Life Sciences Research for Lifelong Health


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Title / Authors / Details Open Access Download

Capture Hi-C reveals novel candidate genes and complex long-range interactions with related autoimmune risk loci.
Martin P, McGovern A, Orozco G, Duffus K, Yarwood A, Schoenfelder S, Cooper NJ, Barton A, Wallace C, Fraser P, Worthington J, Eyre S

Genome-wide association studies have been tremendously successful in identifying genetic variants associated with complex diseases. The majority of association signals are intergenic and evidence is accumulating that a high proportion of signals lie in enhancer regions. We use Capture Hi-C to investigate, for the first time, the interactions between associated variants for four autoimmune diseases and their functional targets in B- and T-cell lines. Here we report numerous looping interactions and provide evidence that only a minority of interactions are common to both B- and T-cell lines, suggesting interactions may be highly cell-type specific; some disease-associated SNPs do not interact with the nearest gene but with more compelling candidate genes (for example, FOXO1, AZI2) often situated several megabases away; and finally, regions associated with different autoimmune diseases interact with each other and the same promoter suggesting common autoimmune gene targets (for example, PTPRC, DEXI and ZFP36L1).

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Nature communications, 6, 2041-1723, 10069, 2015

PMID: 26616563

Open Access

The brown adipocyte protein CIDEA promotes lipid droplet fusion via a phosphatidic acid-binding amphipathic helix.
Barneda D, Planas-Iglesias J, Gaspar ML, Mohammadyani D, Prasannan S, Dormann D, Han GS, Jesch SA, Carman GM, Kagan V, Parker MG, Ktistakis NT, Dixon AM, Klein-Seetharaman J, Henry S, Christian M

Maintenance of energy homeostasis depends on the highly regulated storage and release of triacylglycerol primarily in adipose tissue and excessive storage is a feature of common metabolic disorders. CIDEA is a lipid droplet (LD)-protein enriched in brown adipocytes promoting the enlargement of LDs which are dynamic, ubiquitous organelles specialized for storing neutral lipids. We demonstrate an essential role in this process for an amphipathic helix in CIDEA, which facilitates embedding in the LD phospholipid monolayer and binds phosphatidic acid (PA). LD pairs are docked by CIDEA trans-complexes through contributions of the N-terminal domain and a C-terminal dimerization region. These complexes, enriched at the LD-LD contact site, interact with the cone-shaped phospholipid PA and likely increase phospholipid barrier permeability, promoting LD fusion by transference of lipids. This physiological process is essential in adipocyte differentiation as well as serving to facilitate the tight coupling of lipolysis and lipogenesis in activated brown fat.

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eLife, 4, 2050-084X, , 2015

PMID: 26609809

Open Access

Localizing the lipid products of PI3Kγ in neutrophils.
Norton L, Lindsay Y, Deladeriere A, Chessa T, Guillou H, Suire S, Lucocq J, Walker S, Andrews S, Segonds-Pichon A, Rausch O, Finan P, Sasaki T, Du CJ, Bretschneider T, Ferguson GJ, Hawkins PT, Stephens L

Class I phosphoinositide 3-kinases (PI3Ks) are important regulators of neutrophil migration in response to a range of chemoattractants. Their primary lipid products PtdIns(3,4,5)P3 and PtdIns(3,4)P2 preferentially accumulate near to the leading edge of migrating cells and are thought to act as an important cue organizing molecular and morphological polarization. We have investigated the distribution and accumulation of these lipids independently in mouse neutrophils using eGFP-PH reportersand electron microscopy (EM). We found that authentic mouse neutrophils rapidly polarized their Class I PI3K signalling, as read-out by eGFP-PH reporters, both at the up-gradient leading edge in response to local stimulation with fMLP as well as spontaneously and randomly in response to uniform stimulation. EM studies revealed these events occurred at the plasma membrane, were dominated by accumulation of PtdIns(3,4,5)P3, but not PtdIns(3,4)P2, and were dependent on PI3Kγ and its upstream activation by both Ras and Gβγs.

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Advances in biological regulation, , 2212-4934, , 2015

PMID: 26596865

Open Access

Elf5-centered transcription factor hub controls trophoblast stem cell self-renewal and differentiation through stoichiometry-sensitive shifts in target gene networks.
Latos PA, Sienerth AR, Murray A, Senner CE, Muto M, Ikawa M, Oxley D, Burge S, Cox BJ, Hemberger M

Elf5 is a transcription factor with pivotal roles in the trophoblast compartment, where it reinforces a trophoblast stem cell (TSC)-specific transcriptional circuit. However, Elf5 is also present in differentiating trophoblast cells that have ceased to express other TSC genes such as Cdx2 and Eomes. In the present study, we aimed to elucidate the context-dependent role of Elf5 at the interface between TSC self-renewal and the onset of differentiation. We demonstrate that precise levels of Elf5 are critical for normal expansion of the TSC compartment and embryonic survival, as Elf5 overexpression triggers precocious trophoblast differentiation. Through integration of protein interactome, transcriptome, and genome-wide chromatin immunoprecipitation data, we reveal that this abundance-dependent function is mediated through a shift in preferred Elf5-binding partners; in TSCs, Elf5 interaction with Eomes recruits Tfap2c to triply occupied sites at TSC-specific genes, driving their expression. In contrast, the Elf5 and Tfap2c interaction becomes predominant as their protein levels increase. This triggers binding to double- and single-occupancy sites that harbor the cognate Tfap2c motif, causing activation of the associated differentiation-promoting genes. These data place Elf5 at the center of a stoichiometry-sensitive transcriptional network, where it acts as a molecular switch governing the balance between TSC proliferation and differentiation.

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Genes & development, , 1549-5477, , 2015

PMID: 26584622

Open Access

Dynamic changes in histone modifications precede de novo DNA methylation in oocytes.
Stewart KR, Veselovska L, Kim J, Huang J, Saadeh H, Tomizawa SI, Smallwood SA, Chen T, Kelsey G

Erasure and subsequent reinstatement of DNA methylation in the germline, especially at imprinted CpG islands (CGIs), is crucial to embryogenesis in mammals. The mechanisms underlying DNA methylation establishment remain poorly understood, but a number of post-translational modifications of histones are implicated in antagonizing or recruiting the de novo DNA methylation complex. In mouse oogenesis, DNA methylation establishment occurs on a largely unmethylated genome and in nondividing cells, making it a highly informative model for examining how histone modifications can shape the DNA methylome. Using a chromatin immunoprecipitation (ChIP) and genome-wide sequencing (ChIP-seq) protocol optimized for low cell numbers and novel techniques for isolating primary and growing oocytes, profiles were generated for histone modifications implicated in promoting or inhibiting DNA methylation. CGIs destined for DNA methylation show reduced protective H3K4 dimethylation (H3K4me2) and trimethylation (H3K4me3) in both primary and growing oocytes, while permissive H3K36me3 increases specifically at these CGIs in growing oocytes. Methylome profiling of oocytes deficient in H3K4 demethylase KDM1A or KDM1B indicated that removal of H3K4 methylation is necessary for proper methylation establishment at CGIs. This work represents the first systematic study performing ChIP-seq in oocytes and shows that histone remodeling in the mammalian oocyte helps direct de novo DNA methylation events.

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Genes & development, , 1549-5477, , 2015

PMID: 26584620

Open Access

The cytotoxic T cell proteome and its shaping by the kinase mTOR.
Hukelmann JL, Anderson KE, Sinclair LV, Grzes KM, Murillo AB, Hawkins PT, Stephens LR, Lamond AI, Cantrell DA

We used high-resolution mass spectrometry to map the cytotoxic T lymphocyte (CTL) proteome and the effect of the metabolic checkpoint kinase mTORC1 on CTLs. The CTL proteome was dominated by metabolic regulators and granzymes, and mTORC1 selectively repressed and promoted expression of a subset of CTL proteins (~10%). These included key CTL effector molecules, signaling proteins and a subset of metabolic enzymes. Proteomic data highlighted the potential for negative control of the production of phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) by mTORC1 in CTLs. mTORC1 repressed PtdIns(3,4,5)P3 production and determined the requirement for mTORC2 in activation of the kinase Akt. Our unbiased proteomic analysis thus provides comprehensive understanding of CTL identity and the control of CTL function by mTORC1.

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Nature immunology, , 1529-2916, , 2015

PMID: 26551880

Open Access

Continuous Histone Replacement by Hira Is Essential for Normal Transcriptional Regulation and De Novo DNA Methylation during Mouse Oogenesis.
Nashun B, Hill PW, Smallwood SA, Dharmalingam G, Amouroux R, Clark SJ, Sharma V, Ndjetehe E, Pelczar P, Festenstein RJ, Kelsey G, Hajkova P

The integrity of chromatin, which provides a dynamic template for all DNA-related processes in eukaryotes, is maintained through replication-dependent and -independent assembly pathways. To address the role of histone deposition in the absence of DNA replication, we deleted the H3.3 chaperone Hira in developing mouse oocytes. We show that chromatin of non-replicative developing oocytes is dynamic and that lack of continuous H3.3/H4 deposition alters chromatin structure, resulting in increased DNase I sensitivity, the accumulation of DNA damage, and a severe fertility phenotype. On the molecular level, abnormal chromatin structure leads to a dramatic decrease in the dynamic range of gene expression, the appearance of spurious transcripts, and inefficient de novo DNA methylation. Our study thus unequivocally shows the importance of continuous histone replacement and chromatin homeostasis for transcriptional regulation and normal developmental progression in a non-replicative system in vivo.

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Molecular cell, , 1097-4164, , 2015

PMID: 26549683

Single-cell Hi-C for genome-wide detection of chromatin interactions that occur simultaneously in a single cell.
Nagano T, Lubling Y, Yaffe E, Wingett SW, Dean W, Tanay A, Fraser P

Hi-C is a powerful method that provides pairwise information on genomic regions in spatial proximity in the nucleus. Hi-C requires millions of cells as input and, as genome organization varies from cell to cell, a limitation of Hi-C is that it only provides a population average of genome conformations. We developed single-cell Hi-C to create snapshots of thousands of chromatin interactions that occur simultaneously in a single cell. To adapt Hi-C to single-cell analysis, we modified the protocol to include in-nucleus ligation. This enables the isolation of single nuclei carrying Hi-C-ligated DNA into separate tubes, followed by reversal of cross-links, capture of biotinylated ligation junctions on streptavidin-coated magnetic beads and PCR amplification of single-cell Hi-C libraries. The entire laboratory protocol can be carried out in 1 week, and although we have demonstrated its use in mouse T helper (TH1) cells, it should be applicable to any cell type or species for which standard Hi-C has been successful. We also developed an analysis pipeline to filter noise and assess the quality of data sets in a few hours. Although the interactome maps produced by single-cell Hi-C are sparse, the data provide useful information to understand cellular variability in nuclear genome organization and chromosome structure. Standard wet and dry laboratory skills in molecular biology and computational analysis are required.

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Nature protocols, 10, 1750-2799, 1986-2003, 2015

PMID: 26540590

Oncogenic PI3Kα promotes multipotency in breast epithelial cells.
Okkenhaug K, Roychoudhuri R

The phosphoinositide 3-kinase (PI3K) signaling pathway is among the most frequently altered in cancer. Now, two studies show that a mutated oncogenic PI3Kα, commonly found in breast cancer, leads to dedifferentiation or destabilization of luminal and basal epithelial lineages, which in turn leads to increased cancer cell heterogeneity.

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Science signaling, 8, 1937-9145, pe3, 2015

PMID: 26535006

Open Access

Molecular signatures of plastic phenotypes in two eusocial insect species with simple societies.
Patalano S, Vlasova A, Wyatt C, Ewels P, Camara F, Ferreira PG, Asher CL, Jurkowski TP, Segonds-Pichon A, Bachman M, González-Navarrete I, Minoche AE, Krueger F, Lowy E, Marcet-Houben M, Rodriguez-Ales JL, Nascimento FS, Balasubramanian S, Gabaldon T, Tarver JE, Andrews S, Himmelbauer H, Hughes WO, Guigó R, Reik W, Sumner S

Phenotypic plasticity is important in adaptation and shapes the evolution of organisms. However, we understand little about what aspects of the genome are important in facilitating plasticity. Eusocial insect societies produce plastic phenotypes from the same genome, as reproductives (queens) and nonreproductives (workers). The greatest plasticity is found in the simple eusocial insect societies in which individuals retain the ability to switch between reproductive and nonreproductive phenotypes as adults. We lack comprehensive data on the molecular basis of plastic phenotypes. Here, we sequenced genomes, microRNAs (miRNAs), and multiple transcriptomes and methylomes from individual brains in a wasp (Polistes canadensis) and an ant (Dinoponera quadriceps) that live in simple eusocial societies. In both species, we found few differences between phenotypes at the transcriptional level, with little functional specialization, and no evidence that phenotype-specific gene expression is driven by DNA methylation or miRNAs. Instead, phenotypic differentiation was defined more subtly by nonrandom transcriptional network organization, with roles in these networks for both conserved and taxon-restricted genes. The general lack of highly methylated regions or methylome patterning in both species may be an important mechanism for achieving plasticity among phenotypes during adulthood. These findings define previously unidentified hypotheses on the genomic processes that facilitate plasticity and suggest that the molecular hallmarks of social behavior are likely to differ with the level of social complexity.

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Proceedings of the National Academy of Sciences of the United States of America, , 1091-6490, , 2015

PMID: 26483466

Open Access

A non-proteolytic role for ubiquitin in deadenylation of MHC-I mRNA by the RNA-binding E3-ligase MEX-3C.
Cano F, Rapiteanu R, Sebastiaan Winkler G, Lehner PJ

The regulation of protein and mRNA turnover is essential for many cellular processes. We recently showed that ubiquitin-traditionally linked to protein degradation-directly regulates the degradation of mRNAs through the action of a newly identified family of RNA-binding E3 ubiquitin ligases. How ubiquitin regulates mRNA decay remains unclear. Here, we identify a new role for ubiquitin in regulating deadenylation, the initial and often rate-limiting step in mRNA degradation. MEX-3C, a canonical member of this family of RNA-binding ubiquitin ligases, associates with the cytoplasmic deadenylation complexes and ubiquitinates CNOT7(Caf1), the main catalytic subunit of the CCR4-NOT deadenylation machinery. We establish a new role for ubiquitin in regulating MHC-I mRNA deadenylation as ubiquitination of CNOT7 by MEX-3C regulates its deadenylation activity and is required for MHC-I mRNA degradation. Since neither proteasome nor lysosome inhibitors rescued MEX-3C-mediated MHC-I mRNA degradation, our findings suggest a new non-proteolytic function for ubiquitin in the regulation of mRNA decay.

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Nature communications, 6, 2041-1723, 8670, 2015

PMID: 26471122

Do genome-scale models need exact solvers or clearer standards?
Ebrahim A, Almaas E, Bauer E, Bordbar A, Burgard AP, Chang RL, Dräger A, Famili I, Feist AM, Fleming RM, Fong SS, Hatzimanikatis V, Herrgård MJ, Holder A, Hucka M, Hyduke D, Jamshidi N, Lee SY, Le Novère N, Lerman JA, Lewis NE, Ma D, Mahadevan R, Maranas C, Nagarajan H, Navid A, Nielsen J, Nielsen LK, Nogales J, Noronha A, Pal C, Palsson BO, Papin JA, Patil KR, Price ND, Reed JL, Saunders M, Senger RS, Sonnenschein N, Sun Y, Thiele I

Molecular systems biology, 11, 1744-4292, 831, 2015

PMID: 26467284

Open Access

Perturbations of PIP3 signalling trigger a global remodelling of mRNA landscape and reveal a transcriptional feedback loop.
Kiselev VY, Juvin V, Malek M, Luscombe N, Hawkins P, Novère NL, Stephens L

PIP3 is synthesized by the Class I PI3Ks and regulates complex cell responses, such as growth and migration. Signals that drive long-term reshaping of cell phenotypes are difficult to resolve because of complex feedback networks that operate over extended times. PIP3-dependent modulation of mRNA accumulation is clearly important in this process but is poorly understood. We have quantified the genome-wide mRNA-landscape of non-transformed, breast epithelium-derived MCF10a cells and its response to acute regulation by EGF, in the presence or absence of a PI3Kα inhibitor, compare it to chronic activation of PI3K signalling by cancer-relevant mutations (isogenic cells expressing an oncomutant PI3Kα allele or lacking the PIP3-phosphatase/tumour-suppressor, PTEN). Our results show that whilst many mRNAs are changed by long-term genetic perturbation of PIP3 signalling ('butterfly effect'), a much smaller number do so in a coherent fashion with the different PIP3 perturbations. This suggests a subset of more directly regulated mRNAs. We show that mRNAs respond differently to given aspects of PIP3 regulation. Some PIP3-sensitive mRNAs encode PI3K pathway components, thus suggesting a transcriptional feedback loop. We identify the transcription factor binding motifs SRF and PRDM1 as important regulators of PIP3-sensitive mRNAs involved in cell movement.

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Nucleic acids research, , 1362-4962, , 2015

PMID: 26464442

Reduced number of axonal mitochondria and tau hypophosphorylation in mouse P301L tau knockin neurons.
Rodríguez-Martín T, Pooler AM, Lau DH, Mórotz GM, De Vos KJ, Gilley J, Coleman MP, Hanger DP

Expression of the frontotemporal dementia-related tau mutation, P301L, at physiological levels in adult mouse brain (KI-P301L mice) results in overt hypophosphorylation of tau and age-dependent alterations in axonal mitochondrial transport in peripheral nerves. To determine the effects of P301L tau expression in the central nervous system, we examined the kinetics of mitochondrial axonal transport and tau phosphorylation in primary cortical neurons from P301L knock-in (KI-P301L) mice. We observed a significant 50% reduction in the number of mitochondria in the axons of cortical neurons cultured from KI-P301L mice compared to wild-type neurons. Expression of murine P301L tau did not change the speed, direction of travel or likelihood of movement of mitochondria. Notably, the angle that defines the orientation of the mitochondria in the axon, and the volume of individual moving mitochondria, were significantly increased in neurons expressing P301L tau. We found that murine tau phosphorylation in KI-P301L mouse neurons was diminished and the ability of P301L tau to bind to microtubules was also reduced compared to tau in wild-type neurons. The P301L mutation did not influence the ability of murine tau to associate with membranes in cortical neurons or in adult mouse brain. We conclude that P301L tau is associated with mitochondrial changes and causes an early reduction in murine tau phosphorylation in neurons coupled with impaired microtubule binding of tau. These results support the association of mutant tau with detrimental effects on mitochondria and will be of significance for the pathogenesis of tauopathies.

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Neurobiology of disease, 85, 1095-953X, 1-10, 2015

PMID: 26459111

Open Access

Structure and flexibility of the endosomal Vps34 complex reveals the basis of its function on membranes.
Rostislavleva K, Soler N, Ohashi Y, Zhang L, Pardon E, Burke JE, Masson GR, Johnson C, Steyaert J, Ktistakis NT, Williams RL

Phosphatidylinositol 3-kinase Vps34 complexes regulate intracellular membrane trafficking in endocytic sorting, cytokinesis, and autophagy. We present the 4.4 angstrom crystal structure of the 385-kilodalton endosomal complex II (PIK3C3-CII), consisting of Vps34, Vps15 (p150), Vps30/Atg6 (Beclin 1), and Vps38 (UVRAG). The subunits form a Y-shaped complex, centered on the Vps34 C2 domain. Vps34 and Vps15 intertwine in one arm, where the Vps15 kinase domain engages the Vps34 activation loop to regulate its activity. Vps30 and Vps38 form the other arm that brackets the Vps15/Vps34 heterodimer, suggesting a path for complex assembly. We used hydrogen-deuterium exchange mass spectrometry (HDX-MS) to reveal conformational changes accompanying membrane binding and identify a Vps30 loop that is critical for the ability of complex II to phosphorylate giant liposomes on which complex I is inactive.

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Science (New York, N.Y.), 350, 1095-9203, aac7365, 2015

PMID: 26450213

Purification, characterization and crystallization of the F-ATPase from Paracoccus denitrificans.
Morales-Rios E, Watt IN, Zhang Q, Ding S, Fearnley IM, Montgomery MG, Wakelam MJ, Walker JE

The structures of F-ATPases have been determined predominantly with mitochondrial enzymes, but hitherto no F-ATPase has been crystallized intact. A high-resolution model of the bovine enzyme built up from separate sub-structures determined by X-ray crystallography contains about 85% of the entire complex, but it lacks a crucial region that provides a transmembrane proton pathway involved in the generation of the rotary mechanism that drives the synthesis of ATP. Here the isolation, characterization and crystallization of an integral F-ATPase complex from the α-proteobacterium Paracoccus denitrificans are described. Unlike many eubacterial F-ATPases, which can both synthesize and hydrolyse ATP, the P. denitrificans enzyme can only carry out the synthetic reaction. The mechanism of inhibition of its ATP hydrolytic activity involves a ζ inhibitor protein, which binds to the catalytic F1-domain of the enzyme. The complex that has been crystallized, and the crystals themselves, contain the nine core proteins of the complete F-ATPase complex plus the ζ inhibitor protein. The formation of crystals depends upon the presence of bound bacterial cardiolipin and phospholipid molecules; when they were removed, the complex failed to crystallize. The experiments open the way to an atomic structure of an F-ATPase complex.

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Open biology, 5, 2046-2441, , 2015

PMID: 26423580

Direct Induction of Trophoblast Stem Cells from Murine Fibroblasts.
Kubaczka C, Senner CE, Cierlitza M, Araúzo-Bravo MJ, Kuckenberg P, Peitz M, Hemberger M, Schorle H

Trophoblast stem cells (TSCs) arise from the first cell fate decision in the developing embryo and generate extra-embryonic lineages, giving rise to the fetal portion of the placenta. Mouse embryonic and extra-embryonic lineages are strictly separated by a distinct epigenetic barrier, which is not fully overcome following expression of TSC-determining factors in embryonic stem cells. Here, we show that transient expression of Tfap2c, Gata3, Eomes, and Ets2 is sufficient to reprogram mouse embryonic fibroblasts and post-natal tail-tip-derived fibroblasts into induced TSCs (iTSCs) and surmount the epigenetic barrier separating somatic from extra-embryonic lineages. iTSCs share nearly identical morphological characteristics, gene expression profiles, and DNA methylation patterns with blastocyst-derived TSCs. Furthermore, iTSCs display transgene-independent self-renewal, differentiate along extra-embryonic lineages, and chimerize host placentas following blastocyst injection. These findings provide insights into the transcription factor networks governing TSC identity and opportunities for studying the epigenetic barriers underlying embryonic and extra-embryonic lineage segregation.

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Cell stem cell, 17, 1875-9777, 557-68, 2015

PMID: 26412560

Deep sequencing and de novo assembly of the mouse oocyte transcriptome define the contribution of transcription to the DNA methylation landscape.
Veselovska L, Smallwood SA, Saadeh H, Stewart KR, Krueger F, Maupetit-Méhouas S, Arnaud P, Tomizawa SI, Andrews S, Kelsey G

Previously, a role was demonstrated for transcription in the acquisition of DNA methylation at imprinted control regions in oocytes. Definition of the oocyte DNA methylome by whole genome approaches revealed that the majority of methylated CpG islands are intragenic and gene bodies are hypermethylated. Yet, the mechanisms by which transcription regulates DNA methylation in oocytes remain unclear. Here, we systematically test the link between transcription and the methylome.

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Genome biology, 16, 1474-760X, 209, 0

PMID: 26408185

Open Access

MEK1 and MEK2 inhibitors and cancer therapy: the long and winding road.
Caunt CJ, Sale MJ, Smith PD, Cook SJ

The role of the ERK signalling pathway in cancer is thought to be most prominent in tumours in which mutations in the receptor tyrosine kinases RAS, BRAF, CRAF, MEK1 or MEK2 drive growth factor-independent ERK1 and ERK2 activation and thence inappropriate cell proliferation and survival. New drugs that inhibit RAF or MEK1 and MEK2 have recently been approved or are currently undergoing late-stage clinical evaluation. In this Review, we consider the ERK pathway, focusing particularly on the role of MEK1 and MEK2, the 'gatekeepers' of ERK1/2 activity. We discuss their validation as drug targets, the merits of targeting MEK1 and MEK2 versus BRAF and the mechanisms of action of different inhibitors of MEK1 and MEK2. We also consider how some of the systems-level properties (intrapathway regulatory loops and wider signalling network connections) of the ERK pathway present a challenge for the success of MEK1 and MEK2 inhibitors, discuss mechanisms of resistance to these inhibitors, and review their clinical progress.

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Nature reviews. Cancer, 15, 1474-1768, 577-92, 2015

PMID: 26399658

ACF chromatin-remodeling complex mediates stress-induced depressive-like behavior.
Sun H, Damez-Werno DM, Scobie KN, Shao NY, Dias C, Rabkin J, Koo JW, Korb E, Bagot RC, Ahn FH, Cahill ME, Labonté B, Mouzon E, Heller EA, Cates H, Golden SA, Gleason K, Russo SJ, Andrews S, Neve R, Kennedy PJ, Maze I, Dietz DM, Allis CD, Turecki G, Varga-Weisz P, Tamminga C, Shen L, Nestler EJ

Improved treatment for major depressive disorder (MDD) remains elusive because of the limited understanding of its underlying biological mechanisms. It is likely that stress-induced maladaptive transcriptional regulation in limbic neural circuits contributes to the development of MDD, possibly through epigenetic factors that regulate chromatin structure. We establish that persistent upregulation of the ACF (ATP-utilizing chromatin assembly and remodeling factor) ATP-dependent chromatin-remodeling complex, occurring in the nucleus accumbens of stress-susceptible mice and depressed humans, is necessary for stress-induced depressive-like behaviors. We found that altered ACF binding after chronic stress was correlated with altered nucleosome positioning, particularly around the transcription start sites of affected genes. These alterations in ACF binding and nucleosome positioning were associated with repressed expression of genes implicated in susceptibility to stress. Together, our findings identify the ACF chromatin-remodeling complex as a critical component in the development of susceptibility to depression and in regulating stress-related behaviors.

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Nature medicine, , 1546-170X, , 2015

PMID: 26390241

PI3K Signaling in Normal B Cells and Chronic Lymphocytic Leukemia (CLL).
Okkenhaug K, Burger JA

B cells provide immunity to extracellular pathogens by secreting a diverse repertoire of antibodies with high affinity and specificity for exposed antigens. The B cell receptor (BCR ) is a transmembrane antibody, which facilitates the clonal selection of B cells producing secreted antibodies of the same specificity. The diverse antibody repertoire is generated by V(D)J recombination of heavy and light chain genes, whereas affinity maturation is mediated by activation-induced cytidine deaminase (AID)-mediated mutagenesis. These processes, which are essential for the generation of adaptive humoral immunity, also render B cells susceptible to chromosomal rearrangements and point mutations that in some cases lead to cancer. In this chapter, we will review the central role of PI3K s in mediating signals from the B cell receptor that not only facilitate the development of functional B cell repertoire, but also support the growth and survival of neoplastic B cells, focusing on chronic lymphocytic leukemia (CLL ) B cells. Perhaps because of the central role played by PI3K in BCR signaling, B cell leukemia and lymphomas are the first diseases for which a PI3K inhibitor has been approved for clinical use.

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Current topics in microbiology and immunology, 393, 0070-217X, 123-42, 2016

PMID: 26350103

Open Access

Identification of DYRK1B as a substrate of ERK1/2 and characterisation of the kinase activity of DYRK1B mutants from cancer and metabolic syndrome.
Ashford AL, Dunkley TP, Cockerill M, Rowlinson RA, Baak LM, Gallo R, Balmanno K, Goodwin LM, Ward RA, Lochhead PA, Guichard S, Hudson K, Cook SJ

The dual-specificity tyrosine-phosphorylation-regulated kinase, DYRK1B, is expressed de novo during myogenesis, amplified or mutated in certain cancers and mutated in familial cases of metabolic syndrome. DYRK1B is activated by cis auto-phosphorylation on tyrosine-273 (Y273) within the activation loop during translation but few other DYRK1B phosphorylation sites have been characterised to date. Here, we demonstrate that DYRK1B also undergoes trans-autophosphorylation on serine-421 (S421) in vitro and in cells and that this site contributes to DYRK1B kinase activity. Whilst a DYRK1B(S421A) mutant was completely defective for p-S421 in cells, DYRK1B inhibitors caused only a partial loss of p-S421 suggesting the existence of an additional kinase that could also phosphorylate DYRK1B S421. Indeed, a catalytically inactive DYRK1B(D239A) mutant exhibited very low levels of p-S421 in cells but this was increased by KRAS(G12V). In addition, selective activation of the RAF-MEK1/2-ERK1/2 signalling pathway rapidly increased p-S421 in cells whereas activation of the stress kinases JNK or p38 could not. S421 resides within a Ser-Pro phosphoacceptor motif that is typical for ERK1/2 and recombinant ERK2 phosphorylated DYRK1B at S421 in vitro. Our results show that DYRK1B is a novel ERK2 substrate, uncovering new links between two kinases involved in cell fate decisions. Finally, we show that DYRK1B mutants that have recently been described in cancer and metabolic syndrome exhibit normal or reduced intrinsic kinase activity.

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Cellular and molecular life sciences : CMLS, , 1420-9071, , 2015

PMID: 26346493

Open Access

A novel phosphate-starvation response in fission yeast requires the endocytic function of Myosin I.
Petrini E, Baillet V, Cridge J, Hogan CJ, Guillaume C, Ke H, Brandetti E, Walker S, Koohy H, Spivakov M, Varga-Weisz P

Endocytosis is essential for uptake of many substances into the cell, but how it links to nutritional signalling is poorly understood. Here we show a novel role for endocytosis in regulating the response to low phosphate in Schizosaccharomyces pombe. Loss of function of Myo1, Sla2/End4 or Arp2, proteins involved in the early steps of endocytosis, led to increased proliferation in low phosphate media compared to controls. We show that once cells are deprived of phosphate they undergo a quiescence response that is dependent on the endocytic function of Myo1. Transcriptomic analysis revealed a wide perturbation of gene expression with induction of stress-regulated genes upon phosphate starvation in wildtype but not Δmyo1 cells. Thus, endocytosis plays a pivotal role in mediating the cellular response to nutrients, bridging the external environment and internal molecular functions of the cell.

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Journal of cell science, , 1477-9137, , 2015

PMID: 26345368

Open Access

Cell cycle progression is an essential regulatory component of phospholipid metabolism and membrane homeostasis.
Sanchez-Alvarez M, Zhang Q, Finger F, Wakelam MJ, Bakal C

We show that phospholipid anabolism does not occur uniformly during the metazoan cell cycle. Transition to S-phase is required for optimal mobilization of lipid precursors, synthesis of specific phospholipid species and endoplasmic reticulum (ER) homeostasis. Average changes observed in whole-cell phospholipid composition, and total ER lipid content, upon stimulation of cell growth can be explained by the cell cycle distribution of the population. TORC1 promotes phospholipid anabolism by slowing S/G2 progression. The cell cycle stage-specific nature of lipid biogenesis is dependent on p53. We propose that coupling lipid metabolism to cell cycle progression is a means by which cells have evolved to coordinate proliferation with cell and organelle growth.

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Open biology, 5, 2046-2441, , 2015

PMID: 26333836

Open Access

Polycomb repressive complex PRC1 spatially constrains the mouse embryonic stem cell genome.
Schoenfelder S, Sugar R, Dimond A, Javierre BM, Armstrong H, Mifsud B, Dimitrova E, Matheson L, Tavares-Cadete F, Furlan-Magaril M, Segonds-Pichon A, Jurkowski W, Wingett SW, Tabbada K, Andrews S, Herman B, LeProust E, Osborne CS, Koseki H, Fraser P, Luscombe NM, Elderkin S

The Polycomb repressive complexes PRC1 and PRC2 maintain embryonic stem cell (ESC) pluripotency by silencing lineage-specifying developmental regulator genes. Emerging evidence suggests that Polycomb complexes act through controlling spatial genome organization. We show that PRC1 functions as a master regulator of mouse ESC genome architecture by organizing genes in three-dimensional interaction networks. The strongest spatial network is composed of the four Hox gene clusters and early developmental transcription factor genes, the majority of which contact poised enhancers. Removal of Polycomb repression leads to disruption of promoter-promoter contacts in the Hox gene network. In contrast, promoter-enhancer contacts are maintained in the absence of Polycomb repression, with accompanying widespread acquisition of active chromatin signatures at network enhancers and pronounced transcriptional upregulation of network genes. Thus, PRC1 physically constrains developmental transcription factor genes and their enhancers in a silenced but poised spatial network. We propose that the selective release of genes from this spatial network underlies cell fate specification during early embryonic development.

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Nature genetics, , 1546-1718, , 2015

PMID: 26323060

Open Access