongation inhibits IGF-1R expression and cell transformation.

The insulin-like growth factor 1 receptor (IGF-1R) is involved in transformation, survival, mitogenesis and differentiation. It is overexpressed in many tumors and a validated target for anticancer therapy. In cell-free systems, polypyrimidic peptide nucleic acids (PNAs) can form triplex-like structures with messenger RNAs and halt the ribosomal machinery during the translation elongation. A 17-mer PNA that formed a PNA(2):mRNA complex with a purine-rich sequence located in the coding region of IGF-1R mRNA induced the synthesis of a truncated IGF-1R in vitro. This PNA down-regulated expression of the receptor by 70-80% in prostate cancer cells without affecting insulin receptor expression that exhibits high homology with IGF-1R. Inhibition occurs at the translational level, since the IGF-1R mRNA level measured by quantitative RT-PCR was not affected by PNA treatment. In addition, IGF-1R knockdown by PNA led to an attenuation of phosphorylation of downstream signaling pathways, PI3K/AKT and MAPK, involved in survival and mitogenesis and also to a decrease in cell transformation. Of the steric blockers tested, which included phosphorodiamidate morpholino oligomers and locked nucleic acids, PNA was unique in its ability to form triplex structures with mRNA and to arrest translation elongation.

Sequence-specific targeting of IGF-I and IGF-IR genes by camptothecins.

We and others have clearly demonstrated that a topoisomerase I (Top1) inhibitor, such as camptothecin (CPT), coupled to a triplex-forming oligonucleotide (TFO) through a suitable linker can be used to cause site-specific cleavage of the targeted DNA sequence in in vitro models. Here we evaluated whether these molecular tools induce sequence-specific DNA damage in a genome context. We targeted the insulin-like growth factor (IGF)-I axis and in particular promoter 1 of IGF-I and intron 2 of type 1 insulin-like growth factor receptor (IGF-IR) in cancer cells. The IGF axis molecules represent important targets for anticancer strategies, because of their central role in oncogenic maintenance and metastasis processes. We chemically attached 2 CPT derivatives to 2 TFOs. Both conjugates efficiently blocked gene expression in cells, reducing the quantity of mRNA transcribed by 70-80%, as measured by quantitative RT-PCR. We confirmed that the inhibitory mechanism of these TFO conjugates was mediated by Top1-induced cleavage through the use of RNA interference experiments and a camptothecin-resistant cell line. In addition, induction of phospho-H2AX foci supports the DNA-damaging activity of TFO-CPT conjugates at specific sites. The evaluated conjugates induce a specific DNA damage at the target gene mediated by Top1.

Effective cleavage of structured RNAs by tandems of 10-23 DNAzymes with 3'-modified oligo(2'-O-methylribonucleotide)-effectors.

Catalytic activity of DNAzymes targeted to IGF-I and MDR1 mRNA can be regulated by the combination of 10-23 DNAzyme with 3'-modified oligo(2'-O-methylribonucleotides) that are favorable as effectors due to their high affinity to RNA and nuclease resistance. We have demonstrated that the DNAzyme constructions designed were able to bind and cleave long structured RNA transcripts effectively under simulated physiological conditions.

Global quantitative analysis of protein phosphorylation status in fish exposed to microcystin.

The hepatotoxins, microcystins (MCs) are potent inhibitors of protein phosphatases PP1 and PP2A. These nonribosomal peptides are getting more and more attention because of their acute toxicity and potent tumor-promoting activity. These toxins are produced by freshwater cyanobacteria. Herein, we report a toxicological study conducted on aquatic animal models such as the medaka fish. To date, the detailed mechanisms underlying the toxicity of microcystins are unknown. MC-leucine-arginine (MC-LR) is the most toxic and the most commonly encountered variant of MCs in aquatic environment. It has been used for toxicological investigations on the liver of intoxicated medaka. We performed differential proteome analyses of MC-LR-treated and untreated medaka fish to investigate the mechanisms of establishment of early responses to the toxin. The identification of proteins involved in these early responses might constitute candidates of biomarkers of MC-LR exposure. Cytosolic proteins from livers of exposed or nonexposed medaka were resolved by 2D electrophoresis and detected using stains specific for phosphoproteins and for whole protein content. Overall, 15 spots were found to vary significantly on the proteomic 2D maps or on the phosphoproteomic 2D maps. Of these 15 proteins, only two could not be identified by mass spectrometry. Among the other proteins that were identified, phenylalanine hydroxylase and keratin 18 (type I) showed variations in phoshoryl content in agreement with inhibition of PP2A activity after exposure of the fish to MC-LR. The other identified proteins exhibited variations in their expression level. The identified proteins appear to be involved in cytoskeleton assembly, cell signalling, oxidative stress, and apoptosis. The functional implications of responses to MC-LR exposure of these proteins are discussed. The methodology described in this report should be widely used to a number of tissues and organisms, thus helping in the search for biomarkers of MC-LR contamination.

Modern tools for identification of nucleic acid-binding proteins.

Numerous biological mechanisms depend on nucleic acid--protein interactions. The first step to the understanding of these mechanisms is to identify interacting molecules. Knowing one partner, the identification of other associated molecular species can be carried out using affinity-based purification procedures. When the nucleic acid-binding protein is known, the nucleic acid can be isolated and identified by sensitive techniques such as polymerase chain reaction followed by DNA sequencing or hybridization on chips. The reverse identification procedure is less straightforward in part because interesting nucleic acid-binding proteins are generally of low abundance and there are no methods to amplify amino acid sequences. In this article, we will review the strategies that have been developed to identify nucleic acid-binding proteins. We will focus on methods permitting the identification of these proteins without a priori knowledge of protein candidates.

Temporal variations in the dynamics of potentially microcystin-producing strains in a bloom-forming Planktothrix agardhii (Cyanobacterium) population.

The concentration of microcystins (MCs) produced during blooms depends on variations in both the proportion of strains containing the genes involved in MC production and the MC cell quota (the ratio between the MC concentration and the density of cells with the mcyA genotype) for toxic strains. In order to assess the dynamics of MC-producing and non-MC-producing strains and to identify the impact of environmental factors on the relative proportions of these two subpopulations, we performed a 2-year survey of a perennial bloom of Planktothrix agardhii (cyanobacteria). Applying quantitative real-time PCR to the mcyA and phycocyanin genes, we found that the proportion of cells with the mcyA genotype varied considerably over time (ranging from 30 to 80% of the population). The changes in the proportion of cells with the mcyA genotype appeared to be inversely correlated to changes in the density of P. agardhii cells and also, to a lesser extent, to the availability of certain nutrients and the abundance of cladocerans. Among toxic cells, the MC cell quota varied throughout the survey. However, a negative correlation between the MC cell quota and the mcyA cell number during two short periods characterized by marked changes in the cyanobacterial biomass was found. Finally, only 54% of the variation in the MC concentrations measured in the lake can be explained by the dynamics of the density of cells with the MC producer genotype, suggesting that this measurement is not a satisfactory method for use in monitoring programs intended to predict the toxic risk associated with cyanobacterial proliferation.

Global quantitative analysis of protein expression and phosphorylation status in the liver of the medaka fish (Oryzias latipes) exposed to microcystin-LR I. Balneation study.

Microcystins (MCs) are hepatotoxins with potent inhibitor activity of protein phosphatases PP1 and PP2A. These non-ribosomal peptides are getting more and more attention due to their acute toxicity and potent tumor-promoting activity. These toxins are produced by freshwater cyanobacteria. The most toxic and most commonly encountered variant in aquatic environment is MC-LR (MC Leucine-Arginine). It has been used for toxicological investigations on the liver of intoxicated medaka. Differential proteome as well as differential phosphoproteome analyses have been performed for providing new information on early responses to the toxin. The experiments are also aiming at selecting biomarkers of MC-LR exposure. In the 2D electrophoresis gel protein maps from cytosol of liver cells of animals exposed or non-exposed to the cyanotoxin, 15 spots showed a significant increase or decrease of their stain signal either in specific phosphoprotein stain or total protein stain. Thirteen of these proteins have been identified by mass spectrometry. Among them, phenylalanine hydroxylase (PAH) and keratin 18 type I showed variations in phosphorylation stain in possible agreement with inhibition of PP2A activity. The other identified proteins exhibited variations in their expression level. The identified proteins appear to be involved in cytoskeleton assembly, cell signalling, oxidative stress and apoptosis. Such results confirm that proteomics and phosphoproteomics approaches may become valuable tools to identify signalling pathways implied in MC-LR effects. From accumulated data, specific pools of biomarkers could possibly be selected as specific for toxin exposure.

Analytical biochemistry of DNA--protein assemblies from crude cell extracts.

Purification of specific DNA-protein complexes is a challenging task, as the involved interactions can be both electrostatic/H-bond and hydrophobic. The chromatographic stringency needed to obtain reasonable purifications uses salts and detergents. However, these components elicit the removal of proteins unspecifically bound to the chromatographic support itself, thus contaminating the purification products. In this work, a photocleavable linker connected the target oligonucleotidic sequence to the chromatographic beads so as to allow the irradiation-based release of the purified DNA-protein complexes off the beads. Our bioanalytical conditions were validated by purifying the tetracycline repressor protein onto a specific oligonucleotide. The purification factor was unprecedented, with a single contaminant. The robustness of our method was challenged by applying it to the purification of multiprotein assemblies forming onto DNA damage-mimicking oligonucleotides. The purified components were identified as well-known DNA repair proteins, and were shown to retain their enzymatic activities, as seen by monitoring DNA ligation products. Remarkably, kinase activities, also monitored, were found to be distinct on the beads and on the purified DNA-protein complexes, showing the benefits to uncouple the DNA-protein assemblies from the beads for a proper understanding of biochemical regulatory mechanisms involved in the DNA-protein assemblies.

A hybrid CMV-H1 construct improves efficiency of PEI-delivered shRNA in the mouse brain.

RNA-interference-driven loss of function in specific tissues in vivo should permit analysis of gene function in temporally and spatially defined contexts. However, delivery of efficient short hairpin RNA (shRNA) to target tissues in vivo remains problematic. Here, we demonstrate that efficiency of polyethylenimine (PEI)-delivered shRNA depends on the regulatory sequences used, both in vivo and in vitro. When tested in vivo, silencing of a luciferase target gene by shRNA produced from a hybrid construct composed of the CMV enhancer/promoter placed immediately upstream of an H1 promoter (50%) exceeds that obtained with the H1 promoter alone (20%). In contrast, in NIH 3T3 cells, the H1 promoter was more efficient than the hybrid construct (75 versus 60% inhibition of target gene expression, respectively). To test CMV-H1 shRNA efficiency against an endogenous gene in vivo, we used shRNA against thyroid hormone receptor alpha1 (TRalpha1). When vectorized in the mouse brain, the hybrid construct strongly derepressed CyclinD1-luciferase reporter gene expression, CyclinD1 being a negatively regulated thyroid hormone target gene. We conclude that promoter choice affects shRNA efficiency distinctly in different in vitro and in vivo situations and that a hybrid CMV-H1 construct is optimal for shRNA delivery in the mouse brain.

Unusual DNA conformations: implications for telomeres.

DNA is prone to structural polymorphism: its three-dimensional structure can differ markedly from the classical double helix. Nucleic acid structures composed of more than two strands have also been observed. The guanine-rich sequence of both the telomere and centromere can form a quadruplex based on G-quartets while the complementary cytosine-rich strand can fold into an intercalated tetramer called the i-motif. The G-quartet is a gold mine for structural biologists and the telomere has become a target for anti-cancer drug design since it was observed that deregulation of telomerase favors proliferation of certain tumors. Other DNA sequences may adopt unusual conformations. Polypurine-polypyrimidine sequences capable of forming a triple-stranded structure called H-DNA are found abundantly in the eukaryotic genome and may play a significant role in DNA metabolism, transcription and replication. Triplex-forming oligonucleotides are currently being developed as "anti-gene" agents. Unusual DNA structures may therefore be implicated in fundamental processes such as gene expression and represent unique targets for both structural-specific and sequence-specific agents. In this review, we present work characterizing some of these unusual conformations in terms of structure, stability and formation kinetics and discuss their biological implications.