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1.  Crosstalk between BRCA-Fanconi anemia and mismatch repair pathways prevents MSH2-dependent aberrant DNA damage responses 
The EMBO Journal  2014;33(15):1698-1712.
Several proteins in the BRCA-Fanconi anemia (FA) pathway, such as FANCJ, BRCA1, and FANCD2, interact with mismatch repair (MMR) pathway factors, but the significance of this link remains unknown. Unlike the BRCA-FA pathway, the MMR pathway is not essential for cells to survive toxic DNA interstrand crosslinks (ICLs), although MMR proteins bind ICLs and other DNA structures that form at stalled replication forks. We hypothesized that MMR proteins corrupt ICL repair in cells that lack crosstalk between BRCA-FA and MMR pathways. Here, we show that ICL sensitivity of cells lacking the interaction between FANCJ and the MMR protein MLH1 is suppressed by depletion of the upstream mismatch recognition factor MSH2. MSH2 depletion suppresses an aberrant DNA damage response, restores cell cycle progression, and promotes ICL resistance through a Rad18-dependent mechanism. MSH2 depletion also suppresses ICL sensitivity in cells deficient for BRCA1 or FANCD2, but not FANCA. Rescue by Msh2 loss was confirmed in Fancd2-null primary mouse cells. Thus, we propose that regulation of MSH2-dependent DNA damage response underlies the importance of interactions between BRCA-FA and MMR pathways.
PMCID: PMC4194102  PMID: 24966277
Fanconi anemia; FANCJ; mismatch repair; MLH1; replication stress
2.  Nuclear ARRB1 induces pseudohypoxia and cellular metabolism reprogramming in prostate cancer 
The EMBO Journal  2014;33(12):1365-1382.
Tumour cells sustain their high proliferation rate through metabolic reprogramming, whereby cellular metabolism shifts from oxidative phosphorylation to aerobic glycolysis, even under normal oxygen levels. Hypoxia-inducible factor 1A (HIF1A) is a major regulator of this process, but its activation under normoxic conditions, termed pseudohypoxia, is not well documented. Here, using an integrative approach combining the first genome-wide mapping of chromatin binding for an endocytic adaptor, ARRB1, both in vitro and in vivo with gene expression profiling, we demonstrate that nuclear ARRB1 contributes to this metabolic shift in prostate cancer cells via regulation of HIF1A transcriptional activity under normoxic conditions through regulation of succinate dehydrogenase A (SDHA) and fumarate hydratase (FH) expression. ARRB1-induced pseudohypoxia may facilitate adaptation of cancer cells to growth in the harsh conditions that are frequently encountered within solid tumours. Our study is the first example of an endocytic adaptor protein regulating metabolic pathways. It implicates ARRB1 as a potential tumour promoter in prostate cancer and highlights the importance of metabolic alterations in prostate cancer.
PMCID: PMC4194125  PMID: 24837709
Adaptor; hypoxia; metabolism; prostate; transcription
3.  Synaptic vesicle recycling: steps and principles 
The EMBO Journal  2014;33(8):788-822.
Synaptic vesicle recycling is one of the best-studied cellular pathways. Many of the proteins involved are known, and their interactions are becoming increasingly clear. However, as for many other pathways, it is still difficult to understand synaptic vesicle recycling as a whole. While it is generally possible to point out how synaptic reactions take place, it is not always easy to understand what triggers or controls them. Also, it is often difficult to understand how the availability of the reaction partners is controlled: how the reaction partners manage to find each other in the right place, at the right time. I present here an overview of synaptic vesicle recycling, discussing the mechanisms that trigger different reactions, and those that ensure the availability of reaction partners. A central argument is that synaptic vesicles bind soluble cofactor proteins, with low affinity, and thus control their availability in the synapse, forming a buffer for cofactor proteins. The availability of cofactor proteins, in turn, regulates the different synaptic reactions. Similar mechanisms, in which one of the reaction partners buffers another, may apply to many other processes, from the biogenesis to the degradation of the synaptic vesicle.
PMCID: PMC4194108  PMID: 24596248
clathrin; endocytosis; exocytosis; SNAREs; vesicle pools
4.  Cohesin-mediated interactions organize chromosomal domain architecture 
The EMBO Journal  2013;32(24):3119-3129.
To ensure proper gene regulation within constrained nuclear space, chromosomes facilitate access to transcribed regions, while compactly packaging all other information. Recent studies revealed that chromosomes are organized into megabase-scale domains that demarcate active and inactive genetic elements, suggesting that compartmentalization is important for genome function. Here, we show that very specific long-range interactions are anchored by cohesin/CTCF sites, but not cohesin-only or CTCF-only sites, to form a hierarchy of chromosomal loops. These loops demarcate topological domains and form intricate internal structures within them. Post-mitotic nuclei deficient for functional cohesin exhibit global architectural changes associated with loss of cohesin/CTCF contacts and relaxation of topological domains. Transcriptional analysis shows that this cohesin-dependent perturbation of domain organization leads to widespread gene deregulation of both cohesin-bound and non-bound genes. Our data thereby support a role for cohesin in the global organization of domain structure and suggest that domains function to stabilize the transcriptional programmes within them.
Chromosomal compartmentalization has been recognized as important for genome function. High-resolution techniques such as Hi-C, ChIP- and 4C-seq offer novel insights into cohesin's dynamic role in shaping the nuclear architecture.
PMCID: PMC4489921  PMID: 24185899
chromosome organization; cohesin; transcription
5.  Divergent roles of HDAC1 and HDAC2 in the regulation of epidermal development and tumorigenesis 
The EMBO Journal  2013;32(24):3176-3191.
The histone deacetylases HDAC1 and HDAC2 remove acetyl moieties from lysine residues of histones and other proteins and are important regulators of gene expression. By deleting different combinations of Hdac1 and Hdac2 alleles in the epidermis, we reveal a dosage-dependent effect of HDAC1/HDAC2 activity on epidermal proliferation and differentiation. Conditional ablation of either HDAC1 or HDAC2 in the epidermis leads to no obvious phenotype due to compensation by the upregulated paralogue. Strikingly, deletion of a single Hdac2 allele in HDAC1 knockout mice results in severe epidermal defects, including alopecia, hyperkeratosis, hyperproliferation and spontaneous tumour formation. These mice display impaired Sin3A co-repressor complex function, increased levels of c-Myc protein, p53 expression and apoptosis in hair follicles (HFs) and misregulation of HF bulge stem cells. Surprisingly, ablation of HDAC1 but not HDAC2 in a skin tumour model leads to accelerated tumour development. Our data reveal a crucial function of HDAC1/HDAC2 in the control of lineage specificity and a novel role of HDAC1 as a tumour suppressor in the epidermis.
Divergent roles of HDAC1 and HDAC2 in the regulation of epidermal development and tumorigenesis
Dose-dependent deletion of HDAC1 and -2 in the mouse epidermis reveals non-redundant functions in postnatal skin homeostasis. The novel tumour suppressive function of HDAC1 should caution the application of HDAC inhibitors as therapeutics.
PMCID: PMC3981143  PMID: 24240174
chromatin; epidermis; HDACs
6.  Isogenic human pluripotent stem cell pairs reveal the role of a KCNH2 mutation in long-QT syndrome 
The EMBO Journal  2013;32(24):3161-3175.
Patient-specific induced pluripotent stem cells (iPSCs) will assist research on genetic cardiac maladies if the disease phenotype is recapitulated in vitro. However, genetic background variations may confound disease traits, especially for disorders with incomplete penetrance, such as long-QT syndromes (LQTS). To study the LQT2-associated c.A2987T (N996I) KCNH2 mutation under genetically defined conditions, we derived iPSCs from a patient carrying this mutation and corrected it. Furthermore, we introduced the same point mutation in human embryonic stem cells (hESCs), generating two genetically distinct isogenic pairs of LQTS and control lines. Correction of the mutation normalized the current (IKr) conducted by the HERG channel and the action potential (AP) duration in iPSC-derived cardiomyocytes (CMs). Introduction of the same mutation reduced IKr and prolonged the AP duration in hESC-derived CMs. Further characterization of N996I-HERG pathogenesis revealed a trafficking defect. Our results demonstrated that the c.A2987T KCNH2 mutation is the primary cause of the LQTS phenotype. Precise genetic modification of pluripotent stem cells provided a physiologically and functionally relevant human cellular context to reveal the pathogenic mechanism underlying this specific disease phenotype.
Isogenic human pluripotent stem cell pairs reveal the role of a KCNH2 mutation in long-QT syndrome
Isogenic pairs of patient-derived iPS- and ES-cell lines reveal the molecular problems contributing to cardiac arrest in long-QT syndrome.
PMCID: PMC3981141  PMID: 24213244
gene targeting; HERG; human embryonic stem cells; induced pluripotent stem cells; long-QT syndrome
7.  Endonuclease G mediates α-synuclein cytotoxicity during Parkinson's disease 
The EMBO Journal  2013;32(23):3041-3054.
Malfunctioning of the protein α-synuclein is critically involved in the demise of dopaminergic neurons relevant to Parkinson's disease. Nonetheless, the precise mechanisms explaining this pathogenic neuronal cell death remain elusive. Endonuclease G (EndoG) is a mitochondrially localized nuclease that triggers DNA degradation and cell death upon translocation from mitochondria to the nucleus. Here, we show that EndoG displays cytotoxic nuclear localization in dopaminergic neurons of human Parkinson-diseased patients, while EndoG depletion largely reduces α-synuclein-induced cell death in human neuroblastoma cells. Xenogenic expression of human α-synuclein in yeast cells triggers mitochondria-nuclear translocation of EndoG and EndoG-mediated DNA degradation through a mechanism that requires a functional kynurenine pathway and the permeability transition pore. In nematodes and flies, EndoG is essential for the α-synuclein-driven degeneration of dopaminergic neurons. Moreover, the locomotion and survival of α-synuclein-expressing flies is compromised, but reinstalled by parallel depletion of EndoG. In sum, we unravel a phylogenetically conserved pathway that involves EndoG as a critical downstream executor of α-synuclein cytotoxicity.
The mitochondrial pro-apoptotic nuclease Endonuclease G is a key downstream executor of α-synuclein neurotoxicity in different Parkinson's disease models.
PMCID: PMC3844953  PMID: 24129513
α-synuclein; cell death; endonuclease G; mitochondria; Parkinson's disease
8.  microRNA-17-92 cluster is a direct Nanog target and controls neural stem cell through Trp53inp1 
The EMBO Journal  2013;32(21):2819-2832.
The transcription factor Nanog plays a critical role in the self-renewal of embryonic stem cells as well as in neural stem cells (NSCs). microRNAs (miRNAs) are also involved in stemness regulation. However, the miRNA network downstream of Nanog is still poorly understood. High-throughput screening of miRNA expression profiles in response to modulated levels of Nanog in postnatal NSCs identifies miR-17-92 cluster as a direct target of Nanog. Nanog controls miR-17-92 cluster by binding to the upstream regulatory region and maintaining high levels of transcription in NSCs, whereas Nanog/promoter association and cluster miRNAs expression are lost alongside differentiation. The two miR-17 family members of miR-17-92 cluster, namely miR-17 and miR-20a, target Trp53inp1, a downstream component of p53 pathway. To support a functional role, the presence of miR-17/20a or the loss of Trp53inp1 is required for the Nanog-induced enhancement of self-renewal of NSCs. We unveil an arm of the Nanog/p53 pathway, which regulates stemness in postnatal NSCs, wherein Nanog counteracts p53 signals through miR-17/20a-mediated repression of Trp53inp1.
microRNA-17-92 cluster is a direct Nanog target and controls neural stem cell through Trp53inp1
Direct control of the miRNA-17/92 cluster enables Nanog to restrain p53 activity and thus to maintain pluripotency in neural stem cells.
PMCID: PMC3817465  PMID: 24076654
microRNA; miR-17-92 cluster; Nanog; neural stem cells; Trp53inp1
9.  Endophilin, Lamellipodin, and Mena cooperate to regulate F-actin-dependent EGF-receptor endocytosis 
The EMBO Journal  2013;32(20):2722-2734.
The epidermal growth factor receptor (EGFR) plays an essential role during development and diseases including cancer. Lamellipodin (Lpd) is known to control lamellipodia protrusion by regulating actin filament elongation via Ena/VASP proteins. However, it is unknown whether this mechanism supports endocytosis of the EGFR. Here, we have identified a novel role for Lpd and Mena in clathrin-mediated endocytosis (CME) of the EGFR. We have discovered that endogenous Lpd is in a complex with the EGFR and Lpd and Mena knockdown impairs EGFR endocytosis. Conversely, overexpressing Lpd substantially increases the EGFR uptake in an F-actin-dependent manner, suggesting that F-actin polymerization is limiting for EGFR uptake. Furthermore, we found that Lpd directly interacts with endophilin, a BAR domain containing protein implicated in vesicle fission. We identified a role for endophilin in EGFR endocytosis, which is mediated by Lpd. Consistently, Lpd localizes to clathrin-coated pits (CCPs) just before vesicle scission and regulates vesicle scission. Our findings suggest a novel mechanism in which Lpd mediates EGFR endocytosis via Mena downstream of endophilin.
Endophilin, Lamellipodin, and Mena cooperate to regulate F-actin-dependent EGF-receptor endocytosis
Cooperation between a BAR domain protein and a regulator of actin filament elongation during lamellipodia protrusion reveals actin cytoskeleton roles in endocytic vesicle scission in mammalian cells.
PMCID: PMC3801443  PMID: 24076656
endophilin; EGF-receptor endocytosis; Ena/VASP proteins; Lamellipodin; Mena
10.  TRIAD1 and HHARI bind to and are activated by distinct neddylated Cullin-RING ligase complexes 
The EMBO Journal  2013;32(21):2848-2860.
RING (Really Interesting New Gene)-in-between-RING (RBR) enzymes are a distinct class of E3 ubiquitin ligases possessing a cluster of three zinc-binding domains that cooperate to catalyse ubiquitin transfer. The regulation and biological function for most members of the RBR ligases is not known, and all RBR E3s characterized to date are auto-inhibited for in vitro ubiquitylation. Here, we show that TRIAD1 and HHARI, two members of the Ariadne subfamily ligases, associate with distinct neddylated Cullin-RING ligase (CRL) complexes. In comparison to the modest E3 ligase activity displayed by isolated TRIAD1 or HHARI, binding of the cognate neddylated CRL to TRIAD1 or HHARI greatly stimulates RBR ligase activity in vitro, as determined by auto-ubiquitylation, their ability to stimulate dissociation of a thioester-linked UBCH7∼ubiquitin intermediate, and reactivity with ubiquitin-vinyl methyl ester. Moreover, genetic evidence shows that RBR ligase activity impacts both the levels and activities of neddylated CRLs in vivo. Cumulatively, our work proposes a conserved mechanism of CRL-induced Ariadne RBR ligase activation and further suggests a reciprocal role of this special class of RBRs as regulators of distinct CRLs.
TRIAD1 and HHARI bind to and are activated by distinct neddylated Cullin-RING ligase complexes
Ubiquitin ligases of the distinct Cullin-RING ligase (CRL) and RING-between-RING (RBR) families physically and functionally interact, suggesting how RBR ligase auto-inhibition may be relieved in Ariadne-subfamily members.
PMCID: PMC3817463  PMID: 24076655
auto-inhibition; Cullin-RING ligases; HHARI; RBR E3 ubiquitin ligases; TRIAD1
11.  IFNβ-dependent increases in STAT1, STAT2, and IRF9 mediate resistance to viruses and DNA damage 
The EMBO Journal  2013;32(20):2751-2763.
A single high dose of interferon-β (IFNβ) activates powerful cellular responses, in which many anti-viral, pro-apoptotic, and anti-proliferative proteins are highly expressed. Since some of these proteins are deleterious, cells downregulate this initial response rapidly. However, the expression of many anti-viral proteins that do no harm is sustained, prolonging a substantial part of the initial anti-viral response for days and also providing resistance to DNA damage. While the transcription factor ISGF3 (IRF9 and tyrosine-phosphorylated STATs 1 and 2) drives the first rapid response phase, the related factor un-phosphorylated ISGF3 (U-ISGF3), formed by IFNβ-induced high levels of IRF9 and STATs 1 and 2 without tyrosine phosphorylation, drives the second prolonged response. The U-ISGF3-induced anti-viral genes that show prolonged expression are driven by distinct IFN stimulated response elements (ISREs). Continuous exposure of cells to a low level of IFNβ, often seen in cancers, leads to steady-state increased expression of only the U-ISGF3-dependent proteins, with no sustained increase in other IFNβ-induced proteins, and to constitutive resistance to DNA damage.
IFNβ-dependent increases in STAT1, STAT2, and IRF9 mediate resistance to viruses and DNA damage
IFNβ induces the formation of a novel transcriptional complex, U-ISGF3, which contains un-phosphorylated STATs. U-ISGF3 regulates the expression of a subset of IFNβ-stimulated genes to promote resistance to virus infection and DNA damage.
PMCID: PMC3801437  PMID: 24065129
anti-viral genes; DNA damage resistance; interferon-β; unphosphorylated interferon-stimulated gene factor 3 (U-ISGF3)
12.  Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R-spondin axis 
The EMBO Journal  2013;32(20):2708-2721.
Lgr5 marks adult stem cells in multiple adult organs and is a receptor for the Wnt-agonistic R-spondins (RSPOs). Intestinal, stomach and liver Lgr5+ stem cells grow in 3D cultures to form ever-expanding organoids, which resemble the tissues of origin. Wnt signalling is inactive and Lgr5 is not expressed under physiological conditions in the adult pancreas. However, we now report that the Wnt pathway is robustly activated upon injury by partial duct ligation (PDL), concomitant with the appearance of Lgr5 expression in regenerating pancreatic ducts. In vitro, duct fragments from mouse pancreas initiate Lgr5 expression in RSPO1-based cultures, and develop into budding cyst-like structures (organoids) that expand five-fold weekly for >40 weeks. Single isolated duct cells can also be cultured into pancreatic organoids, containing Lgr5 stem/progenitor cells that can be clonally expanded. Clonal pancreas organoids can be induced to differentiate into duct as well as endocrine cells upon transplantation, thus proving their bi-potentiality.
Unlimited in vitro expansion of adult bi-potent pancreas progenitors through the Lgr5/R-spondin axis
The establishment of conditions for long-term culture and expansion of adult, bi-potent pancreas progenitors may facilitate novel and tailored therapeutic approaches.
PMCID: PMC3801438  PMID: 24045232
beta cell; duct cell; pancreas; Wnt; stem cell
13.  Identification of the missing pluripotency mediator downstream of leukaemia inhibitory factor 
The EMBO Journal  2013;32(19):2561-2574.
Self-renewal of pluripotent mouse embryonic stem (ES) cells is sustained by the cytokine leukaemia inhibitory factor (LIF) acting through the transcription factor Stat3. Several targets of Stat3 have previously been identified, most notably the reprogramming factor Klf4. However, such factors are neither required nor sufficient for the potent effect of LIF. We took advantage of Stat3 null ES cells to confirm that Stat3 mediates the self-renewal response to LIF. Through comparative transcriptome analysis intersected with genome location data, we arrived at a set of candidate transcription factor effectors. Among these, Tfcp2l1 (also known as Crtr-1) was most abundant. Constitutive expression of Tfcp2l1 at levels similar to those induced by LIF effectively substituted for LIF or Stat3 in sustaining clonal self-renewal and pluripotency. Conversely, knockdown of Tfcp2l1 profoundly compromised responsiveness to LIF. We further found that Tfcp2l1 is both necessary and sufficient to direct molecular reprogramming of post-implantation epiblast stem cells to naïve pluripotency. These results establish Tfcp2l1 as the principal bridge between LIF/Stat3 input and the transcription factor core of naïve pluripotency.
Identification of the missing pluripotency mediator downstream of leukaemia inhibitory factor
A genome-wide approach to identify critical effectors of Lif/Stat3 signalling in ESCs reveals Tfcp2I1 as sufficient to sustain self-renewal and direct EpiSCs to naïve pluripotency.
PMCID: PMC3791366  PMID: 23942233
ES cell self-renewal; LIF; pluripotency; reprogramming
14.  Neuronal carbonic anhydrase VII provides GABAergic excitatory drive to exacerbate febrile seizures 
The EMBO Journal  2013;32(16):2275-2286.
Brain carbonic anhydrases (CAs) are known to modulate neuronal signalling. Using a novel CA VII (Car7) knockout (KO) mouse as well as a CA II (Car2) KO and a CA II/VII double KO, we show that mature hippocampal pyramidal neurons are endowed with two cytosolic isoforms. CA VII is predominantly expressed by neurons starting around postnatal day 10 (P10). The ubiquitous isoform II is expressed in neurons at P20. Both isoforms enhance bicarbonate-driven GABAergic excitation during intense GABAA-receptor activation. P13–14 CA VII KO mice show behavioural manifestations atypical of experimental febrile seizures (eFS) and a complete absence of electrographic seizures. A low dose of diazepam promotes eFS in P13–P14 rat pups, whereas seizures are blocked at higher concentrations that suppress breathing. Thus, the respiratory alkalosis-dependent eFS are exacerbated by GABAergic excitation. We found that CA VII mRNA is expressed in the human cerebral cortex before the age when febrile seizures (FS) occur in children. Our data indicate that CA VII is a key molecule in age-dependent neuronal pH regulation with consequent effects on generation of FS.
Neuronal carbonic anhydrase VII provides GABAergic excitatory drive to exacerbate febrile seizures
Carbonic anhydrase VII plays a key role in age-dependent neuronal pH regulation, promoting excitatory GABA transmission and febrile seizures.
PMCID: PMC3746197  PMID: 23881097
carbonic anhydrase expression; chloride accumulation; GABAA receptor; human brain; hyperthermia
15.  A cyclic GMP-dependent signalling pathway regulates bacterial phytopathogenesis 
The EMBO Journal  2013;32(18):2430-2438.
Cyclic guanosine 3′,5′-monophosphate (cyclic GMP) is a second messenger whose role in bacterial signalling is poorly understood. A genetic screen in the plant pathogen Xanthomonas campestris (Xcc) identified that XC_0250, which encodes a protein with a class III nucleotidyl cyclase domain, is required for cyclic GMP synthesis. Purified XC_0250 was active in cyclic GMP synthesis in vitro. The linked gene XC_0249 encodes a protein with a cyclic mononucleotide-binding (cNMP) domain and a GGDEF diguanylate cyclase domain. The activity of XC_0249 in cyclic di-GMP synthesis was enhanced by addition of cyclic GMP. The isolated cNMP domain of XC_0249 bound cyclic GMP and a structure–function analysis, directed by determination of the crystal structure of the holo-complex, demonstrated the site of cyclic GMP binding that modulates cyclic di-GMP synthesis. Mutation of either XC_0250 or XC_0249 led to a reduced virulence to plants and reduced biofilm formation in vitro. These findings describe a regulatory pathway in which cyclic GMP regulates virulence and biofilm formation through interaction with a novel effector that directly links cyclic GMP and cyclic di-GMP signalling.
A cyclic GMP-dependent signalling pathway regulates bacterial phytopathogenesis
In the plant pathogen X. campestris, the second messenger cGMP controls bacterial virulence and biofilm formation through direct regulation of XC_0249, a novel diguanylate cyclase that synthesises the signalling molecule cyclic di-GMP.
PMCID: PMC3770947  PMID: 23881098
biofilm; cyclic di-GMP; signal transduction; virulence; Xanthomonas campestris
16.  Functional determinants of the quorum-sensing non-coding RNAs and their roles in target regulation 
The EMBO Journal  2013;32(15):2158-2171.
Quorum sensing is a chemical communication process that bacteria use to control collective behaviours including bioluminescence, biofilm formation, and virulence factor production. In Vibrio harveyi, five homologous small RNAs (sRNAs) called Qrr1–5, control quorum-sensing transitions. Here, we identify 16 new targets of the Qrr sRNAs. Mutagenesis reveals that particular sequence differences among the Qrr sRNAs determine their target specificities. Modelling coupled with biochemical and genetic analyses show that all five of the Qrr sRNAs possess four stem-loops: the first stem-loop is crucial for base pairing with a subset of targets. This stem-loop also protects the Qrr sRNAs from RNase E-mediated degradation. The second stem-loop contains conserved sequences required for base pairing with the majority of the target mRNAs. The third stem-loop plays an accessory role in base pairing and stability. The fourth stem-loop functions as a rho-independent terminator. In the quorum-sensing regulon, Qrr sRNAs-controlled genes are the most rapid to respond to quorum-sensing autoinducers. The Qrr sRNAs are conserved throughout vibrios, thus insights from this work could apply generally to Vibrio quorum sensing.
Functional determinants of the quorum-sensing non-coding RNAs and their roles in target regulation
Quorum sensing in bacteria is controlled by five small RNAs, Qrr1–5, which exert post-transcriptional control of 16 novel target genes through four conserved stem-loops with distinct roles in target binding and sRNA stability.
PMCID: PMC3730234  PMID: 23838640
quorum sensing; regulation; sRNAs
17.  Threshold-controlled ubiquitination of the EGFR directs receptor fate 
The EMBO Journal  2013;32(15):2140-2157.
How the cell converts graded signals into threshold-activated responses is a question of great biological relevance. Here, we uncover a nonlinear modality of epidermal growth factor receptor (EGFR)-activated signal transduction, by demonstrating that the ubiquitination of the EGFR at the PM is threshold controlled. The ubiquitination threshold is mechanistically determined by the cooperative recruitment of the E3 ligase Cbl, in complex with Grb2, to the EGFR. This, in turn, is dependent on the simultaneous presence of two phosphotyrosines, pY1045 and either one of pY1068 or pY1086, on the same EGFR moiety. The dose–response curve of EGFR ubiquitination correlate precisely with the non-clathrin endocytosis (NCE) mode of EGFR internalization. Finally, EGFR-NCE mechanistically depends on EGFR ubiquitination, as the two events can be simultaneously re-engineered on a phosphorylation/ubiquitination-incompetent EGFR backbone. Since NCE controls the degradation of the EGFR, our findings have implications for how the cell responds to increasing levels of EGFR signalling, by varying the balance of receptor signalling and degradation/attenuation.
The amount of EGF present for binding to its receptor governs an on–off switch of EGFR ubiquitination and hence ligand-controlled non-clathrin-mediated endocytosis and EGFR degradation.
PMCID: PMC3730230  PMID: 23799367
EGFR; endocytosis; ubiquitination
18.  DNA polymerase κ-dependent DNA synthesis at stalled replication forks is important for CHK1 activation 
The EMBO Journal  2013;32(15):2172-2185.
Formation of primed single-stranded DNA at stalled replication forks triggers activation of the replication checkpoint signalling cascade resulting in the ATR-mediated phosphorylation of the Chk1 protein kinase, thus preventing genomic instability. By using siRNA-mediated depletion in human cells and immunodepletion and reconstitution experiments in Xenopus egg extracts, we report that the Y-family translesion (TLS) DNA polymerase kappa (Pol κ) contributes to the replication checkpoint response and is required for recovery after replication stress. We found that Pol κ is implicated in the synthesis of short DNA intermediates at stalled forks, facilitating the recruitment of the 9-1-1 checkpoint clamp. Furthermore, we show that Pol κ interacts with the Rad9 subunit of the 9-1-1 complex. Finally, we show that this novel checkpoint function of Pol κ is required for the maintenance of genomic stability and cell proliferation in unstressed human cells.
DNA polymerase κ-dependent DNA synthesis at stalled replication forks is important for CHK1 activation
A vertebrate translesion synthesis DNA polymerase broadly contributes to checkpoint-activating primer synthesis at stalled replication forks, a role previously ascribed only to replicative polymerases.
PMCID: PMC3730229  PMID: 23799366
DNA polymerase κ; genetic instability; replication checkpoint; replication stress
19.  Bace1 and Neuregulin-1 cooperate to control formation and maintenance of muscle spindles 
The EMBO Journal  2013;32(14):2015-2028.
The protease β-secretase 1 (Bace1) was identified through its critical role in production of amyloid-β peptides (Aβ), the major component of amyloid plaques in Alzheimer's disease. Bace1 is considered a promising target for the treatment of this pathology, but processes additional substrates, among them Neuregulin-1 (Nrg1). Our biochemical analysis indicates that Bace1 processes the Ig-containing β1 Nrg1 (IgNrg1β1) isoform. We find that a graded reduction in IgNrg1 signal strength in vivo results in increasingly severe deficits in formation and maturation of muscle spindles, a proprioceptive organ critical for muscle coordination. Further, we show that Bace1 is required for formation and maturation of the muscle spindle. Finally, pharmacological inhibition and conditional mutagenesis in adult animals demonstrate that Bace1 and Nrg1 are essential to sustain muscle spindles and to maintain motor coordination. Our results assign to Bace1 a role in the control of coordinated movement through its regulation of muscle spindle physiology, and implicate IgNrg1-dependent processing as a molecular mechanism.
Bace1 and Neuregulin-1 cooperate to control formation and maintenance of muscle spindles
Bace1 is required for Nrg1 processing for muscle spindle development. Bace1 inhibition leads to loss of motor coordination even in adult mice, suggesting potentially serious side effects for drugs targeting Bace1 as a treatment for Alzheimer's disease.
PMCID: PMC3715864  PMID: 23792428
Bace1; muscle spindle; Nrg1; proprioception
20.  Sir2 is required for Clr4 to initiate centromeric heterochromatin assembly in fission yeast 
The EMBO Journal  2013;32(17):2321-2335.
Heterochromatin assembly in fission yeast depends on the Clr4 histone methyltransferase, which targets H3K9. We show that the histone deacetylase Sir2 is required for Clr4 activity at telomeres, but acts redundantly with Clr3 histone deacetylase to maintain centromeric heterochromatin. However, Sir2 is critical for Clr4 function during de novo centromeric heterochromatin assembly. We identified new targets of Sir2 and tested if their deacetylation is necessary for Clr4-mediated heterochromatin establishment. Sir2 preferentially deacetylates H4K16Ac and H3K4Ac, but mutation of these residues to mimic acetylation did not prevent Clr4-mediated heterochromatin establishment. Sir2 also deacetylates H3K9Ac and H3K14Ac. Strains bearing H3K9 or H3K14 mutations exhibit heterochromatin defects. H3K9 mutation blocks Clr4 function, but why H3K14 mutation impacts heterochromatin was not known. Here, we demonstrate that recruitment of Clr4 to centromeres is blocked by mutation of H3K14. We suggest that Sir2 deacetylates H3K14 to target Clr4 to centromeres. Further, we demonstrate that Sir2 is critical for de novo accumulation of H3K9me2 in RNAi-deficient cells. These analyses place Sir2 and H3K14 deacetylation upstream of Clr4 recruitment during heterochromatin assembly.
Sir2 is required for Clr4 to initiate centromeric heterochromatin assembly in fission yeast
The demonstration that H3K14 deacetylation promotes recruitment of the Clr4 histone methyltransferase establishes a new function for the Sir2 deacetylase in de novo heterochromatin formation.
PMCID: PMC3770337  PMID: 23771057
centromere; fission yeast; heterochromatin; histone deacetylase; histone methyltransferase
21.  Structure of the human Parkin ligase domain in an autoinhibited state 
The EMBO Journal  2013;32(15):2099-2112.
Mutations in the protein Parkin are associated with Parkinson's disease (PD), the second most common neurodegenerative disease in men. Parkin is an E3 ubiquitin (Ub) ligase of the structurally uncharacterized RING-in-between-RING(IBR)-RING (RBR) family, which, in an HECT-like fashion, forms a catalytic thioester intermediate with Ub. We here report the crystal structure of human Parkin spanning the Unique Parkin domain (UPD, also annotated as RING0) and RBR domains, revealing a tightly packed structure with unanticipated domain interfaces. The UPD adopts a novel elongated Zn-binding fold, while RING2 resembles an IBR domain. Two key interactions keep Parkin in an autoinhibited conformation. A linker that connects the IBR with the RING2 over a 50-Å distance blocks the conserved E2∼Ub binding site of RING1. RING2 forms a hydrophobic interface with the UPD, burying the catalytic Cys431, which is part of a conserved catalytic triad. Opening of intra-domain interfaces activates Parkin, and enables Ub-based suicide probes to modify Cys431. The structure further reveals a putative phospho-peptide docking site in the UPD, and explains many PD-causing mutations.
Structure of the human Parkin ligase domain in an autoinhibited state
The complete structural view of a RING-IBR-RING (RBR) ubiquitin ligase domain reveals an unexpected catalytic triad and explains the effects of various Parkin mutations underlying Parkinson's disease.
PMCID: PMC3730226  PMID: 23727886
E3 ligase; neurodegenerative disease; Parkin; ubiquitin; X-ray crystallography
22.  The nuclear F-actin interactome of Xenopus oocytes reveals an actin-bundling kinesin that is essential for meiotic cytokinesis 
The EMBO Journal  2013;32(13):1886-1902.
Nuclei of Xenopus laevis oocytes grow 100 000-fold larger in volume than a typical somatic nucleus and require an unusual intranuclear F-actin scaffold for mechanical stability. We now developed a method for mapping F-actin interactomes and identified a comprehensive set of F-actin binders from the oocyte nuclei. Unexpectedly, the most prominent interactor was a novel kinesin termed NabKin (Nuclear and meiotic actin-bundling Kinesin). NabKin not only binds microtubules but also F-actin structures, such as the intranuclear actin bundles in prophase and the contractile actomyosin ring during cytokinesis. The interaction between NabKin and F-actin is negatively regulated by Importin-β and is responsive to spatial information provided by RanGTP. Disconnecting NabKin from F-actin during meiosis caused cytokinesis failure and egg polyploidy. We also found actin-bundling activity in Nabkin's somatic paralogue KIF14, which was previously shown to be essential for somatic cell division. Our data are consistent with the notion that NabKin/KIF14 directly link microtubules with F-actin and that such link is essential for cytokinesis.
The presence and role of actin filaments in cell nuclei remains incompletely understood. A proteomics approach now reveals a highly distinct set of F-actin-binding proteins in the nucleus, including a novel kinesin family member.
PMCID: PMC3981176  PMID: 23727888
cytokinesis; kinesins; meiosis; nuclear actin; phalloidin
23.  OPA1 promotes pH flashes that spread between contiguous mitochondria without matrix protein exchange 
The EMBO Journal  2013;32(13):1927-1940.
The chemical nature and functional significance of mitochondrial flashes associated with fluctuations in mitochondrial membrane potential is unclear. Using a ratiometric pH probe insensitive to superoxide, we show that flashes reflect matrix alkalinization transients of ∼0.4 pH units that persist in cells permeabilized in ion-free solutions and can be evoked by imposed mitochondrial depolarization. Ablation of the pro-fusion protein Optic atrophy 1 specifically abrogated pH flashes and reduced the propagation of matrix photoactivated GFP (paGFP). Ablation or invalidation of the pro-fission Dynamin-related protein 1 greatly enhanced flash propagation between contiguous mitochondria but marginally increased paGFP matrix diffusion, indicating that flashes propagate without matrix content exchange. The pH flashes were associated with synchronous depolarization and hyperpolarization events that promoted the membrane potential equilibration of juxtaposed mitochondria. We propose that flashes are energy conservation events triggered by the opening of a fusion pore between two contiguous mitochondria of different membrane potentials, propagating without matrix fusion to equilibrate the energetic state of connected mitochondria.
Mitochondrial fusion events and transient changes in matrix pH linked to membrane depolarization are found to underlie mitochondrial flashes, whose propagation may help equilibrate energy states between connected mitochondria.
PMCID: PMC3981180  PMID: 23714779
bioenergetics; cell signalling; metabolism
24.  The EAL domain protein YciR acts as a trigger enzyme in a c-di-GMP signalling cascade in E. coli biofilm control 
The EMBO Journal  2013;32(14):2001-2014.
C-di-GMP—which is produced by diguanylate cyclases (DGC) and degraded by specific phosphodiesterases (PDEs)—is a ubiquitous second messenger in bacterial biofilm formation. In Escherichia coli, several DGCs (YegE, YdaM) and PDEs (YhjH, YciR) and the MerR-like transcription factor MlrA regulate the transcription of csgD, which encodes a biofilm regulator essential for producing amyloid curli fibres of the biofilm matrix. Here, we demonstrate that this system operates as a signalling cascade, in which c-di-GMP controlled by the DGC/PDE pair YegE/YhjH (module I) regulates the activity of the YdaM/YciR pair (module II). Via multiple direct interactions, the two module II proteins form a signalling complex with MlrA. YciR acts as a connector between modules I and II and functions as a trigger enzyme: its direct inhibition of the DGC YdaM is relieved when it binds and degrades c-di-GMP generated by module I. As a consequence, YdaM then generates c-di-GMP and—by direct and specific interaction—activates MlrA to stimulate csgD transcription. Trigger enzymes may represent a general principle in local c-di-GMP signalling.
The levels of bacterial biofilm regulator c-di-GMP are controlled in E. coli by two diguanylate cyclase/phosphodiesterase pairs acting sequentially, YegE/YhjH and YdaM/YciR. Interestingly, YciR both degrades c-di-GMP and acts as a ‘trigger enzyme' regulating YdaM activity through direct physical interaction.
PMCID: PMC3715855  PMID: 23708798
amyloid; CsgD; curli fibres; cyclic-di-GMP; GGDEF domain
25.  HOT1 is a mammalian direct telomere repeat-binding protein contributing to telomerase recruitment 
The EMBO Journal  2013;32(12):1681-1701.
Telomeres are repetitive DNA structures that, together with the shelterin and the CST complex, protect the ends of chromosomes. Telomere shortening is mitigated in stem and cancer cells through the de novo addition of telomeric repeats by telomerase. Telomere elongation requires the delivery of the telomerase complex to telomeres through a not yet fully understood mechanism. Factors promoting telomerase–telomere interaction are expected to directly bind telomeres and physically interact with the telomerase complex. In search for such a factor we carried out a SILAC-based DNA–protein interaction screen and identified HMBOX1, hereafter referred to as homeobox telomere-binding protein 1 (HOT1). HOT1 directly and specifically binds double-stranded telomere repeats, with the in vivo association correlating with binding to actively processed telomeres. Depletion and overexpression experiments classify HOT1 as a positive regulator of telomere length. Furthermore, immunoprecipitation and cell fractionation analyses show that HOT1 associates with the active telomerase complex and promotes chromatin association of telomerase. Collectively, these findings suggest that HOT1 supports telomerase-dependent telomere elongation.
A homeobox protein binds double-stranded telomeric DNA sequences independent of the shelterin complex, and interacts with active telomerase to positively regulate telomere length.
PMCID: PMC3680732  PMID: 23685356
DNA–protein interaction; HOT1; mass spectrometry; telomeres; telomere length

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