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Pathogens often secrete proteins or nucleic acids that mimic the structure and/or function of molecules expressed in their hosts. Molecular mimicry empowers pathogens to subvert critical host processes and establish infection. We report that the intracellular bacteriumLegionella pneumophilasecretes the toxin SidI (substrate of icm/dot transporter I), which possesses a transfer RNA (tRNA)-like shape and functions as a mannosyl transferase. The 3.1 Å cryo-EM structure of SidI reveals an N-terminal domain that exhibits a characteristic ‘inverted L-shape’ and charge distribution that is present in two other known protein mimics of tRNAs, the bacterial elongation factor EF-G and the mammalian release factor eRF1. In addition, we show that SidI’s C-terminal domain adopts a glycosyl transferase B fold similar to a mannosyl transferase. This molecular coupling of the protein’s fold and enzymatic function allows SidI to bind and glycosylate components of the host translation apparatus, including the ribosome, resulting in a robust block of protein synthesis that is comparable in potency to ricin, one of the most powerful toxins known. Additionally, we find that translational pausing activated by SidI elicits a stress response signature reminiscent of the ribotoxic stress response that is activated by elongation inhibitors that induce ribosome collisions. SidI-mediated effects on the ribosome activate the stress kinases ZAKα and p38, which in turn drive an accumulation of the protein activating transcription factor 3 (ATF3). Intriguingly, ATF3 escapes the translation block imposed by SidI, translocates to the nucleus, and orchestrates the transcription of stress-inducible genes that promote cell death. Thus, usingLegionellaand its effectors as tools, we have unravelled the role of a ribosome-to-nuclear signalling pathway that regulates cell fate.

The UBTF E210K neuroregression syndrome is caused by de novo dominant mutations in UBTF (NM_014233.3:c.628G A, p.Glu210Lys). In humans, onset is typically at 2.5 to 3 years and characterized by slow progression of global motor, cognitive and behavioral dysfunction. Other potentially pathogenic UBTF variants have been reported in humans with severe neurological disease and it remains undetermined if the UBTF E210K mutation operates via gain- and/or loss-of-function. Here we examine the behavioral, cognitive, motor, and molecular effects of Ubtf knockout and knockdown in mice as a means of gauging the role of loss-of-function in humans. Ubtf

SUMMARYRibosomal RNAs (rRNAs) are the most abundant cellular RNAs, and their synthesis from rDNA repeats by RNA Polymerase I accounts for the bulk of all transcription. Despite substantial variation in rRNA transcription rates across cell types, little is known about cell-type-specific factors that bind rDNA and regulate rRNA transcription to meet tissue-specific needs. Using hematopoiesis as a model system, we mapped about 2200 ChIP-Seq datasets for 250 transcription factors (TFs) and chromatin proteins to human and mouse rDNA, and identified robust binding of multiple TF families to canonical TF motifs on rDNA. Using a 47S-FISH-Flow assay developed for nascent rRNA quantification, we demonstrated that targeted degradation of CEBPA (C/EBP alpha), a critical hematopoietic TF with conserved rDNA binding, caused rapid reduction in rRNA transcription due to reduced Pol I occupancy. Our work identifies numerous potential rRNA regulators, and provides a template for dissection of TF roles in rRNA transcription.HIGHLIGHTSMultiple cell-type-specific transcription factors (TFs) bind canonical motifs on rDNA.The hematopoietic TF CEBPA binds to active rDNA alleles at a conserved site.CEBPA promotes Polymerase I occupancy and rRNA transcription in myeloid progenitors.We present ‘47S-FISH-Flow,’ a sensitive assay to quantify nascent rRNA.

Transcription of the ribosomal RNA precursor by RNA polymerase (Pol) I is a major determinant of cellular growth and dysregulation is observed in many cancer types. Here, we present the purification of human Pol I from cells carrying a genomic GFP-fusion on the largest subunit allowing the structural and functional analysis of the enzyme across species. In contrast to yeast, human Pol I carries a single-subunit stalk and in vitro transcription indicates a reduced proofreading activity. Determination of the human Pol I cryo-EM reconstruction in a close-to-native state rationalizes the effects of disease-associated mutations and uncovers an additional domain that is built into the sequence of Pol I subunit RPA1. This ‘dock II’ domain resembles a truncated HMG-box incapable of DNA-binding which may serve as a downstream-transcription factor binding platform in metazoans. Biochemical analysis and ChIP data indicate that Topoisomerase 2a can be recruited to Pol I via the domain and cooperates with the HMG-box domain containing factor UBF. These adaptations of the metazoan Pol I transcription system may allow efficient release of positive DNA supercoils accumulating downstream of the transcription bubble.

The rRNA genes of mouse and human encode the three major RNAs of the ribosome and as such are essential for growth and development. These genes are present in high copy numbers and arranged as direct repeats at the Nucleolar Organizer Regions on multiple chromosomes. Not all the rRNA genes are transcriptionally active, but the molecular mechanisms that determine activity are complex and still poorly understood. Recent studies applying a novel Deconvolution Chromatin Immunoprecipitation (DChIP-Seq) technique in conjunction with conditional gene inactivation provide new insights into the structure of the active rRNA genes and question previous assumptions on the role of chromatin and histone modifications. We suggest an alternative model for the active rRNA gene chromatin and discuss how this structure is determined and maintained.

Transcription of the ∼200 mouse and human ribosomal RNA genes (rDNA) by RNA Polymerase I (RPI/PolR1) accounts for 80% of total cellular RNA, around 35% of all nuclear RNA synthesis, and determines the cytoplasmic ribosome complement. It is therefore a major factor controlling cell growth and its misfunction has been implicated in hypertrophic and developmental disorders. Activation of each rDNA repeat requires nucleosome replacement by the architectural multi-HMGbox factor UBTF to create a 15kbp nucleosome free region (NFR). Formation of this NFR is also essential for recruitment of the TBP-TAFI factor SL1 and for preinitiation complex (PIC) formation at the gene and enhancer-associated promoters of the rDNA. However, these promoters show little sequence commonality and neither UBTF nor SL1 display significant DNA sequence binding specificity, making what drives PIC formation a mystery. Here we show that cooperation between SL1 and the longer UBTF1 splice variant generates the specificity required for rDNA promoter recognition in cell. We find that conditional deletion of the Taf1b subunit of SL1 causes a striking depletion UBTF at both rDNA promoters but not elsewhere across the rDNA. We also find that while both UBTF1 and −2 variants bind throughout the rDNA NFR, only UBTF1 is present with SL1 at the promoters. The data strongly suggest an induced-fit model of RPI promoter recognition in which UBTF1 plays an architectural role. Interestingly, a recurrent UBTF-E210K mutation and the cause of a pediatric neurodegeneration syndrome provides indirect support for this model. E210K knock-in cells show enhanced levels of the UBTF1 splice variant and a concomitant increase in active rDNA copies. In contrast, they also display reduced rDNA transcription and promoter recruitment of SL1. We suggest the underlying cause of the UBTF-E210K syndrome is therefore a reduction in cooperative UBTF1-SL1 promoter recruitment that may be partially compensated by enhanced rDNA activation.

In the search for drugs to effectively treat cancer, the last 10 years have seen a resurgence of interest in targeting ribosome biogenesis. CX-5461 is a potential inhibitor of ribosomal RNA synthesis that is now showing promise in phase I trials as a chemotherapeutic agent for a range of malignancies. Here, we show that CX-5461 irreversibly inhibits ribosomal RNA transcription by arresting RNA polymerase I (RPI/Pol1/PolR1) in a transcription initiation complex. CX-5461 does not achieve this by preventing formation of the pre-initiation complex nor does it affect the promoter recruitment of the SL1 TBP complex or the HMGB-box upstream binding factor (UBF/UBTF). CX-5461 also does not prevent the subsequent recruitment of the initiation-competent RPI–Rrn3 complex. Rather, CX-5461 blocks promoter release of RPI–Rrn3, which remains irreversibly locked in the pre-initiation complex even after extensive drug removal. Unexpectedly, this results in an unproductive mode of RPI recruitment that correlates with the onset of nucleolar stress, inhibition of DNA replication, genome-wide DNA damage and cellular senescence. Our data demonstrate that the cytotoxicity of CX-5461 is at least in part the result of an irreversible inhibition of RPI transcription initiation and hence are of direct relevance to the design of improved strategies of chemotherapy.

SUMMARYThe human pathogenLegionella pneumophila (L.p.)secretes ~330 bacterial effector proteins into the host cell which interfere with numerous cellular pathways and often regulate host cell proteins through post-translational modifications. However, the cellular targets and functions of mostL.p.effectors are not known. In order to obtain a global overview of potential targets of these effectors, we analyzed the host cell proteome, ubiquitinome, and phosphoproteome duringL.p.infection. Our analysis reveals dramatic spatiotemporal changes in the host cell proteome that are dependent on the secretion of bacterial effectors. Strikingly, we show thatL.p.substantially reshapes the mitochondrial proteome and modulates mitochondrial stress response pathways such as the mitochondrial unfolded protein response (UPRmt). To our knowledge, this is the first evidence of manipulation of the UPRmtby a bacterial pathogen in mammalian cells. In addition, we have identified a previously uncharacterizedL.p.effector that is targeted to host cell mitochondria and protects mitochondrial network integrity during mitochondrial stress.

The human pathogen Legionella pneumophila (L.p.) secretes ~330 bacterial effector proteins into the host cell which interfere with numerous cellular pathways and often regulate host cell proteins through post-translational modifications. However, the cellular targets and functions of most L.p. effectors are not known. In order to obtain a global overview of potential targets of these effectors, we analyzed the host cell proteome, ubiquitinome, and phosphoproteome during L.p. infection. Our analysis reveals dramatic spatiotemporal changes in the host cell proteome that are dependent on the secretion of bacterial effectors. Strikingly, we show that L.p. substantially reshapes the mitochondrial proteome and modulates mitochondrial stress response pathways such as the mitochondrial unfolded protein response (UPRmt). To our knowledge, this is the first evidence of manipulation of the UPRmt by a bacterial pathogen in mammalian cells. In addition, we have identified a previously uncharacterized L.p. effector that is targeted to host cell mitochondria and protects mitochondrial network integrity during mitochondrial stress.

UBTF (upstream binding transcription factor) exists as two isoforms; UBTF1 regulates rRNA transcription by RNA polymerase 1, whereas UBTF2 regulates mRNA transcription by RNA polymerase 2. Herein, we describe 4 patients with very similar patterns of neuroregression due to recurrent de novo mutations in UBTF (GRCh37/hg19, NC_000017.10: g.42290219C > T, NM_014233.3: c.628G > A) resulting in the same amino acid change in both UBTF1 and UBTF2 (p.Glu210Lys [p.E210K]). Disease onset in our cohort was at 2.5 to 3 years and characterized by slow progression of global motor, cognitive and behavioral dysfunction. Notable early features included hypotonia with a floppy gait, high-pitched dysarthria and hyperactivity. Later features included aphasia, dystonia, and spasticity. Speech and ambulatory ability were lost by the early teens. Magnetic resonance imaging showed progressive generalized cerebral atrophy (supratentorial > infratentorial) with involvement of both gray and white matter. Patient fibroblasts showed normal levels of UBTF transcripts, increased expression of pre-rRNA and 18S rRNA, nucleolar abnormalities, markedly increased numbers of DNA breaks, defective cell-cycle progression, and apoptosis. Expression of mutant human UBTF1 in Drosophila neurons was lethal. Although no loss-of-function variants are reported in the Exome Aggregation Consortium (ExAC) database and Ubtf−/− is early embryonic lethal in mice, Ubtf+/− mice displayed only mild motor and behavioral dysfunction in adulthood. Our data underscore the importance of including UBTF E210K in the differential diagnosis of neuroregression and suggest that mainly gain-of-function mechanisms contribute to the pathogenesis of the UBTF E210K neuroregression syndrome.

The combination of Chromatin Immunoprecipitation and Massively Parallel Sequencing, or ChIP-Seq, has greatly advanced our genome-wide understanding of chromatin and enhancer structures. However, its resolution at any given genetic locus is limited by several factors. In applying ChIP-Seq to the study of the ribosomal RNA genes, we found that a major limitation to resolution was imposed by the underlying variability in sequence coverage that very often dominates the protein–DNA interaction profiles. Here, we describe a simple numerical deconvolution approach that, in large part, corrects for this variability, and significantly improves both the resolution and quantitation of protein–DNA interaction maps deduced from ChIP-Seq data. This approach has allowed us to determine the in vivo organization of the RNA polymerase I preinitiation complexes that form at the promoters and enhancers of the mouse (Mus musculus) and human (Homo sapiens) ribosomal RNA genes, and to reveal a phased binding of the HMG-box factor UBF across the rDNA. The data identify and map a “Spacer Promoter” and associated stalled polymerase in the intergenic spacer of the human ribosomal RNA genes, and reveal a very similar enhancer structure to that found in rodents and lower vertebrates.

The extended synaptotagmins, E-Syt1, 2 and 3, are multiple C2 domain membrane proteins that are tethered to the endoplasmic reticulum and interact in a calcium dependent manner with plasma membrane phospholipids to form endoplasmic reticulum - plasma membrane junctions. These junctions have been implicated in the exchange of phospholipids between the 2 organelles. The E-Syts have further been implicated in receptor signaling and endocytosis and can interact directly with fibroblast growth factor and other cell surface receptors. Despite these multiple functions, the search for a requirement in vivo has been elusive. Most recently, we found that the genes for E-Syt2 and 3 could be inactivated without effect on mouse development, viability, fertility or morphology. We have now created insertion and deletion mutations in the last of the mouse E-Syt genes. We show that E-Syt1 is specifically expressed throughout the embryonic skeleton during the early stages of chrondrogenesis in a pattern quite distinct from that of E-Syt2 or 3. Despite this, E-Syt1 is also not required for mouse development and propagation. We further show that even the combined inactivation of all 3 E-Syt genes has no effect on mouse viability or fertility in the laboratory. However, this inactivation induces an enhancement in the expression of the genes encoding Orp5/8, Orai1, STIM1 and TMEM110, endoplasmic reticulum - plasma membrane junction proteins that potentially could compensate for E-Syt loss. Given the multiple functions suggested for the E-Syts and their evolutionary conservation, our unexpected findings suggest that they may only provide a survival advantage under specific conditions that have as yet to be identified.

Rett syndrome (RTT) is a severe and rare neurological disorder that is caused by mutations in the X-linked MECP2 (methyl CpG-binding protein 2) gene. MeCP2 protein is an important epigenetic factor in the brain and in neurons. In Mecp2-deficient neurons, nucleoli structures are compromised. Nucleoli are sites of active ribosomal RNA (rRNA) transcription and maturation, a process mainly controlled by nucleolin and mechanistic target of rapamycin (mTOR)–P70S6K signaling. Currently, it is unclear how nucleolin–rRNA–mTOR–P70S6K signaling from RTT cellular model systems translates into human RTT brain. Here, we studied the components of nucleolin–rRNA–mTOR–P70S6K signaling in the brain of RTT patients with common T158M and R255X mutations. Immunohistochemical examination of T158M brain showed disturbed nucleolin subcellular localization, which was absent in Mecp2-deficient homozygous male or heterozygote female mice, compared to wild type (WT). We confirmed by Western blot analysis that nucleolin protein levels are altered in RTT brain, but not in Mecp2-deficient mice. Further, we studied the expression of rRNA transcripts in Mecp2-deficient mice and RTT patients, as downstream molecules that are controlled by nucleolin. By data mining of published ChIP-seq studies, we showed MeCP2-binding at the multi-copy rRNA genes in the mouse brain, suggesting that rRNA might be a direct MeCP2 target gene. Additionally, we observed compromised mTOR–P70S6K signaling in the human RTT brain, a molecular pathway that is upstream of rRNA–nucleolin molecular conduits. RTT patients showed significantly higher phosphorylation of active mTORC1 or mTORC2 complexes compared to age- and sex-matched controls. Correlational analysis of mTORC1/2–P70S6K signaling pathway identified multiple points of deviation from the control tissues that may result in abnormal ribosome biogenesis in RTT brain. To our knowledge, this is the first report of deregulated nucleolin–rRNA–mTOR–P70S6K signaling in the human RTT brain. Our results provide important insight toward understanding the molecular properties of human RTT brain.

// Nourdine Hamdane 1, 2, 3 , Chelsea Herdman 1, 2 , Jean-Clement Mars 1, 2 , Victor Stefanovsky 1 , Michel G. Tremblay 1 , Tom Moss 1, 2 1 Laboratory of Growth and Development, St-Patrick Research Group in Basic Oncology, Cancer Division of the Quebec University Hospital Research Centre, Quebec, QC, Canada 2 Department of Molecular Biology, Medical Biochemistry and Pathology, Faculty of Medicine, Laval University, Quebec, QC, Canada 3 Present address: Inserm, U1110, Institute of Viral and Liver Diseases, Strasbourg, France Correspondence to: Tom Moss, e-mail: Tom.Moss@crhdq.ulaval.ca Keywords: Upstream binding factor (UBF/UBTF), ribosome biogenesis, oncogenic stress, apoptosis, cisplatin Received: January 23, 2015 Accepted: July 27, 2015 Published: August 07, 2015 ABSTRACT Cisplatin-DNA adducts act as strong decoys for the Upstream Binding Factor UBF (UBTF) and have been shown to inhibit transcription of the ribosomal RNA genes by RNA polymerase I. However, it is unclear if this plays a significant role in the chemotherapeutic activity of cis- or carboplatin. We find that cisplatin in fact induces a very rapid displacement of UBF from the ribosomal RNA genes and strong inhibition of ribosomal RNA synthesis, consistent with this being an important factor in its cytotoxicity. Using conditional gene deletion, we recently showed that UBF is an essential factor for transcription of the ribosomal RNA genes and for ribosome biogenesis. We now show that loss of UBF arrests cell proliferation and induces fully penetrant, rapid and synchronous apoptosis, as well as nuclear disruption and cell death, specifically in cells subjected to oncogenic stress. Apoptosis is not affected by homozygous deletion of the p53 gene and occurs equally in cells transformed by SV40 T antigens, by Myc or by a combination of Ras & Myc oncogenes. The data strongly argue that inhibition of UBF function is a major factor in the cytotoxicity of cisplatin. Hence, drug targeting of UBF may be a preferable approach to the use of the highly toxic platins in cancer therapy.

Transcription of the several hundred of mouse and human Ribosomal RNA (rRNA) genes accounts for the majority of RNA synthesis in the cell nucleus and is the determinant of cytoplasmic ribosome abundance, a key factor in regulating gene expression. The rRNA genes, referred to globally as the rDNA, are clustered as direct repeats at the Nucleolar Organiser Regions, NORs, of several chromosomes, and in many cells the active repeats are transcribed at near saturation levels. The rDNA is also a hotspot of recombination and chromosome breakage, and hence understanding its control has broad importance. Despite the need for a high level of rDNA transcription, typically only a fraction of the rDNA is transcriptionally active, and some NORs are permanently silenced by CpG methylation. Various chromatin-remodelling complexes have been implicated in counteracting silencing to maintain rDNA activity. However, the chromatin structure of the active rDNA fraction is still far from clear. Here we have combined a high-resolution ChIP-Seq protocol with conditional inactivation of key basal factors to better understand what determines active rDNA chromatin. The data resolve questions concerning the interdependence of the basal transcription factors, show that preinitiation complex formation is driven by the architectural factor UBF (UBTF) independently of transcription, and that RPI termination and release corresponds with the site of TTF1 binding. They further reveal the existence of an asymmetric Enhancer Boundary Complex formed by CTCF and Cohesin and flanked upstream by phased nucleosomes and downstream by an arrested RNA Polymerase I complex. We find that the Enhancer Boundary Complex is the only site of active histone modification in the 45kbp rDNA repeat. Strikingly, it not only delimits each functional rRNA gene, but also is stably maintained after gene inactivation and the re-establishment of surrounding repressive chromatin. Our data define a poised state of rDNA chromatin and place the Enhancer Boundary Complex as the likely entry point for chromatin remodelling complexes.

Mechanisms to coordinate programs of highly transcribed genes required for cellular homeostasis and growth are unclear. Upstream binding transcription factor (UBTF, also called UBF) is thought to function exclusively in RNA polymerase I (Pol I)-specific transcription of the ribosomal genes. Here, we report that the two isoforms of UBTF (UBTF1/2) are also enriched at highly expressed Pol II-transcribed genes throughout the mouse genome. Further analysis of UBTF1/2 DNA binding in immortalized human epithelial cells and their isogenically matched transformed counterparts reveals an additional repertoire of UBTF1/2-bound genes involved in the regulation of cell cycle checkpoints and DNA damage response. As proof of a functional role for UBTF1/2 in regulating Pol II transcription, we demonstrate that UBTF1/2 is required for recruiting Pol II to the highly transcribed histone gene clusters and for their optimal expression. Intriguingly, lack of UBTF1/2 does not affect chromatin marks or nucleosome density at histone genes. Instead, it results in increased accessibility of the histone promoters and transcribed regions to micrococcal nuclease, implicating UBTF1/2 in mediating DNA accessibility. Unexpectedly, UBTF2, which does not function in Pol I transcription, is sufficient to regulate histone gene expression in the absence of UBTF1. Moreover, depletion of UBTF1/2 and subsequent reduction in histone gene expression is associated with DNA damage and genomic instability independent of Pol I transcription. Thus, we have uncovered a novel role for UBTF1 and UBTF2 in maintaining genome stability through coordinating the expression of highly transcribed Pol I (UBTF1 activity) and Pol II genes (UBTF2 activity). Refereed/Peer-reviewed

The Extended Synaptotagmins (Esyts) are a family of multi-C2 domain membrane proteins with orthologs in organisms from yeast to human. Three Esyt genes exist in mouse and human and these have most recently been implicated in the formation of junctions between endoplasmic reticulum and plasma membrane, as well as the Ca(2+) dependent replenishment of membrane phospholipids. The data are consistent with a function in extracellular signal transduction and cell adhesion, and indeed Esyt2 was previously implicated in both these functions in Xenopus. Despite this, little is known of the function of the Esyts in vivo. We have generated mouse lines carrying homozygous deletions in one or both of the genes encoding the highly homologous Esyt2 and Esyt3 proteins. Surprisingly, esyt2(-/-)/esyt3(-/-) mice develop normally and are both viable and fertile. In contrast, esyt2(-/-)/esyt3(-/-) mouse embryonic fibroblasts display a reduced ability to migrate in standard in vitro assays, and are less resistant to stringent culture conditions and to oxidative stress than equivalent wild type fibroblasts.

RNA metabolic labeling is a method of choice in the study of dynamic changes in the rate of gene transcription and RNA processing. It is particularly applicable to transcription of the ribosomal RNA genes and their processing products due to the very high levels of ribosomal RNA synthesis. Metabolic labeling can detect changes in ribosomal RNA transcription that occur within a few minutes as opposed to the still widely used RT-PCR or Northern blot procedures that measure RNA pool sizes and at best are able to detect changes occurring over several hours or several days. Here, we describe a metabolic labeling technique applicable to the measurement of ribosomal RNA synthesis and processing rates, as well as to the determination of RNA Polymerase I transcription elongation rates.

The nucleolus is the site of ribosome biogenesis and forms around the actively transcribed ribosomal RNA (rRNA) genes. However, the nucleolus is also implicated in cell cycle regulation, tumour suppression and chromosome segregation and nucleolar disfunction is linked to a wide range of human diseases. Interestingly, the nucleolus is also required for genome reprogramming and the establishment of heterochromatin in the mammalian embryo. Mammalian oocytes contain a subnuclear structure that is believed to be the precursor of the functional nucleolus, the Nucleolar Precursor Body (NPB). But the NPB is also required for the organisation of the zygotic heterochromatin and the establishment of pluripotency. We found that disruption of the mouse Upstream Binding Factor (UBF (UBTF)) gene caused disassembly of somatic nucleoli and the accumulation of the key rRNA gene transcription factors into dense subnuclear foci resembling NPBs. Here we show that UBF deletion causes the rRNA genes to collapse onto their centromere-proximal chromosomal sites spatially distinct from NPB-like structures, and that these structures contain rRNA gene transcription factors but not all nucleolar proteins. We further find that embryonic NPBs and their surrounding heterochromatin are both disrupted in UBF-null mouse embryos. These embryos also display subnuclear foci containing the rRNA gene transcription factors and arrest development before completing the forth cleavage division. The data suggest that the rRNA gene transcription factors have an intrinsic ability to interact and form a discrete nuclear compartment even in the absence of any rRNA gene activity and that the formation or maintenance of the zygotic NPB and surrounding heterochromatin requires UBF.

The Extended-Synaptotagmin (E-Syt) membrane proteins were only recently discovered, but have already been implicated in a range of interrelated cellular functions, including calcium and receptor signaling, and membrane lipid transport. However, despite their evolutionary conservation and detailed studies of their molecular actions, we still have little idea of how and when these proteins are required in cellular and organism physiology. Here we review our present understanding of the E-Syts and discuss the molecular functions and in vivo requirements for these proteins.

RNA metabolic labeling is a method of choice in the study of dynamic changes in the rate of gene transcription and RNA processing. It is particularly applicable to transcription of the ribosomal RNA genes and their processing products due to the very high levels of ribosomal RNA synthesis. Metabolic labeling can detect changes in ribosomal RNA transcription that occur within a few minutes as opposed to the still widely used RT-PCR or Northern blot procedures that measure RNA pool sizes and at best are able to detect changes occurring over several hours or several days. Here, we describe a metabolic labeling technique applicable to the measurement of ribosomal RNA synthesis and processing rates, as well as to the determination of RNA Polymerase I transcription elongation rates.

Pak1 is a member of the PAK family of serine/threonine kinases that are downstream effectors of Rac1 and Cdc42 small GTPases and are implicated in cytoskeleton reorganization. Early expression of Pak1 in Xenopus embryos is tissue restricted, suggesting a role in organogenesis and in cranial neural crest (CNC) cell migration. By observing CNC in vivo and after transplantation, we show that a dominant-negative (DN) Pak1 inhibits its migration. DN-Pak1 also specifically modified the expression of several NC markers. Twist expression was decreased and Snail1 expression posteriorized, but Snail2 (Slug), Sox9 and AP2 were unaffected. DN-Pak1 inhibition of CNC migration could be rescued with Snail1 but not with Twist, which, in fact, cooperated with DN-Pak1 in inhibiting migration. The data confirm that neither Snail1 nor Snail2 expression alone is sufficient for Xenopus CNC migration. Furthermore, they show that, in this tissue, Snail1 and Snail2 expression is not interdependent, nor are these factors subject to obligatory co-regulation, and that their expression depends on signal transduction. Our results also represent the first evidence that Pak1 links extracellular signals to the genetic cascade of transcription factors necessary for CNC specification.

"Mutations" in the pattern of CpG methylation imprinting of the human genome have been correlated with a number of diseases including cancer. In particular, aberrant imprinting of tumor suppressor genes by gain of CpG methylation has been observed in many cancers and thus represents an important alternative pathway to gene "mutation" and tumor progression. Inhibitors of DNA methylation display therapeutic effects in the treatment of certain cancers, and it has been assumed these effects are due to the reversal of "mutant" gene imprinting. However, significant reactivation of imprinted tumor suppressor genes is rarely observed in vivo following treatment with DNA methylation inhibitors. A recent study revealed an unexpected requirement for CpG methylation in the synthesis and assembly of the ribosome, an essential function for cell growth and proliferation. As such, the data provide an unforeseen explanation of the action of DNA methylation inhibitors in restricting cancer cell growth.

SummaryTargeting of activated plasma membrane receptors to endocytic pathways is important in determining the outcome of growth factor signaling. However, the molecular mechanisms are still poorly understood. Here, we show that the synaptotagmin-related membrane protein E-Syt2 is essential for rapid endocytosis of the activated FGF receptor and for functional signal transduction during Xenopus development. E-Syt2 depletion prevents an early phase of activated FGF receptor endocytosis that we show is required for ERK activation and the induction of the mesoderm. E-Syt2 interacts selectively with the activated FGF receptor and with Adaptin-2, and is required upstream of Ras activation and of receptor autophosphorylation for ERK activation and the induction of the mesodermal marker Xbra. The data identify E-Syt2 as an endocytic adaptor for the clathrin-mediated pathway whose function is conserved in human and suggest a broader role for the E-Syt subfamily in growth factor signaling.

The p14/p19(ARF) (ARF) product of the CDKN2A gene displays tumor suppressor activity both in the presence and absence of p53/TP53. In p53-negative cells, ARF arrests cell proliferation, at least in part, by suppressing ribosomal RNA synthesis. We show that ARF does this by controlling the subnuclear localization of the RNA polymerase I transcription termination factor, TTF-I. TTF-I shuttles between nucleoplasm and nucleolus with the aid of the chaperone NPM/B23 and a nucleolar localization sequence within its N-terminal regulatory domain. ARF inhibits nucleolar import of TTF-I by binding to this nucleolar localization sequence, causing the accumulation of TTF-I in the nucleoplasm. Depletion of TTF-I recapitulates the effects of ARF on ribosomal RNA synthesis and is rescued by the introduction of a TTF-I transgene. Thus, our data delineate the pathway by which ARF regulates ribosomal RNA synthesis and provide a compelling explanation for the role of NPM.

The mammalian architectural HMGB-Box transcription factor UBF is ubiquitously expressed in two variant forms as the result of a differential splicing event, that in the UBF2 deletes 37 amino acid from the second of six HMGB-boxes. Several attempts to define a function for this shorter UBF2 protein have been less than satisfactory. However, since all mammals appear to display similar levels of the longer and shorter UBF variants, it is unlikely that UBF2 is simply nonfunctional. Previously we showed that phosphorylation of UBF by the MAP-kinase ERK regulates chromatin folding and transcription elongation, explaining the rapid response of the ribosomal RNA genes to growth factors. Here we have investigated the roles the UBF variants play in the response of these genes to ERK activity. We demonstrate that the variant HMGB-box 2 of UBF2 has lost the ability to bind bent DNA and hence to induce chromatin folding. As a result it is significantly less effective than UBF1 at arresting RNAPI elongation but at the same time is more responsive to ERK phosphorylation. Thus, UBF2 functionally simulates a hemi-phosphorylated UBF whose expression may provide a means by which to tune the response of the ribosomal RNA genes to growth factor stimulation.

The mitogen-activated protein kinase kinase kinases of the mixed-lineage kinase (MLK) family have been shown to activate the c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) pathway, and to regulate the other two principal MAPK cascades, p38 and extracellular signal-regulated kinase (ERK). Although there is growing evidence for their involvement in neuronal cell death leading to neurodegenerative disorders, little in vivo data is available for the members of this family of kinases. Here, we report that the inactivation of mouse Mlk1 and Mlk2 genes. Mlk1(-/-) and Mlk2(-/-) mice were found to be viable and healthy. Surprisingly, mice carrying the compound Mlk1/Mlk2 null mutations were also found to be viable, fertile and to have a normal life span. The nervous system, testis and kidney, the major sites of MLK1 and 2 expression, all appear normal, as do other organs where these kinases were found to be more weakly expressed. Surprisingly, developmental neuronal programmed cell death, another potential target for MLK family members, was also found to be unaffected. Our results suggest that there is extensive functional redundancy between MLK1/MLK2 and the other member of the family, MLK3, which is also not required for survival in mouse.

The association of proteins with the DNA double helix can interfere with the accessibility of the latter to nucleases. This is particularly true when using bulky nucleases such as DNase I. The DNase I footprinting method was developed to take advantage of this fact in the study of DNA-protein interactions: it consists in comparing the pattern of fragments generated by the partial digestion of a DNA sequence in the absence of a protein to that produced by its partial digestion in the presence of said protein. Normally, when the two sets of fragments are separated side by side on a gel, the ladder of DNase I-generated fragments produced in the presence of the protein will feature blank regions (devoid of fragments, indicating protection) and/or enhanced cleavage sites (indicating increased availability to the nuclease). This technique can furthermore reveal if multiple sites for a DNA-binding protein are present on a same fragment and in such a case will also allow the comparison of their respective affinities.

So-called architectural DNA-binding proteins such as those of the HMGB-box family induce DNA bending and kinking. However, these proteins often display only a weak sequence preference, making the analysis of their DNA-binding characteristics difficult if not impossible in a standard electrophoretic mobility shift assay (EMSA). In contrast, such proteins often bind prebent DNAs with high affinity and specificity. A synthetic cruciform DNA structure will often provide an ideal binding site for such proteins, allowing their affinities for both bent and linear DNAs to be directly and simply determined by a modified form of EMSA.

The association of proteins with the DNA double helix can interfere with the accessibility of the latter to nucleases. This is particularly true when using bulky nucleases such as DNase I. The DNase I footprinting method was developed to take advantage of this fact in the study of DNA-protein interactions: it consists in comparing the pattern of fragments generated by the partial digestion of a DNA sequence in the absence of a protein to that produced by its partial digestion in the presence of said protein. Normally, when the two sets of fragments are separated side by side on a gel, the ladder of DNase I-generated fragments produced in the presence of the protein will feature blank regions (devoid of fragments, indicating protection) and/or enhanced cleavage sites (indicating increased availability to the nuclease). This technique can furthermore reveal if multiple sites for a DNA-binding protein are present on a same fragment and in such a case will also allow the comparison of their respective affinities.

So-called architectural DNA-binding proteins such as those of the HMGB-box family induce DNA bending and kinking. However, these proteins often display only a weak sequence preference, making the analysis of their DNA-binding characteristics difficult if not impossible in a standard electrophoretic mobility shift assay (EMSA). In contrast, such proteins often bind prebent DNAs with high affinity and specificity. A synthetic cruciform DNA structure will often provide an ideal binding site for such proteins, allowing their affinities for both bent and linear DNAs to be directly and simply determined by a modified form of EMSA.

Growth regulation of the tandemly repeated ribosomal RNA (rRNA) genes in mammals can potentially occur by several distinct mechanisms. Only a fraction of the 200 or so rRNA genes appears to be activated in somatic cells, leaving open the possibility that enhanced transcription could result from gene activation events. Here we have determined the active rRNA gene count after growth stimulation with EGF, direct Raf activation and chromatin hyperacetylation and after inhibiting MAP-kinase signaling. Despite robust changes in rRNA transcription rates, we find no significant variation in active gene number in either mouse fibroblasts or human neuroepithelioma cells. Interestingly, the data also show that rRNA transcription enhancement induced by hyperacetylation is dependent on MEK/ERK signaling. Since ERK and the acetyltransferase CBP both bind the architectural factor UBF, this suggests a mechanism for targeting active CBP to the rRNA genes.

Synthesis of the 45S rRNA by RNA polymerase I limits cell growth. Knowledge of the mechanism of its regulation is therefore key to understanding growth control. rRNA transcription is believed to be regulated solely at initiation/promoter release. However, we found that stimulation of endogenous 45S rRNA synthesis by epidermal growth factor (EGF) and serum failed to induce an increase in RNA polymerase I engagement on the rRNA genes, despite robust enhancement of 45S rRNA synthesis. Further, endogenous transcription elongation rates were measured and found to be directly proportional to 45S rRNA synthesis. Thus, elongation is a rate-limiting step for rRNA synthesis in vivo. ERK phosphorylation of the HMG boxes of UBF, an RNA polymerase I factor essential for transcription enhancement, was shown to directly regulate elongation by inducing the remodeling of ribosomal gene chromatin. The data suggest a mechanism for coordinating the cotranscriptional assembly of preribosomal particles.

Transcription of the ribosomal RNA genes of mammals by RNA polymerase I is rapidly activated by epidermal growth factor via the MAP-kinase (ERK) signaling cascade. This activation is mediated by direct phosphorylation of the HMG box DNA binding domains of the architectural transcription factor UBF. Mutation of the ERK sites of UBF inhibits its normal function and blocks growth factor activation of ribosomal transcription. UBF has little or no DNA sequence selectivity and binds throughout the ribosomal genes, defining a specialized chromatin. Indeed, the HMG boxes of UBF induce looping of the ribosomal DNA to create the enhancesome, a structure somewhat reminiscent of the nucleosome. Here, we show that both ERK phosphorylation and mutations that simulate this phosphorylation decrease the affinity of the individual HMG boxes of UBF for linear ribosomal DNA but have little or no effect on the capacity of these HMG boxes to bind to pre-bent DNA and do not affect the overall binding constant of UBF for the DNA. Electron spectroscopic imaging showed that ERK site UBF mutants do not induce the characteristic DNA looping of the enhancesome and associate with no more than half of the enhancesomal DNA. The data demonstrate that ERK phosphorylation of UBF prevents DNA bending by its first two HMG boxes, leading to a cooperative unfolding of the enhancesome.

Mammalian target of rapamycin (mTOR) is a key regulator of cell growth acting via two independent targets, ribosomal protein S6 kinase 1 (S6K1) and 4EBP1. While each is known to regulate translational efficiency, the mechanism by which they control cell growth remains unclear. In addition to increased initiation of translation, the accelerated synthesis and accumulation of ribosomes are fundamental for efficient cell growth and proliferation. Using the mTOR inhibitor rapamycin, we show that mTOR is required for the rapid and sustained serum-induced activation of 45S ribosomal gene transcription (rDNA transcription), a major rate-limiting step in ribosome biogenesis and cellular growth. Expression of a constitutively active, rapamycin-insensitive mutant of S6K1 stimulated rDNA transcription in the absence of serum and rescued rapamycin repression of rDNA transcription. Moreover, overexpression of a dominant-negative S6K1 mutant repressed transcription in exponentially growing NIH 3T3 cells. Rapamycin treatment led to a rapid dephosphorylation of the carboxy-terminal activation domain of the rDNA transcription factor, UBF, which significantly reduced its ability to associate with the basal rDNA transcription factor SL-1. Rapamycin-mediated repression of rDNA transcription was rescued by purified recombinant phosphorylated UBF and endogenous UBF from exponentially growing NIH 3T3 cells but not by hypophosphorylated UBF from cells treated with rapamycin or dephosphorylated recombinant UBF. Thus, mTOR plays a critical role in the regulation of ribosome biogenesis via a mechanism that requires S6K1 activation and phosphorylation of UBF.

The MLK family of mitogen activated protein kinase kinase kinases (MAPKKK) has been shown to activate Jun N-terminal kinase/stress-activated protein kinase 1 (JNK/SAPK1). However, little is known of the in vivo functions of the MLKs. We have identified a Xenopus laevis MLK that shows highest homology with mammalian MLK2 (62%) and, like MLK2, interacts preferentially with the Rho-family GTPase Rac. xMLK2 was expressed zygotically from late gastrula/early neurula. Surprisingly, this expression was restricted to the cement gland, the brain, and the pronephros. In the differentiating cement gland, xMLK2 expression correlated with cell elongation and the onset of a previously unobserved apoptotic phase, while in the pronephros, expression corresponded with the differentiation and opening of the nephric tubules. Overexpression of xMLK2 in COS7 cells led to a SEK1/MKK4 (MAPKK)-dependent hyperactivation of JNK in response to UV irradiation. xMLK2 was shown to be required for normal cement gland development and pronephric tubule formation using antisense inactivation and a dominant negative xMLK2. The data suggest a novel role for the MLKs as tissue-restricted mediators of signal transduction. They also suggest that tissue-specific responses to common extracellular signals may in part result from the programmed expression of MAPKKKs with differing specificities.

The ribosomal RNA genes encode the enzymatic scaffold of the ribosome and thereby perform perhaps the most basic of all housekeeping functions. However, recent data suggests that they might also control important aspects of cell behavior.

Ribosomal transcription in mammals is regulated in response to growth, differentiation, disease, and aging, but the mechanisms of this regulation have remained unresolved. We show that epidermal growth factor induces immediate, ERK1/2-dependent activation of endogenous ribosomal transcription, while inactivation of ERK1/2 causes an equally immediate reversion to the basal transcription level. ERK1/2 was found to phosphorylate the architectural transcription factor UBF at amino acids 117 and 201 within HMG boxes 1 and 2, preventing their interaction with DNA. Mutation of these sites inhibited transcription activation and abrogated the transcriptional response to ERK1/2. Thus, growth factor regulation of ribosomal transcription likely acts by a cyclic modulation of DNA architecture. The data suggest a central role for ribosome biogenesis in growth regulation.