DAG papers

Group Publications

2020 (1)

Hsu C.; Cairns L.; Hobley L.; Abbott J.; O'Byrne C.; Stanley-Wall N.R. Genomic differences between Listeria monocytogenes EGDe isolates reveals crucial roles for SigB and wall rhamnosylation in biofilm formation. 2020 Journal of Bacteriology

Listeria monocytogenes is a Gram-positive Firmicute that causes food-borne infections, in part due to its ability to use multiple strategies, including biofilm formation, to survive adverse growth conditions. As a potential way to screen for genes required for biofilm formation we harnessed the ability of bacteria to accumulate mutations in the genome over time, diverging the properties of seemingly identical strains. By sequencing the genomes of four laboratory reference strains of the commonly used L. monocytogenes EGDe, we showed that each isolate contains SNPs compared with the reference genome. We discovered that two SNPs, contained in two independent genes within one of the isolates, impacted biofilm formation. Using bacterial genetics and phenotypic assays, we confirmed that rsbU and rmlA influence biofilm formation. RsbU is the upstream regulator of the alternative sigma factor, SigB and mutation of either rsbU or sigB increased biofilm formation. In contrast, deletion of rmlA, which encodes the first enzyme for TDP-L-rhamnose biosynthesis, resulted in a reduction in the amount of biofilm formed. Further analysis of biofilm formation in a strain that still produces TDP-L-rhamnose, but which cannot decorate the wall teichoic acid with rhamnose (rmlT mutant), showed that it is the decorated wall teichoic acid that is required for adhesion of the cells to surfaces. Together these data uncover novel routes by which biofilm formation by L. monocytogenes can be impacted. Importance Biofilms are an important mode of growth in many settings. Here we have looked at small differences in the genomes of the bacteria Listeria monocytogenes isolate EGDe and used them to find out how biofilms form. This is important fundamental information may help new treatments to be developed and also highlights the fact that isolates of the same identity often diverge.

Arnaouteli S.; Matoz-Fernandez D.A.; Porter M.; Kalamara M.; Abbott J.; MacPhee C.E.; Davidson F.A.; Stanley-Wall N.R. Pulcherrimin formation controls growth arrest of the Bacillus subtilis biofilm 2019 Proceedings of the National Academy of Sciences 201903982

Biofilm formation by Bacillus subtilis is a communal process that culminates in the formation of architecturally complex multicellular communities. Here we reveal that the transition of the biofilm into a nonexpanding phase constitutes a distinct step in the process of biofilm development. Using genetic analysis we show that B. subtilis strains lacking the ability to synthesize pulcherriminic acid form biofilms that sustain the expansion phase, thereby linking pulcherriminic acid to growth arrest. However, production of pulcherriminic acid is not sufficient to block expansion of the biofilm. It needs to be secreted into the extracellular environment where it chelates Fe3+ from the growth medium in a nonenzymatic reaction. Utilizing mathematical modeling and a series of experimental methodologies we show that when the level of freely available iron in the environment drops below a critical threshold, expansion of the biofilm stops. Bioinformatics analysis allows us to identify the genes required for pulcherriminic acid synthesis in other Firmicutes but the patchwork presence both within and across closely related species suggests loss of these genes through multiple independent recombination events. The seemingly counterintuitive self-restriction of growth led us to explore if there were any benefits associated with pulcherriminic acid production. We identified that pulcherriminic acid producers can prevent invasion by neighboring communities through the generation of an “iron-free” zone, thereby addressing the paradox of pulcherriminic acid production by B. subtilis.

Elias M.S.; Wright S.C.; Remenyi J.; Abbott J.C.; Bray S.E.; Cole C.; Edwards S.; Gierlinski M.; Glok M.; McGrath J.A.; Nicholson W.V.; Paternoster L.; Prescott A.R.; Have S.T.; Whitfield P.D.; Lamond A.I.; Brown S.J. EMSY expression affects multiple components of skin barrier with relevance to atopic dermatitis 2019 Journal of Allergy and Clinical Immunology

Background Atopic dermatitis (AD) is a common, complex and highly heritable inflammatory skin disease. Genome-wide association studies (GWAS) offer opportunities to identify molecular targets for drug development. A risk locus on chromosome 11q13.5 lies between two candidate genes - EMSY and LRRC32 - but the functional mechanisms affecting risk of AD remain unclear. Objectives To apply a combination of genomic and molecular analytical techniques to investigate which genes are responsible for genetic risk at this locus and to define mechanisms contributing to atopic skin disease. Methods Interrogation of available genomic and chromosome conformation (Hi-C) data in keratinocytes; siRNA-mediated knockdown in skin organotypic culture and functional assessment of barrier parameters; mass-spectrometry global proteomic analysis and quantitative lipid analysis; electron microscopy of organotypic skin and immunohistochemistry of human skin samples. Results Genomic data indicate active promoters in the GWAS locus and upstream of EMSY; EMSY, LRRC32 and the intergenic variants may all be within a single topologically associating domain. siRNA-knockdown of EMSY in organotypic culture leads to enhanced development of barrier function, reflecting increased expression of structural and functional proteins, including filaggrin and filaggrin-2, as well as long-chain ceramides. Conversely, over-expression of EMSY in keratinocytes leads to a reduction in markers of barrier formation. Skin biopsy samples from patients with AD show greater EMSY staining in the nucleus, consistent with increased functional effect of this transcriptional control protein. Conclusion Our findings demonstrate an important role for EMSY in transcriptional regulation and skin barrier formation, supporting EMSY inhibition as a therapeutic approach.

Guo M.; Härtlova A.; Gierliński M.; Prescott A.; Castellvi J.; Losa J.H.; Petersen S.K.; Wenzel U.A.; Dill B.D.; Emmerich C.H.; Cajal S.R.Y.; Russell D.G.; Trost M. Triggering MSR1 promotes JNK‐mediated inflammation in IL‐4‐activated macrophages 2019 The EMBO Journal e100299

Alternatively activated M2 macrophages play an important role in maintenance of tissue homeostasis by scavenging dead cells, cell debris and lipoprotein aggregates via phagocytosis. Using proteomics, we investigated how alternative activation, driven by IL‐4, modulated the phagosomal proteome to control macrophage function. Our data indicate that alternative activation enhances homeostatic functions such as proteolysis, lipolysis and nutrient transport. Intriguingly, we identified the enhanced recruitment of the TAK1/MKK7/JNK signalling complex to phagosomes of IL‐4‐activated macrophages. The recruitment of this signalling complex was mediated through K63 polyubiquitylation of the macrophage scavenger receptor 1 (MSR1). Triggering of MSR1 in IL‐4‐activated macrophages leads to enhanced JNK activation, thereby promoting a phenotypic switch from an anti‐inflammatory to a pro‐inflammatory state, which was abolished upon MSR1 deletion or JNK inhibition. Moreover, MSR1 K63 polyubiquitylation correlated with the activation of JNK signalling in ovarian cancer tissue from human patients, suggesting that it may be relevant for macrophage phenotypic shift in vivo. Altogether, we identified that MSR1 signals through JNK via K63 polyubiquitylation and provides evidence for the receptor's involvement in macrophage polarization. Synopsis \img class="highwire-embed" alt="Embedded Image" src="http://emboj.embopress.org/sites/default/files/highwire/embojnl/early/2019/04/26/embj.2018100299/embed/graphic-1.gif"/\ Macrophage scavenger receptor MSR1 is a key phagocytic receptor for the uptake of lipids and cell debris in macrophages. In IL‐4 activated macrophages MSR1 becomes ubiquitylated, recruits the Tak1/MKK7 kinase complex and signals directly through JNK, which induces pro‐inflammatory cytokine production. Proteomics analysis of phagosomes indicate that alternative activation by IL‐4 enhances phagosomal homeostatic functions in macrophages.Triggering of MSR1 in IL‐4 activated macrophages leads to its ubiquitylation, which recruits the TAK1/MKK7/JNK kinase complex.This leads to pro‐inflammatory signalling, inducing a phenotypic switch of the macrophages.MSR1 ubiquitylation and enhanced JNK signalling is present in human tumour associated macrophages.

Doodhi H.; Kasciukovic T.; Gierlinski M.; Li S.; Clayton L.; Tanaka T.U. In vitro reconstitution of kinetochore–microtubule interface reveals a fundamental error correction mechanism 2019 bioRxiv 455873

\p\For proper chromosome segregation, sister kinetochores must interact with microtubules from opposite spindle poles; this is called bi-orientation. To establish bi-orientation prior to chromosome segregation, any aberrant kinetochore-microtubule interaction must be resolved (error correction) by Aurora B kinase that phosphorylates outer kinetochore components. Aurora B differentially regulates kinetochore attachment to the microtubule plus end and its lateral side (end-on and lateral attachment). However, it is still unknown how kinetochore-microtubule interaction is turned over during error correction. Here we reconstituted the kinetochore-microtubule interface in vitro by attaching Ndc80 protein complexes (Ndc80Cs) to a nanobead that mimics the inner kinetochore. The Ndc80C-nanobeads recapitulated in vitro the lateral and end-on attachments of authentic kinetochores on dynamic microtubules loaded with the Dam1 complex. Using this system, we provide evidence that error correction is driven by direct competition for a kinetochore between the end-on attachment to one microtubule and the lateral attachment to another. We validated this competition-based error correction, using mathematical modelling and live-cell imaging. Our study reveals a fundamental mechanism of error correction, which contributes to efficient establishment of bi-orientation.\\

Eykelenboom J.K.; Gierliński M.; Yue Z.; Hegarat N.; Pollard H.; Fukagawa T.; Hochegger H.; Tanaka T.U. Live imaging of marked chromosome regions reveals their dynamic resolution and compaction in mitosis 2019 J Cell Biol jcb.201807125

When human cells enter mitosis, chromosomes undergo substantial changes in their organization to resolve sister chromatids and compact chromosomes. To comprehend the timing and coordination of these events, we need to evaluate the progression of both sister chromatid resolution and chromosome compaction in one assay. Here we achieved this by analyzing changes in configuration of marked chromosome regions over time, with high spatial and temporal resolution. This assay showed that sister chromatids cycle between nonresolved and partially resolved states with an interval of a few minutes during G2 phase before completing full resolution in prophase. Cohesins and WAPL antagonistically regulate sister chromatid resolution in late G2 and prophase while local enrichment of cohesin on chromosomes prevents precocious sister chromatid resolution. Moreover, our assay allowed quantitative evaluation of condensin II and I activities, which differentially promote sister chromatid resolution and chromosome compaction, respectively. Our assay reveals novel aspects of dynamics in mitotic chromosome resolution and compaction that were previously obscure in global chromosome assays.

Arnaouteli S.; Matoz-Fernandez D.; Porter M.; Kalamara M.; Abbott J.; MacPhee C.E.; Davidson F.A.; Stanley-Wall N.R. Pulcherrimin formation controls growth arrest of the Bacillus subtilis biofilm 2019 bioRxiv 570630

\h3\Abstract\/h3\ \p\Biofilm formation by \textitBacillus subtilis is a communal process that culminates in the formation of architecturally complex multicellular communities. Here we reveal that the transition of the biofilm into a non-expanding phase constitutes a distinct step in the process of biofilm development. Using genetic analysis we show that \textitB. subtilis strains lacking the ability to synthesize pulcherriminic acid form biofilms that sustain the expansion phase, thereby linking pulcherriminic acid to growth arrest. However, production of pulcherriminic acid is not sufficient to block expansion of the biofilm. It needs to be secreted into the extracellular environment where it chelates Fe$^\textrm3+$ from the growth medium in a non-enzymatic reaction. Utilizing mathematical modelling and a series of experimental methodologies we show that when the level of freely available iron in the environment drops below a critical threshold, expansion of the biofilm stops. Bioinformatics analysis allows us to identify the genes required for pulcherriminic acid synthesis in other Firmicutes but the patchwork presence both within and across closely related species suggests loss of these genes through multiple independent recombination events. The seemingly counterintuitive self-restriction of growth led us to explore if there were any benefits associated pulcherriminic acid production. We identified that pulcherriminic acid producers can prevent invasion from neighbouring communities through the generation of an “iron free” zone thereby addressing the paradox of pulcherriminic acid production by \textitB. subtilis.\\\h3\Significance\/h3\ \p\Understanding the processes that underpin the mechanism of biofilm formation, dispersal, and inhibition are critical to allow exploitation and to understand how microbes thrive in the environment. Here, we reveal that the formation of an extracellular iron chelate restricts the expansion of a biofilm. The countering benefit to self-restriction of growth is protection of an environmental niche. These findings highlight the complex options and outcomes that bacteria need to balance in order to modulate their local environment to maximise colonisation, and therefore survival.\\

Froussios K.; Schurch N.J.; Mackinnon K.; Gierliński M.; Duc C.; Simpson G.G.; Barton G.J. How well do RNA-Seq differential gene expression tools perform in a complex eukaryote? A case study in A. thaliana 2019 Bioinformatics

AbstractMotivation. RNA-seq experiments are usually carried out in three or fewer replicates. In order to work well with so few samples, Differential Gene Expr

Guo M.; Hartlova A.; Gierlinski M.; Prescott A.; Castellvi J.; Losa J.H.; Petersen S.K.; Wenzel U.A.; Dill B.D.; Emmerich C.H.; Cajal S.R.y.; Russell D.G.; Trost M. Triggering MSR1 promotes JNK-mediated inflammation in IL-4 activated macrophages 2019 bioRxiv 382853

\p\Alternatively activated M2 macrophages play an important role in maintenance of tissue homeostasis by scavenging dead cells, cell debris and lipoprotein aggregates via phagocytosis. Using proteomics, we investigated how alternative activation, driven by IL-4, modulated the phagosomal proteome to control macrophage function. Our data indicate that alternative activation enhances homeostatic functions such as proteolysis, lipolysis and nutrient transport. Intriguingly, we identified the enhanced recruitment of the TAK1/MKK7/JNK signalling complex to phagosomes of IL-4 activated macrophages. The recruitment of this signalling complex was mediated through K63-polyubiquitylation of the macrophage scavenger receptor 1 (MSR1). Triggering of MSR1 in IL-4 activated macrophages leads to enhanced JNK activation, thereby promoting a phenotypic switch from an anti-inflammatory to a pro-inflammatory state, which was abolished upon MSR1 deletion or JNK inhibition. Moreover, MSR1 K63-polyubiquitylation correlated with the activation of JNK signalling in ovarian cancer tissue from human patients, suggesting that it may be relevant for macrophage phenotypic shift in vivo. Altogether, we identified that MSR1 signals through JNK via K63-polyubiquitylation and provide evidence for the receptor9s involvement in macrophage polarization.\\

Gierlinski M.; Gastaldello F.; Cole C.; Barton G.J. Proteus: an R package for downstream analysis of MaxQuant output 2018 bioRxiv 416511

\p\Proteus is a package for downstream analysis of MaxQuant evidence data in the R environment. It provides tools for peptide and protein aggregation, quality checks, data exploration and visualisation. Interactive analysis is implemented in the Shiny framework, where individual peptides or protein may be examined in the context of a volcano plot. Proteus performs differential expression analysis with the well-established tool limma, which offers robust treatment of missing data, frequently encountered in label-free mass-spectrometry experiments. We demonstrate on real and simulated data that limma results in improved sensitivity over random imputation combined with a t-test as implemented in the popular package Perseus. Embedding Proteus in R provides access to a wide selection of statistical and graphical tools for further analysis and reproducibility by scripting. Availability and implementation: The open-source R package, including example data and tutorials, is available to install from GitHub (https://github.com/bartongrouproteus).\\

Ly T.; Endo A.; Brenes A.; Gierlinski M.; Afzal V.; Pawellek A.; Lamond A.I. Proteome-wide analysis of protein abundance and turnover remodelling during oncogenic transformation of human breast epithelial cells 2018 Wellcome Open Research 3:51

Miettinen T.P.; Peltier J.; Härtlova A.; Gierliński M.; Jansen V.M.; Trost M.; Björklund M. Thermal proteome profiling of breast cancer cells reveals proteasomal activation by CDK4/6 inhibitor palbociclib 2018 The EMBO Journal 37:e98359

Palbociclib is a CDK4/6 inhibitor approved for metastatic estrogen receptor‐positive breast cancer. In addition to G1 cell cycle arrest, palbociclib treatment results in cell senescence, a phenotype that is not readily explained by CDK4/6 inhibition. In order to identify a molecular mechanism responsible for palbociclib‐induced senescence, we performed thermal proteome profiling of MCF7 breast cancer cells. In addition to affecting known CDK4/6 targets, palbociclib induces a thermal stabilization of the 20S proteasome, despite not directly binding to it. We further show that palbociclib treatment increases proteasome activity independently of the ubiquitin pathway. This leads to cellular senescence, which can be counteracted by proteasome inhibitors. Palbociclib‐induced proteasome activation and senescence is mediated by reduced proteasomal association of ECM29. Loss of ECM29 activates the proteasome, blocks cell proliferation, and induces a senescence‐like phenotype. Finally, we find that ECM29 mRNA levels are predictive of relapse‐free survival in breast cancer patients treated with endocrine therapy. In conclusion, thermal proteome profiling identifies the proteasome and ECM29 protein as mediators of palbociclib activity in breast cancer cells. See also: RL de Oliveira \& R Bernards (May 2018) Synopsis \img class="highwire-embed" alt="Embedded Image" src="http://emboj.embopress.org/sites/default/files/highwire/embojnl/37/10/e98359/embed/graphic-1.gif"/\ The breast cancer drug palbociclib arrests cells in G1 phase by CDK4/6 inhibition, but also causes cellular senescence. Thermal proteome profiling shows that the latter is mediated by increased proteasome activation through reduced ECM29 binding. Mass spectrometry‐based cellular thermal shift assay (MS‐CeTSA) analysis of CDK4/6 inhibitor palbociclib targets in MCF7 human breast cancer cells identifies protein complexes including the 20S proteasome.Palbociclib treatment increases proteasome activity independently of the ubiquitin pathway.Palbociclib activates the proteasome by disassociating the proteolysis‐inhibiting scaffold protein ECM29.Proteasome activation is required for palbociclib‐induced cellular senescence, which is sensitive to the proteasome inhibitor bortezomib.

Vasileva V.; Gierlinski M.; Yue Z.; O’Reilly N.; Kitamura E.; Tanaka T.U. Molecular mechanisms facilitating the initial kinetochore encounter with spindle microtubules 2017 J Cell Biol jcb.201608122

The initial kinetochore (KT) encounter with a spindle microtubule (MT; KT capture) is one of the rate-limiting steps in establishing proper KT–MT interaction during mitosis. KT capture is facilitated by multiple factors, such as MT extension in various directions, KT diffusion, and MT pivoting. In addition, KTs generate short MTs, which subsequently interact with a spindle MT. KT-derived MTs may facilitate KT capture, but their contribution is elusive. In this study, we find that Stu1 recruits Stu2 to budding yeast KTs, which promotes MT generation there. By removing Stu2 specifically from KTs, we show that KT-derived MTs shorten the half-life of noncaptured KTs from 48–49 s to 28–34 s. Using computational simulation, we found that multiple factors facilitate KT capture redundantly or synergistically. In particular, KT-derived MTs play important roles both by making a significant contribution on their own and by synergistically enhancing the effects of KT diffusion and MT pivoting. Our study reveals fundamental mechanisms facilitating the initial KT encounter with spindle MTs.

MacGowan S.A.; Madeira F.; Borges T.B.; Schmittner M.S.; Cole C.; Barton G.J. Human Missense Variation is Constrained by Domain Structure and Highlights Functional and Pathogenic Residues 2017 bioRxiv 127050

Human genome sequencing has generated population variant datasets containing millions of variants from hundreds of thousands of individuals. The datasets show the genomic distribution of genetic variation to be influenced on genic and sub-genic scales by gene essentiality, protein domain architecture and the presence of genomic features such as splice donor/acceptor sites. However, the variant data are still too sparse to provide a comparative picture of genetic variation between individual protein residues in the proteome. Here, we overcome this sparsity for ~25,000 human protein domains in 1,291 domain families by aggregating variants over equivalent positions (columns) in multiple sequence alignments of sequence-similar (paralagous) domains. We then compare the resulting variation profiles from the human population to residue conservation across all species and find that the same tertiary structural and functional pressures that affect amino acid conservation during domain evolution constrain missense variant distributions. Thus, depletion of missense variants at a position implies that it is structurally or functionally important. We find such positions are enriched in known disease-associated variants (OR = 2.83, p ≈ 0) while positions that are both missense depleted and evolutionary conserved are further enriched in disease-associated variants (OR = 1.85, p = 3.3x10-17) compared to those that are only evolutionary conserved (OR = 1.29, p = 4.5x10-19). Unexpectedly, a subset of evolutionary Unconserved positions are Missense Depleted in human (UMD positions) and these are also enriched in pathogenic variants (OR = 1.74, p = 0.02). UMD positions are further differentiated from other unconserved residues in that they are enriched in ligand, DNA and protein binding interactions (OR = 1.59, p = 0.003), which suggests this stratification can identify functionally important positions. A different class of positions that are Conserved and Missense Enriched (CME) show an enrichment of ClinVar risk factor variants (OR = 2.27, p = 0.004). We illustrate these principles with the G-Protein Coupled Receptor (GPCR) family, Nuclear Receptor Ligand Binding Domain family and In Between Ring-Finger (IBR) domains and list a total of 343 UMD positions in 211 domain families. This study will have broad applications to: (a) providing focus for functional studies of specific proteins by mutagenesis; (b) refining pathogenicity prediction models; (c) highlighting which residue interactions to target when refining the specificity of small-molecule drugs.

Froussios K.; Schurch N.J.; Mackinnon K.; Gierlinski M.; Duc C.; Simpson G.G.; Barton G.J. How well do RNA-Seq differential gene expression tools perform in a eukaryote with a complex transcriptome? 2017 bioRxiv 090753

RNA-seq experiments are usually carried out in three or fewer replicates. In order to work well with so few samples, Differential Gene Expression (DGE) tools typically assume the form of the underlying distribution of gene expression. A recent highly replicated study revealed that RNA-seq gene expression measurements in yeast are best represented as being drawn from an underlying negative binomial distribution. In this paper, the statistical properties of gene expression in the higher eukaryote Arabidopsis thaliana are shown to be essentially identical to those from yeast despite the large increase in the size and complexity of the transcriptome: Gene expression measurements from this model plant species are consistent with being drawn from an underlying negative binomial or log-normal distribution and the false positive rate performance of nine widely used DGE tools is not strongly affected by the additional size and complexity of the A. thaliana transcriptome. For RNA-seq data, we therefore recommend the use of DGE tools that are based on the negative binomial distribution.

Yue Z.; Komoto S.; Gierlinski M.; Pasquali D.; Kitamura E.; Tanaka T.U. Mechanisms mitigating problems with multiple kinetochores on one microtubule in early mitosis 2017 J Cell Sci jcs.203000

Skip to Next Section Proper chromosome segregation in mitosis relies on correct kinetochore interaction with spindle microtubules. In early mitosis, each kinetochore usually interacts with the lateral side of each microtubule and is subsequently tethered at the microtubule end. However, since eukaryotic cells carry multiple chromosomes, multiple kinetochores could occasionally interact with a single microtubule. The consequence of this is unknown. Here we find that, although two kinetochores (two pairs of sister kinetochores) can interact with the lateral side of one microtubule, only one kinetochore can form sustained attachment to the microtubule end in budding yeast. This leads to detachment of the other kinetochore from the microtubule end or its proximity. Intriguingly, in this context, kinetochore sliding along a microtubule towards a spindle pole delays and diminishes discernible kinetochore detachment. This effect expedites collection of the entire set of kinetochores to a spindle pole. We propose that cells are equipped with the kinetochore sliding mechanism to mitigate the problem with multiple kinetochores on one microtubule in early mitosis.

Singh R.; Lawal H.M.; Schilde C.; Glöckner G.; Barton G.J.; Schaap P.; Cole C. Improved annotation with de novo transcriptome assembly in four social amoeba species 2017 BMC Genomics 18:120

Annotation of gene models and transcripts is a fundamental step in genome sequencing projects. Often this is performed with automated prediction pipelines, which can miss complex and atypical genes or transcripts. RNA sequencing (RNA-seq) data can aid the annotation with empirical data. Here we present de novo transcriptome assemblies generated from RNA-seq data in four Dictyostelid species: D. discoideum, P. pallidum, D. fasciculatum and D. lacteum. The assemblies were incorporated with existing gene models to determine corrections and improvement on a whole-genome scale. This is the first time this has been performed in these eukaryotic species.

Chen Z.; Singh R.; Cole C.; Lawal H.M.; Schilde C.; Febrer M.; Barton G.J.; Schaap P. Adenylate cyclase A acting on PKA mediates induction of stalk formation by cyclic diguanylate at the Dictyostelium organizer 2017 Proceedings of the National Academy of Sciences 201608393

Coordination of cell movement with cell differentiation is a major feat of embryonic development. The Dictyostelium stalk always forms at the organizing tip, by a mechanism that is not understood. We previously reported that cyclic diguanylate (c-di-GMP), synthesized by diguanylate cyclase A (DgcA), induces stalk formation. Here we used transcriptional profiling of dgca− structures to identify target genes for c-di-GMP, and used these genes to investigate the c-di-GMP signal transduction pathway. We found that knockdown of cAMP-dependent protein kinase (PKA) activity in prestalk cells reduced stalk gene induction by c-di-GMP, whereas PKA activation bypassed the c-di-GMP requirement for stalk gene expression. c-di-GMP caused a persistent increase in cAMP, which still occurred in mutants lacking the adenylate cyclases ACG or ACR, or the cAMP phosphodiesterase RegA. However, both inhibition of adenylate cyclase A (ACA) with SQ22536 and incubation of a temperature-sensitive ACA mutant at the restrictive temperature prevented c-di-GMP–induced cAMP synthesis as well as c-di-GMP–induced stalk gene transcription. ACA produces the cAMP pulses that coordinate Dictyostelium morphogenetic cell movement and is highly expressed at the organizing tip. The stalk-less dgca− mutant regained its stalk by expression of a light-activated adenylate cyclase from the ACA promoter and exposure to light, indicating that cAMP is also the intermediate for c-di-GMP in vivo. Our data show that the more widely expressed DgcA activates tip-expressed ACA, which then acts on PKA to induce stalk genes. These results explain why stalk formation in Dictyostelia always initiates at the site of the morphogenetic organizer.

Wilson N.; Cole C.; Kroboth K.; Hunter W.; Mann J.; McLean W.; Kernland Lang K.; Beltraminelli H.; Sabroe R.; Tiffin N.; Sobey G.; Borradori L.; Simpson E.; Smith F. Mutations in POGLUT1 in Galli–Galli/Dowling–Degos disease 2017 British Journal of Dermatology 176:270-274

Tatham M.H.; Cole C.; Scullion P.; Wilkie R.; Westwood N.J.; Stark L.A.; Hay R.T. A proteomic approach to analyse the aspirin-mediated lysine acetylome 2016 Molecular \& Cellular Proteomics mcp.O116.065219

Aspirin, or acetylsalicylic acid is widely used to control pain, inflammation and fever. Important to this function is its ability to irreversibly acetylate cyclooxygenases at active site serines. Aspirin has the potential to acetylate other amino-acid side-chains, leading to the possibility that aspirin-mediated lysine acetylation could explain some of its as-yet unexplained drug actions or side-effects. Using isotopically labeled aspirin-d3, in combination with acetylated lysine purification and LC-MS/MS, we identified over 12000 sites of lysine acetylation from cultured human cells. Although aspirin amplifies endogenous acetylation signals at the majority of detectable endogenous sites, cells tolerate aspirin mediated acetylation very well unless cellular deacetylases are inhibited. Although most endogenous acetylations are amplified by orders of magnitude, lysine acetylation site occupancies remain very low even after high doses of aspirin. This work shows that while aspirin has enormous potential to alter protein function, in the majority of cases aspirin-mediated acetylations do not accumulate to levels likely to elicit biological effects. These findings are consistent with an emerging model for cellular acetylation whereby stoichiometry correlates with biological relevance, and deacetylases act to minimize the biological consequences non-specific chemical acetylations.

Kasparis C.; Reid D.; Wilson N.J.; Okur V.; Cole C.; Hansen C.D.; Bosse K.; Betz R.C.; Khan M.; Smith F.J.D. Isolated recessive nail dysplasia caused by FZD6 mutations: report of three families and review of the literature 2016 Clinical and Experimental Dermatology 41:884-889

Congenital abnormalities of the nail are rare conditions that are most frequently associated with congenital ectodermal syndromes involving several of the epidermal appendages including the skin, teeth, hair and nails. Isolated recessive nail dysplasia (IRND) is much rarer but has recently been recognized as a condition resulting in 20-nail dystrophy in the absence of other cutaneous or extracutaneous findings. A few case reports have identified mutations in the Frizzled 6 (FZD6) gene in families presenting with abnormal nails consistent with IRND. These reports have highlighted the role of Wnt–FZD signalling in the process of nail formation. We report three families presenting with features of IRND, in whom we identified mutations in FZD6, including one previously unreported mutation.

Naeem A.S.; Tommasi C.; Cole C.; Brown S.J.; Zhu Y.; Way B.; Owen S.A.W.; Moffatt M.; Cookson W.O.; Harper J.I.; Wl D.; Brown S.J.; Reinheckel T.; O’Shaughnessy R.F.L. An mTORC1/AKT1/Cathepsin H axis controls filaggrin expression and processing in skin, a novel mechanism for skin barrier disruption in Atopic Dermatitis 2016 Journal of Allergy and Clinical Immunology 0

Dickerson D.; Gierliński M.; Singh V.; Kitamura E.; Ball G.; Tanaka T.U.; Owen-Hughes T. High resolution imaging reveals heterogeneity in chromatin states between cells that is not inherited through cell division 2016 BMC Cell Biology 17:33

Genomes of eukaryotes exist as chromatin, and it is known that different chromatin states can influence gene regulation. Chromatin is not a static structure, but is known to be dynamic and vary between cells. In order to monitor the organisation of chromatin in live cells we have engineered fluorescent fusion proteins which recognize specific operator sequences to tag pairs of syntenic gene loci. The separation of these loci was then tracked in three dimensions over time using fluorescence microscopy.

Ward J.; Cole C.; Febrer M.; Barton G.J. AlmostSignificant: Simplifying quality control of high-throughput sequencing data 2016 Bioinformatics btw559

Motivation: The current generation of DNA sequencing technologies produce a large amount of data quickly. All of these data need to pass some form of quality control (QC) processing and checking before they can be used for any analysis. The large number of samples that are run through Illumina sequencing machines makes the process of QC an onerous and time-consuming task that requires multiple pieces of information from several sources. Results: AlmostSignificant is an open-source platform for aggregating multiple sources of quality metrics as well as run and sample meta-data associated with DNA sequencing runs from Illumina sequencing machines. AlmostSignificant is a graphical platform to streamline the QC of DNA sequencing data, to store these data for future reference together with extra meta-data associated with the sequencing runs not typically retained. This simplifies the challenge of monitoring the volume of data produced by Illumina sequencers. AlmostSignificant has been used to track the quality of over 80 sequencing runs covering over 2500 samples produced over the last three years. Availability: The code and documentation for AlmostSignificant is freely available at https://github.com/bartongroup/AlmostSignificant. Contact: c.cole@dundee.ac.uk, g.j.barton@dundee.ac.uk

Lovgren M.; McAleer M.; Irvine A.; Wilson N.; Tavadia S.; Schwartz M.; Cole C.; Sandilands A.; Smith F.; Zamiri M. Mutations in desmoglein-1 cause diverse inherited palmoplantar keratoderma phenotypes: Implications for genetic screening 2016 British Journal of Dermatology n/a-n/a

Introduction The inherited palmoplantar keratodermas (PPKs) are a heterogeneous group of genodermatoses, characterised by thickening of the epidermis of the palms and soles. No classification system unites satisfactorily clinical presentation, pathology and molecular pathogenesis. There are four patterns of hyperkeratosis: striate, focal, diffuse, and punctate. Mutations in desmoglein-1 (DSG1), a transmembrane glycoprotein, have been reported primarily in striate, but also in focal and diffuse PPKs. Objectives We report seven unrelated pedigrees with dominantly inherited PPK due to mutations in the DSG1 gene, with marked phenotypic variation. Methods Genomic DNA from each family was isolated, and individual exons amplified by polymerase chain reaction (PCR). Sanger sequencing was employed to identify mutations. Results Mutation analysis identified novel mutations in five families (p.Tyr126Hisfs*2, p.Ser521Tyrfs*2, p.Trp3*, p.Asp591Phefs*9 and p.Met249Ilefs*6) with striate palmar involvement and varying focal or diffuse plantar disease, and the recurrent mutation c.76CT, p.Arg26*, in two families with variable PPK patterns. Conclusion We report one recurrent and five novel DSG1 mutations, causing varying patterns of PPK, highlighting the clinical heterogeneity arising from mutations in this gene. This article is protected by copyright. All rights reserved.

Schurch N.J.; Schofield P.; Gierliński M.; Cole C.; Sherstnev A.; Singh V.; Wrobel N.; Gharbi K.; Simpson G.G.; Owen-Hughes T.; Blaxter M.; Barton G.J. How many biological replicates are needed in an RNA-seq experiment and which differential expression tool should you use? 2016 RNA 22:839-851

RNA-seq is now the technology of choice for genome-wide differential gene expression experiments, but it is not clear how many biological replicates are needed to ensure valid biological interpretation of the results or which statistical tools are best for analyzing the data. An RNA-seq experiment with 48 biological replicates in each of two conditions was performed to answer these questions and provide guidelines for experimental design. With three biological replicates, nine of the 11 tools evaluated found only 20%–40% of the significantly differentially expressed (SDE) genes identified with the full set of 42 clean replicates. This rises to 85% for the subset of SDE genes changing in expression by more than fourfold. To achieve 85% for all SDE genes regardless of fold change requires more than 20 biological replicates. The same nine tools successfully control their false discovery rate at ≲5% for all numbers of replicates, while the remaining two tools fail to control their FDR adequately, particularly for low numbers of replicates. For future RNA-seq experiments, these results suggest that at least six biological replicates should be used, rising to at least 12 when it is important to identify SDE genes for all fold changes. If fewer than 12 replicates are used, a superior combination of true positive and false positive performances makes edgeR and DESeq2 the leading tools. For higher replicate numbers, minimizing false positives is more important and DESeq marginally outperforms the other tools.

Singh R.; Lawal H.M.; Schilde C.; Gloeckner G.; Barton G.J.; Schaap P.; Cole C. Improved Annotation with de novo Transcriptome Assembly in Four Social Amoeba Species 2016 bioRxiv 054536

Background: Annotation of gene models and transcripts is a fundamental step in genome sequencing projects. Often this is performed with automated prediction pipelines, which can miss complex and atypical genes or transcripts. RNA-seq data can aid the annotation with empirical data. Here we present de novo transcriptome assemblies generated from RNA-seq data in four Dictyostelid species: D. discoideum, P. pallidum, D. fasciculatum and D. lacteum. The assemblies were incorporated with existing gene models to determine corrections and improvement on a whole-genome scale. This is the first time this has been performed in these eukaryotic species. Results: An initial de novo transcriptome assembly was generated by Trinity for each species and then refined with Program to Assemble Spliced Alignments (PASA). The completeness and quality were assessed with the Core Eukaryotic Genes Mapping Approach (CEGMA) and Transrate tools at each stage of the assemblies. The final datasets of 11,315-12,849 transcripts contained 5,610-7,712 updates and corrections to 50% of existing gene models including changes to hundreds or thousands of protein products. Putative novel genes are also identified and alternative splice isoforms were observed for the first time in P. pallidum, D. lacteum and D. fasciculatum. Conclusions: In taking a whole transcriptome approach to genome annotation with empirical data we have been able to enrich the annotations of four existing genome sequencing projects. In doing so we have identified updates to the majority of the gene annotations across all four species under study and found putative novel genes and transcripts which could be worthy for follow-up. The new transcriptome data we present here will be a valuable resource for genome curators in the Dictyostelia and we propose this effective methodology for use in other genome annotation projects.

Ward J.; Cole C.; Febrer M.; Barton G. AlmostSignificant: Simplifying quality control of high-throughput sequencing data. 2016 bioRxiv 053702

Motivation: The current generation of DNA sequencing technologies produce a large amount of data quickly. All of these data need to pass some form of quality control processing and checking before they can be used for any analysis. The large number of samples that are run through Illumina sequencing machines makes the process of quality control an onerous and time-consuming task that requires multiple pieces of information from several sources. Results: AlmostSignificant is an open-source platform for aggregating multiple sources of quality metrics as well as meta-data associated with DNA sequencing runs from Illumina sequencing machines. AlmostSignificant is a graphical platform to streamline the quality control of DNA sequencing data, to collect and store these data for future reference and to collect extra meta-data associated with the sequencing runs to check for errors and monitor the volume of data produced by the associated machines. AlmostSignificant has been used to track the quality of over 80 sequencing runs covering over 2500 samples produced over the last three years. Availability: The code and documentation for AlmostSignificant is freely available at https://github.com/bartongroup/AlmostSignificant.

Wiechens N.; Singh V.; Gkikopoulos T.; Schofield P.; Rocha S.; Owen-Hughes T. The Chromatin Remodelling Enzymes SNF2H and SNF2L Position Nucleosomes adjacent to CTCF and Other Transcription Factors 2016 PLOS Genet 12:e1005940

Author Summary CTCF is a transcriptional regulator acting as an insulator element interfering with enhancer function and as a boundary between chromatin domains. CTCF has been shown to organise an exquisite array of phased nucleosomes flanking its binding sites. Here we identified SNF2H as the enzyme primarily responsible for organising the extended arrays of nucleosomes adjacent to CTCF sites. We find that SNF2H acts to maintain the occupancy of CTCF at its binding sites, but does not act as a general loading factor for CTCF’s binding partner cohesin. SNF2H’s action at CTCF sites is functionally important as overlapping cohorts of genes are affected by depletion of CTCF or SNF2H. Other transcription factors also organise nucleosomes and we find that the SNF2H and the related enzyme SNF2L contribute to organising nucleosomes at many of these sites.

Gierlinski M. Understanding Statistical Error: A Primer for Biologists 2016 Wiley-Blackwell ISBN:978-1-119-10691-3

This accessible introductory textbook provides a straightforward, practical explanation of how statistical analysis and error measurements should be applied in biological research. Understanding Statistical Error - A Primer for Biologists: * Introduces the essential topic of error analysis to biologists * Contains mathematics at a level that all biologists can grasp * Presents the formulas required to calculate each confidence interval for use in practice * Is based on a successful series of lectures from the author s established course Assuming no prior knowledge of statistics, this book covers the central topics needed for efficient data analysis, ranging from probability distributions, statistical estimators, confidence intervals, error propagation and uncertainties in linear regression, to advice on how to use error bars in graphs properly. Using simple mathematics, all these topics are carefully explained and illustrated with figures and worked examples. The emphasis throughout is on visual representation and on helping the reader to approach the analysis of experimental data with confidence. This useful guide explains how to evaluate uncertainties of key parameters, such as the mean, median, proportion and correlation coefficient. Crucially, the reader will also learn why confidence intervals are important and how they compare against other measures of uncertainty. Understanding Statistical Error - A Primer for Biologists can be used both by students and researchers to deepen their knowledge and find practical formulae to carry out error analysis calculations. It is a valuable guide for students, experimental biologists and professional researchers in biology, biostatistics, computational biology, cell and molecular biology, ecology, biological chemistry, drug discovery, biophysics, as well as wider subjects within life sciences and any field where error analysis is required.

Allen E.H.A.; Courtney D.G.; Atkinson S.D.; Moore J.E.; Mairs L.; Poulsen E.T.; Schiroli D.; Maurizi E.; Cole C.; Hickerson R.P.; James J.; Murgatroyd H.; Smith F.J.D.; MacEwen C.; Enghild J.J.; Nesbit M.A.; Pedrioli D.M.L.; McLean W.H.I.; Moore C.B.T. Keratin 12 missense mutation induces the unfolded protein response and apoptosis in Meesmann epithelial corneal dystrophy 2016 Human Molecular Genetics ddw001

Meesmann epithelial corneal dystrophy (MECD) is a rare autosomal dominant disorder caused by dominant-negative mutations within the KRT3 or KRT12 genes, which encode the cytoskeletal proteins keratin K3 and K12, respectively. To investigate the pathomechanism of this disease we generated and phenotypically characterized a novel knock-in humanised mouse model carrying the severe, MECD-associated, K12-Leu132Pro mutation. Although no overt changes in corneal opacity were detected by slit-lamp examination, the corneas of homozygous mutant mice exhibited histological and ultrastructural epithelial cell fragility phenotypes. An altered keratin expression profile was observed in the cornea of mutant mice, confirmed by Western blot, RNA-seq and qRT-PCR. Mass spectrometry and immunohistochemistry demonstrated a similarly altered keratin profile in corneal tissue from a K12-Leu132Pro MECD patient. The K12-Leu132Pro mutation results in cytoplasmic keratin aggregates. RNA-seq analysis revealed increased chaperone gene expression, while apoptotic unfolded protein response (UPR) markers, CHOP and Caspase 12, were also increased in the MECD mice. Corneal epithelial cell apoptosis was increased 17–fold in the mutant cornea, compared to the wild-type (p0.001). This elevation of UPR marker expression was also observed in the human MECD cornea. This is the first reporting of a mouse model for MECD that recapitulates the human disease, and is a valuable resource in understanding the pathomechanism of the disease. While the most severe phenotype is observed in the homozygous mice, this model will still provide a test-bed for therapies not only for corneal dystrophies but also for other keratinopathies caused by similar mutations.

Wilson N.J.; Cole C.; Milstone L.M.; Kiszewski A.E.; Hansen C.D.; O'Toole E.A.; Schwartz M.E.; McLean W.H.I.; Smith F.J.D. Expanding the Phenotypic Spectrum of Olmsted Syndrome 2015 Journal of Investigative Dermatology 135:2879-2883

McAleer M.A.; Pohler E.; Smith F.J.D.; Wilson N.J.; Cole C.; MacGowan S.; Koetsier J.L.; Godsel L.M.; Harmon R.M.; Gruber R.; Crumrine D.; Elias P.M.; McDermott M.; Butler K.; Broderick A.; Sarig O.; Sprecher E.; Green K.J.; McLean W.H.I.; Irvine A.D. Severe dermatitis, multiple allergies, and metabolic wasting syndrome caused by a novel mutation in the N-terminal plakin domain of desmoplakin 2015 Journal of Allergy and Clinical Immunology 136:1268-1276

Background Severe dermatitis, multiple allergies, and metabolic wasting (SAM) syndrome is a recently recognized syndrome caused by mutations in the desmoglein 1 gene (DSG1). To date, only 3 families have been reported. Objective We studied a new case of SAM syndrome known to have no mutations in DSG1 to detail the clinical, histopathologic, immunofluorescent, and ultrastructural phenotype and to identify the underlying molecular mechanisms in this rare genodermatosis. Methods Histopathologic, electron microscopy, and immunofluorescent studies were performed. Whole-exome sequencing data were interrogated for mutations in desmosomal and other skin structural genes, followed by Sanger sequencing of candidate genes in the patient and his parents. Results No mutations were identified in DSG1; however, a novel de novo heterozygous missense c.1757AC mutation in the desmoplakin gene (DSP) was identified in the patient, predicting the amino acid substitution p.His586Pro in the desmoplakin polypeptide. Conclusions SAM syndrome can be caused by mutations in both DSG1 and DSP. Knowledge of this genetic heterogeneity is important for both analysis of patients and genetic counseling of families. This condition and these observations reinforce the importance of heritable skin barrier defects, in this case desmosomal proteins, in the pathogenesis of atopic disease.

Pohler E.; Cunningham F.; Sandilands A.; Cole C.; Digby S.; McMillan J.; Aristodemou S.; McGrath J.; Smith F.; McLean W.; Munro C.; Zamiri M. Novel autosomal dominant mutation in loricrin presenting as prominent ichthyosis 2015 British Journal of Dermatology 173:1291-1294

Mercier S.; Küry S.; Salort-Campana E.; Magot A.; Agbim U.; Besnard T.; Bodak N.; Bou-Hanna C.; Bréhéret F.; Brunelle P.; Caillon F.; Chabrol B.; Cormier-Daire V.; David A.; Eymard B.; Faivre L.; Figarella-Branger D.; Fleurence E.; Ganapathi M.; Gherardi R.; Goldenberg A.; Hamel A.; Igual J.; Irvine A.D.; Israël-Biet D.; Kannengiesser C.; Laboisse C.; Le Caignec C.; Mahé J.; Mallet S.; MacGowan S.; McAleer M.A.; McLean I.; Méni C.; Munnich A.; Mussini J.; Nagy P.L.; Odel J.; O’Regan G.M.; Péréon Y.; Perrier J.; Piard J.; Puzenat E.; Sampson J.B.; Smith F.; Soufir N.; Tanji K.; Thauvin C.; Ulane C.; Watson R.M.; Khumalo N.P.; Mayosi B.M.; Barbarot S.; Bézieau S. Expanding the clinical spectrum of hereditary fibrosing poikiloderma with tendon contractures, myopathy and pulmonary fibrosis due to FAM111B mutations 2015 Orphanet Journal of Rare Diseases 10:135

Hereditary Fibrosing Poikiloderma (HFP) with tendon contractures, myopathy and pulmonary fibrosis (POIKTMP [MIM 615704]) is a very recently described entity of syndromic inherited poikiloderma. Previously by using whole exome sequencing in five families, we identified the causative gene, FAM111B (NM\_198947.3), the function of which is still unknown. Our objective in this study was to better define the specific features of POIKTMP through a larger series of patients.

Guo M.; Härtlova A.; Dill B.D.; Prescott A.R.; Gierliński M.; Trost M. High-resolution quantitative proteome analysis reveals substantial differences between phagosomes of RAW 264.7 and bone marrow derived macrophages 2015 PROTEOMICS 15:3169-3174

Macrophages are important immune cells operating at the forefront of innate immunity by taking up foreign particles and microbes through phagocytosis. The RAW 264.7 cell line is commonly used for experiments in the macrophage and phagocytosis field. However, little is known how its functions compare to primary macrophages. Here, we have performed an in-depth proteomics characterization of phagosomes from RAW 264.7 and bone marrow derived macrophages by quantifying more than 2500 phagosomal proteins. Our data indicate that there are significant differences for a large number of proteins including important receptors such as mannose receptor 1 and Siglec-1. Moreover, bone marrow derived macrophages phagosomes mature considerably faster by fusion with endosomes and the lysosome which we validated using fluorogenic phagocytic assays. We provide a valuable resource for researcher in the field and recommend careful use of the RAW 264.7 cell line when studying phagosome functions. All MS data have been deposited in the ProteomeXchange with identifier PXD001293 (http:/roteomecentral.proteomexchange.org/dataset/PXD001293).

Seifert A.; Schofield P.; Barton G.J.; Hay R.T. Proteotoxic stress reprograms the chromatin landscape of SUMO modification 2015 Sci. Signal. 8:rs7-rs7

SUMO-2 to the rescue In response to various proteotoxic stresses, such as heat shock or accumulation of misfolded proteins, cells activate protective mechanisms that depend on the production and function of heat shock proteins (HSPs) and other protein chaperones. Seifert et al. found that in response to various proteotoxic stresses, chromatin-associated proteins at sites of active genes were conjugated to SUMO-2 (small ubiquitin-like modifier 2) proteins. Rather than stimulate or repress target gene expression, conjugation with SUMO-2 enhanced the stability of protein complexes at transcription start sites, thus enabling cells to first withstand the initial effects of stress and express genes encoding HSPs and the other factors required for cell survival. The small ubiquitin-like modifier 2 (SUMO-2) is required for survival when cells are exposed to treatments that induce proteotoxic stress by causing the accumulation of misfolded proteins. Exposure of cells to heat shock or other forms of proteotoxic stress induces the conjugation of SUMO-2 to proteins in the nucleus. We investigated the chromatin landscape of SUMO-2 modifications in response to heat stress. Through chromatin immunoprecipitation assays coupled to high-throughput DNA sequencing and mRNA sequencing, we showed that in response to heat shock, SUMO-2 accumulated at nucleosome-depleted, active DNA regulatory elements, which represented binding sites for large protein complexes and were predominantly associated with active genes. However, SUMO did not act as a direct transcriptional repressor or activator of these genes during heat shock. Instead, integration of our results with published proteomics data on heat shock–induced SUMO-2 substrates supports a model in which the conjugation of SUMO-2 to proteins acts as an acute stress response that is required for the stability of protein complexes involved in gene expression and posttranscriptional modification of mRNA. We showed that the conjugation of SUMO-2 to chromatin-associated proteins is an integral component of the proteotoxic stress response, and propose that SUMO-2 fulfills its essential role in cell survival by contributing to the maintenance of protein complex homeostasis. Cells use SUMO-2 to stabilize chromatin-associated proteins and cope with stress. Cells use SUMO-2 to stabilize chromatin-associated proteins and cope with stress.

Gierliński M.; Cole C.; Schofield P.; Schurch N.J.; Sherstnev A.; Singh V.; Wrobel N.; Gharbi K.; Simpson G.; Owen-Hughes T.; Blaxter M.; Barton G.J. Statistical models for RNA-seq data derived from a two-condition 48-replicate experiment 2015 Bioinformatics btv425

Motivation: High-throughput RNA sequencing (RNA-seq) is now the standard method to determine differential gene expression. Identifying differentially expressed genes crucially depends on estimates of read count variability. These estimates are typically based on statistical models such as the negative binomial distribution, which is employed by the tools edgeR, DESeq and cuffdiff. Until now, the validity of these models has usually been tested on either low-replicate RNA-seq data or simulations. Results: A 48-replicate RNA-seq experiment in yeast was performed and data tested against theoretical models. The observed gene read counts were consistent with both log-normal and negative binomial distributions, while the mean-variance relation followed the line of constant dispersion parameter of ~0.01. The high-replicate data also allowed for strict quality control and screening of “bad” replicates, which can drastically affect the gene read-count distribution. Availability: RNA-seq data have been submitted to ENA archive with project ID PRJEB5348. Contact: g.j.barton@dundee.ac.uk

Lin Z.; Zhao J.; Nitoiu D.; Scott C.A.; Plagnol V.; Smith F.J.D.; Wilson N.J.; Cole C.; Schwartz M.E.; McLean W.H.I.; Wang H.; Feng C.; Duo L.; Zhou E.Y.; Ren Y.; Dai L.; Chen Y.; Zhang J.; Xu X.; O’Toole E.A.; Kelsell D.P.; Yang Y. Loss-of-Function Mutations in CAST Cause Peeling Skin, Leukonychia, Acral Punctate Keratoses, Cheilitis, and Knuckle Pads 2015 The American Journal of Human Genetics 96:440-447

Calpastatin is an endogenous specific inhibitor of calpain, a calcium-dependent cysteine protease. Here we show that loss-of-function mutations in calpastatin (CAST) are the genetic causes of an autosomal-recessive condition characterized by generalized peeling skin, leukonychia, acral punctate keratoses, cheilitis, and knuckle pads, which we propose to be given the acronym PLACK syndrome. In affected individuals with PLACK syndrome from three families of different ethnicities, we identified homozygous mutations (c.607dup, c.424AT, and c.1750delG) in CAST, all of which were predicted to encode truncated proteins (p.Ile203Asnfs∗8, p.Lys142∗, and p.Val584Trpfs∗37). Immunohistochemistry shows that staining of calpastatin is reduced in skin from affected individuals. Transmission electron microscopy revealed widening of intercellular spaces with chromatin condensation and margination in the upper stratum spinosum in lesional skin, suggesting impaired intercellular adhesion as well as keratinocyte apoptosis. A significant increase of apoptotic keratinocytes was also observed in TUNEL assays. In vitro studies utilizing siRNA-mediated CAST knockdown revealed a role for calpastatin in keratinocyte adhesion. In summary, we describe PLACK syndrome, as a clinical entity of defective epidermal adhesion, caused by loss-of-function mutations in CAST.

Dill B.D.; Gierlinski M.; Härtlova A.; Arandilla A.G.; Guo M.; Clarke R.G.; Trost M. Quantitative Proteome Analysis of Temporally Resolved Phagosomes Following Uptake Via Key Phagocytic Receptors 2015 Molecular \& Cellular Proteomics 14:1334-1349

Macrophages operate at the forefront of innate immunity and their discrimination of foreign versus “self” particles is critical for a number of responses including efficient pathogen killing, antigen presentation, and cytokine induction. In order to efficiently destroy the particles and detect potential threats, macrophages express an array of receptors to sense and phagocytose prey particles. In this study, we accurately quantified a proteomic time-course of isolated phagosomes from murine bone marrow-derived macrophages induced by particles conjugated to seven different ligands representing pathogen-associated molecular patterns, immune opsonins or apoptotic cell markers. We identified a clear functional differentiation over the three timepoints and detected subtle differences between certain ligand-phagosomes, indicating that triggering of receptors through a single ligand type has mild, but distinct, effects on phagosome proteome and function. Moreover, our data shows that uptake of phosphatidylserine-coated beads induces an active repression of NF-κB immune responses upon Toll-like receptor (TLR)-activation by recruitment of anti-inflammatory regulators to the phagosome. This data shows for the first time a systematic time-course analysis of bone marrow-derived macrophages phagosomes and how phagosome fate is regulated by the receptors triggered for phagocytosis.

Gierliński M.; Cole C.; Schofield P.; Schurch N.J.; Sherstnev A.; Singh V.; Wrobel N.; Gharbi K.; Simpson G.; Owen-Hughes T.; Blaxter M.; Barton G.J. Statistical models for RNA-seq data derived from a two-condition 48-replicate experiment 2015 arXiv:1505.00588 [q-bio]

High-throughput RNA sequencing (RNA-seq) is now the standard method to determine differential gene expression. Identifying differentially expressed genes crucially depends on estimates of read count variability. These estimates are typically based on statistical models such as the negative binomial distribution, which is employed by the tools edgeR, DESeq and cuffdiff. Until now, the validity of these models has usually been tested on either low-replicate RNA-seq data or simulations. A 48-replicate RNA-seq experiment in yeast was performed and data tested against theoretical models. The observed gene read counts were consistent with both log-normal and negative binomial distributions, while the mean-variance relation followed the line of constant dispersion parameter of ~0.01. The high-replicate data also allowed for strict quality control and screening of bad replicates, which can drastically affect the gene read-count distribution. RNA-seq data have been submitted to ENA archive with project ID PRJEB5348.

Schurch N.J.; Schofield P.; Gierliński M.; Cole C.; Sherstnev A.; Singh V.; Wrobel N.; Gharbi K.; Simpson G.G.; Owen-Hughes T.; Blaxter M.; Barton G.J. Evaluation of tools for differential gene expression analysis by RNA-seq on a 48 biological replicate experiment 2015 arXiv:1505.02017 [q-bio]

An RNA-seq experiment with 48 biological replicates in each of 2 conditions was performed to determine the number of biological replicates (\$n\_r\$) required, and to identify the most effective statistical analysis tools for identifying differential gene expression (DGE). When \$n\_r=3\$, seven of the nine tools evaluated give true positive rates (TPR) of only 20 to 40 percent. For high fold-change genes (\$textbarlog\_\2\(FC)textbartextbackslashgt2\$) the TPR is \$textbackslashgt85\$ percent. Two tools performed poorly; over- or under-predicting the number of differentially expressed genes. Increasing replication gives a large increase in TPR when considering all DE genes but only a small increase for high fold-change genes. Achieving a TPR \$textbackslashgt85\$% across all fold-changes requires \$n\_rtextbackslashgt20\$. For future RNA-seq experiments these results suggest \$n\_rtextbackslashgt6\$, rising to \$n\_rtextbackslashgt12\$ when identifying DGE irrespective of fold-change is important. For \$n\_rtextbackslashgt12\$, superior TPR makes edgeR the leading tool tested. For \$n\_r textbackslashge12\$, minimizing false positives is more important and DESeq outperforms the other tools.

Drozdetskiy A.; Cole C.; Procter J.; Barton G.J. JPred4: a protein secondary structure prediction server 2015 Nucleic Acids Research gkv332

JPred4 (http://www.compbio.dundee.ac.uk/jpred4) is the latest version of the popular JPred protein secondary structure prediction server which provides predictions by the JNet algorithm, one of the most accurate methods for secondary structure prediction. In addition to protein secondary structure, JPred also makes predictions of solvent accessibility and coiled-coil regions. The JPred service runs up to 94 000 jobs per month and has carried out over 1.5 million predictions in total for users in 179 countries. The JPred4 web server has been re-implemented in the Bootstrap framework and JavaScript to improve its design, usability and accessibility from mobile devices. JPred4 features higher accuracy, with a blind three-state (α-helix, β-strand and coil) secondary structure prediction accuracy of 82.0% while solvent accessibility prediction accuracy has been raised to 90% for residues 5% accessible. Reporting of results is enhanced both on the website and through the optional email summaries and batch submission results. Predictions are now presented in SVG format with options to view full multiple sequence alignments with and without gaps and insertions. Finally, the help-pages have been updated and tool-tips added as well as step-by-step tutorials.

Madeira F.; Tinti M.; Murugesan G.; Berrett E.; Stafford M.; Toth R.; Cole C.; MacKintosh C.; Barton G.J. 14-3-3-Pred: Improved methods to predict 14-3-3-binding phosphopeptides 2015 Bioinformatics btv133

Motivation: The 14-3-3 family of phosphoprotein-binding proteins regulate many cellular processes by docking onto pairs of phosphorylated Ser and Thr residues in a constellation of intracellular targets. Therefore, there is a pressing need to develop new prediction methods that use an updated set of 14-3-3-binding motifs for the identification of new 14-3-3 targets, and to prioritize the downstream analysis of 2000 potential interactors identified in high-throughput experiments. Results: Here, a comprehensive set of 14-3-3-binding targets from the literature was used to develop 14-3-3-binding phosphosite predictors. Position-specific scoring matrix (PSSM), support vector machines (SVM), and artificial neural network (ANN) classification methods were trained to discriminate experimentally-determined 14-3-3-binding motifs from non-binding phosphopeptides. ANN, PSSM and SVM methods showed best performance for a motif window spanning from -6 to +4 around the binding phosphosite, achieving Matthews correlation coefficient of up to 0.60. Blind prediction showed that all three methods outperform two popular 14-3-3-binding site predictors, Scansite and ELM. The new methods were used for prediction of 14-3-3-binding phosphosites in the human proteome. Experimental analysis of high-scoring predictions in the FAM122A and FAM122B proteins confirms the predictions and suggests the new 14-3-3-predictors will be generally useful. Availability: A standalone prediction webserver is available at http://www.compbio.dundee.ac.uk/1433pred. Human candidate 14-3-3-binding phosphosites were integrated in ANIA: ANnotation and Integrated Analysis of the 14-3-3 interactome database. Contact: cmackintosh@dundee.ac.uk and gjbarton@dundee.ac.uk Supplementary information: Supplementary data are available at Bioinformatics online.

Sternberg J.M.; Gierliński M.; Biéler S.; Ferguson M.A.J.; Ndung'u J.M. Evaluation of the diagnostic accuracy of prototype rapid tests for human African trypanosomiasis 2014 PLoS neglected tropical diseases 8:e3373

BACKGROUND: Diagnosis of human African trypanosomiasis (HAT) remains a challenge both for active screening, which is critical in control of the disease, and in the point-of-care scenario where early and accurate diagnosis is essential. Recently, the first field deployment of a lateral flow rapid diagnostic test (RDT) for HAT, "SD BIOLINE HAT" has taken place. In this study, we evaluated the performance of "SD BIOLINE HAT" and two new prototype RDTs. METHODOLOGY/PRINCIPAL FINDINGS: The performance of "SD BIOLINE HAT" and 2 prototype RDTs was tested using archived plasma from 250 Trypanosoma brucei gambiense patients, and 250 endemic controls. As well as comparison of the sensitivity and specificity of each device, the performance of individual antigens was assessed and the hypothetical performance of novel antigen combinations extrapolated. Neither of the prototype devices were inferior in sensitivity or specificity to "SD BIOLINE HAT" (sensitivity 0.82±0.01, specificity 0.97±0.01, 95% CI) at the 5% margins, while one of the devices (BBI) had significantly superior sensitivity (0.88±0.03). Analysis of the performance of individual antigens was used to model new antigen combinations to be explored in development of the next generation of HAT RDTs. The modelling showed that an RDT using two recombinant antigens (rLiTat1.5 and rISG65) would give a performance similar to the best devices in this study, and would also offer the most robust performance under deteriorating field conditions. CONCLUSIONS/SIGNIFICANCE: Both "SD BIOLINE HAT" and the prototype devices performed comparably well to one another and also to the published performance range of the card agglutination test for trypanosomiasis in sensitivity and specificity. The performance of individual antigens enabled us to predict that an all-recombinant antigen RDT can be developed with an accuracy equivalent to " SD BIOLINE HAT." Such an RDT would have advantages in simplified manufacture, lower unit cost and assured reproducibility.

Speakman C.M.; Domke T.C.; Wongpaiboonwattana W.; Sanders K.; Mudaliar M.; Aalten D.M.; Barton G.J.; Stavridis M.P. Elevated O-GlcNAc Levels Activate Epigenetically Repressed Genes and Delay Mouse ESC Differentiation Without Affecting Naïve to Primed Cell Transition 2014 STEM CELLS 32:2605-2615

The differentiation of mouse embryonic stem cells (ESCs) is controlled by the interaction of multiple signaling pathways, typically mediated by post-translational protein modifications. The addition of O-linked N-acetylglucosamine (O-GlcNAc) to serine and threonine residues of nuclear and cytoplasmic proteins is one such modification (O-GlcNAcylation), whose function in ESCs is only now beginning to be elucidated. Here, we demonstrate that the specific inhibition of O-GlcNAc hydrolase (Oga) causes increased levels of protein O-GlcNAcylation and impairs differentiation of mouse ESCs both in serum-free monolayer and in embryoid bodies (EBs). Use of reporter cell lines demonstrates that Oga inhibition leads to a reduction in the number of Sox1-expressing neural progenitors generated following induction of neural differentiation as well as maintained expression of the ESC marker Oct4 (Pou5f1). In EBs, expression of mesodermal and endodermal markers is also delayed. However, the transition of naïve cells to primed pluripotency indicated by Rex1 (Zfp42), Nanog, Esrrb, and Dppa3 downregulation and Fgf5 upregulation remains unchanged. Finally, we demonstrate that increased O-GlcNAcylation results in upregulation of genes normally epigenetically silenced in ESCs, supporting the emerging role for this protein modification in the regulation of histone modifications and DNA methylation. Stem Cells 2014;32:2605–2615

Olivera-Martinez I.; Schurch N.; Li R.A.; Song J.; Halley P.A.; Das R.M.; Burt D.W.; Barton G.J.; Storey K.G. Major transcriptome re-organisation and abrupt changes in signalling, cell cycle and chromatin regulation at neural differentiation in vivo 2014 Development (Cambridge, England) 141:3266-3276

Here, we exploit the spatial separation of temporal events of neural differentiation in the elongating chick body axis to provide the first analysis of transcriptome change in progressively more differentiated neural cell populations in vivo. Microarray data, validated against direct RNA sequencing, identified: (1) a gene cohort characteristic of the multi-potent stem zone epiblast, which contains neuro-mesodermal progenitors that progressively generate the spinal cord; (2) a major transcriptome re-organisation as cells then adopt a neural fate; and (3) increasing diversity as neural patterning and neuron production begin. Focussing on the transition from multi-potent to neural state cells, we capture changes in major signalling pathways, uncover novel Wnt and Notch signalling dynamics, and implicate new pathways (mevalonate pathway/steroid biogenesis and TGFβ). This analysis further predicts changes in cellular processes, cell cycle, RNA-processing and protein turnover as cells acquire neural fate. We show that these changes are conserved across species and provide biological evidence for reduced proteasome efficiency and a novel lengthening of S phase. This latter step may provide time for epigenetic events to mediate large-scale transcriptome re-organisation; consistent with this, we uncover simultaneous downregulation of major chromatin modifiers as the neural programme is established. We further demonstrate that transcription of one such gene, HDAC1, is dependent on FGF signalling, making a novel link between signals that control neural differentiation and transcription of a core regulator of chromatin organisation. Our work implicates new signalling pathways and dynamics, cellular processes and epigenetic modifiers in neural differentiation in vivo, identifying multiple new potential cellular and molecular mechanisms that direct differentiation.

Cole C.; Kroboth K.; Schurch N.J.; Sandilands A.; Sherstnev A.; O'Regan G.M.; Watson R.M.; Irwin McLean W.H.; Barton G.J.; Irvine A.D.; Brown S.J. Filaggrin-stratified transcriptomic analysis of pediatric skin identifies mechanistic pathways in patients with atopic dermatitis 2014 Journal of Allergy and Clinical Immunology 134:82-91

Background Atopic dermatitis (AD; eczema) is characterized by a widespread abnormality in cutaneous barrier function and propensity to inflammation. Filaggrin is a multifunctional protein and plays a key role in skin barrier formation. Loss-of-function mutations in the gene encoding filaggrin (FLG) are a highly significant risk factor for atopic disease, but the molecular mechanisms leading to dermatitis remain unclear. Objective We sought to interrogate tissue-specific variations in the expressed genome in the skin of children with AD and to investigate underlying pathomechanisms in atopic skin. Methods We applied single-molecule direct RNA sequencing to analyze the whole transcriptome using minimal tissue samples. Uninvolved skin biopsy specimens from 26 pediatric patients with AD were compared with site-matched samples from 10 nonatopic teenage control subjects. Cases and control subjects were screened for FLG genotype to stratify the data set. Results Two thousand four hundred thirty differentially expressed genes (false discovery rate, P \< .05) were identified, of which 211 were significantly upregulated and 490 downregulated by greater than 2-fold. Gene ontology terms for “extracellular space” and “defense response” were enriched, whereas “lipid metabolic processes” were downregulated. The subset of FLG wild-type cases showed dysregulation of genes involved with lipid metabolism, whereas filaggrin haploinsufficiency affected global gene expression and was characterized by a type 1 interferon–mediated stress response. Conclusion These analyses demonstrate the importance of extracellular space and lipid metabolism in atopic skin pathology independent of FLG genotype, whereas an aberrant defense response is seen in subjects with FLG mutations. Genotype stratification of the large data set has facilitated functional interpretation and might guide future therapy development.

Schurch N.J.; Cole C.; Sherstnev A.; Song J.; Duc C.; Storey K.G.; McLean W.H.I.; Brown S.J.; Simpson G.G.; Barton G.J. Improved Annotation of 3′ Untranslated Regions and Complex Loci by Combination of Strand-Specific Direct RNA Sequencing, RNA-Seq and ESTs 2014 PLoS ONE 9:e94270

The reference annotations made for a genome sequence provide the framework for all subsequent analyses of the genome. Correct and complete annotation in addition to the underlying genomic sequence is particularly important when interpreting the results of RNA-seq experiments where short sequence reads are mapped against the genome and assigned to genes according to the annotation. Inconsistencies in annotations between the reference and the experimental system can lead to incorrect interpretation of the effect on RNA expression of an experimental treatment or mutation in the system under study. Until recently, the genome-wide annotation of 3′ untranslated regions received less attention than coding regions and the delineation of intron/exon boundaries. In this paper, data produced for samples in Human, Chicken and A. thaliana by the novel single-molecule, strand-specific, Direct RNA Sequencing technology from Helicos Biosciences which locates 3′ polyadenylation sites to within +/− 2 nt, were combined with archival EST and RNA-Seq data. Nine examples are illustrated where this combination of data allowed: (1) gene and 3′ UTR re-annotation (including extension of one 3′ UTR by 5.9 kb); (2) disentangling of gene expression in complex regions; (3) clearer interpretation of small RNA expression and (4) identification of novel genes. While the specific examples displayed here may become obsolete as genome sequences and their annotations are refined, the principles laid out in this paper will be of general use both to those annotating genomes and those seeking to interpret existing publically available annotations in the context of their own experimental data.

Atrih A.; Mudaliar M.A.V.; Zakikhani P.; Lamont D.J.; Huang J.T.; Bray S.E.; Barton G.; Fleming S.; Nabi G. Quantitative proteomics in resected renal cancer tissue for biomarker discovery and profiling 2014 British Journal of Cancer 110:1622-1633

Saunders S.P.; Goh C.S.M.; Brown S.J.; Palmer C.N.A.; Porter R.M.; Cole C.; Campbell L.E.; Gierlinski M.; Barton G.J.; Schneider G.; Balmain A.; Prescott A.R.; Weidinger S.; Baurecht H.; Kabesch M.; Gieger C.; Lee Y.; Tavendale R.; Mukhopadhyay S.; Turner S.W.; Madhok V.B.; Sullivan F.M.; Relton C.; Burn J.; Meggitt S.; Smith C.H.; Allen M.A.; Barker J.N.W.N.; Reynolds N.J.; Cordell H.J.; Irvine A.D.; McLean W.H.I.; Sandilands A.; Fallon P.G. Tmem79/Matt is the matted mouse gene and is a predisposing gene for atopic dermatitis in human subjects 2013 Journal of Allergy and Clinical Immunology 132:1121-1129

Background Atopic dermatitis (AD) is a major inflammatory condition of the skin caused by inherited skin barrier deficiency, with mutations in the filaggrin gene predisposing to development of AD. Support for barrier deficiency initiating AD came from flaky tail mice, which have a frameshift mutation in Flg and also carry an unknown gene, matted, causing a matted hair phenotype. Objective We sought to identify the matted mutant gene in mice and further define whether mutations in the human gene were associated with AD. Methods A mouse genetics approach was used to separate the matted and Flg mutations to produce congenic single-mutant strains for genetic and immunologic analysis. Next-generation sequencing was used to identify the matted gene. Five independently recruited AD case collections were analyzed to define associations between single nucleotide polymorphisms (SNPs) in the human gene and AD. Results The matted phenotype in flaky tail mice is due to a mutation in the Tmem79/Matt gene, with no expression of the encoded protein mattrin in the skin of mutant mice. Mattft mice spontaneously have dermatitis and atopy caused by a defective skin barrier, with mutant mice having systemic sensitization after cutaneous challenge with house dust mite allergens. Meta-analysis of 4,245 AD cases and 10,558 population-matched control subjects showed that a missense SNP, rs6694514, in the human MATT gene has a small but significant association with AD. Conclusion In mice mutations in Matt cause a defective skin barrier and spontaneous dermatitis and atopy. A common SNP in MATT has an association with AD in human subjects. Atopic dermatitis (AD) is the most common diagnosis in dermatology, affecting approximately 1 in 5 children in the developed world,1 and is frequently associated with atopic asthma and a wide range of allergies.2 AD is a highly heritable complex trait; however, environmental influences also play a role in triggering the atopic diathesis.3 Genome-wide association studies in AD have identified several susceptibility loci4, 5, 6, 7; however, the major and only functionally characterized genetic factor is the filaggrin gene (FLG), which encodes the skin barrier protein filaggrin.3 Prevalent loss-of-function variants in FLG were identified as the cause of the single-gene disorder ichthyosis vulgaris (dry flaky skin).8 Soon thereafter, these variants were shown to be strongly associated with AD,9 with heterozygous odds ratios (ORs) of greater than 7 and homozygous ORs of greater than 150 in case-control studies in which both prevalent and rare variants were analyzed.10 The hypothesis that skin barrier deficiency in the context of FLG mutations is an initiator of AD was confirmed experimentally by using the flaky tail mouse mutant,11 which was shown to carry a frameshift mutation in the murine Flg gene.12 Flaky tail mice have a defective skin barrier, with increased percutaneous transfer of antigens and chemical haptens.12, 13, 14, 15 The ft mutation arose spontaneously in 1958 in the progeny of crosses between heterogeneous stocks of mice with the recessive mutation matted (ma), and these mutations are maintained as a double-mutant (DM) strain known as maft. The matted hair phenotype was used for many years as a surrogate marker for the ft mutation because, remarkably, the ft and ma mutations are closely linked on chromosome 3 in the mouse.16 The DM maft mice have been routinely used for studies of skin barrier–deficient AD in recent years.13, 14, 15, 17 In mice and human subjects the FLG gene resides in the epidermal differentiation complex, a cluster of more than 70 genes encoding proteins involved in skin barrier formation and differentiation of stratified epithelia,18 including those within the hair follicle.18, 19, 20, 21 We suspected the nearby ma gene might also be involved in epithelial barrier function, and in this study we set out to separate this allele from Flgft and identify the causative defect. Back to Article Outline Isolation of the matted mouse strain DM Mattma/maFlgft/ft mice were provided by Dr John P. Sundberg (Jackson Laboratory, Bar Harbor, Me).12 DM mice were crossed with C57BL/6J mice to generate Mattma/+Flgft/+ mice. The Flgft and Mattma mutations were separated and backcrossed to congenic C57BL/6J background in accordance with the breeding strategy outlined (see Fig E1 in this article's Online Repository at www.jacionline.org). C57BL/6J mice were used as wild-type (WT) control animals. B6.CBAGr-ma/J (JAX Mattma/ma) mice were obtained from the Jackson Laboratory. Mice were housed in specific pathogen-free conditions, with irradiated diet and bedding and water ad libitum. All animal experiments were performed in compliance with Irish Department of Health and Children regulations and approved by Trinity College Dublin's BioResources Ethical Review Board. Gene mapping Skin samples were obtained from neonatal mice, and DNA was extracted by using the DNA Purification Kit (Promega, Madison, Wis). Genomic DNA extracted from murine neonatal blood was amplified with the GoTaq Flexi DNA Polymerase kit (Promega). All samples were sequenced by using the ABI 3730 DNA Systems (Applied Biosystems, Foster City, Calif). Mapping primers were used to amplify and sequence murine chromosome 3 (see Table E1, A, in this article's Online Repository at www.jacionline.org). Matted genomic sequences were compared with C57BL/6J for regions of congenicity. Next-generation sequencing and bioinformatics Three replicates from each sample (WT and Mattma/ma) were submitted for next-generation sequencing. The replicates were run multiplexed on an Illumina GAIIx and HiSeq 2000 (Illumina, San Diego, Calif) by using v3 sequencing chemistry and the Roche 454 Titanium workflow (Roche, Mannheim, Germany). For further details on sequencing, bioinformatics, and single nucleotide polymorphism (SNP) and InDel methodologies, see the Methods section in this article's Online Repository at www.jacionline.org. Analysis and identification of murine Tmem79/Matt gene Each of the 4 exons was amplified individually by using PCR with the following conditions for all exons: 1 cycle of 94°C for 5 minutes; 35 cycles of 94°C for 30 seconds, 54°C for 30 seconds, and 72°C for 1 minute; and a final extension at 72°C for 5 minutes. Semiquantitative RT-PCR Mouse tissue samples were lysed with TissueLyser LT (Qiagen, Hilden, Germany), and RNA was extracted with the RNeasy kit (Qiagen). RNA was reverse transcribed with the ImProm-II Reverse Transcription System (Promega). An intron-spanning amplification was carried out on Tmem79/Matt across exons 3 and 4. Krt14 was used as a loading control. RT-PCR primers used are shown in Table E1, B, in this article's Online Repository. Mouse genotyping The 644-bp PCR product of the third Tmem79/Matt exon was digested with the restriction enzyme CviQI (New England Biolabs, Ipswich, Mass), and digested fragments were separated on agarose gel by using electrophoresis. Primers used are shown in Table E1, A. The Flgft mutation was genotyped, as previously described.12 Immunoblotting The epidermis was separated from the dermis of neonatal mice after immersion in 5 mmol/L EDTA at 50°C for 5 minutes, followed by cooling in ice-cold PBS. The separated epidermis was extracted in urea/Tris buffer containing protease inhibitor cocktail (Halt; Thermo Scientific, Erembodegem, Belguim) by means of homogenization. Protein samples were separated on SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes (Millipore, Temecula, Calif), which were probed with rabbit polyclonal antibodies specific for human mattrin (TMEM79; Novus Biologicals, Littleton, Colo). Primary antibodies were detected by means of incubation with a horseradish peroxidase–conjugated goat anti-rabbit secondary antibody (Dako, Stockport, United Kingdom). Immunolabeled proteins were visualized by using chemiluminescence with the ECL detection system (Millipore). Immunofluorescence and Nile Red staining Dorsal murine sections were obtained from 4-day-old neonatal mice and snap-frozen immediately. All frozen samples were then cryosectioned at 4 to 5 μm and stored at 80°C until use. After drying, sections were fixed in 50% methanol acetone, washed in PBS, and incubated with primary antibodies. Nuclei were stained with 4′-6-diamidino-2-phenylindole (Sigma-Aldrich, St Louis, Mo), and slides were fixed in Hydromount (National Diagnostics, Atlanta, Ga). Primary antibodies used in whole mounts were as follows: polyclonal TMEM79 (TMEM79; Novus Biologicals) and monoclonal CK-13 against cytokeratin 17 (Sigma-Aldrich). Frozen mouse skin sections were stained with Nile Red and 6-diamidino-2-phenylindole. Images were collected with a Zeiss LSM700 confocal microscope (Zeiss, Oberkochen, Germany). Quantitative PCR of human cDNA tissue array Expression of TMEM79/MATT cDNA was quantified across a multiple-tissue panel containing cDNA from 48 different human tissues (Origene Technologies, Rockville, Md). This was done with the TaqMan Gene Expression Assay, C\_25986870\_10 (Life Technologies, Carlsbad, Calif), which spans exons 3 to 4 to result in amplicon length of 65 bp in cDNA sequence. The standard used in this assay was a commercially available plasmid containing the full-length WT TMEM79/MATT (Origene Technologies). A standard curve was calculated by using the formulae provided by Applied Biosystems. Phenotypic scoring The severity of inflammation and AD-like pathology was scored by using the macroscopic diagnostic criteria described for the skin inflammation seen in the Nc/Nga mouse model of AD.22 Briefly, a scoring system (0, none; 1, mild; 2, moderate; and 3, severe) was applied to the symptoms of pruritus, edema, erosion, scaling, and erythema. Pruritus (scratch behavior) was observed for 5 minutes. Total scores for each mouse were calculated from the sum of individual scores. Cutaneous challenge with house dust mite antigen Mice were challenged with house dust mite (HDM) to induce allergic skin sensitization, as previously described.13 Briefly, HDM (Greer Laboratories, Lenoir, NC) was topically applied to intact skin of WT and Mattma mice by using Finn chamber patch tests (SmartPractice, Phoenix, Ariz) and secured with Scanpor tape (Bio-Diagnostics, Worcester, United Kingdom). The regimen involved five 1-week exposures to the allergen patch separated by 2-week intervals. Hair was clipped 24 hours before application of HDM that was prepared in endotoxin-free Dulbecco PBS and vehicle to a concentration of 1 mg/mL. Dulbecco PBS was used as the control. Mice were assessed 24 hours after the final HDM challenge. ELISA Serum levels of HDM-specific IgE, IgG1, and IgG2a were detected by means of direct ELISA. Total serum IgE levels were measured with a sandwich ELISA, according to the manufacturer's instructions (BD PharMingen, San Jose, Calif). Histology Dorsal skin sections were removed and fixed in 10% formal saline. Paraffin-embedded sections were stained with hematoxylin and eosin and examined in blinded fashion by 2 observers independently. To quantify dermal cell numbers, we used a previously described scoring system.12 The total number of cells per high-power field (hpf) of view were counted on 20 hpfs per mouse. For skin sections, an arbitrary histologic scoring system was used to quantify acanthosis and hyperkeratosis. Acanthosis scoring was based on the magnitude of epidermal hyperplasia, and hyperkeratosis scoring was based on the magnitude of stratum corneum thickening, with scores (0, basal; 1, mild; 2, moderate; 3, marked; 4, very marked; and 5, extreme pathology) for each parameter based on measurements per hpf in sections. Measurement of transepidermal water loss A Courage and Khazaka Tewameter TM210 (Enviroderm, Evesham, United Kingdom) was used for measurement of transepidermal water loss (TEWL), which was measured on the dorsal flank of mice 24 hours after hair clipping. TEWL was recorded at an ambient temperature of 19°C to 21°C and humidity of 50% ± 5%. In mice sensitized to HDM, TEWL was measured in PBS- and HDM-treated mice before and after the HDM challenge regimen. Electron microscopy For scanning electron microscopy, hair fibers were removed from mice and fixed in cacodylate-buffered glutaraldehyde. Samples were prepared for scanning electron microscopy by using established procedures11 and examined in a Zeiss Supra scanning electron microscope. Statistical analysis of mouse studies GraphPad Prism software (GraphPad Software, La Jolla, Calif) was used for data analysis. Differences between groups were determined by using the Student t test and 2-way ANOVA. Results are presented as means ± SEMs. Differences, indicated as 2-tailed P values, were considered significant at a P value of less than .05. SNP identification in human subjects Human samples from England, Scotland, and Ireland were obtained with consent from patients and Institutional Ethics Committee approval complying with the principles of the Declaration of Helsinki. Human MATT was sequenced by using primers listed in Table E1, C, in this article's Online Repository at www.jacionline.org. Conditions for all exons except exon 2.2 were as follows: 1 cycle of 94°C for 5 minutes; 35 cycles of 94°C for 30 seconds, 54°C for 30 seconds, and 72°C for 1 minute; and a final extension at 72°C for 5 minutes. An annealing temperature of 58°C was used for exon 2.2. SNP rs6684514 (see Table E2 in this article's Online Repository at www.jacionline.org) was genotyped across the case-control study populations by using the TaqMan allelic discrimination genotyping assay (C\_25986870\_10, Life Technologies Corporation). Case-control studies Five independently recruited AD case collections were compared with ethnically matched population control subjects (see the Methods section in this article's Online Repository). Case definitions are described in the Methods section in this article's Online Repository, and demographic data relating to these AD case collections and control subjects are summarized in Table E3 in this article's Online Repository at www.jacionline.org. Genotype imputation For in silico replication, we used the 2 German AD case-control selections (see Table E3), which had been genotyped on Illumina 300k and Affymetrix 6.0p (Affymetrix, Santa Clara, Calif), respectively, and imputed based on the 1000 Genomes database (phase I integrated variant set release, March 2012). Previous imputation data sets were curated, and samples with a greater than 5% missing rate, excess of heterozygosity (±5 SDs away from the sample mean), and unexpected relatedness (PI\_HAT0.1875, halfway between expected IBD for third- and second-degree relatives) were excluded. Further outliers from the multidimensional scaling of the pairwise IBS matrix were removed. Imputation was carried out with SHAPEIT and IMPUTE2. The SNP rs6684514 investigated in silico had an imputation quality of 0.996 (proper info) and a CR of 0.997. Statistical genetics Case-control comparisons for each case collection and population control subjects were performed by using logistic regression analysis in Stata 12.0 (StataCorp, College Station, Tex). Meta-analysis (under fixed- and random-effects models) of the resulting log ORs and their SEs was performed with the “metan” function in Stata 12.0. Back to Article Outline Identification of Matt as the gene mutation in matted mice To separate the 2 mutations in the DM maft mice, we used the backcross-intercross protocol outlined in Fig E1. Mice with the Flgft mutant allele (Flg c.5303delA) were genotyped, as previously described.12 Progeny from putative heterozygous intercrosses were checked for the recessive matted hair phenotype to identify ma/ma homozygotes. By this means, we separated both alleles. Because SNP analysis showed that the initial DM maft mouse was maintained on a mixed-strain background, we generated single-mutant N12 mice congenic on the C57BL/6J background. By using this backcross-intercross strategy to generate congenic ma/ma homozygous mice (see Fig E1), the recessive disease–causing locus should be ultimately visible as a small area of homozygosity flanked by 2 unequal heterozygous regions (see Fig E2, A, in this article's Online Repository at www.jacionline.org). To map this, we sequenced several short PCR fragments across the region from C57BL/6J congenic ma/ma homozygotes (see Table E1, A) to identify SNPs. By this means, a 1.05-Mb critical interval was defined (Fig 1, A). In parallel, we used epidermal transcriptomic sequencing to compare WT C57BL/6J and congenic ma/ma homozygotes. A nonsense mutation, p.Y280*, in the Tmem79 gene, located within the interval (Fig 1, A), was identified in the RNAseq data (not shown). The mutation in Tmem79 was confirmed by using conventional sequencing of genomic PCR products (Fig 1, B) and restriction digests (see Fig E2, B). This mutant transcript was expressed at approximately 30% of the WT levels in ma/ma epidermis (Fig 1, C). By using a commercially available antibody against the C-terminus of the encoded protein, a single band of approximately 49 kDa was seen in epidermal extracts from WT and congenic Flgft/ft mice, with the protein absent in congenic ma/ma and DM mice (Fig 1, D), indicating the presence of a truncated protein or an absence of protein in these mice. Interestingly, epidermal extracts from Flgft/ft mice had increased expression of protein relative to WT extracts. Tmem79 was renamed Matt, encoding the protein mattrin; similarly, the human ortholog was renamed MATT (approved by the Genome Nomenclature Committee). By convention, after identification of the gene, the ma mutant allele was redesignated as Mattma. Mattrin is expressed in mouse and human skin Matt is a compact 5-exon gene on mouse 3qF1 spanning 5781 bp of genomic DNA, encoding a 391-amino-acid protein with a calculated molecular weight of 43.5 kDa. The human ortholog, MATT, which is syntenic on 1q23.1, has an identical organization. Mattrin is a transmembrane domain protein with no previously ascribed function (Fig 1, E). Bioinformatics analysis predicts a long internal N-terminal domain, 5 transmembrane domains, and a short C-terminus (Fig 1, E, and see Fig E3 in this article's Online Repository at www.jacionline.org). The mutation p.Y280* occurs before the third transmembrane domain (Fig 1, E). Immunohistochemistry revealed that mattrin was present in epidermal granular layer keratinocytes in WT mice (Fig 2, A) but was absent from Mattma/ma animals (Fig 2, B). Similarly, the protein was observed in granular layer cells in human epidermis (see Fig E4, A and B, in this article's Online Repository at www.jacionline.org) and was also found in the hair follicles of mice (Fig 2, A) and human subjects (see Fig E4, C and D). In addition, the mRNA is widely expressed in human tissues and, consistent with immunohistochemistry, is strongly expressed in skin (see Fig E4, E). Expression quantitative trait locus analysis23 showed that Matt is in a network of proteins expressed late in epidermal differentiation, with an expression quantitative trait locus profile closely matching that of Rhbg (transporter protein) and Rab25 (membrane trafficking, data not shown). Mattrin shows distant sequence homology to the Membrane-Associated Proteins in Eicosanoid and Glutathione metabolism (MAPEG) protein family (see Fig E3),24 members of which have roles in lipid catabolism.25 Immunohistochemistry with the lipophilic dye Nile Red revealed highly organized stacks of cornified cell envelopes in the stratum corneum of WT animals (Fig 2, C) that were highly disorganized in Mattma/ma mice, with discontinuous, uneven, and highly disorganized cornified cell envelopes (Fig 2, D). Mattma/ma mice have spontaneous dermatitis Gross examination of adult Flgft/ft and Mattma/ma mice demonstrates that the 2 strains differ considerably from each other (Fig 3, A). Macroscopic clinical scoring22 demonstrated Mattma/ma and DM mice spontaneously having progressive dermatitis-like skin inflammation, which did not occur in adult Flgft/ft animals (see Fig E5, A, in this article's Online Repository at www.jacionline.org). Although all Mattma/ma and DM mice had marked skin inflammation, there was a broad spectrum of pathology, with some animals exhibiting profound lesions and excoriation and occasional blepharitis and eyelid dermatitis (see Fig E6, B, in this article's Online Repository at www.jacionline.org). Interestingly, both neonatal Flgft/ft and DM mice have significant ichthyosis (P .001) compared with WT mice, but Mattma/ma mice did not, indicating the postnatal importance of filaggrin (see Fig E5, A, and E6, A). With respect to the matted hair phenotype,11, 26 both Mattma/ma and DM animals had the keratinization defect, with hairs forming clumps11, 26 and hair breakage and occasional alopecia evident at 32 weeks (Fig 3, A, and see Fig E6, B). Scanning electron microscopy confirmed Mattma/ma and DM mice had fragile hairs prone to longitudinal splitting and breakage with defective cuticle morphology (see Fig E7, A, in this article's Online Repository at www.jacionline.org). Furthermore, Mattma/ma mice had distorted hair follicle morphogenesis (see Fig E7, B). Skin histology (Fig 3, B) showed that Mattma/ma and DM mice have marked acanthosis (P .001) and prominent orthokeratosis (P .001) with dermal inflammatory infiltrates (P .001; see Fig E5, B). In contrast, Flgft/ft mice had occasional foci of acanthosis, mild diffuse orthokeratosis, and increased dermal cell infiltration relative to WT mice (P .001). However, the phenotype in Flgft/ft mice was subclinical, with no overt dermatitis (Fig 3, A, and see Fig E5, A). Mattma/ma mutant mice are atopic and have a defective skin barrier All mutant strains spontaneously had increased IgE levels relative to WT mice, which was markedly more pronounced in Mattma/ma and DM animals relative to Flgft/ft mice (P .001; see Fig E5, D). Because the severity of skin inflammation in AD parallels barrier permeability,27, 28 TEWL was analyzed to quantify skin barrier dysregulation in mouse strains. TEWL was significantly increased in adult Mattma/ma and DM mice (P .001; see Fig E5, C). In contrast, TEWL was unaltered in Flgft/ft mice relative to that seen in WT animals (see Fig E5, C). HDM allergen was applied to the intact skin of Mattma/ma and WT mice to address whether the altered skin barrier in Mattma/ma mice influences allergen sensitization. Percutaneous sensitization with allergen led to enhanced skin inflammation in Mattma/ma mice (Fig 4, A-C). Consistent with the defective barrier, TEWL was significantly upregulated (P .001) in HDM-treated Mattma/ma mice after allergen application (Fig 4, D). Crucially, HDM-specific IgE responses were significantly (P .05) evoked in the sera of HDM-treated Mattma/ma mice, as well as HDM-specific IgG1 and IgG2a responses (Fig 4, E). In contrast, the skin of WT (Fig 4) and Flgft/ft (data not shown) mice was refractory to cutaneous HDM exposure. Collectively, these data demonstrate that Mattma/ma mice have spontaneous AD and a defective skin barrier that facilitates percutaneous allergic sensitization. A mutation in MATT is associated with AD To investigate whether MATT is associated with human AD, all exons of human MATT were sequenced in 55 Irish AD cases who were negative for FLG null mutations. Several noncoding SNPs and 1 known missense SNP, rs6684514, were identified (see Table E2 in this article's Online Repository at www.jacionline.org). Case-control analyses conducted on 2 independent AD case collections, English adult AD and United Kingdom pediatric AD, with separate population-matched control subjects (see Table E3) showed a significant association between rs6684514 and AD (Table I). The minor allele (A) is protective for disease in both the English adult AD (OR, approximately 0.791; P = .0038) and UK pediatric AD (OR, approximately 0.770; P = .0143) populations (Table I). Notably, when controlling for the strong and significant FLG null mutations, it was shown that the association of rs6684514 with AD is independent of FLG (Table I and see Table E4 in this article's Online Repository at www.jacionline.org). The association of rs6684514 with AD was further replicated in a German AD case-control analysis in which the OR after controlling for FLG mutations was 0.86 (95% CI, 0.76-0.97; P = .0161; Table I). However, this association was not replicated in Irish or Scottish case-control analyses (Table I). A meta-analysis using a fixed-effects model of all 4,245 AD cases from England, Scotland, Ireland, and Germany with 10,558 population-matched control subjects did not show significant heterogeneity between the study groups (P = .078). A small but significant effect of rs6684514 on AD risk was confirmed in the meta-analysis (OR, 0.91; 95% CI, 0.86-0.96; P = .001; Fig 5; see Table E4). A random-effects meta-analysis also produced similar results (OR, 0.90; 95% CI, 0.82-0.98; P = .015; see Fig E8 in this article's Online Repository at www.jacionline.org). English adult severe AD is defined as early-onset, persistent, and severe disease. UK mild-moderate pediatric AD includes cases collected from an English population birth cohort (n = 177) and a Scottish General Practice collection (n = 161); the English pediatric control subjects were ascertained not to have AD at the age of 7 to 9 years. Irish pediatric AD cases were collected in secondary and tertiary care clinics in Ireland; Irish population control subjects are healthy adult blood donors. Scottish asthma cases with AD are subjects with physician-diagnosed asthma and parent-reported AD. The German case and control rs6684514 genotypes were ascertained by imputation from genome-wide SNP analysis data; all other SNP genotypes and FLG null mutations were analyzed by using TaqMan allelic discrimination assays. In the English, United Kingdom, and Irish collections, 4 prevalent FLG null mutations were analyzed (R501X, 2282del4, R2447X, and S3247X), whereas in the German population 2 FLG null mutations were analyzed (R501X and 2282del4). Statistical analysis was performed through logistic regression in Stata 10.0 software (StataCorp). NA, FLG genotype data not available. Back to Article Outline Previously, we demonstrated that FLG loss-of-function mutations have a very strong relevance for the common inflammatory skin disease AD and associated atopic phenotypes3, 9, 10 and identified an analogous mutation in the murine homolog Flg in the spontaneously occurring flaky tail DM (maft) mouse.12 Subsequently, these DM mice have been widely used as a model of heritable skin barrier deficiency and spontaneous dermatitis13, 14, 15, 17 and as a model of filaggrin deficiency–associated AD pathogenesis in patients.29 In this study we have identified the matted phenotype in the DM mouse as arising from a second mutation in Matt (Tmem79). The Mattma mutation results in defective expression of the transmembrane protein mattrin, which is highly expressed in the upper granular layer of epidermal keratinocytes, with a predicted role in lipid homeostasis. These studies have elucidated the relative contributions of the Flg and Matt mutations in the DM mouse. The Mattma mutation led to the striking development of spontaneous AD-like skin pathology and atopy in adult mice. These data indicate that the DM mouse is not a true model of filaggrin deficiency–associated AD-like skin inflammation. It is notable that the Flgft mutation has a neonatal influence, whereas the Mattma mutation has a progressive influence with age. This indicates the polygenic nature of the DM mouse as an AD model and indicates the differential influence of both the Flgft and Mattma mutations. Recently, Flg−/− mice were shown to have no overt dermatitis with age,30 with the authors unable to sensitize Flg−/− mice by means of application of OVA, an allergen commonly used in models of skin inflammation,31 to the intact skin barrier, results similar to our data. Notably, Flgft/ft mice were not sensitized to HDM (data not shown), indicating an impermissibility of the Flgft/ft skin barrier to protein antigen ingress. These data indicate that the DM mouse is not a true model of filaggrin deficiency–associated AD-like skin inflammation. In this study we adopted a translational approach and also addressed whether mattrin was implicated in human AD. A missense SNP in MATT was shown to have a small but significant effect on the risk for human AD. Because mattrin shows distant sequence homology to MAPEG family members,24 which are known to catalyze glutathione-dependent transformations of lipophilic substrates at the lipid bilayer, sequence homology suggests a possible role for mattrin in the biology of lipids or lipid-like molecules.25 Epidermal transcriptomic analysis of WT versus Mattma/ma mice has revealed changes in several transcripts involved in fatty acid and lipid biology (data not shown). However, 1-dimensional and 2-dimensional thin-layer chromatography of epidermal lipid extracts did not reveal any differences in the migration or abundance of ceramides, phospholipids, or polar or nonpolar lipids between WT and Mattma/ma mice (data not shown). Future work will require analysis of the glutathione binding of mattrin, elucidation of lipophilic substrates of mattrin, and an investigation of the downstream signaling pathways and local immunologic milieu in Mattma mice. In summary, using a translational mouse-patient strategy, we have identified a new gene mutation that leads to dermatitis in mice and have further demonstrated that a variant in the human gene is associated with AD. Back to Article Outline We thank the patients affected by eczema and their families for their interest and participation. We also thank Masa Amagai and his research group from Keio University, Tokyo, Japan, for their open and enthusiastic collaboration, discussion, and sharing of data. Back to Article Outline Back to Article Outline

Preston G.C.; Feijoo-Carnero C.; Schurch N.; Cowling V.H.; Cantrell D.A. The Impact of KLF2 Modulation on the Transcriptional Program and Function of CD8 T Cells 2013 PLoS ONE 8:e77537

Krüppel-like factor 2 (KLF2) is a transcription factor that is highly expressed in quiescent T lymphocytes and downregulated in effector T cells. We now show that antigen receptor engagement downregulates KLF2 expression in a graded response determined by the affinity of T cell antigen receptor (TCR) ligand and the integrated activation of protein kinase B and the MAP kinases ERK1/2. The present study explores the importance of KLF2 downregulation and reveals that the loss of KLF2 controls a select portion of the CD8 effector T cell transcriptional program. In particular, KLF2 loss is required for CD8 T cells to express the inflammatory chemokine receptor CXCR3 and for maximum clonal expansion of T cells. KLF2 thus negatively controls the ability of CD8 T cells to respond to the CXCR3 ligand CXCL10. Strikingly, the KLF2 threshold for restraining expression of CXCR3 is very low and quite distinct to the KLF2 threshold for restraining T cell proliferation. KLF2 is thus an analogue (tunable) not a digital (on/off) cellular switch where the magnitude of KLF2 expression differentially modifies the T cell responses.

Duc C.; Sherstnev A.; Cole C.; Barton G.J.; Simpson G.G. Transcription Termination and Chimeric RNA Formation Controlled by Arabidopsis thaliana FPA 2013 PLoS Genet 9:e1003867

Author SummaryThe ends of almost all eukaryotic protein-coding genes are defined by a poly(A) signal. When genes are transcribed into mRNA by RNA polymerase II, the poly(A) signal guides cleavage of the precursor mRNA at a particular site; this is accompanied by the addition of a poly(A) tail to the mRNA and termination of transcription. Many genes have more than one poly(A) signal and the regulated choice of which to select can effectively determine what the gene will code for, how the gene can be regulated and where transcription termination occurs. We discovered a rare example of a regulator of poly(A) site choice, called FPA, while studying flower development in the model plant Arabidopsis thaliana. Studying FPA therefore provides an opportunity to understand not only its roles in plant biology but also the generic consequences of disrupting alternative polyadenylation. In this study, we use a technique called direct RNA sequencing to quantify genome-wide shifts in poly(A) site selection in plants that lack FPA function. One of our most striking findings is that in the absence of FPA we detect chimeric RNAs formed between two otherwise separate and well-characterised genes.

Saner N.; Karschau J.; Natsume T.; Gierlinski M.; Retkute R.; Hawkins M.; Nieduszynski C.A.; Blow J.J.; Moura A.P.S.; Tanaka T.U. Stochastic association of neighboring replicons creates replication factories in budding yeast 2013 The Journal of Cell Biology 202:1001-1012

Natsume T.; Müller C.A.; Katou Y.; Retkute R.; Gierliński M.; Araki H.; Blow J.J.; Shirahige K.; Nieduszynski C.A.; Tanaka T.U. Kinetochores coordinate pericentromeric cohesion and early DNA replication by Cdc7-Dbf4 kinase recruitment 2013 Molecular Cell 50:661-674

Centromeres play several important roles in ensuring proper chromosome segregation. Not only do they promote kinetochore assembly for microtubule attachment, but they also support robust sister chromatid cohesion at pericentromeres and facilitate replication of centromeric DNA early in S phase. However, it is still elusive how centromeres orchestrate all these functions at the same site. Here, we show that the budding yeast Dbf4-dependent kinase (DDK) accumulates at kinetochores in telophase, facilitated by the Ctf19 kinetochore complex. This promptly recruits Sld3-Sld7 replication initiator proteins to pericentromeric replication origins so that they initiate replication early in S phase. Furthermore, DDK at kinetochores independently recruits the Scc2-Scc4 cohesin loader to centromeres in G1 phase. This enhances cohesin loading and facilitates robust pericentromeric cohesion in S phase. Thus, we have found the central mechanism by which kinetochores orchestrate early S phase DNA replication and robust sister chromatid cohesion at microtubule attachment sites.

MacKenzie K.F.; Clark K.; Naqvi S.; McGuire V.A.; Noehren G.; Kristariyanto Y.; Bosch M.; Mudaliar M.; McCarthy P.C.; Pattison M.J.; Pedrioli P.G.A.; Barton G.J.; Toth R.; Prescott A.; Arthur J.S.C. PGE2 Induces Macrophage IL-10 Production and a Regulatory-like Phenotype via a Protein Kinase A-SIK-CRTC3 Pathway 2013 The Journal of Immunology 190:565-577

Pohler E.; Mamai O.; Hirst J.; Zamiri M.; Horn H.; Nomura T.; Irvine A.D.; Moran B.; Wilson N.J.; Smith F.J.D.; Goh C.S.M.; Sandilands A.; Cole C.; Barton G.J.; Evans A.T.; Shimizu H.; Akiyama M.; Suehiro M.; Konohana I.; Shboul M.; Teissier S.; Boussofara L.; Denguezli M.; Saad A.; Gribaa M.; Dopping-Hepenstal P.J.; McGrath J.A.; Brown S.J.; Goudie D.R.; Reversade B.; Munro C.S.; McLean W.H.I. Haploinsufficiency for AAGAB causes clinically heterogeneous forms of punctate palmoplantar keratoderma 2012 Nature Genetics 44:1272-1276

Palmoplantar keratodermas (PPKs) are a group of disorders that are diagnostically and therapeutically problematic in dermatogenetics. Punctate PPKs are characterized by circumscribed hyperkeratotic lesions on the palms and soles with considerable heterogeneity. In 18 families with autosomal dominant punctate PPK, we report heterozygous loss-of-function mutations in AAGAB, encoding α- and γ-adaptin–binding protein p34, located at a previously linked locus at 15q22. α- and γ-adaptin–binding protein p34, a cytosolic protein with a Rab-like GTPase domain, was shown to bind both clathrin adaptor protein complexes, indicating a role in membrane trafficking. Ultrastructurally, lesional epidermis showed abnormalities in intracellular vesicle biology. Immunohistochemistry showed hyperproliferation within the punctate lesions. Knockdown of AAGAB in keratinocytes led to increased cell division, which was linked to greatly elevated epidermal growth factor receptor (EGFR) protein expression and tyrosine phosphorylation. We hypothesize that p34 deficiency may impair endocytic recycling of growth factor receptors such as EGFR, leading to increased signaling and cellular proliferation.

Sherstnev A.; Duc C.; Cole C.; Zacharaki V.; Hornyik C.; Ozsolak F.; Milos P.M.; Barton G.J.; Simpson G.G. Direct sequencing of Arabidopsis thaliana RNA reveals patterns of cleavage and polyadenylation 2012 Nature Structural \& Molecular Biology 19:845-852

It has recently been shown that RNA 3′-end formation plays a more widespread role in controlling gene expression than previously thought. To examine the impact of regulated 3′-end formation genome-wide, we applied direct RNA sequencing to A. thaliana. Here we show the authentic transcriptome in unprecedented detail and describe the effects of 3′-end formation on genome organization. We reveal extreme heterogeneity in RNA 3′ ends, discover previously unrecognized noncoding RNAs and propose widespread reannotation of the genome. We explain the origin of most poly(A)+ antisense RNAs and identify cis elements that control 3′-end formation in different registers. These findings are essential to understanding what the genome actually encodes, how it is organized and how regulated 3′-end formation affects these processes. View full text

Klotz-Noack K.; McIntosh D.; Schurch N.; Pratt N.; Blow J.J. Re-replication induced by geminin depletion occurs from G2 and is enhanced by checkpoint activation 2012 Journal of Cell Science 125:2436-2445

To prevent re-replication of DNA in a single cell cycle, the licensing of replication origins by Mcm2-7 is prevented during S and G2 phases. Animal cells achieve this by cell-cycle-regulated proteolysis of the essential licensing factor Cdt1 and inhibition of Cdt1 by geminin. Here we investigate the consequences of ablating geminin in synchronised human U2OS cells. Following geminin loss, cells complete an apparently normal S phase, but a proportion arrest at the G2-M boundary. When Cdt1 accumulates in these cells, DNA re-replicates, suggesting that the key role of geminin is to prevent re-licensing in G2. If cell cycle checkpoints are inhibited in cells lacking geminin, cells progress through mitosis and less re-replication occurs. Checkpoint kinases thereby amplify re-replication into an all-or-nothing response by delaying geminin-depleted cells in G2. Deep DNA sequencing revealed no preferential re-replication of specific genomic regions after geminin depletion. This is consistent with the observation that cells in G2 have lost their replication timing information. By contrast, when Cdt1 is overexpressed or is stabilised by the neddylation inhibitor MLN4924, re-replication can occur throughout S phase.

Muramoto T.; Cannon D.; Gierlinski M.; Corrigan A.; Barton G.J.; Chubb J.R. Live imaging of nascent RNA dynamics reveals distinct types of transcriptional pulse regulation 2012 Proceedings of the National Academy of Sciences of the United States of America 109:7350-7355

Transcription of genes can be discontinuous, occurring in pulses or bursts. It is not clear how properties of transcriptional pulses vary between different genes. We compared the pulsing of five housekeeping and five developmentally induced genes by direct imaging of single gene transcriptional events in individual living Dictyostelium cells. Each gene displayed its own transcriptional signature, differing in probability of firing and pulse duration, frequency, and intensity. In contrast to the prevailing view from both prokaryotes and eukaryotes that transcription displays binary behavior, strongly expressed housekeeping genes altered the magnitude of their transcriptional pulses during development. These nonbinary "tunable" responses may be better suited than stochastic switch behavior for housekeeping functions. Analysis of RNA synthesis kinetics using fluorescence recovery after photobleaching implied modulation of housekeeping-gene pulse strength occurs at the level of transcription initiation rather than elongation. In addition, disparities between single cell and population measures of transcript production suggested differences in RNA stability between gene classes. Analysis of stability using RNAseq revealed no major global differences in stability between developmental and housekeeping transcripts, although strongly induced RNAs showed unusually rapid decay, indicating tight regulation of expression.

Boisvert F.; Ahmad Y.; Gierliński M.; Charrière F.; Lamont D.; Scott M.; Barton G.; Lamond A.I. A quantitative spatial proteomics analysis of proteome turnover in human cells 2012 Molecular \& cellular proteomics: MCP 11:M111.011429

Measuring the properties of endogenous cell proteins, such as expression level, subcellular localization, and turnover rates, on a whole proteome level remains a major challenge in the postgenome era. Quantitative methods for measuring mRNA expression do not reliably predict corresponding protein levels and provide little or no information on other protein properties. Here we describe a combined pulse-labeling, spatial proteomics and data analysis strategy to characterize the expression, localization, synthesis, degradation, and turnover rates of endogenously expressed, untagged human proteins in different subcellular compartments. Using quantitative mass spectrometry and stable isotope labeling with amino acids in cell culture, a total of 80,098 peptides from 8,041 HeLa proteins were quantified, and their spatial distribution between the cytoplasm, nucleus and nucleolus determined and visualized using specialized software tools developed in PepTracker. Using information from ion intensities and rates of change in isotope ratios, protein abundance levels and protein synthesis, degradation and turnover rates were calculated for the whole cell and for the respective cytoplasmic, nuclear, and nucleolar compartments. Expression levels of endogenous HeLa proteins varied by up to seven orders of magnitude. The average turnover rate for HeLa proteins was ~20 h. Turnover rate did not correlate with either molecular weight or net charge, but did correlate with abundance, with highly abundant proteins showing longer than average half-lives. Fast turnover proteins had overall a higher frequency of PEST motifs than slow turnover proteins but no general correlation was observed between amino or carboxyl terminal amino acid identities and turnover rates. A subset of proteins was identified that exist in pools with different turnover rates depending on their subcellular localization. This strongly correlated with subunits of large, multiprotein complexes, suggesting a general mechanism whereby their assembly is controlled in a different subcellular location to their main site of function.

Tibarewal P.; Zilidis G.; Spinelli L.; Schurch N.; Maccario H.; Gray A.; Perera N.M.; Davidson L.; Barton G.J.; Leslie N.R. PTEN protein phosphatase activity correlates with control of gene expression and invasion, a tumor-suppressing phenotype, but not with AKT activity 2012 Science Signaling 5:ra18

The tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) has a well-characterized lipid phosphatase activity and a poorly characterized protein phosphatase activity. We show that both activities are required for PTEN to inhibit cellular invasion and to mediate most of its largest effects on gene expression. PTEN appears to dephosphorylate itself at threonine 366, and mutation of this site makes lipid phosphatase activity sufficient for PTEN to inhibit invasion. We propose that the dominant role for PTEN's protein phosphatase activity is autodephosphorylation-mediated regulation of its lipid phosphatase activity. Because PTEN's regulation of invasion and these changes in gene expression required lipid phosphatase activity, but did not correlate with the total cellular abundance of its phosphatidylinositol 3,4,5-trisphosphate (PIP₃) lipid substrate or AKT activity, we propose that localized PIP₃ signaling may play a role in those PTEN-mediated processes that depend on both its protein and lipid phosphatase activities. Finally, we identified a tumor-derived PTEN mutant selectively lacking protein phosphatase activity, indicating that in some circumstances the regulation of invasion and not that of AKT can correlate with PTEN-mediated tumor suppression.

Gandhi S.R.; Gierliński M.; Mino A.; Tanaka K.; Kitamura E.; Clayton L.; Tanaka T.U. Kinetochore-dependent microtubule rescue ensures their efficient and sustained interactions in early mitosis 2011 Developmental Cell 21:920-933

How kinetochores regulate microtubule dynamics to ensure proper kinetochore-microtubule interactions is unknown. Here, we studied this during early mitosis in Saccharomyces cerevisiae. When a microtubule shrinks and its plus end reaches a kinetochore bound to its lateral surface, the microtubule end attempts to tether the kinetochore. This process often fails and, responding to this failure, microtubule rescue (conversion from shrinkage to growth) occurs, preventing kinetochore detachment from the microtubule end. This rescue is promoted by Stu2 transfer (ortholog of vertebrate XMAP215/ch-TOG) from the kinetochore to the microtubule end. Meanwhile, microtubule rescue distal to the kinetochore is also promoted by Stu2, which is transported by a kinesin-8 motor Kip3 along the microtubule from the kinetochore. Microtubule extension following rescue facilitates interaction with other widely scattered kinetochores, diminishing long delays in collecting the complete set of kinetochores by microtubules. Thus, kinetochore-dependent microtubule rescue ensures efficient and sustained kinetochore-microtubule interactions in early mitosis.

Sugden C.; Ross S.; Annesley S.J.; Cole C.; Bloomfield G.; Ivens A.; Skelton J.; Fisher P.R.; Barton G.; Williams J.G. A Dictyostelium SH2 adaptor protein required for correct DIF-1 signaling and pattern formation 2011 Developmental Biology 353:290-301

Gkikopoulos T.; Singh V.; Tsui K.; Awad S.; Renshaw M.J.; Schofield P.; Barton G.J.; Nislow C.; Tanaka T.U.; Owen-Hughes T. The SWI/SNF complex acts to constrain distribution of the centromeric histone variant Cse4 2011 The EMBO journal 30:1919-1927

In order to gain insight into the function of the Saccharomyces cerevisiae SWI/SNF complex, we have identified DNA sequences to which it is bound genomewide. One surprising observation is that the complex is enriched at the centromeres of each chromosome. Deletion of the gene encoding the Snf2 subunit of the complex was found to cause partial redistribution of the centromeric histone variant Cse4 to sites on chromosome arms. Cultures of snf2Δ yeast were found to progress through mitosis slowly. This was dependent on the mitotic checkpoint protein Mad2. In the absence of Mad2, defects in chromosome segregation were observed. In the absence of Snf2, chromatin organisation at centromeres is less distinct. In particular, hypersensitive sites flanking the Cse4 containing nucleosomes are less pronounced. Furthermore, SWI/SNF complex was found to be especially effective in the dissociation of Cse4 containing chromatin in vitro. This suggests a role for Snf2 in the maintenance of point centromeres involving the removal of Cse4 from ectopic sites.

Navarro M.N.; Goebel J.; Feijoo-Carnero C.; Morrice N.; Cantrell D.A. Phosphoproteomic analysis reveals an intrinsic pathway for the regulation of histone deacetylase 7 that controls the function of cytotoxic T lymphocytes 2011 Nature Immunology 12:352-361

Koningsbruggen S.; Gierlinski M.; Schofield P.; Martin D.; Barton G.J.; Ariyurek Y.; Dunnen J.T.; Lamond A.I. High-resolution whole-genome sequencing reveals that specific chromatin domains from most human chromosomes associate with nucleoli 2010 Molecular Biology of the Cell 21:3735-3748

The nuclear space is mostly occupied by chromosome territories and nuclear bodies. Although this organization of chromosomes affects gene function, relatively little is known about the role of nuclear bodies in the organization of chromosomal regions. The nucleolus is the best-studied subnuclear structure and forms around the rRNA repeat gene clusters on the acrocentric chromosomes. In addition to rDNA, other chromatin sequences also surround the nucleolar surface and may even loop into the nucleolus. These additional nucleolar-associated domains (NADs) have not been well characterized. We present here a whole-genome, high-resolution analysis of chromatin endogenously associated with nucleoli. We have used a combination of three complementary approaches, namely fluorescence comparative genome hybridization, high-throughput deep DNA sequencing and photoactivation combined with time-lapse fluorescence microscopy. The data show that specific sequences from most human chromosomes, in addition to the rDNA repeat units, associate with nucleoli in a reproducible and heritable manner. NADs have in common a high density of AT-rich sequence elements, low gene density and a statistically significant enrichment in transcriptionally repressed genes. Unexpectedly, both the direct DNA sequencing and fluorescence photoactivation data show that certain chromatin loci can specifically associate with either the nucleolus, or the nuclear envelope.

Remenyi J.; Hunter C.J.; Cole C.; Ando H.; Impey S.; Monk C.E.; Martin K.J.; Barton G.J.; Hutvagner G.; Arthur J.S.C. Regulation of the miR-212/132 locus by MSK1 and CREB in response to neurotrophins 2010 Biochemical Journal 428:281-291

Cole C.; Sobala A.; Lu C.; Thatcher S.R.; Bowman A.; Brown J.W.S.; Green P.J.; Barton G.J.; Hutvagner G. Filtering of deep sequencing data reveals the existence of abundant Dicer-dependent small RNAs derived from tRNAs 2009 RNA 15:2147-2160

Deep sequencing technologies such as Illumina, SOLiD, and 454 platforms have become very powerful tools in discovering and quantifying small RNAs in diverse organisms. Sequencing small RNA fractions always identifies RNAs derived from abundant RNA species such as rRNAs, tRNAs, snRNA, and snoRNA, and they are widely considered to be random degradation products. We carried out bioinformatic analysis of deep sequenced HeLa RNA and after quality filtering, identified highly abundant small RNA fragments, derived from mature tRNAs that are likely produced by specific processing rather than from random degradation. Moreover, we showed that the processing of small RNAs derived from tRNAGln is dependent on Dicer in vivo and that Dicer cleaves the tRNA in vitro.

Golebiowski F.; Matic I.; Tatham M.H.; Cole C.; Yin Y.; Nakamura A.; Cox J.; Barton G.J.; Mann M.; Hay R.T. System-Wide Changes to SUMO Modifications in Response to Heat Shock 2009 Sci. Signal. 2:ra24

Cole C.; Barber J.D.; Barton G.J. The Jpred 3 secondary structure prediction server 2008 Nucleic Acids Research 36:W197-W201

Jpred (http://www.compbio.dundee.ac.uk/jpred) is a secondary structure prediction server powered by the Jnet algorithm. Jpred performs over 1000 predictions per week for users in more than 50 countries. The recently updated Jnet algorithm provides a three-state (α-helix, β-strand and coil) prediction of secondary structure at an accuracy of 81.5%. Given either a single protein sequence or a multiple sequence alignment, Jpred derives alignment profiles from which predictions of secondary structure and solvent accessibility are made. The predictions are presented as coloured HTML, plain text, PostScript, PDF and via the Jalview alignment editor to allow flexibility in viewing and applying the data. The new Jpred 3 server includes significant usability improvements that include clearer feedback of the progress or failure of submitted requests. Functional improvements include batch submission of sequences, summary results via email and updates to the search databases. A new software pipeline will enable Jnet/Jpred to continue to be updated in sync with major updates to SCOP and UniProt and so ensures that Jpred 3 will maintain high-accuracy predictions.