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Recent clinical trials showed that targeting of inhibitory receptors on T cells induces durable responses in a subset of cancer patients, despite advanced disease. However, the regulatory switches controlling T-cell function in immunosuppressive tumours are not well understood. Here we show that such inhibitory mechanisms can be systematically discovered in the tumour microenvironment. We devised an in vivo pooled short hairpin RNA (shRNA) screen in which shRNAs targeting negative regulators became highly enriched in murine tumours by releasing a block on T-cell proliferation upon tumour antigen recognition. Such shRNAs were identified by deep sequencing of the shRNA cassette from T cells infiltrating tumour or control tissues. One of the target genes was Ppp2r2d, a regulatory subunit of the PP2A phosphatase family. In tumours, Ppp2r2d knockdown inhibited T-cell apoptosis and enhanced T-cell proliferation as well as cytokine production. Key regulators of immune function can therefore be discovered in relevant tissue microenvironments., Recent work has shown that cytotoxic T cells have a central role in immune-mediated control of cancer (1-7). T cells are able to specifically detect and eliminate cancer cells following [...]

Cullin-RING ligases (CRLs) are a family of E3 ubiquitin ligase complexes that rely on either RING-box 1 (RBX1) or sensitive to apoptosis gene (SAG), also known as RBX2, for activity. RBX1 and SAG are both overexpressed in human lung cancer; however, their contribution to patient survival and lung tumorigenesis is unknown. Here, we report that overexpression of SAG, but not RBX1, correlates with poor patient prognosis and more advanced disease. We found that SAG is overexpressed in murine [Kras.sup.G12D]-driven lung tumors and that Sag deletion suppressed lung tumorigenesis and extended murine life span. Using cultured lung cancer cells, we showed that SAG knockdown suppressed growth and survival, inactivated both NF-κB and mTOR pathways, and resulted in accumulation of tumor suppressor substrates, including p21, p27, NOXA, and BIM. Importantly, growth suppression by SAG knockdown was partially rescued by simultaneous knockdown of p21 or the mTOR inhibitor DEPTOR. Treatment with MLN4924, a small molecule inhibitor of CRL E3s, also inhibited the formation of [Kras.sup.G12D]-induced lung tumors through a similar mechanism involving inactivation of NF-κB and mTOR and accumulation of tumor suppressor substrates. Together, our results demonstrate that Sag is a Kras-cooperating oncogene that promotes lung tumorigenesis and suggest that targeting SAG-CRL E3 ligases may be an effective therapeutic approach for Kras-driven lung cancers., Introduction CRL (Cullin-RING ligase) complexes consist of a scaffold protein cullin (8 family members), an adaptor protein (few family members), a substrate-recognizing protein (many family members), and a RING component [...]

Synthetic lethality provides a potential mechanistic framework for the therapeutic targeting of genetic and functional deficiencies in cancers and is now being explored widely. The first clinical exemplification of synthetic [...]

Activated oncogenes and anticancer chemotherapy induce cellular senescence, a terminal growth arrest of viable cells characterized by S-phase entry-blocking histone 3 lysine 9 trimethylation (H3K9me3) (1,2). Although therapy-induced senescence (TIS) improves long-term outcomes (3), potentially harmful properties of senescent tumour cells make their quantitative elimination a therapeutic priority. Here we use the Eμ-myc transgenic mouse lymphoma model in which TIS depends on the H3K9 histone methyltransferase Suv39h[1.sup.-]to show the mechanism and therapeutic exploitation of senescence-related metabolic reprogramming in vitro and in vivo. After senescence-inducing chemotherapy, TIS-competent lymphomas but not TIS-incompetent Suv39h[1.sup.-] lymphomas show increased glucose utilization and much higher ATP production. We demonstrate that this is linked to massive proteotoxic stress, which is a consequence of the senescence-associated secretory phenotype (SASP) described previously (4-6). SASP-producing TIS cells exhibited endoplasmic reticulum stress, an unfolded protein response (UPR), and increased ubiquitination, thereby targeting toxic proteins for autophagy in an acutely energy-consuming fashion. Accordingly, TIS lymphomas, unlike senescence models that lack a strong SASP response, were more sensitive to blocking glucose utilization or autophagy, which led to their selective elimination through caspase-12-and caspase-3-mediated endoplasmic-reticulum-related apoptosis. Consequently, pharmacological targeting of these metabolic demands on TIS induction in vivo prompted tumour regression and improved treatment outcomes further. These findings unveil the hypercatabolic nature of TIS that is therapeutically exploitable by synthetic lethal metabolic targeting., Anticancer agents are known to induce cellular senescence, but diagnostic procedures to non-invasively detect a senescent proliferation arrest of cancer lesions in response to chemotherapy in vivo remain to be [...]

Transcription factors bind in a combinatorial fashion to specify the on-and-off states of genes; the ensemble of these binding events forms a regulatory network, constituting the wiring diagram for a cell. To examine the principles of the human transcriptional regulatory network, we determined the genomic binding information of 119 transcription-related factors in over 450 distinct experiments. We found the combinatorial, co-association of transcription factors to be highly context specific: distinct combinations of factors bind at specific genomic locations. In particular, there are significant differences in the binding proximal and distal to genes. We organized all the transcription factor binding into a hierarchy and integrated it with other genomic information (for example, microRNA regulation), forming a dense meta-network. Factors at different levels have different properties; for instance, top-level transcription factors more strongly influence expression and middle-level ones co-regulate targets to mitigate information-flow bottlenecks. Moreover, these co-regulations give rise to many enriched network motifs (for example, noise-buffering feed-forward loops). Finally, more connected network components are under stronger selection and exhibit a greater degree of allele-specific activity (that is, differential binding to the two parental alleles). The regulatory information obtained in this study will be crucial for interpreting personal genome sequences and understanding basic principles of human biology and disease., A central goal in biology is to understand how a limited cohort of transcription factors is able to organize the large diversity of gene-expression patterns in different cell types and [...]

Plasmodium falciparum is the major cause of malaria globally and is transmitted by mosquitoes. During parasitic development, P. falciparum-infected erythrocytes (P. falciparum-IEs) express multiple polymorphic proteins known as variant surface antigens (VSAs), including the P. falciparum erythrocyte membrane protein 1 (PfEMP1). VSA-specific antibodies are associated with protection from symptomatic and severe malaria. However, the importance of the different VSA targets of immunity to malaria remains unclear, which has impeded an understanding of malaria immunity and vaccine development. In this study, we developed assays using transgenic P. falciparum with modified PfEMP1 expression to quantify serum antibodies to VSAs among individuals exposed to malaria. We found that the majority of the human antibody response to the IE targets PfEMP1. Furthermore, our longitudinal studies showed that individuals with PfEMP1-specific antibodies had a significantly reduced risk of developing symptomatic malaria, whereas antibodies to other surface antigens were not associated with protective immunity. Using assays that measure antibody-mediated phagocytosis of IEs, an important mechanism in parasite clearance, we identified PfEMP1 as the major target of these functional antibodies. Taken together, these data demonstrate that PfEMP1 is a key target of humoral immunity. These findings advance our understanding of the targets and mediators of human immunity to malaria and have major implications for malaria vaccine development., Introduction Malaria caused by the protozoan parasite Plasmodium falciparum is a major burden of disease globally, causing an estimated 225 million illness episodes and around 800,000 deaths per year (1). [...]

Cells of the immune system have evolved various molecular mechanisms to sense their environment and react to alterations of self. NK cells are lymphocytes with effector and regulatory functions, which are remarkably adaptable to changes in self. In a study published in this issue of the JCI, Tarek and colleagues report the clinical benefits of manipulating NK cell adaptation to self in an innovative mAb-based therapy against neuroblastoma (NB). This novel therapeutic strategy should stimulate further research on NK cell therapies., NK cells are involved in the elimination of tumor cells and infected cells (1); they can kill their cellular targets via cytotoxic granule exocytosis and also secrete cytokines such as [...]

Peroxisomes are eukaryotic organelles important for the metabolism of long-chain fatty acids (1,2). Here we show that in numerous fungal species, several core enzymes of glycolysis, including glyceraldehyde3-phosphate dehydrogenase (GAPDH) and 3-phosphoglycerate kinase (PGK), reside in both the cytoplasm and peroxisomes. We detected in these enzymes cryptic type 1 peroxisomal targeting signals (PTS1) (3), which are activated by post-transcriptional processes. Notably, the molecular mechanisms that generate the peroxisomal isoforms vary considerably among different species. In the basidiomycete plant pathogen Ustilago maydis, peroxisomal targeting of Pgk1 results from ribosomal read-through, whereas alternative splicing generates the PTS1 of Gapdh. In the filamentous ascomycete Aspergillus nidulans, peroxisomal targeting of these enzymes is achieved by exactly the opposite mechanisms. We also detected PTS1 motifs in the glycolytic enzymes triose-phosphate isomerase and fructose-bisphosphate aldolase. U. maydis mutants lacking the peroxisomal isoforms of Gapdh or Pgk1 showed reduced virulence. In addition, mutational analysis suggests that GAPDH, together with other peroxisomal NADH-dependent dehydrogenases, has a role in redox homeostasis. Owing to its hidden nature, partial peroxisomal targeting of well-studied cytoplasmic enzymes has remained undetected. Thus, we anticipate that further bona fide cytoplasmic proteins exhibit similar dual targeting., Peroxisomes are highly versatile organelles involved in many biological processes in addition to their common function in fatty-acid metabolism and hydrogen peroxide degradation (4). In plants, specialized peroxisomes (glyoxysomes) are [...]

Merkel cell polyomavirus (MCV) is the recently discovered cause of most Merkel cell carcinomas (MCCs), an aggressive form of nonmelanoma skin cancer. Although MCV is known to integrate into the tumor cell genome and to undergo mutation, the molecular mechanisms used by this virus to cause cancer are unknown. Here, we show that MCV small T (sT) antigen is expressed in most MCC tumors, where it is required for tumor cell growth. Unlike the closely related SV40 sT, MCV sT transformed rodent fibroblasts to anchorage-and contact-independent growth and promoted serum-free proliferation of human cells. These effects did not involve protein phosphatase 2A (PP2A) inhibition. MCV sT was found to act downstream in the mammalian target of rapamycin (mTOR) signaling pathway to preserve eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) hyperphosphorylation, resulting in dysregulated cap-dependent translation. MCV sT-associated 4E-BP1 serine 65 hyperphosphorylation was resistant to mTOR complex (mTORC1) and mTORC2 inhibitors. Steady-state phosphorylation of other downstream Akt-mTOR targets, including S6K and 4E-BP2, was also increased by MCV sT. Expression of a constitutively active 4E-BP1 that could not be phosphorylated antagonized the cell transformation activity of MCV sT. Taken together, these experiments showed that 4E-BP1 inhibition is required for MCV transformation. Thus, MCV sT is an oncoprotein, and its effects on dysregulated cap-dependent translation have clinical implications for the prevention, diagnosis, and treatment of MCV-related cancers., Introduction Polyomavirus research has been central to cancer biology (1). Studies on simian vacuolating virus 40 (SV40) T antigen led to the discovery of p53 and uncovered functions for the [...]

Puberty onset is initiated by activation of neurons that secrete gonadotropin-releasing hormone (GnRH). The timing and progression of puberty may depend upon temporal coordination of two opposing central mechanisms--a restraint of GnRH secretion before puberty onset, followed by enhanced stimulation of GnRH release to complete reproductive maturation during puberty. Neuronal estrogen receptor [alpha] (ER[alpha]) has been implicated in both controls; however, the underlying neural circuits are not well understood. Here we test whether these mechanisms are mediated by neurons that express kisspeptin, a neuropeptide that modulates GnRH neurosecretion. Strikingly, conditional ablation of ER[alpha] in kisspeptin neurons results in a dramatic advancement of puberty onset in female mice. Furthermore, subsequent pubertal maturation is arrested in these animals, as they fail to acquire normal ovulatory cyclicity. We show that the temporal coordination of juvenile restraint and subsequent pubertal activation is likely mediated by ER[alpha] in two separate kisspeptin neuronal populations in the hypothalamus. estradiol-17[beta] | conditional gene targeting | kiss1 | arcuate nucleus | anteroventral periventricular nucleus doi/ 10.1073/pnas.1012406108

Trophoblast invasion likely depends on complex cross talk between the fetal and maternal tissues and may involve the modulation of phosphatidylinositol 3-kinase (PI3K)/AKT signaling activity in maternal decidual cells. In this report, we studied implantation in [Pten.sup.tm1Hwu/tm1Hwu];[Amhr2.sup.tm3(cre)Bhr/+] mice, which lack the PI3K signaling antagonist gene Pten in myometrial and stromal/decidual cells. Primiparous [Pten.sup.tm1Hwu/tm1Hwu]; [Amhr2.sup.tm3(cre)Bhr/+] mice were found to be subfertile because of increased fetal mortality at e 11.5. Histopathological analyses revealed a failure of decidual regression in these mice, accompanied by reduced or absent invasion of fetal trophoblast glycogen cells and giant cells, abnormal development of the placental labyrinth, and frequent apparent intrauterine fetal growth restriction. Unexpectedly, the loss of phosphate and tensin homolog deleted on chromosome l0 (PTEN) expression in [Pten.sup.tm1Hwu/tm1Hwu];[Amhr2.sup.tm3(cre)Bhr/+] decidual cells was not accompanied by a detectable increase in AKT phosphorylation or altered expression or activation of PI3K/AKT downstream effectors such as mammalian target of rapamycin or glycogen synthase kinase-3[beta]. Terminal deoxynucleotidyl transferase-mediated nick end labeling and bromodeoxyuridine incorporation analyses attributed to the lack of decidual regression mainly to decreased apoptosis in [Pten.sup.tm1Hwu/tm1Hwu];[Amhr2.sup.tm3(cre)Bhr/+] decidual cells, rather than to increased proliferation. Remodeling of the maternal vasculature was delayed in [Pten.sup.tm1Hwu/tm1Hwu];[Amhr2.sup.tm3(cre)Bhr/+] uteri at e1 1.5, as evidenced by persistence of vascular smooth muscle and decreased infiltration of uterine natural killer cells. In addition, thickening of the myometrium and disorganization of the muscle fibers were observed before and throughout gestation. Almost all [Pten.sup.tm1Hwu/tm1Hwu];[Amhr2.sup.tm3(cre)Bhr/+] mice failed to carry a second litter to term, apparently attributable to endometrial hyperplasia and uterine infections. Together, these data demonstrate novel roles of PTEN in the mammalian uterus and its requirement for proper trophoblast invasion and decidual regression. phosphate and tensin homolog deleted on chromosome 10; AKT; decidua; myometrium; conditional gene targeting doi: 10.1152/ajpendo.00255.2010.

In the absence of treatment, most HIV-1-infected humans develop AIDS. However, a minority are long-term nonprogressors, and resistance is associated with the presence of particular HLA-[B.sup.*]27/ [B.sup.*]57 molecules. In contrast, most HIV-1-infected chimpanzees do not contract AIDS. In comparison with humans, chimpanzees experienced an ancient selective sweep affecting the MHC class I repertoire. We have determined the peptide-binding properties of frequent chimpanzee MHC class I molecules, and show that, like HLA-[B.sup.*]27/[B.sup.*]57, they target similar conserved areas of HIV-1/[SIV.sub.cpz]. In addition, many animals appear to possess multiple molecules targeting various conserved areas of the HIV-1/[SIV.sub.cpz] Gag protein, a quantitative aspect of the immune response that may further minimize the chance of viral escape. The functional characteristics of the contemporary chimpanzee MHC repertoire suggest that the selective sweep was caused by a lentiviral pandemic. HIV/SIV | peptide-binding | selection | selective sweep doi/ 10.1073/pnas.1009136107

At present, genetically modified rats have not been generated from ES cells because stable ES cells and a suitable injection method are not available. To monitor the pluripotency of rat ES cells, we generated Oct4-Venus transgenic (Tg) rats via a conventional method, in which Venus is expressed by the Oct4 promoter/enhancer. This monitoring system enabled us to define a significant condition of culture to establish authentic rat ES cells based on a combination of 20% FBS and cell signaling inhibitors for Rho-associated kinase, mitogen-activated protein kinase, TGF-[beta], and glycogen synthase kinase-3. The rat ES cells expressed ES cell markers such as Oct4, Nanog, Sox2, and Rex1 and retained a normal karyotype. Embryoid bodies and teratomas were also produced from the rat ES cells. All six ES cell lines derived from three different rat strains successfully achieved germline transmission, which strongly depended on the presence of the inhibitors during the injection process. Most importantly, high-quality Tg rats possessing a correct transgene expression pattern were successfully generated via the selection of gene-manipulated ES cell clones through germ-line transmission. Our rat ES cells should be sufficiently able to receive gene targeting as well as Tg manipulation, thus providing valuable animal models for the study of human diseases. genetic engineering | rat | embryonic stem cells doi/ 10.1073/pnas.1009582107

It is commonly assumed but not proven that microRNAs (miRNAs) and their targets coevolve. Under this assumption, miRNAs and targets from different species may interact adversely, resulting in reduced fitness. However, the strength of the adverse interactions may not be detectable because even outright deletions of miRNAs often manifest only subtle fitness effects. We tested and measured the strength of heterospecific interactions by carrying out transgenic experiments across Drosophila species by overexpressing the miR310s cluster of Drosophila melanogaster (Dm310s) and Drosophila pseudoobscura (Dp310s) in D. melanogaster. Flies overexpressing the heterospecific Dp310s are only one-third as viable as those overexpressing the conspecific Dm310s. The viability effect is easily detectable in comparison to the effect of the deletion of miR310s. The number of genes significantly misexpressed under the influence of Dp310s is 3-10 times greater than under Dm310s. Importantly, the numbers of predicted targets are similar between them. Expression analysis of the predicted target genes suggests that miRNAs may sometimes function to buffer fluctuations in the transcriptome output. After the buffering function has evolved, heterospecific combinations may cause adverse effects, microRNAs | coevolution | canalization | genetic buffering doi/ 10.1073/pnas.1007591107

In Drosophila, microRNAs (miRNAs) typically guide Argonaute1 to repress messenger RNA (mRNA), whereas small interfering RNAs (siRNAs) guide Argonaute2 to destroy viral and transposon RNA. Unlike siRNAs, miRNAs rarely form extensive numbers of base pairs to the mRNAs they regulate. We find that extensive complementarity between a target RNA and an Argonaute1-bound miRNA triggers miRNA tailing and 3'-to-5' trimming. In flies, Argonaute2-bound small RNAs--but not those bound to Argonautel--bear a 2'-O-methyl group at their 3' ends. This modification blocks target-directed small RNA remodeling: In flies lacking Hen1, the enzyme that adds the 2'-O-methyl group, Argonaute2-associated siRNAs are tailed and trimmed. Target complementarity also affects small RNA stability in human ceils. These results provide an explanation for the partial complementarity between animal miRNAs and their targets. doi: 10.1126/science.1187058

More than 200 direct CodY target genes in Staphylococcus aureus were identified by genome-wide analysis of in vitro DNA binding. This analysis, which was confirmed for some genes by DNase I footprinting assays, revealed that CodY is a direct regulator of numerous transcription units associated with amino acid biosynthesis, transport of macromolecules, and virulence. The virulence genes regulated by CodY fell into three groups. One group was dependent on the Agr system for its expression; these genes were indirectly regulated by CodY through its repression of the agr locus. A second group was regulated directly by CodY. The third group, which includes genes for alpha-toxin and capsule synthesis, was regulated by CodY in two ways, i.e., by direct repression and by repression of the agr locus. Since S. aureus CodY was activated in vitro by the branched chain amino acids and GTP, CodY appears to link changes in intracellular metabolite pools with the induction of numerous adaptive responses, including virulence. doi: 10.1128/JB.00220-10

We present a computational method for identifying potential targets of a transcription factor (TF) using wild-type gene expression time series data. For each putative target gene we fit a simple differential equation model of transcriptional regulation, and the model likelihood serves as a score to rank targets. The expression profile of the TF is modeled as a sample from a Gaussian process prior distribution that is integrated out using a nonparametric Bayesian procedure. This results in a parsimonious model with relatively few parameters that can be applied to short time series datasets without noticeable overfitting. We assess our method using genome-wide chromatin immunoprecipitation (ChiP-chip) and loss-of-function mutant expression data for two TFs, Twist, and Mef2, controlling mesoderm development in Drosophila. Lists of topranked genes identified by our method are significantly enriched for genes close to bound regions identified in the ChiP-chip data and for genes that are differentially expressed in loss-of-function mutants. Targets of Twist display diverse expression profiles, and in this case a model-based approach performs significantly better than scoring based on correlation with TF expression. Our approach is found to be comparable or superior to ranking based on mutant differential expression scores, Also, we show how integrating complementary wild-type spatial expression data can further improve target ranking performance. Bayesian inference | Gaussian process inference | gene regulation | gene regulatory network | systems biology doi/ 10.1073/pnas.0914285107

A bioengineering method for self-assembly of multifunctional superstructures with in-advance programmable properties has been proposed. The method employs two unique proteins, barnase and barstar, to rapidly join the structural components together directly in water solutions. The properties of the superstructures can be designed on demand by linking different agents of various sizes and chemical nature, designated for specific goals. As a proof of concept, colloidally stable trifunctional structures have been assembled by binding together magnetic particles, quantum dots, and antibodies using barnase and barstar. The assembly has demonstrated that the bonds between these proteins are strong enough to hold macroscopic (5 nm-3 [micro]m) particles together. Specific interaction of such superstructures with cancer cells resulted in fluorescent labeling of the cells and their responsiveness to magnetic field. The method can be used to join inorganic moieties, organic particles, and single biomolecules for synergistic use in different applications such as biosensors, photonics, and nanomedicine. superstructures | cancer cells | targeting | fusion proteins | magnetic nanoparticles doi/10.1073/pnas.1001142107

Autophagy is a catabolic process by which cells remove long-lived proteins and damaged organelles for recycling. Viral infections may also induce autophagic response. Here we show that hepatitis B virus (HBV), a pathogen that chronically infects [approximately equal to]350 million people globally, can enhance autophagic response in cell cultures, mouse liver, and during natural infection. This enhancement of the autophagic response is not coupled by an increase of autophagic protein degradation and is dependent on the viral X protein, which binds to and enhances the enzymatic activity of phosphatidylinositol 3-kinase class III, an enzyme critical for the initiation of autophagy. Further analysis indicates that autophagy enhances HBV DNA replication, with minimal involvement of late autophagic vacuoles in this process. Our studies thus demonstrate that a DNA virus can use autophagy to enhance its own replication and indicate the possibility of targeting the autophagic pathway for the treatment of HBV patients. autophagy | hepatitis B virus DNA replication | hepatitis B virus X protein | PI3KC3 doi/10.1073/pnas.0911373107

Caenorhabditis elegans fern-3 binding factor (FBF) is a founding member of the PUMILIO/FBF (PUF) family of mRNA regulatory proteins. It regulates multiple mRNAs critical for stem cell maintenance and germline development. Here, we report crystal structures of FBF in complex with 6 different 9-nt RNA sequences, including elements from 4 natural mRNAs. These structures reveal that FBF binds to conserved bases at positions 1-3 and 7-8. The key specificity determinant of FBF vs. other PUF proteins lies in positions 4-6. In FBF/RNA complexes, these bases stack directly with one another and turn away from the RNA-binding surface. A short region of FBF is sufficient to impart its unique specificity and lies directly opposite the flipped bases. We suggest that this region imposes a flattened curvature on the protein; hence, the requirement for the additional nucleotide. The principles of FBF/RNA recognition suggest a general mechanism by which PUF proteins recognize distinct families of RNAs yet exploit very nearly identical atomic contacts in doing so. crystal structure | RNA | translational regulation | fern-3 binding factor | base flipping www.pnas.org./cgi/doi/10.1073/pnas.0812076106

The moss Physcomitrella is unique among plants in that it permits efficient gene targeting by homologous recombination. Furthermore, transformed DNA can replicate episomally in Physcomitrella. Here we show that episomally replicating DNA can be rescued back into Escherichia coil, and we use such rescue to study the fate of the transformed DNA. Significantly, plasmids rescued from moss transformed with circular DNA are identical to the original plasmid, whereas plasmids rescued from moss transformed with linearized DNA frequently have deletions created by direct repeat recombination. These events are highly predictable in that they target the longest direct repeat on the plasmid if this repeat is at least 12 bp. Episomal transformants obtained with linearized DNA show a more than 1,000-fold amplification of the DNA whereas transformants obtained with circular DNA have much lower copy numbers. Most episomal transformants quickly lose the plasmid in the absence of selection, but a semistable type of transformant that loses the plasmid at a much lower frequency was also observed. The consistent rescue of the original plasmid, or of predictable derivatives thereof, suggests that molecular genetics methods which rely on shuttle plasmids are feasible in Physcomitrella. episomal replication | plasmid rescue | recombination | shuttle plasmid www.pnas.org/cgi/doi/10.1073/pnas.0908037106

Reversible insertion of IS492 at a site within epsG on the Pseudoalteromonas atlantica chromosome controls peripheral extracellular polysaccharide production and biofilm formation by P. atlantica. High-frequency precise excision of IS492 from epsG requires 5 and 7 bp of flanking DNA, suggesting that IS492 transposition involves a site-specific recombination mechanism. The site specificity of IS492 insertion was examined in P. atlantica and shown to be specific for a 7-bp target, 5'-CTTGTTA-3'. Characterization of numerous insertion events at the target site in epsG indicated that insertion is also orientation specific. The frequency of IS492 insertion at the epsG target site (2.7 x [10.sup.-7]/cell/generation), determined by quantitative PCR, is 4 to 5 orders of magnitude lower than the frequency of IS492 precise excision from the same site. Comparison of insertion sites for IS492 and the highly related ISPtu2 from Pseudoalteromonas tunicata suggests DNA sequence and/or structural features that may contribute to site recognition and recombination by the transposase of IS492. doi: 10.1128/JB.00771-09

Heterochromatic gene silencing results from the establishment of a repressive chromatin structure over reporter genes. Gene silencing is often variegated, implying that chromatin may stochastically switch from repressive to permissive structures as cells divide. To identify remodeling enzymes involved in reorganizing heterochromatin, we tested 11 SNF2-type chromatin remodelers in Drosophila for effects on gene silencing. Overexpression of five remodelers affects gene silencing, and the most potent de-repressor is the [alpha]-thalassaemia mental retardation syndrome X-linked (ATRX) homolog X-linked nuclear protein (XNP). Although the mammalian ATRX protein localizes to heterochromatin, Drosophila XNP is not a general component of heterochromatin. Instead, XNP localizes to active genes and to a major focus near the heterochromatin of the X chromosome. The XNP focus corresponds to an unusual decondensed satellite DNA block, and both active genes and the XNP focus are sites of ongoing nucleosome replacement. We suggest that the XNP remodeler modulates nucleosome dynamics at its target sites to limit chromatin accessibility. Although XNP at active genes may contribute to gene silencing, we find that a single focus is present across Drosophila species and that perturbation of this site cripples heterochromatic gene silencing. Thus, the XNP focus appears to be a functional genetic element that can contribute to gene silencing throughout the nucleus. heterochromatin | nucleosome dynamics | chromatin remodelers

WT1, a critical regulator of kidney development, is a tumor suppressor for nephroblastoma but in some contexts functions as an oncogene. A limited number of direct transcriptional targets of WT1 have been identified to explain its complex roles in tumorigenesis and organogenesis. In this study we performed genome-wide screening for direct WT1 targets, using a combination of ChIP-ChIP and expression arrays. Promoter regions bound by WT1 were highly G-rich and resembled the sites for a number of other widely expressed transcription factors such as SP1, MAZ, and ZNF219. Genes directly regulated by WT1 were implicated in MAPK signaling, axon guidance, and Wnt pathways. Among directly bound and regulated genes by Wr1, nine were identified in the Wnt signaling pathway, suggesting that WT1 modulates a subset of Wnt components and responsive genes by direct binding. To prove the biological importance of the interplay between WT1 and Wnt signaling, we showed that WT1 blocked the ability of Wnt8 to induce a secondary body axis during Xenopus embryonic development. WT1 inhibited TCF-mediated transcription activated by Wnt ligand, wild type and mutant, stabilized [beta]-catenin by preventing TCF4 loading onto a promoter. This was neither due to direct binding of WT1 to the TCF binding site nor to interaction between WT1 and TCF4, but by competition of WT1 and TCF4 for CBP. WT1 interference with Writ signaling represents an important mode of its action relevant to the suppression of tumor growth and guidance of development. ChIP-ChIP | microarray | tumor suppressor

Using zinc-finger nucleases (ZFNs) to cleave the chromosomal target, we have achieved high frequencies of gene targeting in the Drosophila germline. Both local mutagenesis through non-homologous end joining (NHEJ) and gene replacement via homologous recombination (HR) are stimulated by target cleavage. In this study we investigated the mechanisms that underlie these processes, using materials for the rosy (ry) locus. The frequency of HR dropped significantly in flies homozygous for mutations in spnA (Rad51) or okr (Rad54), two components of the invasion-mediated synthesis-dependent strand annealing (SDSA) pathway. When single-strand annealing (SSA) was also blocked by the use of a circular donor DNA, HR was completely abolished. This indicates that the majority of HR proceeds via SDSA, with a minority mediated by SSA. In flies deficient in lig4 (DNA ligase IV), a comportent of the major NHEJ pathway, the proportion of HR products rose significantly. This indicates that most NHEJ products are produced in a lig4-dependent process. When both spnA and lig4 were mutated and a circular donor was provided, the frequency of ry mutations was still high and no HR products were recovered. The local mutations produced in these circumstances must have arisen through an alternative, lig4-independent end-joining mechanism. These results show what repair pathways operate on double-strand breaks in this gene targeting system. They also demonstrate that the outcome can be biased toward gene replacement by disabling the major NHEJ pathway and toward simple mutagenesis by interfering with the major HR process. DOI: 10.1534/genetics.109.101329

A variety of viruses establish latency by integrating their genome into the host genome. The integration event generally occurs in a nonspecific manner, precluding the prediction of functional consequences from resulting disruptions of affected host genes. The nonpathogenic adeno-associated virus (AAV) is unique in its ability to stably integrate in a site-specific manner into the human MBS85 gene. To gain a better understanding of the integration mechanism and the consequences of MBS85 disruption, we analyzed the molecular structure of AAV integrants in various latently infected human cell lines. Our study led to the observation that AAV integration causes an extensive but partial duplication of the target gene. Intriguingly, the molecular organization of the integrant leaves the possibility that a functional copy of the disrupted target gene could potentially be preserved despite the resulting rearrangements. A latently infected, Mbs85-targeted mouse ES cell line was generated to study the functional consequences of the observed duplication-based integration mechanism. AAV-modified ES cell lines continued to self-renew, maintained their multilineage differentiation potential and contributed successfully to mouse development when injected into blastocysts. Thus, our study reveals a viral strategy for targeted genome addition with the apparent absence of functional consequences. embryonic stem cells | MBS85 | gene targeting | Rep | AAVS1

The 'GTPase switch' paradigm, in which a GTPase switches between an active, GTP-bound state and an inactive, GDP-bound state through the recruitment of nucleotide exchange factors (GEFs) or GTPase activating proteins (GAPs), has been used to interpret the regulatory mechanism of many GTPases. A notable exception to this paradigm is provided by two GTPases in the signal recognition particle (SRP) and the SRP receptor (SR) that control the co-translational targeting of proteins to cellular membranes. Instead of the classical 'GTPase switch,' both the SRP and SR undergo a series of discrete conformational rearrangements during their interaction with one another, culminating in their reciprocal GTPase activation. Here, we show that this series of rearrangements during SRP-SR binding and activation provide important control points to drive and regulate protein targeting. Using real-time fluorescence, we showed that the cargo for SRP-ribosomes translating nascent polypeptides with signal sequences--accelerates SRP*SR complex assembly over 100-fold, thereby driving rapid delivery of cargo to the membrane. A series of subsequent rearrangements in the SRP*SR GTPase complex provide important driving forces to unload the cargo during late stages of protein targeting. Further, the cargo delays GTPase activation in the SRP*SR complex by 8-12 fold, creating an important time window that could further improve the efficiency and fidelity of protein targeting. Thus, the SRP and SR GTPases, without recruiting external regulatory factors, constitute a self-sufficient system that provides exquisite spatial and temporal control of a complex cellular process. conformational change | fluorescence spectroscopy | protein targeting and translocation | signal recognition particle

To cleave DNA, Type III restriction enzymes must communicate the relative orientation of two asymmetric recognition sites over hundreds of base pairs. The basis of this long-distance communication, for which ATP hydrolysis by their helicase domains is required, is poorly understood. Several conflicting DNA-Looping mechanisms have been proposed, driven either by active DNA translocation or passive 3D diffusion. Using single-molecule DNA stretching in combination with bulk-solution assays, we provide evidence that looping is both highly unlikely and unnecessary, and that communication is strictly confined to a 1D route. Integrating our results with previous data, a simple communication scheme is concluded based on 1D diffusion along DNA. 1D diffusion | ATPase | helicase | single molecule | motor protein

A subtype of diffuse large B-cell lymphoma (DLBCL), termed activated B-cell-like (ABC) DLBCL, depends on constitutive nuclear factor-[kappa]B (NF-[kappa]B) signaling for survival. Small molecule inhibitors of I[kappa]B kinase [beta] (IKK[beta]), a key regulator of the NF-[kappa]B pathway, kill ABC DLBCL cells and hold promise for the treatment of this lymphoma type. We conducted an RNA interference genetic screen to investigate potential mechanisms of resistance of ABC DLBCL cells to IKK[beta] inhibitors. We screened a library of small hairpin RNAs (shRNAs) targeting 500 protein kinases for shRNAs that would increase the killing of an ABC DLBCL cell line in the presence of a small molecule IKK[beta] inhibitor. Two independent shRNAs targeting IKK[alpha] synergized with the IKK[beta] inhibitor to kill three different ABC DLBCL cell lines but were not toxic by themselves. Surprisingly, IKK[alpha] shRNAs blocked the classical rather than the alternative NF-[kappa]B pathway in ABC DLBCL cells, as judged by inhibition of I[kappa]B[alpha] phosphorylation. IKK[alpha] shRNA toxicity was reversed by coexpression of wild-type but not kinase inactive forms of IKK[alpha], suggesting that IKK[alpha] may directly phosphorylate I[kappa]B[alpha] under conditions of IKK[beta] inhibition. In models of physiologic NF-[kappa]B pathway activation by CARD11 or tumor necrosis factor-[alpha], compensatory IKK[alpha] activity was also observed with IKK[beta] inhibition. These results suggest that therapy for ABC DLBCL may be improved by targeting both IKK[alpha] and IKK[beta], possibly through CARD11 inhibition. Ikka | Ikkb | Nf-kB | RNAi

Chk1 is widely known as a DNA damage checkpoint signaling protein. Unlike many other checkpoint proteins, Chk1 also plays an essential but poorly defined role in the proliferation of unperturbed cells. Activation of Chk1 after DNA damage is known to require the phosphorylation of several C-terminal residues, including the highly conserved S317 and S345 sites. To evaluate the respective roles of these individual sites and assess their contribution to the functions of Chk1, we used a gene targeting approach to introduce point mutations into the endogenous human CHK1 locus. We report that the essential and nonessential functions of Chk1 are regulated through distinct phosphorylation events and can be genetically uncoupled. The DNA damage response function of Chk1 was nonessential. Targeted mutation of S317 abrogated [G.sub.2]/M checkpoint activation, prevented subsequent phosphorylation of Chk1, impaired efficient progression of DNA replication forks, and increased fork stalling, but did not impact viability. Thus, the nonessential DNA damage response function of Chk1 could be unambiguously linked to its role in DNA replication control. In contrast, a CHK1 allele with mutated S345 did not support viability, indicating an essential role for this residue during the unperturbed cell cycle. A distinct, physiologic mode of S345 phosphorylation, initiated at the centrosome during unperturbed mitosis was independent of codon 317 status and mechanistically distinct from the ordered and sequential phosphorylation of serine residues on Chk1 induced by DNA damage. Our findings suggest an essential regulatory role for Chk1 phosphorylation during mitotic progression. ATR | centrosome | phosphorylation | mitosis | gene targeting

We report very high gene targeting frequencies in Drosophila by direct embryo injection of mRNAs encoding specific zinc-finger nucleases (ZFNs). Both local mutagenesis via nonhomologous end joining (NHEJ) and targeted gene replacement via homologous recombination (HR) have been achieved in up to 10% of all targets at a given locus. In embryos that are wild type for DNA repair, the products are dominated by NHEJ mutations. In recipients deficient in the NHEJ component, DNA ligase IV, the majority of products arise by HR with a coinjected donor DNA, with no loss of overall efficiency in target modification. We describe the application of the ZFN injection procedure to mutagenesis by NHEJ of 2 new genes in Drosophila melanogaster: coil and pask. Pairs of novel ZFNs designed for targets within those genes led to the production of null mutations at each locus. The injection procedure is much more rapid than earlier approaches and makes possible the generation and recovery of targeted gene alterations at essentially any locus within 2 fly generations. coilin | DNA ligase IV | DNA repair | PAS kinase | targeted mutagenesis

Previous studies have shown that a dominant negative form of c-Jun (TAM67) suppresses mouse skin carcinogenesis both in vitro and in vivo. The current study identifies Sulfiredoxin (Srx) as a unique target of activator protein-1 (AP-1) activation and TAM67 inhibition. Manipulation of Srx levels by ShRNA or over-expression demonstrates that Srx is critical for redox homeostasis through reducing hyperoxidized peroxiredoxins. In JB6 cells, knockdown of Srx abolishes tumor promoter-induced transformation and enhances cell sensitivity to oxidative stress. Knockdown of Srx also impairs c-Jun phosphorylation, implicating a role for Srx in the feedback regulation of AP-1 activity. Screening of patient tissues by tissue microarray reveals elevated Srx expression in several types of human skin cancers. Our study indicates that Srx is a functionally significant target of AP-1 blockade that may have value in cancer prevention or treatment. skin tumor | tumor promotion | peroxiredoxin

Translocation of the tRNA x mRNA complex through the bacterial ribosome is driven by the multidomain guanosine triphosphatase elongation factor G (EF-G). We have used isothermal titration calorimetry to characterize the binding of GDP and GTP to free EF-G at 4[degrees]C, 20[degrees]C, and 37[degrees]C. The binding affinity of EF-G is higher to GDP than to GTP at 4[degrees]C, but lower at 37[degrees]C. The binding enthalpy and entropy change little with temperature in the case of GDP binding but change greatly in the case of GTP binding. These observations are compatible with a large decrease in the solvent-accessible hydrophobic surface area of EF-G on GTP, but not GDP, binding. The explanation we propose is the locking of the switch 1 and switch 2 peptide loops in the G domain of EF-G to the [gamma]-phosphate of GTP. From these data, in conjunction with previously reported structural data on guanine nucleotide-bound EF-G, we suggest that EF-G enters the pretranslocation ribosome as an 'activity chimera,' with the G domain activated by the presence of GTP but the overall factor conformation in the inactive form typical of a GDP-bound multidomain guanosine triphosphatase. We propose that the active overall conformation of EF-G is attained only in complex with the ribosome in its 'ratcheted state,' with hybrid tRNA binding sites. GTPase | guanine nucleotide binding | isothermal titration calorimetry | thermodynamic parameters of interaction

Recognition sites for microRNAs (miRNAs) have been reported to be located in the 3' untranslated regions of transcripts. In a computational screen for highly conserved motifs within coding regions, we found an excess of sequences conserved at the nucleotide level within coding regions in the human genome, the highest scoring of which are enriched for miRNA target sequences. To validate our results, we experimentally demonstrated that the let-7 miRNA directly targets the miRNA-processing enzyme Dicer within its coding sequence, thus establishing a mechanism for a miRNA/Dicer autoregulatory negative feedback loop. We also found computational evidence to suggest that miRNA target sites in coding regions and 3' UTRs may differ in mechanism. This work demonstrates that miRNAs can directly target transcripts within their coding region in animals, and it suggests that a complete search for the regulatory targets of miRNAs should be expanded to include genes with recognition sites within their coding regions. As more genomes are sequenced, the methodological approach that we used for identifying motifs with high sequence conservation will be increasingly valuable for detecting functional sequence motifs within coding regions. computational biology | posttranscriptional regulation | comparative genomics | multiple-sequence alignment | evolutionary conservation

A functional RNase P ribozyme (M 1GS RNA) was constructed to target the overlapping mRNA region of two murine cytomegalovirus (MCMV) capsid proteins essential for viral replication: the assembly protein (mAP) and M80. The customized ribozyme efficiently cleaved the target mRNA sequence in vitro. Moreover, 80% reduction in the expression of mAP and M80 and a 2,000-fold reduction in viral growth were observed in cells expressing the ribozyme. In contrast, there was no significant reduction in viral gene expression and growth in cells that either did not express the ribozyme or produced a 'disabled' ribozyme carrying mutations that abolished its catalytic activity. When the ribozyme-expressing constructs were delivered into MCMV-infected SCID mice via a modified 'hydrodynamic transfection' procedure, expression of ribozymes was observed in the livers and spleens. Compared with the control animals that did not receive any MIGS constructs or received the disabled ribozyme construct, animals receiving the functional ribozyme construct exhibited a significant reduction of viral gene expression and infection. Viral titers in the spleens, livers, lungs, and salivary glands of the functional ribozyme-treated SaD mice at 21 days after infection were 200- to 2,000-fold lower than those in the control animals. Moreover, survival of the infected animals significantly improved upon receiving the functional ribozyme construct. Our study examines the use of MIGS ribozymes for inhibition of gene expression in animals and demonstrates the utility of RNase P ribozymes for gene targeting applications in vivo. cytomegalovirus | gene targeting | herpesvirus | antisense

The Saccharomyces cerevisiae RAD54 gene has critical roles in DNA double-strand break repair, homologous recombination, and gene targeting. Previous results show that the yeast gene enhances gene targeting when expressed in Arabidopsis thaliana. In this work we address the trans-species compatibility of Rad54 functions. We show that overexpression of yeast RAD54 in Arabidopsis enhances DNA damage resistance several fold. Thus, the yeast gene is active in the Arabidopsis homologous-recombination repair system. Moreover, we have identified an A. thaliana ortholog of yeast RAD54, named AtRAD54. This gene, with close sequence similarity to RAD54, complements methylmethane sulfonate (MMS) sensitivity but not UV sensitivity or gene targeting defects of rad54[DELTA] mutant yeast cells. Overexpression of AtRAD54 in Arabidopsis leads to enhanced resistance to DNA damage. This gene's assignment as a RAD54 ortholog is further supported by the interaction of AtRad54 with AtRad51 and the interactions between alien proteins (i.e., yeast Rad54 with AtRAD51 and yeast Rad51 with AtRad54) in a yeast two-hybrid experiment. These interactions hint at the molecular nature of this interkingdom complementation, although the stronger effect of the yeast Rad54 in plants than AtRad54 in yeast might be explained by an ability of the Rad54 protein to act alone, independently of its interaction with Rad51.

Oxygen-regulated protein 150 (ORP150) is an inducible endoplasmic reticulum (ER) chaperone molecule that is upregulated after numerous cellular insults and has a cytoprotective role in renal, neural, and cardiac models of ischemiareperfusion injury. ORP150 also has been shown to play a role in cellular [Ca.sup.2+] homeostasis, and in turn, regulating calpain activity. In this study, we identified ORP 150 in whole rat renal cortical mitochondria and matrix fractions, demonstrated the targeting of an ORP150GFP construct to the mitochondria of NIH-3T3 cells, and showed that the NH2-terminal 13 amino acids of ORP150 are sufficient for this translocation. ORP150 expression was found to be regulated by the anti-C/enhancer-binding protein homologous protein (CHOP)/ GADD153 transcription factor and ORP150 levels increased in the mitochondria and ER of COS-7 cells after diverse stresses, including hypoxia, serum starvation, prolyl hydroxylase inhibition with dimethyloxaloylglycine, and exposure to tunicamycin, ethidium, bromide, and 2-deoxyglucose. Induction of the mitochondrial specific stress response in COS-7 cells through expression of an ornithine transcarbamylase mutant (AOTC) increased mitochondrial ORPI50 levels and mitochondrial ca/pain activity. To determine whether mitochondrial ORP150 and mitochondrial calpain 10 interact, rat cortical mitochondria exposed to [Ca.sup.2+] resulted in ORP150 cleavage in a calpain inhibitor-dependent manner, revealing that ORP150 is a substrate and may be regulated by calpain 10. These data reveal a novel cellular localization for ORP150 and that mitochondrial ORP150 is upregulated by CHOP/GADD153 in response to mitochondrial and ER stress. Our data also reveal that ORP150 is a substrate for mitochondrial calpain 10. calpain; endoplasmic reticulum

Like Bcl-2, Mcl-1 is an important survival factor for many cancers, its expression contributing to chemo-resistance and disease relapse. However, unlike other prosurvival Bcl-2-like proteins, Mcl-1 stability is acutely regulated. For example, the Bcl-2 homology 3 (BH3)-only protein Noxa, which preferentially binds to Mcl-1, also targets it for proteasomal degradation. In this paper, we describe the discovery and characterization of a novel BH3-like ligand derived from Bim, Bims2A, which is highly selective for Mcl-1. Unlike Noxa, Bims2A is unable to trigger Mcl-1 degradation, yet, like Noxa, Bims2A promotes cell killing only when Bcl-[x.sub.L] is absent or neutralized. Furthermore, killing by endogenous Bim is not associated with Mcl-1 degradation. Thus, functional inactivation of Mcl-1 does not always require its elimination. Rather, it can be efficiently antagonized by a BH3-like ligand tightly engaging its binding groove, which is confirmed here with a structural study. Our data have important implications for the discovery of compounds that might kill cells whose survival depends on Mcl-1.

Members of the SLC4 bicarbonate transporter family are involved in solute transport and pH homeostasis. Here we report that disrupting the Sic4a10 gene, which encodes the [Na.sup.+]-coupled [Cl.sup.-]-HC[O.sub.3]- exchanger Slc4al0 (NCBE), drastically reduces brain ventricle volume and protects against fatal epileptic seizures in mice. In choroid plexus epithelial cells, Slc4al0 localizes to the basolateral membrane. These cells displayed a diminished recovery from an acid load in KO mice. Slc4al0 also was expressed in neurons. Within the hippocampus, the Slc4al0 protein was abundant in CA3 pyramidal cells. In the CA3 area, propionate-induced intracellular acidification and attenuation of 4-aminopyridine-induced network activity were prolonged in KO mice. Our data indicate that Slc4a10 is involved in the control of neuronal pH and excitability and may contribute to the secretion of cerebrospinal fluid. Hence, Slc4a10 is a promising pharmacological target for the therapy of epilepsy or elevated intracranial pressure. cerebrospinal fluid | epilepsy | ion transport | knockout mouse | pH regulation

Development of the vascular system depends on the highly coordinated actions of a variety of angiogenic regulators. Several of these regulators are members of the tyrosine kinase superfamily, including VEGF receptors and angiopoietin receptors, Tie1 and Tie2. Tyrosine kinase signaling is counter-regulated by the activity of tyrosine phosphatases, including vascular endothelial protein tyrosine phosphatase (VE-PTP), which has previously been shown to modulate Tie2 activity. We generated mice in which VE-PTP is replaced with a reporter gene. We confirm that VE-PTP is expressed in endothelium and also show that VE-PTP is highly expressed in the developing outflow tract of the heart and later is expressed in developing heart valves. Vasculogenesis occurs normally in mice lacking VE-PTP; however, angiogenesis is abnormal. Angiogenic defects in VE-PTP-null mice were most pronounced in the yolk sac and include a complete failure to elaborate the primitive vascular scaffold into higher-order branched arteries, veins, and capillaries. VE-PTP continues to be expressed into adulthood in the vasculature and heart valves, suggesting later roles in vascular development or homeostasis. VE-PTP is also expressed in the vasculature of growing tumors, suggesting that VE-PTP may be a new potential target for angiogenic therapies. gene targeting | tyrosine kinase | Tie2

Author(s): David Cyranoski A heated dispute over gene-editing that began in blogs and social media is now playing out in the scientific literature. Six months ago, Chinese researchers reported that [...]