ALBERT

Description: The dihedral angle formed at junctions between two plagioclase grains and a grain of augite is only very rarely in textural equilibrium in gabbros from kilometre-scale crustal layered intrusions. The median of a population of these disequilibrium angles, cpp , varies systematically within a single layered intrusion, remaining constant over large stretches of stratigraphy with significant increases and decreases associated with the addition or reduction respectively of the number of phases on the liquidus of the bulk magma. The stepwise changes in cpp are present in the Upper Zone of the Bushveld Complex, the Megacyclic Unit I of the Sept Iles Intrusion, and the Layered Series of the Skaergaard intrusion. The plagioclase-bearing cumulates of Rum have a bimodal distribution of cpp , dependent on whether the cumulus assemblage includes clinopyroxene. The presence of the stepwise changes is independent of the order of arrival of cumulus phases and of the composition of either the cumulus phases or the inferred composition of the interstitial liquid. The only parameter that behaves in an exactly analogous manner to cpp is the rate of change in enthalpy with temperature ( H / T ) during crystallization. Both H / T and cpp increase with the addition of a liquidus phase, and decrease with the removal of a liquidus phase. The replacement of one phase by another has little effect on H / T and no discernible effect on cpp . An increase of H / T results in an increase in the fraction of the total enthalpy budget that is the latent heat of crystallization (the fractional latent heat). It also increases the mass crystallized in each incremental temperature drop (the crystal productivity). These increases of both fractional latent heat and crystal productivity are likely to cause an increase in the time taken to form three-grain junctions in the mush via thermal buffering of a thickened mushy layer. We suggest these are the underlying causes of stepwise increases in cpp . Stepwise changes in the geometry of three-grain junctions in fully solidified gabbros thus provide a clear microstructural marker for the progress of fractionation down the liquid line of descent in layered intrusions.

Description: High-resolution sampling in monogenetic fields has the potential to reveal fine-scale heterogeneity of the mantle, a feature that may be overwhelmed by larger fluxes of magma, or missed by under-sampling. The Quaternary Auckland Volcanic Field (AVF) in northern New Zealand is a basaltic field of 51 small-volume volcanic centres, and is one of the best-sampled examples of a monogenetic volcanic field. We present data for 12 centres in the volcanic field. These show the large compositional variations between volcanoes as well as through single eruptive sequences. Whole-rock compositions range from subalkaline basalt in the larger centres, through alkali basalt to nephelinite in the smallest centres. Fractional crystallization has had a limited effect in many of the centres, but high-pressure clinopyroxene crystallization may have occurred in others. Three end-members are observed in Pb isotope space, indicating that distinct mantle source components are involved in the petrogenesis of the magmas. Whole-rock multi-element patterns show that the larger centres have prominent positive Sr anomalies and lack K anomalies, whereas the smaller centres have prominent negative K anomalies and lack Sr anomalies. The melting parameters and compositions of the sources involved are modelled using trace element ratios and multi-element patterns, and three components are characterized: (1) fertile peridotite with a Pb-isotope composition similar to Pacific mid-ocean ridge basalt; (2) eclogite domains with a HIMU-like isotope composition dispersed within the fertile peridotite; (3) slightly depleted subduction-metasomatized peridotitic lithospheric mantle (containing c . 3% subduction fluids). Modelling shows that melting in the AVF begins in garnet-bearing fertile asthenosphere (with preferential melting of eclogite domains) and that melts are variably diluted by melts of the lithospheric source. The U–Th isotope compositions of the end-members in the AVF show 230 Th excess [( 230 Th/ 232 Th) ratios of 1·11–1·38], with the samples of lower ( 230 Th/ 232 Th) exhibiting higher ( 238 U/ 232 Th), which we attribute to the dilution effect of the melts from the lithospheric mantle source. Modelling reveals a correlation between melting in the asthenosphere, the degree of melting and incorporation of the metasomatized lithospheric mantle source, and the resultant size of the volcanic centre. This suggests that the scale of the eruption may essentially be controlled by asthenospheric mantle dynamics.

Description: The origin of mafic and ultramafic sills exhibiting different whole-rock compositional profiles (e.g. I-, C-, D-, M- and S-shaped profiles) remains controversial. We have addressed this issue by revisiting three ~100 m thick Siberian dolerite sills (Vavukansky, Kuz’movsky and Vilyuysky) that display remarkable internal differentiation. The Vavukansky sill has an M-shaped profile with prominent basal and top reversals showing inward increases in whole-rock MgO, Mg-number [100Mg/(Mg + Fe)] and normative An content [100An/(An + Ab)], followed by the Layered and Upper Border Series with inward decreases in these indices. The Kuz’movsky and Vilyuysky sills both show S-shaped profiles similar to the Vavukansky sill, but lack a top reversal. These whole-rock M- and S-shaped profiles are accompanied by similar profiles in mineral compositions. Plagioclase and, to a lesser extent, olivine show systematic inward increases in An content and Mg-number, respectively, across basal and top reversals. These compositional trends are followed by inward decreases in these ratios in the interiors of the Vavukansky and Kuz’movsky sills. Currently accepted models attribute whole-rock M- and S-shaped compositional profiles to crystal settling, compositional convection or compaction operating in closed systems. Our observations challenge these traditional interpretations because variations in mineral compositions observed in marginal reversals cannot result from closed-system fractionation. We suggest instead that initially the sills evolved as open systems that were slowly inflated by magmas that became gradually more primitive with time. The inflation was accompanied by in situ crystallization that preserved the preceding fractionation history of the injected magmas by forming basal and top reversals with minerals becoming more primitive inwards. This process culminated with rapid inflation of the sills to their current size owing to a major influx of primitive magma. Subsequently, magma flow through the sills ceased and they evolved as closed systems by fractional crystallization. This resulted in the Layered and Upper Border Series with minerals becoming more evolved inwards. This model can be extended to explain other compositional profiles and petrological features in mafic and ultramafic sills.

Description: We have developed a new screening methodology for identifying all genes that control the expression of a target gene through genetic or metabolic interactions. The screen combines mutant libraries with luciferase reporter constructs, whose expression can be monitored in vivo and over time in different environmental conditions. We apply the method to identify the genes that control the expression of the gene acs , encoding the acetyl coenzyme A synthetase, in Escherichia coli . We confirm most of the known genetic regulators, including CRP–cAMP, IHF and components of the phosphotransferase system. In addition, we identify new regulatory interactions, many of which involve metabolic intermediates or metabolic sensing, such as the genes pgi, pfkA , sucB and lpdA , encoding enzymes in glycolysis and the TCA cycle. Some of these novel interactions were validated by quantitative reverse transcriptase-polymerase chain reaction. More generally, we observe that a large number of mutants directly or indirectly influence acs expression, an effect confirmed for a second promoter, sdhC . The method is applicable to any promoter fused to a luminescent reporter gene in combination with a deletion mutant library.

Description: Tandem repeats (TRs) are often present in proteins with crucial functions, responsible for resistance, pathogenicity and associated with infectious or neurodegenerative diseases. This motivates numerous studies of TRs and their evolution, requiring accurate multiple sequence alignment. TRs may be lost or inserted at any position of a TR region by replication slippage or recombination, but current methods assume fixed unit boundaries, and yet are of high complexity. We present a new global graph-based alignment method that does not restrict TR unit indels by unit boundaries. TR indels are modeled separately and penalized using the phylogeny-aware alignment algorithm. This ensures enhanced accuracy of reconstructed alignments, disentangling TRs and measuring indel events and rates in a biologically meaningful way. Our method detects not only duplication events but also all changes in TR regions owing to recombination, strand slippage and other events inserting or deleting TR units. We evaluate our method by simulation incorporating TR evolution, by either sampling TRs from a profile hidden Markov model or by mimicking strand slippage with duplications. The new method is illustrated on a family of type III effectors, a pathogenicity determinant in agriculturally important bacteria Ralstonia solanacearum. We show that TR indel rate variation contributes to the diversification of this protein family.

Description: A renewed interest in non-coding RNA (ncRNA) has led to the discovery of novel RNA species and post-transcriptional ribonucleoside modifications, and an emerging appreciation for the role of ncRNA in RNA epigenetics. Although much can be learned by amplification-based analysis of ncRNA sequence and quantity, there is a significant need for direct analysis of RNA, which has led to numerous methods for purification of specific ncRNA molecules. However, no single method allows purification of the full range of cellular ncRNA species. To this end, we developed a multidimensional chromatographic platform to resolve, isolate and quantify all canonical ncRNAs in a single sample of cells or tissue, as well as novel ncRNA species. The applicability of the platform is demonstrated in analyses of ncRNA from bacteria, human cells and plasmodium-infected reticulocytes, as well as a viral RNA genome. Among the many potential applications of this platform are a system-level analysis of the dozens of modified ribonucleosides in ncRNA, characterization of novel long ncRNA species, enhanced detection of rare transcript variants and analysis of viral genomes.

Description: The synthesis of protein from messenger RNA during translation is a highly dynamic process that plays a key role in controlling the efficiency and fidelity of genome-wide protein expression. The availability of aminoacylated transfer RNA (tRNA) is a major factor influencing the speed of ribosomal movement, which depending on codon choices, varies considerably along a transcript. Furthermore, it has been shown experimentally that tRNA availability can vary significantly under different growth and stress conditions, offering the cell a way to adapt translational dynamics across the genome. Existing models of translation have neglected fluctuations of tRNA pools, instead assuming fixed tRNA availabilities over time. This has lead to an incomplete understanding of this process. Here, we show for the entire Escherichia coli genome how and to what extent translational speed profiles, which capture local aspects of translational elongation, respond to measured shifts in tRNA availability. We find that translational profiles across the genome are affected to differing degrees, with genes that are essential or related to fundamental processes such as translation, being more robust than those linked to regulation. Furthermore, we reveal how fluctuating tRNA availability influences profiles of specific sequences known to play a significant role in translational control of gene expression.

Description: Central to Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas systems are repeated RNA sequences that serve as Cas-protein–binding templates. Classification is based on the architectural composition of associated Cas proteins, considering repeat evolution is essential to complete the picture. We compiled the largest data set of CRISPRs to date, performed comprehensive, independent clustering analyses and identified a novel set of 40 conserved sequence families and 33 potential structure motifs for Cas-endoribonucleases with some distinct conservation patterns. Evolutionary relationships are presented as a hierarchical map of sequence and structure similarities for both a quick and detailed insight into the diversity of CRISPR-Cas systems. In a comparison with Cas-subtypes, I-C, I-E, I-F and type II were strongly coupled and the remaining type I and type III subtypes were loosely coupled to repeat and Cas1 evolution, respectively. Subtypes with a strong link to CRISPR evolution were almost exclusive to bacteria; nevertheless, we identified rare examples of potential horizontal transfer of I-C and I-E systems into archaeal organisms. Our easy-to-use web server provides an automated assignment of newly sequenced CRISPRs to our classification system and enables more informed choices on future hypotheses in CRISPR-Cas research: http://rna.informatik.uni-freiburg.de/CRISPRmap .

Description: Escherichia coli Exonuclease IX (ExoIX), encoded by the xni gene, was the first identified member of a novel subfamily of ubiquitous flap endonucleases (FENs), which possess only one of the two catalytic metal-binding sites characteristic of other FENs. We have solved the first structure of one of these enzymes, that of ExoIX itself, at high resolution in DNA-bound and DNA-free forms. In the enzyme–DNA cocrystal, the single catalytic site binds two magnesium ions. The structures also reveal a binding site in the C-terminal domain where a potassium ion is directly coordinated by five main chain carbonyl groups, and we show this site is essential for DNA binding. This site resembles structurally and functionally the potassium sites in the human FEN1 and exonuclease 1 enzymes. Fluorescence anisotropy measurements and the crystal structures of the ExoIX:DNA complexes show that this potassium ion interacts directly with a phosphate diester in the substrate DNA.

Description: One of the obstacles hindering a better understanding of cancer is its heterogeneity. However, computational approaches to model cancer heterogeneity have lagged behind. To bridge this gap, we have developed a new probabilistic approach that models individual cancer cases as mixtures of subtypes. Our approach can be seen as a meta-model that summarizes the results of a large number of alternative models. It does not assume predefined subtypes nor does it assume that such subtypes have to be sharply defined. Instead given a measure of phenotypic similarity between patients and a list of potential explanatory features, such as mutations, copy number variation, microRNA levels, etc., it explains phenotypic similarities with the help of these features. We applied our approach to Glioblastoma Multiforme (GBM). The resulting model Prob_GBM, not only correctly inferred known relationships but also identified new properties underlining phenotypic similarities. The proposed probabilistic framework can be applied to model relations between similarity of gene expression and a broad spectrum of potential genetic causes.

Description: Estrogen regulates over a thousand genes, with an equal number of them being induced or repressed. The distinct mechanisms underlying these dual transcriptional effects remain largely unknown. We derived comprehensive views of the transcription machineries assembled at estrogen-responsive genes through integrating multiple types of genomic data. In the absence of estrogen, the majority of genes formed higher-order chromatin structures, including DNA loops tethered to protein complexes involving RNA polymerase II (Pol II), estrogen receptor alpha (ERα) and ERα-pioneer factors. Genes to be ‘repressed’ by estrogen showed active transcription at promoters and throughout the gene bodies; genes to be ‘induced’ exhibited active transcription initiation at promoters, but with transcription paused in gene bodies. In the presence of estrogen, the majority of estrogen-induced genes retained the original higher-order chromatin structures, whereas most estrogen-repressed genes underwent a chromatin reconfiguration. For estrogen-induced genes, estrogen enhances transcription elongation, potentially through recruitment of co-activators or release of co-repressors with unique roles in elongation. For estrogen-repressed genes, estrogen treatment leads to chromatin structure reconfiguration, thereby disrupting the originally transcription-efficient chromatin structures. Our in silico studies have shown that estrogen regulates gene expression, at least in part, through modifying previously assembled higher-order complexes, rather than by facilitating de novo assembly of machineries.

Description: The mineralocorticoid receptor (MR) is a ligand-induced transcription factor belonging to the steroid receptor family and involved in water-electrolyte homeostasis, blood pressure regulation, inflammation and fibrosis in the renocardiovascular system. The MR shares a common hormone-response-element with the glucocorticoid receptor but nevertheless elicits MR-specific effects including enhanced epidermal growth factor receptor (EGFR) expression via unknown mechanisms. The EGFR is a receptor tyrosine kinase that leads to activation of MAP kinases, but that can also function as a signal transducer for other signaling pathways. In the present study, we mechanistically investigate the interaction between a newly discovered MR- but not glucocorticoid receptor- responsive-element (=MRE1) of the EGFR promoter, specificity protein 1 (SP1) and MR to gain general insights into MR-specificity. Biological relevance of the interaction for EGFR expression and consequently for different signaling pathways in general is demonstrated in human, rat and murine vascular smooth muscle cells and cells of EGFR knockout mice. A genome-wide promoter search for identical binding regions followed by quantitative PCR validation suggests that the identified MR-SP1–MRE1 interaction might be applicable to other genes. Overall, a novel principle of MR-specific gene expression is explored that applies to the pathophysiologically relevant expression of the EGFR and potentially also to other genes.

Description: Selective inhibitory crosstalk has been known to occur within the signaling pathways of the dioxin (AhR) and estrogen (ERα) receptors. More specifically, ERα represses a cytochrome P450-encoding gene ( CYP1A1 ) that converts cellular estradiol into a metabolite that inhibits the cell cycle, while it has no effect on a P450-encoding gene ( CYP1B1 ) that converts estrodiol into a genotoxic product. Here we show that ERα represses CYP1A1 by targeting the Dnmt3B DNA methyltransferase and concomitant DNA methylation of the promoter. We also find that histone H2A.Z can positively contribute to CYP1A1 gene expression, and its presence at that gene is inversely correlated with DNA methylation. Taken together, our results provide a framework for how ERα can repress transcription, and how that impinges on the production of an enzyme that generates genotoxic estradiol metabolites, and potential breast cancer progression. Finally, our results reveal a new mechanism for how H2A.Z can positively influence gene expression, which is by potentially competing with DNA methylation events in breast cancer cells.

Description: TFIIIB and TFIIIC are multi-subunit factors required for transcription by RNA polymerase III. We present a genome-wide high-resolution footprint map of TFIIIB–TFIIIC complexes in Saccharomyces cerevisiae , obtained by paired-end sequencing of micrococcal nuclease-resistant DNA. On tRNA genes, TFIIIB and TFIIIC form stable complexes with the same distinctive occupancy pattern but in mirror image, termed ‘bootprints’. Global analysis reveals that the TFIIIB–TFIIIC transcription complex exhibits remarkable structural elasticity: tRNA genes vary significantly in length but remain protected by TFIIIC. Introns, when present, are markedly less protected. The RNA polymerase III transcription terminator is flexibly accommodated within the transcription complex and, unexpectedly, plays a major structural role by delimiting its 3'-boundary. The ETC sites, where TFIIIC binds without TFIIIB, exhibit different bootprints, suggesting that TFIIIC forms complexes involving other factors. We confirm six ETC sites and report a new site ( ETC10 ). Surprisingly, TFIIIC, but not TFIIIB, interacts with some centromeric nucleosomes, suggesting that interactions between TFIIIC and the centromere may be important in the 3D organization of the nucleus.

Description: The process of DNA mismatch repair is initiated when MutS recognizes mismatched DNA bases and starts the repair cascade. The Escherichia coli MutS protein exists in an equilibrium between dimers and tetramers, which has compromised biophysical analysis. To uncouple these states, we have generated stable dimers and tetramers, respectively. These proteins allowed kinetic analysis of DNA recognition and structural analysis of the full-length protein by X-ray crystallography and small angle X-ray scattering. Our structural data reveal that the tetramerization domains are flexible with respect to the body of the protein, resulting in mostly extended structures. Tetrameric MutS has a slow dissociation from DNA, which can be due to occasional bending over and binding DNA in its two binding sites. In contrast, the dimer dissociation is faster, primarily dependent on a combination of the type of mismatch and the flanking sequence. In the presence of ATP, we could distinguish two kinetic groups: DNA sequences where MutS forms sliding clamps and those where sliding clamps are not formed efficiently. Interestingly, this inability to undergo a conformational change rather than mismatch affinity is correlated with mismatch repair.

Description: Phosphorylation of histone H4 serine 47 (H4S47ph) is catalyzed by Pak2, a member of the p21-activated serine/threonine protein kinase (Pak) family and regulates the deposition of histone variant H3.3. However, the phosphatase(s) involved in the regulation of H4S47ph levels was unknown. Here, we show that three phosphatases (PP1α, PP1β and Wip1) regulate H4S47ph levels and H3.3 deposition. Depletion of each of the three phosphatases results in increased H4S47ph levels. Moreover, PP1α, PP1β and Wip1 bind H3-H4 in vitro and in vivo , whereas only PP1α and PP1β, but not Wip1, interact with Pak2 in vivo . These results suggest that PP1α, PP1β and Wip1 regulate the levels of H4S47ph through directly acting on H4S47ph, with PP1α and PP1β also likely regulating the activity of Pak2. Finally, depletion of PP1α, PP1β and Wip1 leads to increased H3.3 occupancy at candidate genes tested, elevated H3.3 deposition and enhanced association of H3.3 with its chaperones HIRA and Daxx. These results reveal a novel role of three phosphatases in chromatin dynamics in mammalian cells.

Description: Previously we identified Rrp1 and Rrp2 as two proteins required for the Sfr1/Swi5-dependent branch of homologous recombination (HR) in Schizosaccharomyces pombe . Here we use a yeast two-hybrid approach to demonstrate that Rrp1 and Rrp2 can interact with each other and with Swi5, an HR mediator protein. Rrp1 and Rrp2 form co-localizing methyl methanesulphonate–induced foci in nuclei, further suggesting they function as a complex. To place the Rrp1/2 proteins more accurately within HR sub-pathways, we carried out extensive epistasis analysis between mutants defining Rrp1/2, Rad51 (recombinase), Swi5 and Rad57 (HR-mediators) plus the anti-recombinogenic helicases Srs2 and Rqh1. We confirm that Rrp1 and Rrp2 act together with Srs2 and Swi5 and independently of Rad57 and show that Rqh1 also acts independently of Rrp1/2. Mutants devoid of Srs2 are characterized by elevated recombination frequency with a concomitant increase in the percentage of conversion-type recombinants. Strains devoid of Rrp1 or Rrp2 did not show a change in HR frequency, but the number of conversion-type recombinants was increased, suggesting a possible function for Rrp1/2 with Srs2 in counteracting Rad51 activity. Our data allow us to propose a model placing Rrp1 and Rrp2 functioning together with Swi5 and Srs2 in a synthesis-dependent strand annealing HR repair pathway.

Description: In eukaryotic cells, gene expression is mediated by enhancer activation of RNA polymerase at distant promoters. Recently, distinctions between enhancers and promoters have been blurred by the discovery that enhancers are associated with RNA polymerase and are sites of RNA synthesis. Here, we present an analysis of the insulin-like growth factor 2/H19 muscle enhancer. This enhancer includes a short conserved core element that is organized into chromatin typical of mammalian enhancers, binds tissue-specific transcription factors and functions on its own in vitro to activate promoter transcription. However, in a chromosomal context, this element is not sufficient to activate distant promoters. Instead, enhancer function also requires transcription in cis of a long non-coding RNA, Nctc1 . Thus, the insulin-like growth factor 2/H19 enhancer is an active transcriptional complex whose own transcription is essential to its function.

Description: Evolutionarily conserved RNA secondary structures are a robust indicator of purifying selection and, consequently, molecular function. Evaluating their genome-wide occurrence through comparative genomics has consistently been plagued by high false-positive rates and divergent predictions. We present a novel benchmarking pipeline aimed at calibrating the precision of genome-wide scans for consensus RNA structure prediction. The benchmarking data obtained from two refined structure prediction algorithms, RNAz and SISSIz, were then analyzed to fine-tune the parameters of an optimized workflow for genomic sliding window screens. When applied to consistency-based multiple genome alignments of 35 mammals, our approach confidently identifies 〉4 million evolutionarily constrained RNA structures using a conservative sensitivity threshold that entails historically low false discovery rates for such analyses (5–22%). These predictions comprise 13.6% of the human genome, 88% of which fall outside any known sequence-constrained element, suggesting that a large proportion of the mammalian genome is functional. As an example, our findings identify both known and novel conserved RNA structure motifs in the long noncoding RNA MALAT1 . This study provides an extensive set of functional transcriptomic annotations that will assist researchers in uncovering the precise mechanisms underlying the developmental ontologies of higher eukaryotes.

Description: The p53 core domain binds to response elements (REs) that contain two continuous half-sites as a cooperative tetramer, but how p53 recognizes discontinuous REs is not well understood. Here we describe the crystal structure of the p53 core domain bound to a naturally occurring RE located at the promoter of the Bcl-2-associated X protein (BAX) gene, which contains a one base-pair insertion between the two half-sites. Surprisingly, p53 forms a tetramer on the BAX-RE that is nearly identical to what has been reported on other REs with a 0-bp spacer. Each p53 dimer of the tetramer binds in register to a half-site and maintains the same protein–DNA interactions as previously observed, and the two dimers retain all the protein–protein contacts without undergoing rotation or translation. To accommodate the additional base pair, the DNA is deformed and partially disordered around the spacer region, resulting in an apparent unwinding and compression, such that the interactions between the dimers are maintained. Furthermore, DNA deformation within the p53-bound BAX-RE is confirmed in solution by site-directed spin labeling measurements. Our results provide a structural insight into the mechanism by which p53 binds to discontinuous sites with one base-pair spacer.

Description: RNA-based therapeutic approaches using splice-switching oligonucleotides have been successfully applied to rescue dystrophin in Duchenne muscular dystrophy (DMD) preclinical models and are currently being evaluated in DMD patients. Although the modular structure of dystrophin protein tolerates internal deletions, many mutations that affect nondispensable domains of the protein require further strategies. Among these, trans -splicing technology is particularly attractive, as it allows the replacement of any mutated exon by its normal version as well as introducing missing exons or correcting duplication mutations. We have applied such a strategy in vitro by using cotransfection of pre– trans -splicing molecule (PTM) constructs along with a reporter minigene containing part of the dystrophin gene harboring the stop-codon mutation found in the mdx mouse model of DMD. Optimization of the different functional domains of the PTMs allowed achieving accurate and efficient trans -splicing of up to 30% of the transcript encoded by the cotransfected minigene. Optimized parameters included mRNA stabilization, choice of splice site sequence, inclusion of exon splice enhancers and artificial intronic sequence. Intramuscular delivery of adeno-associated virus vectors expressing PTMs allowed detectable levels of dystrophin in mdx and mdx4Cv , illustrating that a given PTM can be suitable for a variety of mutations.

Description: We show the existence and uniqueness of a solution for the nonlocal vector-valued Allen–Cahn variational inequality in a formulation involving Lagrange multipliers for local and nonlocal constraints. Furthermore, we propose and analyse a primal–dual active set (PDAS) method for local and nonlocal vector-valued Allen–Cahn variational inequalities. The local convergence behaviour of the PDAS algorithm is studied by interpreting the approach as a semismooth Newton method and numerical simulations are presented demonstrating its efficiency.

Description: In this paper, we will present a generalized convolution quadrature for solving linear parabolic and hyperbolic evolution equations. The original convolution quadrature method by Lubich works very nicely for equidistant time steps while the generalization of the method and its analysis to nonuniform time stepping is by no means obvious. We will introduce the generalized convolution quadrature allowing for variable time steps and develop a theory for its error analysis. This method opens the door for further development towards adaptive time stepping for evolution equations. As the main application of our new theory, we will consider the wave equation in exterior domains which is formulated as a retarded boundary integral equation.

Description: We address the error control of Galerkin discretization (in space) of linear second-order hyperbolic problems. More specifically, we derive a posteriori error bounds in the L ( L 2 ) norm for finite element methods for the linear wave equation, under minimal regularity assumptions. The theory is developed for both the space-discrete case and for an implicit fully discrete scheme. The derivation of these bounds relies crucially on carefully constructed space and time reconstructions of the discrete numerical solutions, in conjunction with a technique introduced by Baker (1976, Error estimates for finite element methods for second-order hyperbolic equations. SIAM J. Numer. Anal. , 13 , 564–576) in the context of a priori error analysis of Galerkin discretization of the wave problem in weaker-than-energy spatial norms.

Description: The surface finite element method can be used to approximate curvatures on embedded hypersurfaces and to discretize geometric partial differential equations. In this paper, we present a definition of discrete Ricci curvature on polyhedral hypersurfaces of arbitrary dimension based on the discretization of a weak formulation with isoparametric finite elements. We prove that for a piecewise quadratic approximation of a two- or three-dimensional hypersurface R n +1 , this definition approximates the Ricci curvature of with a linear order of convergence in the L 2 ( ) norm. By using a smoothing scheme in the case of a piecewise linear approximation of , we still get a convergence of order 2/3 in the L 2 ( ) norm and of order 1/3 in the W 1, 2 ( ) norm.

Description: We give general conditions which guarantee that the sequence generated by a descent algorithm converges to an equilibrium point. The convergence result is based on the Lojasiewicz gradient inequality; optimal convergence rates are also derived, as well as a stability result. We show how our results apply to a large variety of standard time discretizations of gradient-like flows. Schemes with variable time step are considered and optimal conditions on the maximal step size are derived. Applications to time and space discretizations of the Allen–Cahn equation, the sine–Gordon equation and a damped wave equation are given.

Description: This paper presents quadratic finite-volume methods for elliptic and parabolic problems on quadrilateral meshes that use Barlow points (optimal stress points) for dual partitions. Introducing Barlow points into the finite-volume formulations results in better approximation properties at the cost of loss of symmetry. The novel ‘symmetrization’ technique adopted in this paper allows us to derive optimal-order error estimates in the H 1 - and L 2 -norms for elliptic problems and in the L ( H 1 )- and L ( L 2 )-norms for parabolic problems. Superconvergence of the difference between the gradients of the finite-volume solution and the interpolant can also be derived. Numerical results confirm the proved error estimates.

Description: A linear parabolic differential equation on a moving surface is discretized in space by evolving-surface finite elements and in time by backward difference formulas (BDFs). Using results from Dahlquist's G-stability theory and Nevanlinna & Odeh's multiplier technique together with properties of the spatial semidiscretization, stability of the full discretization is proved for BDF methods up to order 5 and optimal-order convergence is shown. Numerical experiments illustrate the behaviour of the fully discrete method.

Description: In this article, we develop the a priori and a posteriori error analysis of hp -version interior penalty discontinuous Galerkin finite element methods for strongly monotone quasi-Newtonian fluid flows in a bounded Lipschitz domain R d , d = 2, 3. In the latter case, computable upper and lower bounds on the error are derived in terms of a natural energy norm, which are explicit in the local mesh size and local polynomial degree of the approximating finite element method. A series of numerical experiments illustrate the performance of the proposed a posteriori error indicators within an automatic hp -adaptive refinement algorithm.

Description: In recent years, it has become increasingly clear that the critical issue in gradient methods is the choice of the step length, whereas using gradient as the search direction may lead to very effective algorithms, whose surprising behaviour has only been partially explained, mostly in terms of the spectrum of the Hessian matrix. On the other hand, the convergence of the classical Cauchy steepest descent (SD) method has been analysed extensively and related to the spectral properties of the Hessian matrix, but the connection with the spectrum of the Hessian has not been exploited much to modify the method in order to improve its behaviour. In this work, we show how, for convex quadratic problems, moving from some theoretical properties of the SD method, second-order information provided by the step length can be exploited to dramatically improve the usually poor practical behaviour of this method. This allows us to achieve computational results comparable with those of the Barzilai and Borwein algorithm, with the further advantage of monotonic behaviour.

Description: Magma dynamics and time scales during the VEI 5, 2000 bp eruption of El Misti volcano, southern Peru (EM2000BP) are investigated to address cyclic explosive activity at this hazardous volcano. The 1·4 km 3 of pumice falls and flows have abundant mingled pumice of high-K, calc-alkaline rhyolite and andesite composition. Phenocryst zoning and compositions reveal mutual exchange of plagioclase between the two magmas; amphibole in the rhyolite was derived from the andesite. Amphiboles in the andesite are predominantly unrimmed crystals whereas those in the rhyolite mostly exhibit reaction rims. Phase equilibria indicate that the andesite formed at ~900–950°C and 2–3 kbar pressure and was water-saturated with 5·1–6·0 wt % H 2 O, broadly similar to El Misti magmas overall. Amphibole, plagioclase, Ti-magnetite, and two pyroxenes were the crystallizing phases. A separate rhyolite magma existed higher in the crust at a temperature of 816 ± 30°C and ~5% H 2 O in which only plagioclase and Fe–Ti oxides were stable. The lack of cognate amphibole in the rhyolite despite H 2 O saturation requires that it staged above the stability limit of amphibole (〈100 MPa). Exchange reactions in amphibole (dominantly pargasitic) and trace element partitioning in plagioclase indicate that both andesite and rhyolite magmas were broadly constant in temperature and H 2 O content. These constraints suggest that the initially separate rhyolite and deeper andesite magmas interacted by an initial andesite recharge event that resulted in mingling and crystal exchange. A period of 50–60 days is required for amphibole introduced into the rhyolite to develop reaction rims owing to decompression. These rims are dominated by plagioclase, a consequence of the Al-rich nature of the amphibole. The lack of reaction rims on amphibole in the andesite implicates a second, more-forceful and voluminous eruption-triggering recharge event during which andesite rose rapidly from source to surface in ≤5 days at ascent rates of at least 0·023 m s –1 . Further decompression-driven crystallization is recorded in plagioclase rims and microlite growth that may have contributed to a rapid increase in viscosity leading to explosive eruption. This VEI 5 plinian eruption shares characteristics with other explosive events at El Misti on a time scale of 2000–4000 years, suggesting periodic recharge-driven explosive activity.

Description: We report structural evidence of ductile strain localization in mantle pyroxenite from the spinel to plagioclase websterite transition in the Ronda Peridotite (southern Spain). Mapping shows that, in this domain, small-scale shear zones occurring at the base of the lithospheric section are systematically located within thin pyroxenite layers, suggesting that the pyroxenite was locally weaker than the host peridotite. Strain localization is associated with a sudden decrease of grain size and increasing volume fractions of plagioclase and amphibole as a result of a spinel to plagioclase phase transformation reaction during decompression. This reaction also fostered hydrogen extraction (‘dehydroxylation’) from clinopyroxene producing effective fluid saturation that catalyzed the synkinematic net-transfer reaction. This reaction produced fine-grained olivine and plagioclase, allowing the onset of grain-size sensitive creep and further strain localization in these pyroxenite bands. The strain localization in the pyroxenites is thus explained by their more fertile composition, which allowed earlier onset of the phase transition reactions. Geothermobarometry undertaken on compositionally zoned constituent minerals suggests that this positive feedback between reactions and deformation is associated with cooling from at least 1000°C to 700°C and decompression from 1·0 to 0·5 GPa.

Description: In reverse genetics, a gene’s function is elucidated through targeted modifications in the coding region or associated DNA cis -regulatory elements. To this purpose, recently developed customizable transcription activator-like effector nucleases (TALENs) have proven an invaluable tool, allowing introduction of double-strand breaks at predetermined sites in the genome. Here we describe a practical and efficient method for the targeted genome engineering in Drosophila . We demonstrate TALEN-mediated targeted gene integration and efficient identification of mutant flies using a traceable marker phenotype. Furthermore, we developed an easy TALEN assembly (easyT) method relying on simultaneous reactions of DNA Bae I digestion and ligation, enabling construction of complete TALENs from a monomer unit library in a single day. Taken together, our strategy with easyT and TALEN-plasmid microinjection simplifies mutant generation and enables isolation of desired mutant fly lines in the F 1 generation.

Description: High-throughput sequencing technologies have allowed for the cataloguing of variation in personal human genomes. In this manuscript, we present alu-detect , a tool that combines read-pair and split-read information to detect novel Alus and their precise breakpoints directly from either whole-genome or whole-exome sequencing data while also identifying insertions directly in the vicinity of existing Alus. To set the parameters of our method, we use simulation of a faux reference, which allows us to compute the precision and recall of various parameter settings using real sequencing data. Applying our method to 100 bp paired Illumina data from seven individuals, including two trios, we detected on average 1519 novel Alus per sample. Based on the faux-reference simulation, we estimate that our method has 97% precision and 85% recall. We identify 808 novel Alus not previously described in other studies. We also demonstrate the use of alu-detect to study the local sequence and global location preferences for novel Alu insertions.

Description: DT40 cells derived from chicken B lymphocytes exhibit exceptionally high homologous recombination rates. Therefore, they can be used as a convenient tool and model for gene targeting experiments. However, lack of efficient cloning strategies, protein purification protocols and a well annotated protein database limits the utility of these cells for proteomic studies. Here we describe a fast and inexpensive experimental pipeline for protein localization, quantification and mass spectrometry–based interaction studies using DT40 cells. Our newly designed set of pQuant vectors and a sequence- and ligation-independent cloning (SLIC) strategy allow for simple and efficient generation of gene targeting constructs, facilitating homologous-recombination–based protein tagging on a multi-gene scale. We also report proof of principle results using the key proteins involved in RNA decay, namely EXOSC8, EXOSC9, CNOT7 and UPF1.

Description: Regulation of eukaryotic gene transcription is often combinatorial in nature, with multiple transcription factors (TFs) regulating common target genes, often through direct or indirect mutual interactions. Many individual examples of cooperative binding by directly interacting TFs have been identified, but it remains unclear how pervasive this mechanism is during animal development. Cooperative TF binding should be manifest in genomic sequences as biased arrangements of TF-binding sites. Here, we explore the extent and diversity of such arrangements related to gene regulation during Drosophila embryogenesis. We used the DNA-binding specificities of 322 TFs along with chromatin accessibility information to identify enriched spacing and orientation patterns of TF-binding site pairs. We developed a new statistical approach for this task, specifically designed to accurately assess inter-site spacing biases while accounting for the phenomenon of homotypic site clustering commonly observed in developmental regulatory regions. We observed a large number of short-range distance preferences between TF-binding site pairs, including examples where the preference depends on the relative orientation of the binding sites. To test whether these binding site patterns reflect physical interactions between the corresponding TFs, we analyzed 27 TF pairs whose binding sites exhibited short distance preferences. In vitro protein–protein binding experiments revealed that 〉65% of these TF pairs can directly interact with each other. For five pairs, we further demonstrate that they bind cooperatively to DNA if both sites are present with the preferred spacing. This study demonstrates how DNA-binding motifs can be used to produce a comprehensive map of sequence signatures for different mechanisms of combinatorial TF action.

Description: HU is a protein that plays a role in various bacterial processes including compaction, transcription and replication of the genome. Here, we use atomic force microscopy to study the effect of HU on the stiffness and supercoiling of double-stranded DNA. First, we measured the persistence length, height profile, contour length and bending angle distribution of the DNA–HU complex after different incubation times of HU with linear DNA. We found that the persistence and contour length depend on the incubation time. At high concentrations of HU, DNA molecules first become stiff with a larger value of the persistence length. The persistence length then decreases over time and the molecules regain the flexibility of bare DNA after ~2 h. Concurrently, the contour length shows a slight increase. Second, we measured the change in topology of closed circular relaxed DNA following binding of HU. Here, we observed that HU induces supercoiling over a similar time span as the measured change in persistence length. Our observations can be rationalized in terms of the formation of a nucleoprotein filament followed by a structural rearrangement of the bound HU on DNA. The rearrangement results in a change in topology, an increase in bending flexibility and an increase in contour length through a decrease in helical pitch of the duplex.

Description: Magnetic isotope and magnetic field effects on the rate of DNA synthesis catalysed by polymerases β with isotopic ions 24 Mg 2+ , 25 Mg 2+ and 26 Mg 2+ in the catalytic sites were detected. No difference in enzymatic activity was found between polymerases β carrying 24 Mg 2+ and 26 Mg 2+ ions with spinless, non-magnetic nuclei 24 Mg and 26 Mg. However, 25 Mg 2+ ions with magnetic nucleus 25 Mg were shown to suppress enzymatic activity by two to three times with respect to the enzymatic activity of polymerases β with 24 Mg 2+ and 26 Mg 2+ ions. Such an isotopic dependence directly indicates that in the DNA synthesis magnetic mass-independent isotope effect functions. Similar effect is exhibited by polymerases β with Zn 2+ ions carrying magnetic 67 Zn and non-magnetic 64 Zn nuclei, respectively. A new, ion–radical mechanism of the DNA synthesis is suggested to explain these effects. Magnetic field dependence of the magnesium-catalysed DNA synthesis is in a perfect agreement with the proposed ion–radical mechanism. It is pointed out that the magnetic isotope and magnetic field effects may be used for medicinal purposes (trans-cranial magnetic treatment of cognitive deceases, cell proliferation, control of the cancer cells, etc).

Description: Translation of the sigma factor RpoS is activated by DsrA, RprA and ArcA, three small non-coding sRNAs (sRNA) that expose the ribosome-binding site (RBS) by opening up an inhibitory loop. In the RpoS network, no sRNAs have been found to pair with the RBS, a most common sRNA target site in bacteria. Here, we generate Ribo-0, an artificial sRNA, which represses rpoS translation by pairing with the RBS. Ribo-0 bypasses the RNA chaperon Hfq but requires the RBS to be loosely blocked. Ribo-0 interacts with DsrA and reshapes the RpoS network. Specifically, in the intact RpoS network, DsrA activates rpoS translation by freeing up the RBS. In the modified RpoS network where Ribo-0 is introduced, the DsrA-caused RBS exposure facilitates Ribo-0 binding, thereby strengthening Ribo-0 inhibition. In other words, Ribo-0 changes DsrA from an activator to an accomplice for repressing rpoS translation. This work presents an artificial mechanism of rpoS regulation, reveals mutual effects of native and synthetic players and demonstrates genetic context-dependency of their functions.

Description: Revealing the clonal composition of a single tumor is essential for identifying cell subpopulations with metastatic potential in primary tumors or with resistance to therapies in metastatic tumors. Sequencing technologies provide only an overview of the aggregate of numerous cells. Computational approaches to de-mix a collective signal composed of the aberrations of a mixed cell population of a tumor sample into its individual components are not available. We propose an evolutionary framework for deconvolving data from a single genome-wide experiment to infer the composition, abundance and evolutionary paths of the underlying cell subpopulations of a tumor. We have developed an algorithm (TrAp) for solving this mixture problem. In silico analyses show that TrAp correctly deconvolves mixed subpopulations when the number of subpopulations and the measurement errors are moderate. We demonstrate the applicability of the method using tumor karyotypes and somatic hypermutation data sets. We applied TrAp to Exome-Seq experiment of a renal cell carcinoma tumor sample and compared the mutational profile of the inferred subpopulations to the mutational profiles of single cells of the same tumor. Finally, we deconvolve sequencing data from eight acute myeloid leukemia patients and three distinct metastases of one melanoma patient to exhibit the evolutionary relationships of their subpopulations.

Description: The nonsense-mediated mRNA decay (NMD) pathway selectively degrades mRNAs harboring premature termination codons but also regulates the abundance of cellular RNAs. We sought to identify transcripts that are regulated by two novel NMD factors, DHX34 and neuroblastoma amplified sequence (NBAS), which were identified in a genome-wide RNA interference screen in Caenorhabditis elegans and later shown to mediate NMD in vertebrates. We performed microarray expression profile analysis in human cells, zebrafish embryos and C. elegans that were individually depleted of these factors. Our analysis revealed that a significant proportion of genes are co-regulated by DHX34, NBAS and core NMD factors in these three organisms. Further analysis indicates that NMD modulates cellular stress response pathways and membrane trafficking across species . Interestingly, transcripts encoding different NMD factors were sensitive to DHX34 and NBAS depletion, suggesting that these factors participate in a conserved NMD negative feedback regulatory loop, as was recently described for core NMD factors. In summary, we find that DHX34 and NBAS act in concert with core NMD factors to co-regulate a large number of endogenous RNA targets. Furthermore, the conservation of a mechanism to tightly control NMD homeostasis across different species highlights the importance of the NMD response in the control of gene expression.

Description: Artificial DNA looping peptides were engineered to study the roles of protein and DNA flexibility in controlling the geometry and stability of protein-mediated DNA loops. These LZD (leucine zipper dual-binding) peptides were derived by fusing a second, C-terminal, DNA-binding region onto the GCN4 bZip peptide. Two variants with different coiled-coil lengths were designed to control the relative orientations of DNA bound at each end. Electrophoretic mobility shift assays verified formation of a sandwich complex containing two DNAs and one peptide. Ring closure experiments demonstrated that looping requires a DNA-binding site separation of 310 bp, much longer than the length needed for natural loops. Systematic variation of binding site separation over a series of 10 constructs that cyclize to form 862-bp minicircles yielded positive and negative topoisomers because of two possible writhed geometries. Periodic variation in topoisomer abundance could be modeled using canonical DNA persistence length and torsional modulus values. The results confirm that the LZD peptides are stiffer than natural DNA looping proteins, and they suggest that formation of short DNA loops requires protein flexibility, not unusual DNA bendability. Small, stable, tunable looping peptides may be useful as synthetic transcriptional regulators or components of protein–DNA nanostructures.

Description: In recent years, there has been an enormous interest in developing methods for the approximation of manifold-valued functions. In this paper, we focus on the manifold of symmetric positive-definite (SPD) matrices. We investigate the use of SPD-matrix means to adapt linear positive approximation methods to SPD-matrix-valued functions. Specifically, we adapt corner-cutting subdivision schemes and Bernstein operators. We present the concept of admissible matrix means and study the adapted approximation schemes based on them. Two important cases of admissible matrix means are treated in detail: the exp–log and the geometric matrix means. We derive special properties of the approximation schemes based on these means. The geometric mean is found to be superior in the sense of preserving more properties of the data, such as monotonicity and convexity. Furthermore, we give error bounds for the approximation of univariate SPD-matrix-valued functions by the adapted operators.

Description: We present a mass-preserving scheme for the stochastic nonlinear Schrödinger equation with multiplicative noise of Stratonovich type. It is a splitting scheme and we present an explicit formula for solving the sub-step related to the nonlinear part. The scheme is unconditionally stable in the L 2 norm. For the linear stochastic Schrödinger equation, we prove that the scheme has a strong convergence rate in time equal to 1, which is not common for stochastic partial differential equations with noise depending on space and time.

Description: A numerical scheme for the approximation of the elastic flow of inextensible curves is devised and convergence of approximations to exact solutions of the nonlinear time-dependent partial differential equation is proved. The nonlinear, pointwise constraint of local length preservation is linearized about a previous solution in each time step which leads to a sequence of linear saddle-point problems. The spatial discretization is based on piecewise Bézier curves and the resulting semiimplicit scheme is unconditionally stable and convergent.

Description: We study the coercivity properties and the norm dependence on the wave-number k of certain regularized combined field boundary integral operators that we recently introduced for the solution of two- and three-dimensional acoustic scattering problems with Neumann boundary conditions. We show that in the case of circular and spherical boundaries, our regularized combined field boundary integral operators are L 2 coercive for large enough values of the coupling parameter, and that the norms of these operators are bounded by constant multiples of the coupling parameter. We establish that the norms of the regularized combined field boundary integral operators grow modestly with the wave-number k for smooth boundaries and we provide numerical evidence that these operators are L 2 coercive for two-dimensional starlike boundaries. We present and analyse a fully discrete collocation (Nyström) method for the solution of two-dimensional acoustic scattering problems with Neumann boundary conditions based on regularized combined field integral equations. In particular, for analytic boundaries and boundary data, we establish pointwise superalgebraic convergence rates of the discrete solutions.

Description: The classical theory of Gaussian quadrature assumes a positive weight function. We will show that in some cases Gaussian rules can be constructed with respect to an oscillatory weight, yielding methods with complex quadrature nodes and positive weights. These rules are well suited to highly oscillatory integrals because they attain optimal asymptotic order. We show that, for the Fourier oscillator, this approach yields the numerical method of steepest descent, a method with optimal asymptotic order that has previously been proposed for this class of integrals. However, the approach readily extends to more general kernels, such as Bessel functions that appear as the kernel of the Hankel transform.

Description: We consider anisotropic Allen–Cahn equations with interfacial energy induced by an anisotropic surface energy density . Assuming that is positive, positively homogeneous of degree 1, strictly convex in tangential directions to the unit sphere and sufficiently smooth, we show the stability of various time discretizations. In particular, we consider a fully implicit and a linearized time discretization of the interfacial energy combined with implicit and semiimplicit time discretizations of the double-well potential. In the semiimplicit variant, concave terms are taken explicitly. The arising discrete spatial problems are solved by globally convergent truncated nonsmooth Newton multigrid methods. Numerical experiments show the accuracy of the different discretizations. We also illustrate that pinch-off under anisotropic mean curvature flow is no longer invariant under rotation of the initial configuration for a fixed orientation of the anisotropy.

Description: Pnkp is the end-healing and end-sealing component of an RNA repair system present in diverse bacteria from many phyla. Pnkp is composed of three catalytic modules: an N-terminal polynucleotide 5' kinase, a central 2',3' phosphatase and a C-terminal ligase. The phosphatase module is a Mn 2+ -dependent phosphodiesterase–monoesterase that dephosphorylates 2',3'-cyclic phosphate RNA ends. Here we report the crystal structure of the phosphatase domain of Clostridium thermocellum Pnkp with Mn 2+ and citrate in the active site. The protein consists of a core binuclear metallo-phosphoesterase fold (exemplified by bacteriophage phosphatase) embellished by distinctive secondary structure elements. The active site contains a single Mn 2+ in an octahedral coordination complex with Asp187, His189, Asp233, two citrate oxygens and a water. The citrate fills the binding site for the scissile phosphate, wherein it is coordinated by Arg237, Asn263 and His264. The citrate invades the site normally occupied by a second metal (engaged by Asp233, Asn263, His323 and His376), and thereby dislocates His376. A continuous tract of positive surface potential flanking the active site suggests an RNA binding site. The structure illuminates a large body of mutational data regarding the metal and substrate specificity of Clostridium thermocellum Pnkp phosphatase.

Description: The formation of the open promoter complex (RPo) in which the melted DNA containing the transcription start site is located at the RNA polymerase (RNAP) catalytic centre is an obligatory step in the transcription of DNA into RNA catalyzed by RNAP. In the RPo, an extensive network of interactions is established between DNA, RNAP and the -factor and the formation of functional RPo occurs via a series of transcriptional intermediates (collectively ‘RPi’). A single tryptophan is ideally positioned to directly engage with the flipped out base of the non-template strand at the +1 site. Evidence suggests that this tryptophan (i) is involved in either forward translocation or DNA scrunching and (ii) in 54 -regulated promoters limits the transcription activity of at least one intermediate complex (RPi) before the formation of a fully functional RPo. Limiting RPi activity may be important in preventing the premature synthesis of abortive transcripts, suggesting its involvement in a general mechanism driving the RPi to RPo transition for transcription initiation.

Description: Guanine-rich RNA sequences can fold into non-canonical, four stranded helical structures called G-quadruplexes that have been shown to be widely distributed within the mammalian transcriptome, as well as being key regulatory elements in various biological mechanisms. That said, their role within the 3'-untranslated region (UTR) of mRNA remains to be elucidated and appreciated. A bioinformatic analysis of the 3'-UTRs of mRNAs revealed enrichment in G-quadruplexes. To shed light on the role(s) of these structures, those found in the LRP5 and FXR1 genes were characterized both in vitro and in cellulo . The 3'-UTR G-quadruplexes were found to increase the efficiencies of alternative polyadenylation sites, leading to the expression of shorter transcripts and to possess the ability to interfere with the miRNA regulatory network of a specific mRNA. Clearly, G-quadruplexes located in the 3'-UTRs of mRNAs are cis -regulatory elements that have a significant impact on gene expression.

Description: One of the primary aims of synthetic biology is to (re)design metabolic pathways towards the production of desired chemicals. The fast pace of developments in molecular biology increasingly makes it possible to experimentally redesign existing pathways and implement de novo ones in microbes or using in vitro platforms. For such experimental studies, the bottleneck is shifting from implementation of pathways towards their initial design. Here, we present an online tool called ‘Metabolic Tinker’, which aims to guide the design of synthetic metabolic pathways between any two desired compounds. Given two user-defined ‘target’ and ‘source’ compounds, Metabolic Tinker searches for thermodynamically feasible paths in the entire known metabolic universe using a tailored heuristic search strategy. Compared with similar graph-based search tools, Metabolic Tinker returns a larger number of possible paths owing to its broad search base and fast heuristic, and provides for the first time thermodynamic feasibility information for the discovered paths. Metabolic Tinker is available as a web service at http://osslab.ex.ac.uk/tinker.aspx . The same website also provides the source code for Metabolic Tinker, allowing it to be developed further or run on personal machines for specific applications.

Description: COHCAP (City of Hope CpG Island Analysis Pipeline) is an algorithm to analyze single-nucleotide resolution DNA methylation data produced by either an Illumina methylation array or targeted bisulfite sequencing. The goal of the COHCAP algorithm is to identify CpG islands that show a consistent pattern of methylation among CpG sites. COHCAP is currently the only DNA methylation package that provides integration with gene expression data to identify a subset of CpG islands that are most likely to regulate downstream gene expression, and it can generate lists of differentially methylated CpG islands with ~50% concordance with gene expression from both cell line data and heterogeneous patient data. For example, this article describes known breast cancer biomarkers (such as estrogen receptor) with a negative correlation between DNA methylation and gene expression. COHCAP also provides visualization for quality control metrics, regions of differential methylation and correlation between methylation and gene expression. This software is freely available at https://sourceforge.net/projects/cohcap/ .

Description: Tristetraprolin (TTP) and let-7 microRNA exhibit suppressive effects on cell growth through down-regulation of oncogenes. Both TTP and let-7 are often repressed in human cancers, thereby promoting oncogenesis by derepressing their target genes. However, the precise mechanism of this repression is unknown. We here demonstrate that p53 stimulated by the DNA-damaging agent doxorubicin (DOX) induced the expression of TTP in cancer cells. TTP in turn increased let-7 levels through down-regulation of Lin28a . Correspondingly, cancer cells with mutations or inhibition of p53 failed to induce the expression of both TTP and let-7 on treatment with DOX. Down-regulation of TTP by small interfering RNAs attenuated the inhibitory effect of DOX on let-7 expression and cell growth. Therefore, TTP provides an important link between p53 activation induced by DNA damage and let-7 biogenesis. These novel findings provide a mechanism for the widespread decrease in TTP and let-7 and chemoresistance observed in human cancers.

Description: The observation that long tracts of RNA are associated with replicating molecules of mitochondrial DNA (mtDNA) suggests that the mitochondrial genome of mammals is copied by an unorthodox mechanism. Here we show that these RNA-containing species are present in living cells and tissue, based on interstrand cross-linking. Using DNA synthesis in organello , we demonstrate that isolated mitochondria incorporate radiolabeled RNA precursors, as well as DNA precursors, into replicating DNA molecules. RNA-containing replication intermediates are chased into mature mtDNA, to which they are thus in precursor–product relationship. While a DNA chain terminator rapidly blocks the labeling of mitochondrial replication intermediates, an RNA chain terminator does not. Furthermore, processed L-strand transcripts can be recovered from gel-extracted mtDNA replication intermediates. Therefore, instead of concurrent DNA and RNA synthesis, respectively, on the leading and lagging strands, preformed processed RNA is incorporated as a provisional lagging strand during mtDNA replication. These findings indicate that RITOLS is a physiological mechanism of mtDNA replication, and that it involves a ‘bootlace' mechanism, in which processed transcripts are successively hybridized to the lagging-strand template, as the replication fork advances.

Description: Escherichia coli Exonuclease I (ExoI) digests single-stranded DNA (ssDNA) in the 3'-5' direction in a highly processive manner. The crystal structure of ExoI, determined previously in the absence of DNA, revealed a C-shaped molecule with three domains that form a central positively charged groove. The active site is at the bottom of the groove, while an extended loop, proposed to encircle the DNA, crosses over the groove. Here, we present crystal structures of ExoI in complex with four different ssDNA substrates. The structures all have the ssDNA bound in essentially the predicted manner, with the 3'-end in the active site and the downstream end under the crossover loop. The central nucleotides of the DNA form a prominent bulge that contacts the SH3-like domain, while the nucleotides at the downstream end of the DNA form extensive interactions with an ‘anchor’ site. Seven of the complexes are similar to one another, but one has the ssDNA bound in a distinct conformation. The highest-resolution structure, determined at 1.95 Å, reveals an Mg 2+ ion bound to the scissile phosphate in a position corresponding to Mg B in related two-metal nucleases. The structures provide new insights into the mechanism of processive digestion that will be discussed.

Description: The rpoS mRNA, which encodes the master regulator S of general stress response, requires Hfq-facilitated base pairing with DsrA small RNA for efficient translation at low temperatures. It has recently been proposed that one mechanism underlying Hfq action is to bridge a transient ternary complex by simultaneously binding to rpoS and DsrA. However, no structural evidence of Hfq simultaneously bound to different RNAs has been reported. We detected simultaneous binding of Hfq to rpoS and DsrA fragments. Crystal structures of AU6A•Hfq•A7 and Hfq•A7 complexes were resolved using 1.8- and 1.9-Å resolution, respectively. Ternary complex has been further verified in solution by NMR. In vivo , activation of rpoS translation requires intact Hfq, which is capable of bridging rpoS and DsrA simultaneously into ternary complex. This ternary complex possibly corresponds to a meta-stable transition state in Hfq-facilitated small RNA–mRNA annealing process.

Description: The functions of RNAs, like proteins, are determined by their structures, which, in turn, are determined by their sequences. Comparison/alignment of RNA molecules provides an effective means to predict their functions and understand their evolutionary relationships. For RNA sequence alignment, most methods developed for protein and DNA sequence alignment can be directly applied. RNA 3-dimensional structure alignment, on the other hand, tends to be more difficult than protein structure alignment due to the lack of regular secondary structures as observed in proteins. Most of the existing RNA 3D structure alignment methods use only the backbone geometry and ignore the sequence information. Using both the sequence and backbone geometry information in RNA alignment may not only produce more accurate classification, but also deepen our understanding of the sequence–structure–function relationship of RNA molecules. In this study, we developed a new RNA alignment method based on elastic shape analysis (ESA). ESA treats RNA structures as three dimensional curves with sequence information encoded on additional dimensions so that the alignment can be performed in the joint sequence–structure space. The similarity between two RNA molecules is quantified by a formal distance, geodesic distance. Based on ESA, a rigorous mathematical framework can be built for RNA structure comparison. Means and covariances of full structures can be defined and computed, and probability distributions on spaces of such structures can be constructed for a group of RNAs. Our method was further applied to predict functions of RNA molecules and showed superior performance compared with previous methods when tested on benchmark datasets. The programs are available at http://stat.fsu.edu/ ~jinfeng/ESA.html.

Description: DNA methylation is a mechanism for long-term transcriptional regulation and is required for normal cellular differentiation. Failure to properly establish or maintain DNA methylation patterns leads to cell dysfunction and diseases such as cancer. Identifying DNA methylation signatures in complex tissues can be challenging owing to inaccurate cell enrichment methods and low DNA yields. We have developed a technique called laser capture microdissection-reduced representation bisulfite sequencing (LCM-RRBS) for the multiplexed interrogation of the DNA methylation status of cytosine–guanine dinucleotide islands and promoters. LCM-RRBS accurately and reproducibly profiles genome-wide methylation of DNA extracted from microdissected fresh frozen or formalin-fixed paraffin-embedded tissue samples. To demonstrate the utility of LCM-RRBS, we characterized changes in DNA methylation associated with gonadectomy-induced adrenocortical neoplasia in the mouse. Compared with adjacent normal tissue, the adrenocortical tumors showed reproducible gains and losses of DNA methylation at genes involved in cell differentiation and organ development. LCM-RRBS is a rapid, cost-effective, and sensitive technique for analyzing DNA methylation in heterogeneous tissues and will facilitate the investigation of DNA methylation in cancer and organ development.

Description: Double-strand break (DSB) repair pathways are critical for the maintenance of genomic integrity and the prevention of tumorigenesis in mammalian cells. Here, we present the development and validation of a novel assay to measure mutagenic non-homologous end-joining (NHEJ) repair in living cells, which is inversely related to canonical NHEJ and is based on the sequence-altering repair of a single site-specific DSB at an intrachromosomal locus. We have combined this mutagenic NHEJ assay with an established homologous recombination (HR) assay such that both pathways can be monitored simultaneously. In addition, we report the development of a ligand-responsive I-SceI protein, in which the timing and kinetics of DSB induction can be precisely controlled by regulating protein stability and cellular localization in cells. Using this system, we report that mutagenic NHEJ repair is suppressed in growth-arrested and serum-deprived cells, suggesting that end-joining activity in proliferating cells is more likely to be mutagenic. Collectively, the novel DSB repair assay and inducible I-SceI will be useful tools to further elucidate the complexities of NHEJ and HR repair.

Description: The core promoter is the region in which RNA polymerase II is recruited to the DNA and acts to initiate transcription, but the extent to which the core promoter sequence determines promoter activity levels is largely unknown. Here, we identified several base content and k-mer sequence features of the yeast core promoter sequence that are highly predictive of maximal promoter activity. These features are mainly located in the region 75 bp upstream and 50 bp downstream of the main transcription start site, and their associations hold for both constitutively active promoters and promoters that are induced or repressed in specific conditions. Our results unravel several architectural features of yeast core promoters and suggest that the yeast core promoter sequence downstream of the TATA box (or of similar sequences involved in recruitment of the pre-initiation complex) is a major determinant of maximal promoter activity. We further show that human core promoters also contain features that are indicative of maximal promoter activity; thus, our results emphasize the important role of the core promoter sequence in transcriptional regulation.

Description: Basic helix–loop–helix/Per–Arnt–Sim (bHLH/PAS) transcription factors function broadly in development, homeostasis and stress response. Active bHLH/PAS heterodimers consist of a ubiquitous signal-regulated subunit (e.g., hypoxia-inducible factors, HIF-1α/2α/3α; the aryl hydrocarbon receptor, AhR) or tissue-restricted subunit (e.g., NPAS1/3/4, Single Minded 1/2), paired with a general partner protein, aryl hydrocarbon receptor nuclear translocator (Arnt or Arnt2). We have investigated regulation of the neuron-enriched Arnt paralogue, Arnt2. We find high Arnt/Arnt2 ratios in P19 embryonic carcinoma cells and ES cells are dramatically reversed to high Arnt2/Arnt on neuronal differentiation. mRNA half-lives of Arnt and Arnt2 remain similar in both parent and neuronal differentiated cells. The GC-rich Arnt2 promoter, while heavily methylated in Arnt only expressing hepatoma cells, is methylation free in P19 and ES cells, where it is bivalent with respect to active H3K4me3 and repressive H3K27me3 histone marks. Typical of a ‘transcription poised’ developmental gene, H3K27me3 repressive marks are removed from Arnt2 during neuronal differentiation. Our data are consistent with a switch to predominant Arnt2 expression in neurons to allow specific functions of neuronal bHLH/PAS factors and/or to avoid neuronal bHLH/PAS factors from interfering with AhR/Arnt signalling.

Description: The conserved pre-mRNA retention and splicing (RES) complex, which in yeast consists of Bud13p, Snu17p and Pml1p, is thought to promote nuclear retention of unspliced pre-mRNAs and enhance splicing of a subset of transcripts. Here, we find that the absence of Bud13p or Snu17p causes greatly reduced levels of the modified nucleoside N 4 -acetylcytidine (ac 4 C) in tRNA and that a lack of Pml1p reduces ac 4 C levels at elevated temperatures. The ac 4 C nucleoside is normally found at position 12 in the tRNA species specific for serine and leucine. We show that the tRNA modification defect in RES-deficient cells is attributable to inefficient splicing of TAN1 pre-mRNA and the effects of reduced Tan1p levels on formation of ac 4 C. Analyses of cis -acting elements in TAN1 pre-mRNA showed that the intron sequence between the 5' splice site and branchpoint is necessary and sufficient to mediate RES dependency. We also show that in RES-deficient cells, the TAN1 pre-mRNA is targeted for degradation by the cytoplasmic nonsense-mediated mRNA decay pathway, indicating that poor nuclear retention may contribute to the tRNA modification defect. Our results demonstrate that TAN1 pre-mRNA processing has an unprecedented requirement for RES factors and that the complex controls the formation of ac 4 C in tRNA.

Description: The regulation of skeletal muscle gene expression during myogenesis is mediated by lineage-specific transcription factors in combination with numerous cofactors, many of which modify chromatin structure. However, the involvement of scaffolding proteins that organize chromatin and chromatin-associated regulatory proteins has not extensively been explored in myogenic differentiation. Here, we report that Scaffold attachment factor b1 (Safb1), primarily associated with transcriptional repression, functions as a positive regulator of myogenic differentiation. Knockdown of Safb1 inhibited skeletal muscle marker gene expression and differentiation in cultured C2C12 myoblasts. In contrast, over-expression resulted in the premature expression of critical muscle structural proteins and formation of enlarged thickened myotubes. Safb1 co-immunoprecipitated with MyoD and was co-localized on myogenic promoters. Upon Safb1 knockdown, the repressive H3K27me3 histone mark and binding of the Polycomb histone methyltransferase Ezh2 persisted at differentiation-dependent gene promoters. In contrast, the appearance of histone marks and regulators associated with myogenic gene activation, such as myogenin and the SWI/SNF chromatin remodelling enzyme ATPase, Brg1, was blocked. These results indicate that the scaffold protein Safb1 contributes to the activation of skeletal muscle gene expression during myogenic differentiation by facilitating the transition of promoter sequences from a repressive chromatin structure to one that is transcriptionally permissive.

Description: Transcription activator-like effector nucleases (TALENs) are a powerful new approach for targeted gene disruption in various animal models, but little is known about their activities in Mus musculus, the widely used mammalian model organism. Here, we report that direct injection of in vitro transcribed messenger RNA of TALEN pairs into mouse zygotes induced somatic mutations, which were stably passed to the next generation through germ-line transmission. With one TALEN pair constructed for each of 10 target genes, mutant F0 mice for each gene were obtained with the mutation rate ranged from 13 to 67% and an average of ~40% of total healthy newborns with no significant differences between C57BL/6 and FVB/N genetic background. One TALEN pair with single mismatch to their intended target sequence in each side failed to yield any mutation. Furthermore, highly efficient germ-line transmission was obtained, as all the F0 founders tested transmitted the mutations to F1 mice. In addition, we also observed that one bi-allele mutant founder of Lepr gene, encoding Leptin receptor, had similar diabetic phenotype as db/db mouse. Together, our results suggest that TALENs are an effective genetic tool for rapid gene disruption with high efficiency and heritability in mouse with distinct genetic background.

Description: We describe a new cell-free protein synthesis (CFPS) method for site-specific incorporation of non-natural amino acids (nnAAs) into proteins in which the orthogonal tRNA (o-tRNA) and the modified protein (i.e. the protein containing the nnAA) are produced simultaneously. Using this method, 0.9–1.7 mg/ml of modified soluble super-folder green fluorescent protein (sfGFP) containing either p -azido- l -phenylalanine (pAzF) or p -propargyloxy- l -phenylalanine (pPaF) accumulated in the CFPS solutions; these yields correspond to 50–88% suppression efficiency. The o-tRNA can be transcribed either from a linearized plasmid or from a crude PCR product. Comparison of two different o-tRNAs suggests that the new platform is not limited by Ef-Tu recognition of the acylated o-tRNA at sufficiently high o-tRNA template concentrations. Analysis of nnAA incorporation across 12 different sites in sfGFP suggests that modified protein yields and suppression efficiencies (i.e. the position effect) do not correlate with any of the reported trends. Sites that were ineffectively suppressed with the original o-tRNA were better suppressed with an optimized o-tRNA (o-tRNA opt ) that was evolved to be better recognized by Ef-Tu. This new platform can also be used to screen scissile ribozymes for improved catalysis.

Description: The DNA damage-binding protein 2 (DDB2) is an adapter protein that can direct a modular Cul4-DDB1-RING E3 Ligase complex to sites of ultraviolet light-induced DNA damage to ubiquitinate substrates during nucleotide excision repair. The DDB2 transcript is ultraviolet-inducible; therefore, its regulation is likely important for its function. Curiously, the DDB2 mRNA is reportedly short-lived, but the transcript does not contain any previously characterized cis -acting determinants of mRNA stability in its 3' untranslated region (3'UTR). Here, we used a tetracycline regulated d2EGFP reporter construct containing specific 3'UTR sequences from DDB2 to identify novel cis -acting elements that regulate mRNA stability. Synthetic 3'UTRs corresponding to sequences as short as 25 nucleotides from the central region of the 3'UTR of DDB2 were sufficient to accelerate decay of the heterologous reporter mRNA. Conversely, these same 3'UTRs led to more rapid induction of the reporter mRNA, export of the message to the cytoplasm and the subsequent accumulation of the encoded reporter protein, indicating that this newly identified cis -acting element affects transcriptional and post-transciptional processes. These results provide clear evidence that nuclear and cytoplasmic processing of the DDB2 mRNA is inextricably linked.

Description: Despite increasing data on the properties of replication origins, molecular mechanisms underlying origin recognition complex (ORC) positioning in the genome are still poorly understood. The Su(Hw) protein accounts for the activity of best-studied Drosophila insulators. Here, we show that Su(Hw) recruits the histone acetyltransferase complex SAGA and chromatin remodeler Brahma to Su(Hw)-dependent insulators, which gives rise to regions with low nucleosome density and creates conditions for ORC binding. Depletion in Su(Hw) leads to a dramatic drop in the levels of SAGA, Brahma and ORC subunits and a significant increase in nucleosome density on Su(Hw)-dependent insulators, whereas artificial Su(Hw) recruitment itself is sufficient for subsequent SAGA, Brahma and ORC binding. In contrast to the majority of replication origins that associate with promoters of active genes, Su(Hw)-binding sites constitute a small proportion (6%) of ORC-binding sites that are localized preferentially in transcriptionally inactive chromatin regions termed BLACK and BLUE chromatin. We suggest that the key determinants of ORC positioning in the genome are DNA-binding proteins that constitute different DNA regulatory elements, including insulators, promoters and enhancers. Su(Hw) is the first example of such a protein.

Description: PIWI-interacting RNAs (piRNAs) provide defence against transposable element (TE) expansion in the germ line of metazoans. piRNAs are processed from the transcripts encoded by specialized heterochromatic clusters enriched in damaged copies of transposons. How these regions are recognized as a source of piRNAs is still elusive. The aim of this study is to determine how transgenes that contain a fragment of the Long Interspersed Nuclear Elements (LINE)-like I transposon lead to an acquired TE resistance in Drosophila . We show that such transgenes, being inserted in unique euchromatic regions that normally do not produce small RNAs, become de novo bidirectional piRNA clusters that silence I- element activity in the germ line. Strikingly, small RNAs of both polarities are generated from the entire transgene and flanking genomic sequences—not only from the transposon fragment. Chromatin immunoprecipitation analysis shows that in ovaries, the trimethylated histone 3 lysine 9 (H3K9me3) mark associates with transgenes producing piRNAs. We show that transgene-derived hsp70 piRNAs stimulate in trans cleavage of cognate endogenous transcripts with subsequent processing of the non-homologous parts of these transcripts into piRNAs.

Description: The DNA damage response (DDR) involves both the control of DNA damage repair and signaling to cell cycle checkpoints. Therefore, unraveling the underlying mechanisms of the DDR is important for understanding tumor suppression and cellular resistance to clastogenic cancer therapeutics. Because the DDR is likely to be influenced by chromatin regulation at the sites of DNA damage, we investigated the role of heterochromatin protein 1 (HP1) during the DDR process. We monitored double-strand breaks (DSBs) using the H2AX foci marker and found that depleting cells of HP1 caused genotoxic stress, a delay in the repair of DSBs and elevated levels of apoptosis after irradiation. Furthermore, we found that these defects in repair were associated with impaired BRCA1 function. Depleting HP1 reduced recruitment of BRCA1 to DSBs and caused defects in two BRCA1-mediated DDR events: (i) the homologous recombination repair pathway and (ii) the arrest of cell cycle at the G 2 /M checkpoint. In contrast, depleting HP1 from cells did not affect the non-homologous end-joining (NHEJ) pathway: instead it elevated the recruitment of the 53BP1 NHEJ factor to DSBs. Notably, all three subtypes of HP1 seemed to be almost equally important for these DDR functions. We suggest that the dynamic interaction of HP1 with chromatin and other DDR factors could determine DNA repair choice and cell fate after DNA damage. We also suggest that compromising HP1 expression could promote tumorigenesis by impairing the function of the BRCA1 tumor suppressor.

Description: All orthobunyaviruses possess three genome segments of single-stranded negative sense RNA that are encapsidated with the virus-encoded nucleocapsid (N) protein to form a ribonucleoprotein (RNP) complex, which is uncharacterized at high resolution. We report the crystal structure of both the Bunyamwera virus (BUNV) N–RNA complex and the unbound Schmallenberg virus (SBV) N protein, at resolutions of 3.20 and 2.75 Å, respectively. Both N proteins crystallized as ring-like tetramers and exhibit a high degree of structural similarity despite classification into different orthobunyavirus serogroups. The structures represent a new RNA-binding protein fold. BUNV N possesses a positively charged groove into which RNA is deeply sequestered, with the bases facing away from the solvent. This location is highly inaccessible, implying that RNA polymerization and other critical base pairing events in the virus life cycle require RNP disassembly. Mutational analysis of N protein supports a correlation between structure and function. Comparison between these crystal structures and electron microscopy images of both soluble tetramers and authentic RNPs suggests the N protein does not bind RNA as a repeating monomer; thus, it represents a newly described architecture for bunyavirus RNP assembly, with implications for many other segmented negative-strand RNA viruses.

Description: Infected cell protein 8 (ICP8) from herpes simplex virus 1 was first identified as a single-strand (ss) DNA-binding protein. It is essential for, and abundant during, viral replication. Studies in vitro have shown that ICP8 stimulates model replication reactions, catalyzes annealing of complementary ssDNAs and, in combination with UL12 exonuclease, will catalyze ssDNA annealing homologous recombination. DNA annealing and strand transfer occurs within large oligomeric filaments of ssDNA-bound ICP8. We present the first 3D reconstruction of a novel ICP8–ssDNA complex, which seems to be the basic unit of the DNA annealing machine. The reconstructed volume consists of two nonameric rings containing ssDNA stacked on top of each other, corresponding to a molecular weight of 2.3 MDa. Fitting of the ICP8 crystal structure suggests a mechanism for the annealing reaction catalyzed by ICP8, which is most likely a general mechanism for protein-driven DNA annealing.

Description: Genome-wide prediction of transcription factor binding sites is notoriously difficult. We have developed and applied a logistic regression approach for prediction of binding sites for the p53 transcription factor that incorporates sequence information and chromatin modification data. We tested this by comparison of predicted sites with known binding sites defined by chromatin immunoprecipitation (ChIP), by the location of predictions relative to genes, by the function of nearby genes and by analysis of gene expression data after p53 activation. We compared the predictions made by our novel model with predictions based only on matches to a sequence position weight matrix (PWM). In whole genome assays, the fraction of known sites identified by the two models was similar, suggesting that there was little to be gained from including chromatin modification data. In contrast, there were highly significant and biologically relevant differences between the two models in the location of the predicted binding sites relative to genes, in the function of nearby genes and in the responsiveness of nearby genes to p53 activation. We propose that these contradictory results can be explained by PWM and ChIP data reflecting primarily biophysical properties of protein–DNA interactions, whereas chromatin modification data capture biologically important functional information.

Description: 5-Aza-2'-deoxycytidine (5-azadC) is a DNA methyltransferase (DNMT) inhibitor increasingly used in treatments of hematological diseases and works by being incorporated into DNA and trapping DNMT. It is unclear what DNA lesions are caused by 5-azadC and if such are substrates for DNA repair. Here, we identify that 5-azadC induces DNA damage as measured by -H2AX and 53BP1 foci. Furthermore, 5-azadC induces radial chromosomes and chromatid breaks that depend on active replication, which altogether suggest that trapped DNMT collapses oncoming replication forks into double-strand breaks. We demonstrate that RAD51-mediated homologous recombination (HR) is activated to repair 5-azadC collapsed replication forks. Fanconi anemia (FA) is a rare autosomal recessive disorder, and deaths are often associated with leukemia. Here, we show that FANCG -deficient cells fail to trigger HR-mediated repair of 5-azadC-induced lesions, leading to accumulation of chromatid breaks and inter-chromosomal radial fusions as well as hypersensitivity to the cytotoxic effects of 5-azadC. These data demonstrate that the FA pathway is important to protect from 5-azadC-induced toxicity. Altogether, our data demonstrate that cytotoxicity of the epigenetic drug 5-azadC can, at least in part, be explained by collapsed replication forks requiring FA-mediated HR for repair.

Description: Replicative DNA polymerases require an RNA primer for leading and lagging strand DNA synthesis, and primase is responsible for the de novo synthesis of this RNA primer. However, the archaeal primase from Pyrococcus furiosus (Pfu) frequently incorporates mismatched nucleoside monophosphate, which stops RNA synthesis. Pfu DNA polymerase (PolB) cannot elongate the resulting 3'-mismatched RNA primer because it cannot remove the 3'-mismatched ribonucleotide. This study demonstrates the potential role of a RecJ-like protein from P. furiosus (PfRecJ) in proofreading 3'-mismatched ribonucleotides. PfRecJ hydrolyzes single-stranded RNA and the RNA strand of RNA/DNA hybrids in the 3'–5' direction, and the kinetic parameters (K m and K cat ) of PfRecJ during RNA strand digestion are consistent with a role in proofreading 3'-mismatched RNA primers. Replication protein A, the single-stranded DNA–binding protein, stimulates the removal of 3'-mismatched ribonucleotides of the RNA strand in RNA/DNA hybrids, and Pfu DNA polymerase can extend the 3'-mismatched RNA primer after the 3'-mismatched ribonucleotide is removed by PfRecJ. Finally, we reconstituted the primer-proofreading reaction of a 3'-mismatched ribonucleotide RNA/DNA hybrid using PfRecJ, replication protein A, Proliferating cell nuclear antigen (PCNA) and PolB. Given that PfRecJ is associated with the GINS complex, a central nexus in archaeal DNA replication fork, we speculate that PfRecJ proofreads the RNA primer in vivo .

Description: The Ntg1 and Mhr1 proteins initiate rolling-circle mitochondrial (mt) DNA replication to achieve homoplasmy, and they also induce homologous recombination to maintain mitochondrial genome integrity. Although replication and recombination profoundly influence mitochondrial inheritance, the regulatory mechanisms that determine the choice between these pathways remain unknown. In Saccharomyces cerevisiae , double-strand breaks (DSBs) introduced by Ntg1 at the mitochondrial replication origin ori5 induce homologous DNA pairing by Mhr1, and reactive oxygen species (ROS) enhance production of DSBs. Here, we show that a mitochondrial nuclease encoded by the nuclear gene DIN7 (DNA damage inducible gene) has 5'-exodeoxyribonuclease activity. Using a small – mtDNA bearing ori5 (hypersuppressive; HS) as a model mtDNA, we revealed that DIN7 is required for ROS-enhanced mtDNA replication and recombination that are both induced at ori5 . Din7 overproduction enhanced Mhr1-dependent mtDNA replication and increased the number of residual DSBs at ori5 in HS- – cells and increased deletion mutagenesis at the ori5 region in + cells. However, simultaneous overproduction of Mhr1 suppressed all of these phenotypes and enhanced homologous recombination. Our results suggest that after homologous pairing, the relative activity levels of Din7 and Mhr1 modulate the preference for replication versus homologous recombination to repair DSBs at ori5 .

Description: Transcription and mRNA export are linked processes. However, the molecular mechanisms of this coordination are not clear. Sus1 (hENY2) participates in this coordination as part of two protein complexes: SAGA, a transcriptional co-activator; TREX-2, which functions in mRNA biogenesis and export. Here, we investigate the coordinated action of SAGA and TREX-2 required for gene expression. We demonstrate that TREX-2 subunit Sem1 also participates in transcription activation. Like Sus1, Sem1 is required for the induction of ARG1 and GAL1 , these being SAGA-regulated genes. Chromatin immunoprecipitations show that proper recruitment of certain SAGA subunits to the GAL1 promoter depends on Sem1. Notably, both in vivo and in vitro analyses reveal that Sem1 influences SAGA-dependent histone H2B deubiquitylation. Most of these phenotypes are also found to depend on another TREX-2 subunit, Thp1. These results unveil a new role for Sem1 in the activation of the SAGA-dependent gene GAL1 and influencing H2B deubiquitylation. Our work provides insights into a novel functional relationship between Sem1 and the SAGA complex.

Description: The introduction of next generation sequencing methods in genome studies has made it possible to shift research from a gene-centric approach to a genome wide view. Although methods and tools to detect single nucleotide polymorphisms are becoming more mature, methods to identify and visualize structural variation (SV) are still in their infancy. Most genome browsers can only compare a given sequence to a reference genome; therefore, direct comparison of multiple individuals still remains a challenge. Therefore, the implementation of efficient approaches to explore and visualize SVs and directly compare two or more individuals is desirable. In this article, we present a visualization approach that uses space-filling Hilbert curves to explore SVs based on both read-depth and pair-end information. An interactive open-source Java application, called Meander , implements the proposed methodology, and its functionality is demonstrated using two cases. With Meander , users can explore variations at different levels of resolution and simultaneously compare up to four different individuals against a common reference. The application was developed using Java version 1.6 and Processing.org and can be run on any platform. It can be found at http://homes.esat.kuleuven.be/~bioiuser/meander .

Description: Restriction endonucleases are highly specific in recognizing the particular DNA sequence they act on. However, their activity is affected by sequence context, enzyme concentration and buffer composition. Changes in these factors may lead to either ineffective cleavage at the cognate restriction site or relaxed specificity allowing cleavage of degenerate ‘star’ sites. Additionally, uncharacterized restriction endonucleases and engineered variants present novel activities. Traditionally, restriction endonuclease activity is assayed on simple substrates such as plasmids and synthesized oligonucleotides. We present and use high-throughput Illumina sequencing-based strategies to assay the sequence specificity and flanking sequence preference of restriction endonucleases. The techniques use fragmented DNA from sequenced genomes to quantify restriction endonuclease cleavage on a complex genomic DNA substrate in a single reaction. By mapping millions of restriction site–flanking reads back to the Escherichia coli and Drosophila melanogaster genomes we were able to quantitatively characterize the cognate and star site activity of EcoRI and MfeI and demonstrate genome-wide decreases in star activity with engineered high-fidelity variants EcoRI-HF and MfeI-HF, as well as quantify the influence on MfeI cleavage conferred by flanking nucleotides. The methods presented are readily applicable to all type II restriction endonucleases that cleave both strands of double-stranded DNA.

Description: Accurately characterizing transcription factor (TF)-DNA affinity is a central goal of regulatory genomics. Although thermodynamics provides the most natural language for describing the continuous range of TF-DNA affinity, traditional motif discovery algorithms focus instead on classification paradigms that aim to discriminate ‘bound’ and ‘unbound’ sequences. Moreover, these algorithms do not directly model the distribution of tags in ChIP-seq data. Here, we present a new algorithm named T hermodynamic M odeling of ChIP- s eq (TherMos), which directly estimates a position-specific binding energy matrix (PSEM) from ChIP-seq/exo tag profiles. In cross-validation tests on seven genome-wide TF-DNA binding profiles, one of which we generated via ChIP-seq on a complex developing tissue, TherMos predicted quantitative TF-DNA binding with greater accuracy than five well-known algorithms. We experimentally validated TherMos binding energy models for Klf4 and Esrrb, using a novel protocol to measure PSEMs in vitro . Strikingly, our measurements revealed strong non-additivity at multiple positions within the two PSEMs. Among the algorithms tested, only TherMos was able to model the entire binding energy landscape of Klf4 and Esrrb. Our study reveals new insights into the energetics of TF-DNA binding in vivo and provides an accurate first-principles approach to binding energy inference from ChIP-seq and ChIP-exo data.

Description: The circular chromosome of Escherichia coli has been suggested to fold into a collection of sequentially consecutive domains, genes in each of which tend to be co-expressed. It has also been suggested that such domains, forming a partition of the genome, are dynamic with respect to the physiological conditions. However, little is known about which DNA segments of the E. coli genome form these domains and what determines the boundaries of these domain segments. We present a computational model here to partition the circular genome into consecutive segments, theoretically suggestive of the physically folded supercoiled domains, along with a method for predicting such domains under specified conditions. Our model is based on a hypothesis that the genome of E. coli is partitioned into a set of folding domains so that the total number of unfoldings of these domains in the folded chromosome is minimized, where a domain is unfolded when a biological pathway, consisting of genes encoded in this DNA segment, is being activated transcriptionally. Based on this hypothesis, we have predicted seven distinct sets of such domains along the E. coli genome for seven physiological conditions, namely exponential growth, stationary growth, anaerobiosis, heat shock, oxidative stress, nitrogen limitation and SOS responses. These predicted folding domains are highly stable statistically and are generally consistent with the experimental data of DNA binding sites of the nucleoid-associated proteins that assist the folding of these domains, as well as genome-scale protein occupancy profiles, hence supporting our proposed model. Our study established for the first time a strong link between a folded E. coli chromosomal structure and the encoded biological pathways and their activation frequencies.

Description: RNA is a major drug target, but the design of small molecules that modulate RNA function remains a great challenge. In this context, a series of structurally homologous ‘polyamide amino acids’ (PAA) was studied as HIV-1 trans-activating response (TAR) RNA ligands. An extensive thermodynamic study revealed the occurence of an enthalpy–entropy compensation phenomenon resulting in very close TAR affinities for all PAA. However, their binding modes and their ability to compete with the Tat fragment strongly differ according to their structure. Surprisingly, PAA that form loose complexes with TAR were shown to be stronger Tat competitors than those forming tight ones, and thermal denaturation studies demonstrated that loose complexes are more stable than tight ones. This could be correlated to the fact that loose and tight ligands induce distinct RNA conformational changes as revealed by circular dichroism experiments, although nuclear magnetic resonance (NMR) experiments showed that the TAR binding site is the same in all cases. Finally, some loose PAA also display promising inhibitory activities on HIV-infected cells. Altogether, these results lead to a better understanding of RNA interaction modes that could be very useful for devising new ligands of relevant RNA targets.

Description: RbpA is a small non–DNA-binding transcription factor that associates with RNA polymerase holoenzyme and stimulates transcription in actinobacteria, including Streptomyces coelicolor and Mycobacterium tuberculosis. RbpA seems to show specificity for the vegetative form of RNA polymerase as opposed to alternative forms of the enzyme. Here, we explain the basis of this specificity by showing that RbpA binds directly to the principal subunit in these organisms, but not to more diverged alternative factors. Nuclear magnetic resonance spectroscopy revealed that, although differing in their requirement for structural zinc, the RbpA orthologues from S. coelicolor and M. tuberculosis share a common structural core domain, with extensive, apparently disordered, N- and C-terminal regions. The RbpA– interaction is mediated by the C-terminal region of RbpA and domain 2, and S. coelicolor RbpA mutants that are defective in binding are unable to stimulate transcription in vitro and are inactive in vivo . Given that RbpA is essential in M. tuberculosis and critical for growth in S. coelicolor , these data support a model in which RbpA plays a key role in the cycle in actinobacteria.

Description: Differentiation of skeletal muscle cells is accompanied by drastic changes in gene expression programs that depend on activation and repression of genes at defined time points. Here we identify the serine/threonine kinase homeodomain-interacting protein kinase 2 (HIPK2) as a corepressor that inhibits myocyte enhancer factor 2 (MEF2)-dependent gene expression in undifferentiated myoblasts. Downregulation of HIPK2 expression by shRNAs results in elevated expression of muscle-specific genes, whereas overexpression of the kinase dampens transcription of these genes. HIPK2 is constitutively associated with a multi-protein complex containing histone deacetylase (HDAC)3 and HDAC4 that serves to silence MEF2C-dependent transcription in undifferentiated myoblasts. HIPK2 interferes with gene expression on phosphorylation and HDAC3-dependent deacetylation of MEF2C. Ongoing muscle differentiation is accompanied by elevated caspase activity, which results in caspase-mediated cleavage of HIPK2 following aspartic acids 916 and 977 and the generation of a C-terminally truncated HIPK2 protein. The short form of the kinase loses its affinity to the repressive multi-protein complex and its ability to bind HDAC3 and HDAC4, thus alleviating its repressive function for expression of muscle genes. This study identifies HIPK2 as a further protein that determines the threshold and kinetics of gene expression in proliferating myoblasts and during the initial steps of myogenesis.

Description: The structure of nucleosomes that contain the cenH3 histone variant has been controversial. In budding yeast, a single right-handed cenH3/H4/H2A/H2B tetramer wraps the ~80-bp Centromere DNA Element II (CDE II) sequence of each centromere into a ‘hemisome’. However, attempts to reconstitute cenH3 particles in vitro have yielded exclusively ‘octasomes’, which are observed in vivo on chromosome arms only when Cse4 (yeast cenH3) is overproduced. Here, we show that Cse4 octamers remain intact under conditions of low salt and urea that dissociate H3 octamers. However, particles consisting of two DNA duplexes wrapped around a Cse4 octamer and separated by a gap efficiently split into hemisomes. Hemisome dimensions were confirmed using a calibrated gel-shift assay and atomic force microscopy, and their identity as tightly wrapped particles was demonstrated by gelFRET. Surprisingly, Cse4 hemisomes were stable in 4 M urea. Stable Cse4 hemisomes could be reconstituted using either full-length or tailless histones and with a 78-bp CDEII segment, which is predicted to be exceptionally stiff. We propose that CDEII DNA stiffness evolved to favor Cse4 hemisome over octasome formation. The precise correspondence between Cse4 hemisomes resident on CDEII in vivo and reconstituted on CDEII in vitro without any other factors implies that CDEII is sufficient for hemisome assembly.

Description: The triazine derivative 12459 is a potent G-quadruplex ligand that triggers apoptosis or delayed growth arrest, telomere shortening and G-overhang degradation, as a function of its concentration and time exposure to the cells. We have investigated here the DNA damage response induced by 12459 in A549 cells. Submicromolar concentrations of 12459 triggers a delayed Chk1-ATR–mediated DNA damage response associated with a telomeric dysfunction and a G2/M arrest. Surprisingly, increasing concentrations of 12459 leading to cell apoptosis induced a mechanism that bypasses the DNA damage signaling and leads to the dephosphorylation of Chk1 and -H2AX. We identified the phosphatase Protein Phosphatase Magnesium dependent 1D/Wild-type P53-Induced Phosphatase (PPM1D/WIP1) as a factor responsible for this dephosphorylation. SiRNA-mediated depletion of PPM1D/WIP1 reactivates the DNA damage signaling by 12459. In addition, PPM1D/WIP1 is activated by reactive oxygen species (ROS) induced by 12459. ROS generated by 12459 are sufficient to trigger an early DNA damage in A549 cells when PPM1D/WIP1 is depleted. However, ROS inactivation by N -acetyl cysteine (NAC) treatment does not change the apoptotic response induced by 12459. Because PPM1D expression was recently reported to modulate the recruitment of DNA repair molecules, our data would suggest a cycle of futile protection against 12459, thus leading to a delayed mechanism of cell death.

Description: The current progress in the study of the spatial organization of interphase chromosomes became possible owing to the development of the chromosome conformation capture (3C) protocol. The crucial step of this protocol is the proximity ligation—preferential ligation of DNA fragments assumed to be joined within nuclei by protein bridges and solubilized as a common complex after formaldehyde cross-linking and DNA cleavage. Here, we show that a substantial, and in some cases the major, part of DNA is not solubilized from cross-linked nuclei treated with restriction endonuclease(s) and sodium dodecyl sulphate and that this treatment neither causes lysis of the nucleus nor drastically affects its internal organization. Analysis of the ligation frequencies of the mouse β-globin gene domain DNA fragments demonstrated that the previously reported 3C signals were generated predominantly, if not exclusively, in the insoluble portion of the 3C material. The proximity ligation thus occurs within the cross-linked chromatin cage in non-lysed nuclei. The finding does not compromise the 3C protocol but allows the consideration of an active chromatin hub as a folded chromatin domain or a nuclear compartment rather than a rigid complex of regulatory elements.

Description: MicroRNAs (miRNAs) constitute an important class of small regulatory RNAs that are derived from distinct hairpin precursors (pre-miRNAs). In contrast to mature miRNAs, which have been characterized in numerous genome-wide studies of different organisms, research on global profiling of pre-miRNAs is limited. Here, using massive parallel sequencing, we have performed global characterization of both mouse mature and precursor miRNAs. In total, 87 369 704 and 252 003 sequencing reads derived from 887 mature and 281 precursor miRNAs were obtained, respectively. Our analysis revealed new aspects of miRNA/pre-miRNA processing and modification, including eight Ago2-cleaved pre-miRNAs, eight new instances of miRNA editing and exclusively 5' tailed mirtrons. Furthermore, based on the sequences of both mature and precursor miRNAs, we developed a miRNA discovery pipeline, miRGrep, which does not rely on the availability of genome reference sequences. In addition to 239 known mouse pre-miRNAs, miRGrep predicted 41 novel ones with high confidence. Similar as known ones, the mature miRNAs derived from most of these novel loci showed both reduced abundance following Dicer knockdown and the binding with Argonaute2. Evaluation on data sets obtained from Caenorhabditis elegans and Caenorhabditis sp.11 demonstrated that miRGrep could be widely used for miRNA discovery in metazoans, especially in those without genome reference sequences.

Description: Thousands of novel transcripts have been identified using deep transcriptome sequencing. This discovery of large and ‘hidden’ transcriptome rejuvenates the demand for methods that can rapidly distinguish between coding and noncoding RNA. Here, we present a novel alignment-free method, Coding Potential Assessment Tool (CPAT), which rapidly recognizes coding and noncoding transcripts from a large pool of candidates. To this end, CPAT uses a logistic regression model built with four sequence features: open reading frame size, open reading frame coverage, Fickett TESTCODE statistic and hexamer usage bias. CPAT software outperformed (sensitivity: 0.96, specificity: 0.97) other state-of-the-art alignment-based software such as Coding-Potential Calculator (sensitivity: 0.99, specificity: 0.74) and Phylo Codon Substitution Frequencies (sensitivity: 0.90, specificity: 0.63). In addition to high accuracy, CPAT is approximately four orders of magnitude faster than Coding-Potential Calculator and Phylo Codon Substitution Frequencies, enabling its users to process thousands of transcripts within seconds. The software accepts input sequences in either FASTA- or BED-formatted data files. We also developed a web interface for CPAT that allows users to submit sequences and receive the prediction results almost instantly.

Description: Base excision repair (BER) is a frontline repair system that is responsible for maintaining genome integrity and thus preventing premature aging, cancer and many other human diseases by repairing thousands of DNA lesions and strand breaks continuously caused by endogenous and exogenous mutagens. This fundamental and essential function of BER not only necessitates tight control of the continuous availability of basic components for fast and accurate repair, but also requires temporal and spatial coordination of BER and cell cycle progression to prevent replication of damaged DNA. The major goal of this review is to critically examine controversial and newly emerging questions about mammalian BER pathways, mechanisms regulating BER capacity, BER responses to DNA damage and their links to checkpoint control of DNA replication.

Description: The attainment of strong pharmacological effects with oligonucleotides is hampered by inefficient access of these molecules to their sites of action in the cytosol or nucleus. Attempts to address this problem with lipid or polymeric delivery systems have been only partially successful. Here, we describe a novel alternative approach involving the use of a non-toxic small molecule to enhance the pharmacological effects of oligonucleotides. The compound Retro-1 was discovered in a screen for small molecules that reduce the actions of bacterial toxins and has been shown to block the retrograde trafficking pathway. We demonstrate that Retro-1 can also substantially enhance the effectiveness of antisense and splice switching oligonucleotides in cell culture. This effect occurs at the level of intracellular trafficking or processing and is correlated with increased oligonucleotide accumulation in the nucleus but does not involve the perturbation of lysosomal compartments. We also show that Retro-1 can alter the effectiveness of splice switching oligonucleotides in the in vivo setting. These observations indicate that it is possible to enhance the pharmacological actions of oligonucleotides using non-toxic and non-lysosomotropic small molecule adjuncts.

Description: Major histocompatibility complex class II (MHCII) molecules are heterodimeric surface proteins involved in the presentation of exogenous antigens during the adaptive immune response. We demonstrate the existence of a fine level of regulation, coupling the transcription and processing of mRNAs encoding α and β chains of MHCII molecules, mediated through binding of their Untraslated Regions (UTRs) to the same ribonucleoproteic complex (RNP). We propose a dynamic model, in the context of the ‘MHCII RNA operon’ in which the increasing levels of DRA and DRB mRNAs are docked by the RNP acting as a bridge between 5'- and 3'-UTR of the same messenger, building a loop structure and, at the same time, joining the two chains, thanks to shared common predicted secondary structure motifs. According to cell needs, as during immune surveillance, this RNP machinery guarantees a balanced synthesis of DRA and DRB mRNAs and a consequent balanced surface expression of the heterodimer.

Description: The RNA exosome is an essential ribonuclease complex involved in RNA processing and decay. It consists of a 9-subunit catalytically inert ring composed of six RNase PH-like proteins forming a central channel and three cap subunits with KH/S1 domains located at the top. The yeast exosome catalytic activity is supplied by the Dis3 (also known as Rrp44) protein, which has both endo- and exoribonucleolytic activities and the nucleus-specific exonuclease Rrp6. In vitro studies suggest that substrates reach the Dis3 exonucleolytic active site following passage through the ring channel, but in vivo support is lacking. Here, we constructed an Rrp41 ring subunit mutant with a partially blocked channel that led to thermosensitivity and synthetic lethality with Rrp6 deletion. Rrp41 mutation caused accumulation of nuclear and cytoplasmic exosome substrates including the non-stop decay reporter, for which degradation is dependent on either endonucleolytic or exonucleolytic Dis3 activities. This suggests that the central channel also controls endonucleolytic activity. In vitro experiments performed using Chaetomium thermophilum exosomes reconstituted from recombinant subunits confirmed this notion. Finally, we analysed the impact of a lethal mutation of conserved basic residues in Rrp4 cap subunit and found that it inhibits digestion of single-stranded and structured RNA substrates.

Description: Single-cell genomics is revolutionizing basic genome research and clinical genetic diagnosis. However, none of the current research or clinical methods for single-cell analysis distinguishes between the analysis of a cell in G1-, S- or G2/M-phase of the cell cycle. Here, we demonstrate by means of array comparative genomic hybridization that charting the DNA copy number landscape of a cell in S-phase requires conceptually different approaches to that of a cell in G1- or G2/M-phase. Remarkably, despite single-cell whole-genome amplification artifacts, the log2 intensity ratios of single S-phase cells oscillate according to early and late replication domains, which in turn leads to the detection of significantly more DNA imbalances when compared with a cell in G1- or G2/M-phase. Although these DNA imbalances may, on the one hand, be falsely interpreted as genuine structural aberrations in the S-phase cell’s copy number profile and hence lead to misdiagnosis, on the other hand, the ability to detect replication domains genome wide in one cell has important applications in DNA-replication research. Genome-wide cell-type-specific early and late replicating domains have been identified by analyses of DNA from populations of cells, but cell-to-cell differences in DNA replication may be important in genome stability, disease aetiology and various other cellular processes.

Description: As researchers begin probing deep coverage sequencing data for increasingly rare mutations and subclonal events, the fidelity of next generation sequencing (NGS) laboratory methods will become increasingly critical. Although error rates for sequencing and polymerase chain reaction (PCR) are well documented, the effects that DNA extraction and other library preparation steps could have on downstream sequence integrity have not been thoroughly evaluated. Here, we describe the discovery of novel C 〉 A/G 〉 T transversion artifacts found at low allelic fractions in targeted capture data. Characteristics such as sequencer read orientation and presence in both tumor and normal samples strongly indicated a non-biological mechanism. We identified the source as oxidation of DNA during acoustic shearing in samples containing reactive contaminants from the extraction process. We show generation of 8-oxoguanine (8-oxoG) lesions during DNA shearing, present analysis tools to detect oxidation in sequencing data and suggest methods to reduce DNA oxidation through the introduction of antioxidants. Further, informatics methods are presented to confidently filter these artifacts from sequencing data sets. Though only seen in a low percentage of reads in affected samples, such artifacts could have profoundly deleterious effects on the ability to confidently call rare mutations, and eliminating other possible sources of artifacts should become a priority for the research community.

Description: Transgenic animals are extensively used to model human disease. Typically, the transgene copy number is estimated, but the exact integration site and configuration of the foreign DNA remains uncharacterized. When transgenes have been closely examined, some unexpected configurations have been found. Here, we describe a method to recover transgene insertion sites and assess structural rearrangements of host and transgene DNA using microarray hybridization and targeted sequence capture. We used information about the transgene insertion site to develop a polymerase chain reaction genotyping assay to distinguish heterozygous from homozygous transgenic animals. Although we worked with a bacterial artificial chromosome transgenic mouse line, this method can be used to analyse the integration site and configuration of any foreign DNA in a sequenced genome.