bluuebunny/crossref_metadata_2025 huggingface dataset:
Xe-p9, a Xenopus Suc1/Cks protein, is essential for the Cdc2-dependent phosphorylation of the anaphase- promoting complex at mitosis
Authors: Patra Debabrata , Dunphy William G.
Published: February 2008

Degradation of mitotic cyclins on exit from M phase occurs by ubiquitin-mediated proteolysis. The ubiquitination of mitotic cyclins is regulated by the anaphase-promoting complex (APC) or cyclosome. Xe-p9, the Xenopus homolog of the Suc1/Cks protein, is required for some step in mitotic cyclin destruction inXenopus egg extracts. Specifically, if p9 is removed from interphase egg extracts, these p9-depleted extracts are unable to carry out the proteolysis of cyclin B after entry into mitosis and thus remain arrested in M phase. To explore the molecular basis of this defect, we depleted p9 from extracts that had already entered M phase and thus contained an active APC. We found that ubiquitin-mediated proteolysis of cyclin B was not compromised under these circumstances, suggesting that p9 is not directly required for ubiquitination or proteolysis. Further analysis of extracts from which p9 had been removed during interphase showed that, at the beginning of mitosis, these extracts are unable to carry out the hyperphosphorylation of the Cdc27 component of the APC, which coincides with the initial activation of the APC. p9 can be found in a complex with a small fraction of the Cdc27 protein during M phase but not interphase. The phosphorylation of the Cdc27 protein (either associated with the APC or in an isolated, bacterially expressed form) by recombinant Cdc2/cyclin B is strongly enhanced by p9. Our results indicate that p9 directly regulates the phosphorylation of the APC by Cdc2/cyclin B. These studies indicate that the Suc1/Cks protein modulates substrate recognition by a cyclin-dependent kinase.
Human and mouse homologs ofSchizosaccharomyces pombe rad1+andSaccharomyces cerevisiae RAD17:linkage to checkpoint control and mammalian meiosis
Authors: Freire Raimundo , Murguía Jose R. , Tarsounas Madalina , Lowndes Noel F. , Moens Peter B. , Jackson Stephen P.
Published: February 2008

Preventing or delaying progress through the cell cycle in response to DNA damage is crucial for eukaryotic cells to allow the damage to be repaired and not incorporated irrevocably into daughter cells. Several genes involved in this process have been discovered in fission and budding yeast. Here, we report the identification of human and mouse homologs of theSchizosaccharomyces pombeDNA damage checkpoint control generad1+and itsSaccharomyces cerevisiaehomologRAD17.The human geneHRAD1is located on chromosome 5p13 and is most homologous toS. pombe rad1+. This gene encodes a 382-amino-acid residue protein that is localized mainly in the nucleus and is expressed at high levels in proliferative tissues. This human gene significantly complements the sensitivity to UV light of aS. pombestrain mutated inrad1+. Moreover,HRAD1complements the checkpoint control defect of this strain after UV exposure. In addition to functioning in DNA repair checkpoints,S. cerevisiae RAD17plays a role during meiosis to prevent progress through prophase I when recombination is interrupted. Consistent with a similar role in mammals, Rad1 protein is abundant in testis, and is associated with both synapsed and unsynapsed chromosomes during meiotic prophase I of spermatogenesis, with a staining pattern distinct from that of the recombination proteins Rad51 and Dmc1. Together, these data imply an important role for hRad1 both in the mitotic DNA damage checkpoint and in meiotic checkpoint mechanisms, and suggest that these events are highly conserved from yeast to humans.
Cdc34 and the F-box protein Met30 are required for degradation of the Cdk-inhibitory kinase Swe1
Authors: Kaiser Peter , Sia Rey A.L. , Bardes Elaine G.S. , Lew Daniel J. , Reed Steven I.
Published: February 2008

Ubiquitin-mediated proteolysis controls the abundance of many cell cycle regulatory proteins. Recent work in Saccharomyces cerevisiae suggests that a complex consisting of Cdc53, Skp1, and a third component known as an F-box protein (termed SCF) in combination with Cdc34 specifically targets regulatory proteins for degradation, and that substrate specificity is likely to be mediated by the F-box subunit. A screen for genetic interactions with a cdc34mutation yielded MET30, which encodes an F-box protein.MET30 is an essential gene required for cell cycle progression and met30 mutations interact genetically with mutations in SCF components. Furthermore, physical interactions between Met30, Cdc53, Cdc34, and Skp1 in vivo provide evidence for an SCFMet30complex. We demonstrate the involvement of Met30 in the degradation of the Cdk-inhibitory kinase Swe1. Swe1 is stabilized in met30mutants and GST-Met30 pull-down experiments reveal that Met30 specifically binds Swe1 in vivo. Furthermore, extracts prepared fromcdc34 or met30 mutants are defective in polyubiquitination of Swe1. Taken together, these data suggest that SCF-mediated proteolysis may contribute to the regulation of entry into mitosis. Our data, in combination with previously published results, also provide evidence for distinct SCF complexes in vivo and support the idea that their F-box subunits mediate SCF substrate specificity.
Analysis of a Caenorhabditis elegans Twist homolog identifies conserved and divergent aspects of mesodermal patterning
Authors: Harfe Brian D. , Gomes Ana Vaz , Kenyon Cynthia , Liu Jun , Krause Michael , Fire Andrew
Published: February 2008

Mesodermal development is a multistep process in which cells become increasingly specialized to form specific tissue types. InDrosophila and mammals, proper segregation and patterning of the mesoderm involves the bHLH factor Twist. We investigated the activity of a Twist-related factor, CeTwist, during Caenorhabditis elegans mesoderm development. Embryonic mesoderm in C. elegans derives from a number of distinct founder cells that are specified during the early lineages; in contrast, a single blast cell (M) is responsible for all nongonadal mesoderm formation during postembryonic development. Using immunofluorescence and reporter fusions, we determined the activity pattern of the gene encoding CeTwist. No activity was observed during specification of mesodermal lineages in the early embryo; instead, the gene was active within the M lineage and in a number of mesodermal cells with nonstriated muscle fates. A role for CeTwist in postembryonic mesodermal cell fate specification was indicated by ectopic expression and genetic interference assays. These experiments showed that CeTwist was responsible for activating two target genes normally expressed in specific subsets of nonstriated muscles derived from the M lineage. In vitro and in vivo assays suggested that CeTwist cooperates with theC. elegans E/Daughterless homolog in directly activating these targets. The two target genes that we have studied,ceh-24 and egl-15, encode an NK-2 class homeodomain and an FGF receptor (FGFR) homolog, respectively. Twist activates FGFR and NK-homeodomain target genes during mesodermal patterning ofDrosophila and similar target interactions have been proposed to modulate mesenchymal growth during closure of the vertebrate skull. These results suggest the possibility that a conserved pathway may be used for diverse functions in mesodermal specification.
Activin is an essential early mesenchymal signal in tooth development that is required for patterning of the murine dentition
Authors: Ferguson Christine A. , Tucker Abigail S. , Christensen Lars , Lau Anthony L. , Matzuk Martin M. , Sharpe Paul T.
Published: February 2008

Development of the mammalian tooth has been intensively studied as a model system for epithelial/mesenchymal interactions during organogenesis, and progress has been made in identifying key molecules involved in this signaling. We show that activin βA is expressed in presumptive tooth-germ mesenchyme and is thus a candidate for a signaling molecule in tooth development. Analysis of tooth development in activin βAmutant embryos shows that incisor and mandibular molar teeth fail to develop beyond the bud stage. Activin βA is thus an essential component of tooth development. Development of maxillary molars, however, is unaffected in the mutants. Using tissue recombination experiments we show that activin is required in the mesenchyme prior to bud formation and that although activin signaling from mesenchyme to epithelium takes place, mutant epithelium retains its ability to support tooth development. Implantation of beads soaked in activin A, into developing mandibles, is able to completely rescue tooth development from E11.5, but not E12.5 or E13.5, confirming that activin is an early, essential mesenchyme signal required before tooth bud formation. Normal development of maxillary molars in the absence of activin shows a position specific role for this pathway in development of dentition. Functional redundancy with activin B or other TGFβ family members that bind to activin receptors cannot explain development of maxillary molars in the mutants since the activin-signaling pathway appears not to be active in these tooth germs. The early requirement for activin signaling in the mesenchyme in incisor and mandibular molar tooth germs must be carried-out in maxillary molar mesenchyme by other independent signaling pathways.
okra and spindle-B encode components of the RAD52 DNA repair pathway and affect meiosis and patterning in Drosophila oogenesis
Authors: Ghabrial Amin , Ray Robert P. , Schüpbach Trudi
Published: February 2008

okra (okr), spindle-B (spnB), andspindle-D (spnD) are three members of a group of female sterile loci that produce defects in oocyte and egg morphology, including variable dorsal–ventral defects in the eggshell and embryo, anterior–posterior defects in the follicle cell epithelium and in the oocyte, and abnormalities in oocyte nuclear morphology. Many of these phenotypes reflect defects in grk-Egfr signaling processes, and can be accounted for by a failure to accumulate wild-type levels of Gurken and Fs(1)K10. We have cloned okr and spnB, and show that okr encodes the Drosophila homolog of the yeast DNA-repair protein Rad54, and spnB encodes a Rad51-like protein related to the meiosis-specific DMC1 gene. In functional tests of their role in DNA repair, we find that okrbehaves like its yeast homolog in that it is required in both mitotic and meiotic cells. In contrast, spnB and spnD appear to be required only in meiosis. The fact that genes involved in meiotic DNA metabolism have specific effects on oocyte patterning implies that the progression of the meiotic cell cycle is coordinated with the regulation of certain developmental events during oogenesis.
Amino acid–amino acid contacts at the cooperativity interface of the bacteriophage λ and P22 repressors
Authors: Whipple Frederick W. , Hou Emmeline F. , Hochschild Ann
Published: February 2008

The bacteriophage λ repressor and its relatives bind cooperatively to adjacent as well as artificially separated operator sites. This cooperativity is mediated by a protein–protein interaction between the DNA-bound dimers. Here we use a genetic approach to identify two pairs of amino acids that interact at the dimer–dimer interface. One of these pairs is nonconserved in the aligned sequences of the λ and P22 repressors; we show that a λ repressor variant bearing the P22 residues at these two positions interacts specifically with the P22 repressor. The other pair consists of a conserved ion pair; we reverse the charges at these two positions and demonstrate that, whereas the individual substitutions abolish the interaction of the DNA-bound dimers, these changes in combination restore the interaction of both λcI and P22c2 dimers.
Mitotic inactivation of a human SWI/SNF chromatin remodeling complex
Authors: Sif Saïd , Stukenberg P. Todd , Kirschner Marc W. , Kingston Robert E.
Published: February 2008

During mitosis, chromatin is condensed into mitotic chromosomes and transcription is inhibited, processes that might be opposed by the chromatin remodeling activity of the SWI/SNF complexes. Brg1 and hBrm, which are components of human SWI/SNF (hSWI/SNF) complexes, were recently shown to be phosphorylated during mitosis. This suggested that phosphorylation might be used as a switch to modulate SWI/SNF activity. Using an epitope-tag strategy, we have purified hSWI/SNF complexes at different stages of the cell cycle, and found that hSWI/SNF was inactive in cells blocked in G2–M. Mitotic hSWI/SNF contained Brg1 but not hBrm, and was phosphorylated on at least two subunits, hSWI3 and Brg1. In vitro, active hSWI/SNF from asynchronous cells can be phosphorylated and inactivated by ERK1, and reactivated by dephosphorylation. hSWI/SNF isolated as cells traversed mitosis regained activity when its subunits were dephosphorylated either in vitro or in vivo. We propose that this transitional inactivation and reactivation of hSWI/SNF is required for formation of a repressed chromatin structure during mitosis and reformation of an active chromatin structure as cells leave mitosis.
Developmentally programmed assembly of higher order telomerase complexes with distinct biochemical and structural properties
Authors: Greene Eric C. , Shippen Dorothy E.
Published: February 2008

In Euplotes crassus, telomerase is responsible for telomere maintenance during vegetative growth and de novo telomere synthesis during macronuclear development. Here we show that telomerase in the vegetative stage of the life cycle exists as a 280-kD complex that can add telomeric repeats only onto telomeric DNA primers. Following the initiation of macronuclear development, telomerase assembles into larger complexes of 550 kD, 1600 kD, and 5 MD. In the 1600-kDa and 5-MDa complexes, telomerase is more processive than in the two smaller complexes and can add telomeres de novo onto nontelomeric 3′ ends. Assembly of higher order telomerase complexes is accompanied by an extended region of RNase V1 and RNase T1 protection in the telomerase RNA subunit that is not observed with telomerase from vegetatively growing cells. The protected residues encompass a highly conserved region previously proposed to serve as a platform for formation of higher order structures. These findings provide the first direct demonstration of developmentally regulated higher order telomerase complexes with unique biochemical and structural properties.
mei-W68 in Drosophila melanogaster encodes a Spo11 homolog: evidence that the mechanism for initiating meiotic recombination is conserved
Authors: McKim Kim S. , Hayashi-Hagihara Aki
Published: February 2008

Meiotic recombination requires the action of several gene products in both Saccharomyces cerevisiae and Drosophila melanogaster. Genetic studies in D. melanogaster have shown that the mei-W68 gene is required for all meiotic gene conversion and crossing-over. We cloned mei-W68 using a new genetic mapping method in which P elements are used to promote crossing-over at their insertion sites. This resulted in the high-resolution mapping of mei-W68 to a <18-kb region that contains a homolog of the S. cerevisiae spo11 gene. Molecular analysis of several mutants confirmed that mei-W68 encodes anspo11 homolog. Spo11 and MEI-W68 are members of a family of proteins similar to a novel type II topoisomerase. On the basis of this and other lines of evidence, Spo11 has been proposed to be the enzymatic activity that creates the double-strand breaks needed to initiate meiotic recombination. This raises the possibility that recombination in Drosophila is also initiated by double-strand breaks. Although these homologous genes are required absolutely for recombination in both species, their roles differ in other respects. In contrast to spo11, mei-W68 is not required for synaptonemal complex formation and does have a mitotic role.
Saccharomyces cerevisiae cAMP-dependent protein kinase controls entry into stationary phase through the Rim15p protein kinase
Authors: Reinders Anke , Bürckert Niels , Boller Thomas , Wiemken Andres , De Virgilio Claudio
Published: February 2008

The Saccharomyces cerevisiae protein kinase Rim15p was identified previously as a stimulator of meiotic gene expression. Here, we show that loss of Rim15p causes an additional pleiotropic phenotype in cells grown to stationary phase on rich medium; this phenotype includes defects in trehalose and glycogen accumulation, in transcriptional derepression of HSP12, HSP26, andSSA3, in induction of thermotolerance and starvation resistance, and in proper G1 arrest. These phenotypes are commonly associated with hyperactivity of the Ras/cAMP pathway. Tests of epistasis suggest that Rim15p may act in this pathway downstream of the cAMP-dependent protein kinase (cAPK). Accordingly, deletion of RIM15 suppresses the growth defect of a temperature-sensitive adenylate-cyclase mutant and, most importantly, renders cells independent of cAPK activity. Conversely, overexpression of RIM15 suppresses phenotypes associated with a mutation in the regulatory subunit of cAPK, exacerbates the growth defect of strains compromised for cAPK activity, and partially induces a starvation response in logarithmically growing wild-type cells. Biochemical analyses reveal that cAPK-mediated in vitro phosphorylation of Rim15p strongly inhibits its kinase activity. Taken together, these results place Rim15p immediately downstream and under negative control of cAPK and define a positive regulatory role of Rim15p for entry into both meiosis and stationary phase.
The HIV-1 Tat cellular coactivator Tat–SF1 is a general transcription elongation factor
Authors: Li Xiao-Yong , Green Michael R
Published: February 2008

The human immunodeficiency virus type 1 (HIV-1) Tat protein strongly and specifically stimulates transcription elongation from the HIV-1 LTR and provides an important in vitro model system to study this process. Here we use protein-affinity chromatography to identify cellular factors involved in transcription elongation. A Tat-affinity column bound one transcription factor, Tat–SF1, efficiently and selectively. Tat–SF1 was identified originally as a Tat-specific coactivator, but we show it is a general transcription elongation factor. Our results also reveal the existence of an ATP-inactivatable general elongation factor (AIEF) required for Tat–SF1 activity and for which Tat can substitute functionally.
Senescence of human fibroblasts induced by oncogenic Raf
Authors: Zhu Jiyue , Woods Douglas , McMahon Martin , Bishop J. Michael
Published: February 2008

The oncogenes RAS and RAF came to view as agents of neoplastic transformation. However, in normal cells, these genes can have effects that run counter to oncogenic transformation, such as arrest of the cell division cycle, induction of cell differentiation, and apoptosis. Recent work has demonstrated that RAS elicits proliferative arrest and senescence in normal mouse and human fibroblasts. Because the Raf/MEK/MAP kinase signaling cascade is a key effector of signaling from Ras proteins, we examined the ability of conditionally active forms of Raf-1 to elicit cell cycle arrest and senescence in human cells. Activation of Raf-1 in nonimmortalized human lung fibroblasts (IMR-90) led to the prompt and irreversible arrest of cellular proliferation and the premature onset of senescence. Concomitant with the onset of cell cycle arrest, we observed the induction of the cyclin-dependent kinase (CDK) inhibitors p21Cip1 and p16Ink4a. Ablation of p53 and p21Cip1 expression by use of the E6 oncoprotein of HPV16 demonstrated that expression of these proteins was not required for Raf-induced cell cycle arrest or senescence. Furthermore, cell cycle arrest and senescence were elicited in IMR-90 cells by the ectopic expression of p16Ink4a alone. Pharmacological inhibition of the Raf/MEK/MAP kinase cascade prevented Raf from inducing p16Ink4a and also prevented Raf-induced senescence. We conclude that the kinase cascade initiated by Raf can regulate the expression of p16Ink4a and the proliferative arrest and senescence that follows. Induction of senescence may provide a defense against neoplastic transformation when the MAP kinase signaling cascade is inappropriately active.
Chromatin, TAFs, and a novel multiprotein coactivator are required for synergistic activation by Sp1 and SREBP-1a in vitro
Authors: Näär Anders M. , Beaurang Pierre A. , Robinson Karen M. , Oliner Jon D. , Avizonis Daina , Scheek Sigrid , Zwicker Jörk , Kadonaga James T. , Tjian Robert
Published: February 2008

The promoter selectivity factor Sp1 often cooperates with other enhancer-binding proteins to activate transcription. To study the molecular underpinnings of these regulatory events, we have reconstituted in vitro the synergy observed in vivo between Sp1 and the sterol-regulated factor SREBP-1a at the low density lipoprotein receptor (LDLR) promoter. Using a highly purified human transcription system, we found that chromatin, TAFs, and a novel SREBP-binding coactivator activity, which includes CBP, are all required to mediate full synergistic activation by Sp1 and SREBP-1a. The SREBP-binding domain of CBP inhibits activation by SREBP-1a and Sp1 in a dominant-negative fashion that is both chromatin- and activator-specific. Whereas recombinant CBP alone is not sufficient to mediate activation, a human cellular fraction containing CBP can support high levels of chromatin-dependent synergistic activation. Purification of this activity to near homogeneity resulted in the identification of a multiprotein coactivator, including CBP, that selectively binds to the SREBP-1a activation domain and is capable of mediating high levels of synergistic activation by SREBP/Sp1 on chromatin templates. The development of a reconstituted chromatin transcription system has allowed us to isolate a novel coactivator that is recruited by the SREBP-1a activation domain and that functions in concert with TFIID to coordinate the action of multiple activators at complex promoters in the context of chromatin.
Yeast telomeres exert a position effect on recombination between internal tracts of yeast telomeric DNA
Authors: Stavenhagen Jeffrey B. , Zakian Virginia A.
Published: February 2008

In Saccharomyces cerevisiae, proximity to a telomere affects both transcription and replication of adjacent DNA. In this study, we show that telomeres also impose a position effect on mitotic recombination. The rate of recombination between directly repeated tracts of telomeric C1–3A/TG1–3 DNA was reduced severely by proximity to a telomere. In contrast, recombination of two control substrates was not affected by telomere proximity. Thus, unlike position effects on transcription or replication, inhibition of recombination was sequence specific. Moreover, the repression of recombination was not under the same control as transcriptional repression (telomere position effect; TPE), as mutations in genes essential for TPE did not alleviate telomeric repression of recombination. The reduction in recombination between C1–3A/TG1–3 tracts near the telomere was caused by an absence of Rad52p-dependent events as well as a reduction in Rad1p-dependent events. The sequence-specific repression of recombination near the telomere was eliminated in cells that overexpressed the telomere-binding protein Rap1p, a condition that also increased recombination between C1–3A/TG1–3 tracts at internal positions on the chromosome. We propose that the specific inhibition between C1–3A/TG1–3 tracts near the telomere occurs through the action of a telomere-specific end-binding protein that binds to the single-strand TG1–3 tail generated during the processing of recombination intermediates. The recombination inhibitor protein may also block recombination between endogenous telomeres.
Synapse-specific and neuregulin-induced transcription require an Ets site that binds GABPα/GABPβ
Authors: Fromm Larry , Burden Steven J.
Published: February 2008

Localization of acetylcholine receptors (AChRs) to neuromuscular synapses is mediated by multiple pathways. Agrin, which is the signal for one pathway, stimulates a redistribution of previously unlocalized AChRs to synaptic sites. The signal for a second pathway is not known, but this signal stimulates selective transcription of AChRgenes in myofiber nuclei located near the synaptic site. Neuregulin (NRG) is a good candidate for the extracellular signal that induces synapse-specific gene expression, since NRG is concentrated at synaptic sites and activates AChR gene expression in cultured muscle cells. Previous studies have demonstrated that 181 bp of 5′ flanking DNA from the AChR δ-subunit gene are sufficient to confer synapse-specific transcription in transgenic mice and NRG responsiveness in cultured muscle cells, but the critical sequences within this cis-acting regulatory region have not been identified. We transfected AChR δ-subunit–hGH gene fusions into a muscle cell line, and we show that a potential binding site for Ets proteins is required for NRG-induced gene expression. Furthermore, we produced transgenic mice carrying AChR δ-subunit–hGH gene fusions with a mutation in this NRG-response element (NRE), and we show that this NRE is necessary for synapse-specific transcription in mice. The NRE binds proteins in myotube nuclear extracts, and nucleotides that are important for NRG responsiveness are likewise critical for formation of the protein–DNA complex. This complex contains GABPα, an Ets protein, and GABPβ, a protein that lacks an Ets domain but dimerizes with GABPα, because formation of the protein–DNA complex is inhibited by antibodies to either GABPα or GABPβ. These results demonstrate that synapse-specific and NRG-induced gene expression require an Ets-binding site and suggest that GABPα/GABPβ mediates the transcriptional response of the AChR δ-subunit gene to synaptic signals, including NRG.
HIV-1 Vpr interacts with the nuclear transport pathway to promote macrophage infection
Authors: Vodicka Marie A. , Koepp Deanna M. , Silver Pamela A. , Emerman Michael
Published: February 2008

HIV-1 Vpr promotes nuclear entry of viral nucleic acids in nondividing macrophages and also causes a G2 cell-cycle arrest. Consistent with its role in nuclear transport, we show Vpr localizes to the nuclear envelope in both human and yeast cells. Like the importin-β subunit of the nuclear import receptor, Vpr also interacts with the yeast importin-α subunit and nucleoporins. Moreover, overexpression of either Vpr or importin-β in yeast blocks nuclear transport of mRNAs. A mutant form of Vpr (Vpr F34I) that does not localize at the nuclear envelope, or bind to importin-α and nucleoporins, renders HIV-1 incapable of infecting macrophages efficiently. Vpr F34I, however, still causes a G2 arrest, demonstrating that the dual functions of Vpr are genetically separable. Our data suggest Vpr functionally resembles importin-β in nuclear import of the HIV-1 pre-integration complex and this function is essential for the role of Vpr in macrophage infection, but not G2 arrest.
Chromatin assembly factor I contributes to the maintenance, but not the re-establishment, of silencing at the yeast silent mating loci
Authors: Enomoto Shinichiro , Berman Judith
Published: February 2008

CAC1/RLF2 encodes the largest subunit of chromatin assembly factor I (CAF-I), a complex that assembles newly synthesized histones onto recently replicated DNA in vitro. In vivo,cac1/rlf2 mutants are defective in telomeric silencing and mislocalize Rap1p, a telomere-binding protein. Here, we report that in cells lacking CAF-I the silent mating loci are derepressed partially. MATa cac1 cells exhibit an unusual response to α-factor: They arrest and form mating projections (shmoos) initially, but are unable to sustain the arrest state, giving rise to clusters of shmooing cells. cac1 MATa HMLaHMRa strains do not form these shmoo clusters, indicating that derepression of HMLα causes the shmoo cluster phenotype in cac1 cells. When SIR3 is reintroduced intosir1 sir3 cells, HML remains derepressed indicating that SIR1 is required for the re-establishment of silencing atHML. In contrast, when SIR3 is reintroduced intocac1 sir3 cells, silencing is restored to HML,indicating that CAF-I is not required for the re-establishment of silencing. Loss of the other CAF-I subunits (Cac2p and Cac3p/Msi1p) also results in the shmoo cluster phenotype, implying that loss of CAF-I activity gives rise to this unstable repression of HML. Strains carrying certain mutations in the amino terminus of histone H4 and strains with limiting amounts of Sir2p or Sir3p also form shmoo clusters, implying that the shmoo cluster phenotype is indicative of defects in maintenance of the structural integrity of silent chromatin. MATa cac− sir1 double mutants have a synergistic mating defect, suggesting that the two silencing mechanisms, establishment and maintenance, function cooperatively. We propose a model to explain the distinctions between the establishment and the maintenance of silent chromatin.
Functional and regulatory interactions between Hox andextradenticle genes
Authors: Azpiazu Natalia , Morata Ginés
Published: February 2008

The homeobox gene extradenticle (exd) acts as a cofactor of Hox function both in Drosophila and vertebrates. It has been shown that the distribution of the Exd protein is developmentally regulated at the post-translational level; in the regions where exd is not functional Exd is present only in the cell cytoplasm, whereas it accumulates in the nuclei of cells requiringexd function. We show that the subcellular localization of Exd is regulated by the BX-C genes and that each BX-C gene can prevent or reduce nuclear translocation of Exd to different extents. In spite of this negative regulation, two BX-C genes, Ultrabithorax andabdominal-A, require exd activity for their maintenance and function. We propose that mutual interactions between Exd and BX-C proteins ensure the correct amounts of interacting molecules. As theHoxd10 gene has the same properties as Drosophila BX-C genes, we suggest that the control mechanism of subcellular distribution of Exd found in Drosophila probably operates in other organisms as well.
DRhoGEF2 encodes a member of the Dbl family of oncogenes and controls cell shape changes during gastrulation in Drosophila
Authors: Häcker Udo , Perrimon Norbert
Published: February 2008

We have identified a gene, DRhoGEF2, which encodes a putative guanine nucleotide exchange factor belonging to the Dbl family of oncogenes. DRhoGEF2 function is essential for the coordination of cell shape changes during gastrulation. In the absence of maternalDRhoGEF2 gene activity, mesodermal and endodermal primordia fail to invaginate. The phenotype seen in DRhoGEF2 mutants is more severe than the defects associated with mutations in two previously identified gastrulation genes, folded gastrulationand concertina, suggesting that DRhoGEF2 acts in a signaling pathway independent of these genes. Expression of dominant-negative DRhoA during gastrulation results in phenocopies of the DRhoGEF2 mutant, suggesting that a signaling cascade involving DRhoGEF2 and the small GTPase DRhoA is responsible for the regulation of cell shape changes during early Drosophilamorphogenesis.
Insulin resistance and diabetes mellitus in transgenic mice expressing nuclear SREBP-1c in adipose tissue: model for congenital generalized lipodystrophy
Authors: Shimomura Iichiro , Hammer Robert E. , Richardson James A. , Ikemoto Shinji , Bashmakov Yuriy , Goldstein Joseph L. , Brown Michael S.
Published: February 2008

Overexpression of the nuclear form of sterol regulatory element-binding protein-1c (nSREBP-1c/ADD1) in cultured 3T3-L1 preadipocytes was shown previously to promote adipocyte differentiation. Here, we produced transgenic mice that overexpress nSREBP-1c in adipose tissue under the control of the adipocyte-specific aP2 enhancer/promoter. A syndrome with the following features was observed: (1) Disordered differentiation of adipose tissue. White fat failed to differentiate fully, and the size of white fat depots was markedly decreased. Brown fat was hypertrophic and contained fat-laden cells resembling immature white fat. Levels of mRNA encoding adipocyte differentiation markers (C/EBPα, PPARγ, adipsin, leptin, UCP1) were reduced, but levels of Pref-1 and TNFα were increased. (2) Marked insulin resistance with 60-fold elevation in plasma insulin. (3) Diabetes mellitus with elevated blood glucose (>300 mg/dl) that failed to decline when insulin was injected. (4) Fatty liver from birth and elevated plasma triglyceride levels later in life. These mice exhibit many of the features of congenital generalized lipodystrophy (CGL), an autosomal recessive disorder in humans.
Drosophila unpaired encodes a secreted protein that activates the JAK signaling pathway
Authors: Harrison Douglas A. , McCoon Patricia E. , Binari Richard , Gilman Michael , Perrimon Norbert
Published: February 2008

In vertebrates, many cytokines and growth factors have been identified as activators of the JAK/STAT signaling pathway. In Drosophila, JAK and STAT molecules have been isolated, but no ligands or receptors capable of activating the pathway have been described. We have characterized the unpaired(upd) gene, which displays the same distinctive embryonic mutant defects as mutations in the Drosophila JAK (hopscotch) and STAT (stat92E) genes. Upd is a secreted protein, associated with the extracellular matrix, that activates the JAK pathway. We propose that Upd is a ligand that relies on JAK signaling to stimulate transcription of pair-rule genes in a segmentally restricted manner in the early Drosophila embryo.
Myoblast city, the Drosophila homolog of DOCK180/CED-5, is required in a Rac signaling pathway utilized for multiple developmental processes
Authors: Nolan Katherine M. , Barrett Kathy , Lu Yu , Hu Kang-Quan , Vincent Sylvie , Settleman Jeffrey
Published: February 2008

The Rac and Cdc42 GTPases share several regulators and effectors, yet perform distinct biological functions. The factors determining such specificity in vivo have not been identified. In a mutational screen inDrosophila to identify Rac-specific signaling components, we isolated 11 alleles of myoblast city (mbc). mbcmutant embryos exhibit defects in dorsal closure, myogenesis, and neural development. DOCK180, the mammalian homolog of Mbc, associates with Rac, but not Cdc42, in a nucleotide-independent manner. These results suggest that Mbc is a specific upstream regulator of Rac activity that mediates several morphogenetic processes inDrosophila embryogenesis.
Allosteric interactions between capping enzyme subunits and the RNA polymerase II carboxy-terminal domain
Authors: Cho Eun-Jung , Rodriguez Christine R. , Takagi Toshimitsu , Buratowski Stephen
Published: February 2008

mRNA capping is a cotranscriptional event mediated by the association of capping enzyme with the phosphorylated carboxy-terminal domain (CTD) of RNA polymerase II. In the yeast Saccharomyces cerevisiae, capping enzyme is composed of two subunits, the mRNA 5′-triphosphatase (Cet1) and the mRNA guanylyltransferase (Ceg1). Here we map interactions between Ceg1, Cet1, and the CTD. Although the guanylyltransferase subunit can bind alone to the CTD, it cannot be guanylylated unless the triphosphatase subunit is also present. Therefore, the yeast mRNA guanylyltransferase is regulated by allosteric interactions with both the triphosphatase and CTD.
Ligand-independent recruitment of steroid receptor coactivators to estrogen receptor by cyclin D1
Authors: Zwijsen Renate M.L. , Buckle Robin S. , Hijmans E. Marielle , Loomans Cindy J.M. , Bernards René
Published: February 2008

The estrogen receptor (ER) is an important regulator of growth and differentiation of breast epithelium. Transactivation by ER depends on a leucine-rich motif, which constitutes a ligand-regulated binding site for steroid receptor coactivators (SRCs). Cyclin D1 is frequently amplified in breast cancer and can activate ER through direct binding. We show here that cyclin D1 also interacts in a ligand-independent fashion with coactivators of the SRC-1 family through a motif that resembles the leucine-rich coactivator binding motif of nuclear receptors. By acting as a bridging factor between ER and SRCs, cyclin D1 can recruit SRC-family coactivators to ER in the absence of ligand. A cyclin D1 mutant that binds to ER but fails to recruit coactivators preferentially interferes with ER activation in breast cancer cells that have high levels of cyclin D1. These data support that cyclin D1 contributes significantly to ER activation in breast cancers in which the protein is overexpressed. Our present results reveal a novel route of coactivator recruitment to ER and establish a direct role for cyclin D1 in regulation of transcription.
Synaptonemal complex morphogenesis and sister-chromatid cohesion require Mek1-dependent phosphorylation of a meiotic chromosomal protein
Authors: Bailis Julie M. , Roeder G. Shirleen
Published: February 2008

Development of yeast meiotic chromosome cores into full-length synaptonemal complexes requires the MEK1 gene product, a meiosis-specific protein kinase homolog. The Mek1 protein associates with meiotic chromosomes and colocalizes with the Red1 protein, which is a component of meiotic chromosome cores. Mek1 and Red1 interact physically in meiotic cells, as demonstrated by coimmunoprecipitation and the two-hybrid protein system. Hop1, another protein associated with meiotic chromosome cores, also interacts with Mek1 but only in the presence of Red1. Red1 displays Mek1-dependent phosphorylation, both in vitro and in vivo, and Mek1 kinase activity is necessary for Mek1 function in vivo. Fluorescent in situ hybridization analysis indicates that Mek1-mediated phosphorylation of Red1 is required for meiotic sister-chromatid cohesion, raising the possibility that cohesion is regulated by protein phosphorylation.
A novel, putative MEK kinase controls developmental timing and spatial patterning in Dictyostelium and is regulated by ubiquitin-mediated protein degradation
Authors: Chung Chang Y. , Reddy T.B.K. , Zhou Kemin , Firtel Richard A.
Published: February 2008

We have identified a developmentally regulated, putative MEK kinase (MEKKα) that contains an F-box and WD40 repeats and plays a complex role in regulating cell-type differentiation and spatial patterning. Cells deficient in MEKKα develop precociously and exhibit abnormal cell-type patterning with an increase in one of the prestalk compartments (pstO), a concomitant reduction in the prespore domain, and a loss of the sharp compartment boundaries, resulting in overlapping prestalk and prespore domains. Overexpression of MEKKα or MEKKα lacking the WD40 repeats results in very delayed development and a severe loss of compartment boundaries. Prespore and prestalk cells are interspersed throughout the slug. Analysis of chimeric organisms suggests that MEKKα function is required for the proper induction and maintenance of prespore cell differentiation. We show that the WD40 repeats target MEKKα to the cortical region of the cell, whereas the F-box/WD40 repeats direct ubiquitin-mediated MEKKα degradation. We identify a UBC and a UBP (ubiquitin hydrolase) that interact with the F-box/WD40 repeats. Our findings indicate that cells lacking the ubiquitin hydrolase have phenotypes similar to those ofMEKKα null (mekkα−) cells, further supporting a direct genetic and biochemical interaction between MEKKα, the UBC, and the UBP. We demonstrate that UBC and UBP differentially control MEKKα ubiquitination/deubiquitination and degradation through the F-box/WD40 repeats in a cell-type-specific and temporally regulated manner. Our results represent a novel mechanism that includes targeted protein degradation by which MAP kinase cascade components can be controlled. More importantly, our findings suggest a new paradigm of spatial and temporal control of the kinase activity controlling spatial patterning during multicellular development, which parallels the temporally regulated degradation of proteins required for cell-cycle progression.
Dorsoventral patterning in the Drosophila central nervous system: the intermediate neuroblasts defective homeobox gene specifies intermediate column identity
Authors: Weiss Joseph B. , Von Ohlen Tonia , Mellerick Dervla M. , Dressler Gregory , Doe Chris Q. , Scott Matthew P.
Published: February 2008

One of the first steps in neurogenesis is the diversification of cells along the dorsoventral axis. In Drosophila the central nervous system develops from three longitudinal columns of cells: ventral cells that express the vnd/nk2 homeobox gene, intermediate cells, and dorsal cells that express the mshhomeobox gene. Here we describe a new Drosophila homeobox gene,intermediate neuroblasts defective (ind), which is expressed specifically in the intermediate column cells. ind is essential for intermediate column development: Null mutants have a transformation of intermediate to dorsal column neuroectoderm fate, and only 10% of the intermediate column neuroblasts develop. The establishment of dorsoventral column identity involves negative regulation: Vnd represses ind in the ventral column, whereas ind represses msh in the intermediate column. Vertebrate genes closely related to vnd (Nkx2.1 andNkx2.2), ind (Gsh1 and Gsh2), andmsh (Msx1 and Msx3) are expressed in corresponding ventral, intermediate, and dorsal domains during vertebrate neurogenesis, raising the possibility that dorsoventral patterning within the central nervous system is evolutionarily conserved.
Formation and specification of ventral neuroblasts is controlled by vnd in Drosophila neurogenesis
Authors: Chu Hsin , Parras Carlos , White Kalpana , Jiménez Fernando
Published: February 2008

During Drosophila neural development, neuroblasts delaminate from the neuroectoderm of each hemisegment in a stereotypic orthogonal array of five rows and three columns (ventral, intermediate, and dorsal). Prevailing evidence indicates that the individual neuroblast fate is determined by the domain-specific expression of genes along the dorsoventral and anteroposterior axis. Here, we analyze the role of Vnd, a NK-2 homeodomain protein, expressed initially in the ventral neuroectoderm adjacent to the ventral midline, in the dorsoventral patterning of the neuroectoderm and the neuroblasts. We show that invnd null mutants most ventral neuroblasts do not form and the few that form do not develop ventral fates, but instead develop intermediate-like fates. Furthermore, we demonstrate that Vnd influences the gene expression patterns in the ventral proneural clusters and neuroectoderm, and that its action in neuroblast formation includes, but is not exclusive to the activation of proneuralAS-C genes. Through the use of GAL4/UAS gene-expression system we show that ectopic Vnd expression can promote ventral-like fates in intermediate and dorsal neuroblasts and can suppress certain normal characteristics of the intermediate and dorsal neuroectoderm. Our results are discussed in the context of the current evidence in dorsoventral patterning in the Drosophilaneuroectoderm.
Modeling mutations in the G1 arrest pathway in human gliomas: overexpression of CDK4 but not loss of INK4a–ARF induces hyperploidy in cultured mouse astrocytes
Authors: Holland Eric C. , Hively Wendy P. , Gallo Vittorio , Varmus Harold E.
Published: February 2008

Nearly all human gliomas exhibit alterations in one of three genetic loci governing G1 arrest: INK4a–ARF,CDK4, or RB. To discern the roles of CDK4amplification and INK4a–ARF loss in gliomagenesis, we compared the behavior of astrocytes lacking a functional INK4a–ARFlocus with astrocytes overexpressing CDK4. Either a deficiency of p16INK4a and p19ARF or an increase in Cdk4 allows cultured astrocytes to grow without senescence. Astrocytes overexpressing CDK4 grow more slowly thanINK4a–ARF-deficient astrocytes and convert to a tetraploid state at high efficiency; in contrast, INK4a–ARF-deficient cells remain pseudodiploid, consistent with properties observed in human gliomas with corresponding lesions in these genes.
c-Cbl/Sli-1 regulates endocytic sorting and ubiquitination of the epidermal growth factor receptor
Authors: Levkowitz Gil , Waterman Hadassa , Zamir Eli , Kam Zvi , Oved Shlomo , Langdon Wallace Y. , Beguinot Laura , Geiger Benjamin , Yarden Yosef
Published: February 2008

Ligand-induced down-regulation of two growth factor receptors, EGF receptor (ErbB-1) and ErbB-3, correlates with differential ability to recruit c-Cbl, whose invertebrate orthologs are negative regulators of ErbB. We report that ligand-induced degradation of internalized ErbB-1, but not ErbB-3, is mediated by transient mobilization of a minor fraction of c-Cbl into ErbB-1-containing endosomes. This recruitment depends on the receptor’s tyrosine kinase activity and an intact carboxy-terminal region. The alternative fate is recycling of internalized ErbBs to the cell surface. Cbl-mediated receptor sorting involves covalent attachment of ubiquitin molecules, and subsequent lysosomal and proteasomal degradation. The oncogenic viral form of Cbl inhibits down-regulation by shunting endocytosed receptors to the recycling pathway. These results reveal an endosomal sorting machinery capable of controlling the fate, and, hence, signaling potency, of growth factor receptors.
A constitutively active epidermal growth factor receptor cooperates with disruption of G1 cell-cycle arrest pathways to induce glioma-like lesions in mice
Authors: Holland Eric C. , Hively Wendy P. , DePinho Ronald A. , Varmus Harold E.
Published: February 2008

The epidermal growth factor receptor (EGFR) gene is amplified or mutated in 30%–50% of human gliobastoma multiforme (GBM). These mutations are associated usually with deletions of theINK4a–ARF locus, which encodes two gene products (p16INK4a and p19ARF) involved in cell-cycle arrest and apoptosis. We have investigated the role of EGFR mutation in gliomagenesis, using avian retroviral vectors to transfer a mutantEGFR gene to glial precursors and astrocytes in transgenic mice expressing tv-a, a gene encoding the retrovirus receptor. TVA, under control of brain cell type-specific promoters. We demonstrate that expression of a constitutively active, mutant form of EGFR in cells in the glial lineage can induce lesions with many similarities to human gliomas. These lesions occur more frequently with gene transfer to mice expressing tv-a from the progenitor-specific nestin promoter than to mice expressing tv-a from the astrocyte-specific glial fibrillary acidic protein (GFAP) promoter, suggesting that tumors arise more efficiently from immature cells in the glial lineage. Furthermore, EGFR-induced gliomagenesis appears to require additional mutations in genes encoding proteins involved in cell-cycle arrest pathways. We have produced these combinations by simultaneously infecting tv-a transgenic mice with vectors carrying cdk4 and EGFR or by infectingtv-a transgenic mice bearing a disrupted INK4a–ARFlocus with the EGFR-carrying vector alone. Moreover,EGFR-induced gliomagenesis does not occur in conjunction withp53 deficiency, unless the mice are also infected with a vector carrying cdk4. The gliomagenic combinations of genetic lesions required in mice are similar to those found in human gliomas.