bluuebunny/crossref_metadata_2025 huggingface dataset:
Calcineurin acts through theCRZ1/TCN1-encoded transcription factor to regulate gene expression in yeast
Authors: Stathopoulos Angelike M. , Cyert Martha S.
Published: February 2008

Calcineurin is a conserved Ca2+/calmodulin-dependent protein phosphatase that plays a critical role in Ca2+ signaling. We describe new components of a calcineurin-mediated response in yeast, the Ca2+-induced transcriptional activation ofFKS2, which encodes a β-1,3 glucan synthase. A 24-bp region of the FKS2 promoter was defined as sufficient to confer calcineurin-dependent transcriptional induction on a minimal promoter in response to Ca2+ and was named CDRE (forcalcineurin-dependentresponse element). The product ofCRZ1 (YNL027w) was identified as an activator of CDRE-driven transcription. Crz1p contains zinc finger motifs and binds specifically to the CDRE. Genetic analysis revealed that crz1Δ mutant cells exhibit several phenotypes similar to those of calcineurin mutants and that overexpression of CRZ1 in calcineurin mutants suppressed these phenotypes. These results suggest that Crz1p functions downstream of calcineurin to effect multiple calcineurin-dependent responses. Moreover, the calcineurin-dependent transcriptional induction of FKS2 in response to Ca2+, α-factor, and Na+ was found to require CRZ1. In addition, we found that the calcineurin-dependent transcriptional regulation ofPMR2 and PMC1 required CRZ1. However, transcription of PMR2 and PMC1 was activated by only a subset of the treatments that activated FKS2 transcription. Thus, in response to multiple signals, calcineurin acts through the Crz1p transcription factor to differentially regulate the expression of several target genes in yeast.
windbeutel,a gene required for dorsoventral patterning inDrosophila,encodes a protein that has homologies to vertebrate proteins of the endoplasmic reticulum
Authors: Konsolaki Mary , Schüpbach Trudi
Published: February 2008

The formation of the dorsoventral axis of theDrosophilaembryo depends on cell–cell interactions that take place in the female ovary and involve the activation of transmembrane receptors by secreted ligands. The genewindbeutelfunctions in the somatic follicle cells of the ovary and is required for the generation of a signal that will determine the ventral side of the embryo. This signal originates in the follicle cells during oogenesis, but its actions are only manifested after fertilization, when the egg has already been laid. We have performed a molecular analysis ofwindbeutel.We have found thatwindbeutelencodes a putative resident protein of the endoplasmic reticulum, and has homologs in rats and humans. The gene is expressed for a brief period of time in the follicle cells of the ovary, at around the time when the dorsoventral axis of the egg chamber is first established. We propose that Windbeutel is responsible for the folding and/or modification of a specific factor that is secreted from the follicle cells and participates in the activation of the ventralizing signal.
Phosphorylation of spliceosomal protein SAP 155 coupled with splicing catalysis
Authors: Wang Changyu , Chua Katrin , Seghezzi Wolfgang , Lees Emma , Gozani Or , Reed Robin
Published: February 2008

The U2 snRNP component SAP 155 contacts pre-mRNA on both sides of the branch site early in spliceosome assembly and is therefore positioned near or at the spliceosome catalytic center. We have isolated a cDNA encoding human SAP 155 and identified its highly related Saccharomyces cerevisiae homolog (50% identity). The carboxy-terminal two-thirds of SAP 155 shows the highest conservation and is remarkably similar to the regulatory subunit A of the phosphatase PP2A. Significantly, SAP 155 is phosphorylated concomitant with or just after catalytic step one, making this the first example of a protein modification tightly regulated with splicing catalysis.
The ATP-dependent PIM1 protease is required for the expression of intron-containing genes in mitochondria
Authors: van Dyck Luc , Neupert Walter , Langer Thomas
Published: February 2008

The ATP-dependent PIM1 protease, a Lon-like protease localized in the mitochondrial matrix, is required for mitochondrial genome integrity in yeast. Cells lacking PIM1 accumulate lesions in the mitochondrial DNA (mtDNA) and therefore lose respiratory competence. The identification of a multicopy suppressor, which stabilizes mtDNA in the absence of PIM1, enabled us to characterize novel functions of PIM1 protease during mitochondrial biogenesis. The synthesis of mitochondrially encoded cytochrome c oxidase subunit I (CoxI) and cytochrome b (Cob) is impaired in pim1 mutants containing mtDNA. PIM1-mediated proteolysis is required for the translation of mature COXI mRNA. Moreover, deficiencies in the splicing of COXI and COB transcripts, which appear to be restricted to introns encoding mRNA maturases, were observed in cells lacking the PIM1 gene. Transcripts of COXI andCOB genes harboring multiple introns are degraded in the absence of PIM1. These results establish multiple, essential functions of the ATP-dependent PIM1 protease during mitochondrial gene expression.
Spalten, a protein containing Gα-protein-like and PP2C domains, is essential for cell-type differentiation in Dictyostelium
Authors: Aubry Laurence , Firtel Richard A.
Published: February 2008

We have identified a novel gene, Spalten (Spn) that is essential for Dictyostelium multicellular development. Spn encodes a protein with an amino-terminal domain that shows very high homology to Gα-protein subunits, a highly charged inter-region, and a carboxy-terminal domain that encodes a functional PP2C. Spn is essential for development past the mound stage, being required cell autonomously for prestalk gene expression and nonautonomously for prespore cell differentiation. Mutational analysis demonstrates that the PP2C domain is the Spn effector domain and is essential for Spn function, whereas the Gα-like domain is required for membrane targeting and regulation of Spn function. Moreover, Spn carrying mutations in the Gα-like domain that do not affect membrane targeting but affect specificity of guanine nucleotide binding in known GTP-binding proteins are unable to fully complement the spn−phenotype, suggesting that the Gα-like domain regulates Spn function either directly or indirectly by mediating its interactions with other proteins. Our results suggest that Spn encodes a signaling molecule with a novel Gα-like regulatory domain.
The competence transcription factor of Bacillus subtilis recognizes short A/T-rich sequences arranged in a unique, flexible pattern along the DNA helix
Authors: Hamoen Leendert W. , Van Werkhoven Aske F. , Bijlsma Jetta J.E. , Dubnau David , Venema Gerard
Published: February 2008

The development of genetic competence in Bacillus subtilisis regulated by a complex signal transduction cascade, which leads to the synthesis of the competence transcription factor (CTF). Previous studies suggested that CTF is encoded by comK. ComK is required for the transcription of comK itself, as well as of the late competence genes encoding the DNA uptake machinery and of genes required for homologous recombination. Here, we used purified ComK to study its role in transcription and to determine the DNA recognition sequence for ComK. In vitro transcription from the comGpromoter, which depends on ComK in vivo, was observed on the addition of purified ComK together with Bacillus subtilis RNA polymerase, proving that ComK is CTF. To determine the DNA sequences involved in ComK recognition, footprinting analysis was performed with promoter fragments of the CTF-dependent genes: comC, comE, comF, comG, comK, and addAB. The ComK binding sites determined by DNase I protection experiments were unusually long, with average lengths of ∼65 bp, and displayed only weak sequence similarities. Hydroxy-radical footprinting, performed with the addABpromoter, revealed a unique arrangement of four short A/T-rich sequences. Gel retardation experiments indicated that four molecules of ComK bound the addAB promoter and the dyad symmetrical arrangement of the four A/T-rich sequences implied that ComK functions as a tetramer composed of two dimers each recognizing the motif AAAAN5TTTT. Comparable A/T-rich sequences were identified in all six DNase I footprints and could be used to predict ComK targets in the B. subtilis genome. On the basis of the variability in distance between the ComK–dimer binding sites, ComK-regulated promoters could be divided into three classes, demonstrating a remarkable flexibility in the binding of ComK.The pattern of hydroxy-radical protections suggested that ComK binds at one face of the DNA helix through the minor groove. This inference was strengthened by the observation that minor groove binding drugs inhibited the binding of ComK.
Smad2 transduces common signals from receptor serine–threonine and tyrosine kinases
Authors: de Caestecker Mark P. , Parks W. Tony , Frank Chistopher J. , Castagnino Paola , Bottaro Donald P. , Roberts Anita B. , Lechleider Robert J.
Published: February 2008

SMAD proteins mediate signals from receptor serine–threonine kinases (RSKs) of the TGF-β superfamily. We demonstrate here that HGF and EGF, which signal through RTKs, can also mediate SMAD-dependent reporter gene activation and induce rapid phosphorylation of endogenous SMAD proteins by kinase(s) downstream of MEK1. HGF induces phosphorylation and nuclear translocation of epitope-tagged Smad2 and a mutation that blocks TGF-β signaling also blocks HGF signal transduction. Smad2 may thus act as a common positive effector of TGF-β- and HGF-induced signals and serve to modulate cross talk between RTK and RSK signaling pathways.
p107 is a suppressor of retinoblastoma development in pRb-deficient mice
Authors: Robanus-Maandag Els , Dekker Marleen , van der Valk Martin , Carrozza Maria-Luisa , Jeanny Jean-Claude , Dannenberg Jan-Hermen , Berns Anton , te Riele Hein
Published: February 2008

Hemizygosity for the retinoblastoma gene RB in man strongly predisposes to retinoblastoma. In the mouse, however,Rb hemizygosity leaves the retina normal, whereas inRb−/− chimeras pRb-deficient retinoblasts undergo apoptosis. To test whether concomitant inactivation of the Rb-related gene p107 is required to unleash the oncogenic potential of pRb deficiency in the mouse retina, we inactivated both Rb and p107 by homologous recombination in embryonic stem cells and generated chimeric mice. Retinoblastomas were found in five out of seven adult pRb/p107-deficient chimeras. The retinal tumors showed amacrine cell differentiation, and therefore originated from cells committed to the inner but not the outer nuclear layer. Retinal lesions were already observed at embryonic day 17.5. At this stage, the primitive nuclear layer exhibited severe dysplasia, including rosette-like arrangements, and apoptosis. These findings provide formal proof for the role of loss of Rb in retinoblastoma development in the mouse and the first in vivo evidence that p107 can exert a tumor suppressor function.
Localization-dependent translation requires a functional interaction between the 5′ and 3′ ends ofoskar mRNA
Authors: Gunkel Niki , Yano Tamaki , Markussen Finn-Hugo , Olsen Lisbeth C. , Ephrussi Anne
Published: February 2008

The precise restriction of proteins to specific domains within a cell plays an important role in early development and differentiation. An efficient way to localize and concentrate proteins is by localization of mRNA in a translationally repressed state, followed by activation of translation when the mRNA reaches its destination. A central issue is how localized mRNAs are derepressed. In this study we demonstrate that, when oskar mRNA reaches the posterior pole of the Drosophila oocyte, its translation is derepressed by an active process that requires a specific element in the 5′ region of the mRNA. We demonstrate that this novel type of element is a translational derepressor element, whose functional interaction with the previously identified repressor region in the oskar 3′ UTR is required for activation of oskar mRNA translation at the posterior pole. The derepressor element only functions at the posterior pole, suggesting that a locally restricted interaction betweentrans-acting factors and the derepressor element may be the link between mRNA localization and translational activation. We also show specific interaction of two proteins with the oskar mRNA 5′ region; one of these also recognizes the 3′ repressor element. We discuss the possible involvement of these factors as well as known genes in the process of localization-dependent translation.
Notochord repression of endodermal Sonic hedgehog permits pancreas development
Authors: Hebrok Matthias , Kim Seung K. , Melton Douglas A.
Published: February 2008

Notochord signals to the endoderm are required for development of the chick dorsal pancreas. Sonic hedgehog (SHH) is normally absent from pancreatic endoderm, and we provide evidence that notochord, in contrast to its effects on adjacent neuroectoderm where SHH expression is induced, represses SHH expression in adjacent nascent pancreatic endoderm. We identify activin-βB and FGF2 as notochord factors that can repress endodermal SHH and thereby permit expression of pancreas genes including Pdx1 and insulin. Endoderm treatment with antibodies that block hedgehog activity also results in pancreatic gene expression. Prevention of SHH expression in prepancreatic dorsal endoderm by intercellular signals, like activin and FGF, may be critical for permitting early steps of chick pancreatic development.
Miranda mediates asymmetric protein and RNA localization in the developing nervous system
Authors: Schuldt Alison J. , Adams Jan H.J. , Davidson Catherine M. , Micklem David R. , Haseloff Jim , Johnston Daniel St , Brand Andrea H.
Published: February 2008

Neuroblasts undergo asymmetric stem cell divisions to generate a series of ganglion mother cells (GMCs). During these divisions, the cell fate determinant Prospero is asymmetrically partitioned to the GMC by Miranda protein, which tethers it to the basal cortex of the dividing neuroblast. Interestingly,prosperomRNA is similarly segregated by the dsRNA binding protein, Staufen. Here we show that Staufen interacts in vivo with a segment of theprospero3′ UTR. Staufen protein andprosperoRNA colocalize to the apical side of the neuroblast at interphase, but move to the basal side during prophase. Both the apical and basal localization of Staufen are abolished by the removal of a conserved domain from the carboxyl terminus of the protein, which interacts in a yeast two-hybrid screen with Miranda protein. Furthermore, Miranda colocalizes with Staufen protein andprosperomRNA during neuroblast divisions, and neither Staufen norprosperoRNA are localized inmirandamutants. Thus Miranda, which localizes Prospero protein, also localizesprosperoRNA through its interaction with Staufen protein.
A bacterial ATP-dependent, enhancer binding protein that activates the housekeeping RNA polymerase
Authors: Bowman William C. , Kranz Robert G.
Published: February 2008

A commonly accepted view of gene regulation in bacteria that has emerged over the last decade is that promoters are transcriptionally activated by one of two general mechanisms. The major type involves activator proteins that bind to DNA adjacent to where the RNA polymerase (RNAP) holoenzyme binds, usually assisting in recruitment of the RNAP to the promoter. This holoenzyme uses the housekeeping ς70 or a related factor, which directs the core RNAP to the promoter and assists in melting the DNA near the RNA start site. A second type of mechanism involves the alternative sigma factor (called ς54 or ςN) that directs RNAP to highly conserved promoters. In these cases, an activator protein with an ATPase function oligomerizes at tandem sites far upstream from the promoter. The nitrogen regulatory protein (NtrC) from enteric bacteria has been the model for this family of activators. Activation of the RNAP/ς54 holoenzyme to form the open complex is mediated by the activator, which is tethered upstream. Hence, this class of protein is sometimes called the enhancer binding protein family or the NtrC class. We describe here a third system that has properties of each of these two types. The NtrC enhancer binding protein from the photosynthetic bacterium, Rhodobacter capsulatus, is shown in vitro to activate the housekeeping RNAP/ς70 holoenzyme. Transcriptional activation by this NtrC requires ATP binding but not hydrolysis. Oligomerization at distant tandem binding sites on a supercoiled template is also necessary. Mechanistic and evolutionary questions of these systems are discussed.
Renal agenesis in mice homozygous for a gene trap mutation in the gene encoding heparan sulfate 2-sulfotransferase
Authors: Bullock Simon L. , Fletcher Judy M. , Beddington Rosa S.P. , Wilson Valerie A.
Published: February 2008

Heparan sulfate proteoglycans have been implicated in the presentation of a number of secreted signaling molecules to their signal-transducing receptors. We have characterized a gene trap mutation in the gene encoding a heparan sulfate biosynthetic enzyme, heparan sulfate 2-sulfotransferase (HS2ST). Transgenic mice were generated from embryonic stem cells harboring this insertion.lacZ reporter gene activity in heterozygous embryos demonstrates that the gene is expressed differentially during embryogenesis, presumably directing dynamic changes in heparan sulfate structure. Moreover, mice homozygous for the Hs2st gene trap allele die in the neonatal period, exhibiting bilateral renal agenesis and defects of the eye and the skeleton. Analysis of kidney development in Hs2st mutants reveals that the gene is not required for two early events—ureteric bud outgrowth from the Wolffian duct and initial induction of Pax-2 expression in the metanephric mesenchyme. It is required, however, for mesenchymal condensation around the ureteric bud and initiation of branching morphogenesis. Because 2-O-sulfation has been shown to influence the functional interactions of ligands with heparan sulfate in vitro, we discuss the possibility that the Hs2st mutant phenotype is a consequence of compromised interactions between growth factors and their signal-transducing receptors. These data provide the first genetic evidence that the regulated synthesis of differentially glycosylated proteoglycans can affect morphogenesis during vertebrate development.
C/EBPβ, but not C/EBPα, is essential for ductal morphogenesis, lobuloalveolar proliferation, and functional differentiation in the mouse mammary gland
Authors: Seagroves Tiffany N. , Krnacik Susanne , Raught Brian , Gay Jason , Burgess-Beusse Bonnie , Darlington Gretchen J. , Rosen Jeffrey M.
Published: February 2008

The CCAAT/enhancer binding proteins (C/EBPs) are differentially expressed throughout mammary gland development and interact with binding sites within the promoter of a milk protein gene, β-casein. The specific roles of C/EBPβ and C/EBPα in mouse mammary gland development and differentiation have been investigated in mice that carry targeted deletions of these genes. C/EBPβ−/− virgin mice exhibited cystic, enlarged mammary ducts with decreased secondary branching. Transplantation of C/EBPβ−/− mammary epithelium into the cleared mammary fat pads of nude mice confirmed that this defect in ductal morphogenesis was intrinsic to the epithelium. When treated with estrogen/progesterone (E+P) to simulate pregnancy, C/EBPβ−/− mammary glands displayed only limited lobuloalveolar development and ductal side branching. Primary mammary epithelial cells obtained from E+P-treated C/EBPβ−/−mice that were cultured on extracellular matrix gels did not functionally differentiate in response to lactogenic hormones despite their organization into three-dimensional structures. Expression of β-casein protein was inhibited 85%–100% and whey acidic protein (WAP) was undetectable. In contrast, no detectable alterations in mammary development or β-casein expression were observed in mammary outgrowths derived from newborn C/EBPα−/− mammary epithelium transplanted into the cleared mammary fat pads of syngeneic hosts. These results demonstrate that C/EBPβ, but not C/EBPα, is required for ductal morphogenesis, lobuloalveolar development, and functional differentiation of mammary epithelial cells.
pha-4, anHNF-3 homolog, specifies pharyngeal organ identity inCaenorhabditis elegans
Authors: Horner Michael A. , Quintin Sophie , Domeier Mary Ellen , Kimble Judith , Labouesse Michel , Mango Susan E.
Published: February 2008

To build complex organs, embryos have evolved mechanisms that integrate the development of cells unrelated to one another by cell type or ancestry. Here we show that the pha-4 locus establishes organ identity for the Caenorhabditis elegans pharynx. Inpha-4 mutants, pharyngeal cells are transformed into ectoderm. Conversely, ectopic pha-4 expression produces excess pharyngeal cells. pha-4 encodes an HNF-3 homolog selectively expressed in the nascent digestive tract, including all pharynx precursors at the time they are restricted to a pharyngeal fate. We suggest that pha-4 is a key component of a transcription-based mechanism to endow cells with pharyngeal organ identity.
Identification of functional exonic splicing enhancer motifs recognized by individual SR proteins
Authors: Liu Hong-Xiang , Zhang Michael , Krainer Adrian R.
Published: February 2008

Using an in vitro randomization and functional selection procedure, we have identified three novel classes of exonic splicing enhancers (ESEs) recognized by human SF2/ASF, SRp40, and SRp55, respectively. These ESEs are functional in splicing and are highly specific. For SF2/ASF and SRp55, in most cases, only the cognate SR protein can efficiently recognize an ESE and activate splicing. In contrast, the SRp40-selected ESEs can function with either SRp40 or SRp55, but not with SF2/ASF. UV cross-linking/competition and immunoprecipitation experiments showed that SR proteins recognize their cognate ESEs in nuclear extract by direct and specific binding. A motif search algorithm was used to derive consensus sequences for ESEs recognized by these SR proteins. Each SR protein yielded a distinct 5- to 7-nucleotide degenerate consensus. These three consensus sequences occur at higher frequencies in exons than in introns and may thus help define exon–intron boundaries. They occur in clusters within regions corresponding to naturally occurring, mapped ESEs. We conclude that a remarkably diverse set of sequences can function as ESEs. The degeneracy of these motifs is consistent with the fact that exonic enhancers evolved within extremely diverse protein coding sequences and are recognized by a small number of SR proteins that bind RNA with limited sequence specificity.
Functional intertwining of Dpp and EGFR signaling during Drosophilaendoderm induction
Authors: Szüts Dávid , Eresh Salih , Bienz Mariann
Published: February 2008

Endoderm induction in Drosophila is mediated by the extracellular signals Decapentaplegic (Dpp) and Wingless (Wg). We discovered a secondary signal with a permissive role in this process, namely Vein, a neuregulin-like ligand that stimulates the epidermal growth factor receptor (EGFR) and Ras signaling. Dpp and Wg up-regulatevein expression in the midgut mesoderm in two regions overlapping the Dpp sources. Experiments based on lack of function and ectopic stimulation of Dpp and EGFR signaling show that these two pathways are functionally interdependent and that they synergize with each other, revealing functional intertwining. The transcriptional response elements for the Dpp signal in midgut enhancers from homeotic target genes are bipartite, comprising CRE sites as well as binding sites for the Dpp signal-transducing protein Mad. Of these sites, the CRE seems to function primarily in the response to Ras, the secondary signal of Dpp. We discuss the potential significance of why an inductive process might use a secondary signal whose function is intertwined with that of the primary signal.
New POU dimer configuration mediates antagonistic control of an osteopontin preimplantation enhancer by Oct-4 and Sox-2
Authors: Botquin Valérie , Hess Heike , Fuhrmann Guy , Anastassiadis Constantinos , Gross Michael K. , Vriend Gerrit , Schöler Hans R.
Published: February 2008

The POU transcription factor Oct-4 is expressed specifically in the germ line, pluripotent cells of the pregastrulation embryo and stem cell lines derived from the early embryo. Osteopontin (OPN) is a protein secreted by cells of the preimplantation embryo and contains a GRGDS motif that can bind to specific integrin subtypes and modulate cell adhesion/migration. We show thatOct-4 and OPN are coexpressed in the preimplantation mouse embryo and during differentiation of embryonal cell lines. Immunoprecipitation of the first intron of OPN (i-opn) from covalently fixed chromatin of embryonal stem cells by Oct-4-specific antibodies indicates that Oct-4 binds to this fragment in vivo. The i-opn fragment functions as an enhancer in cell lines that resemble cells of the preimplantation embryo. Furthermore, it contains a novel palindromic Oct factor recognition element (PORE) that is composed of an inverted pair of homeodomain-binding sites separated by exactly 5 bp (ATTTG +5 CAAAT). POU proteins can homo- and heterodimerize on the PORE in a configuration that has not been described previously. Strong transcriptional activation of the OPN element requires an intact PORE. In contrast, the canonical octamer overlapping with the downstream half of the PORE is not essential. Sox-2 is a transcription factor that contains an HMG box and is coexpressed with Oct-4 in the early mouse embryo. Sox-2 represses Oct-4 mediated activation of i-opn by way of a canonical Sox element that is located close to the PORE. Repression depends on a carboxy-terminal region of Sox-2 that is outside of the HMG box. Expression, DNA binding, and transactivation data are consistent with the hypothesis that OPN expression is regulated by Oct-4 and Sox-2 in preimplantation development.
Identification of a novel class of genomic DNA-binding sites suggests a mechanism for selectivity in target gene activation by the tumor suppressor protein p53
Authors: Resnick-Silverman Lois , St. Clair Selvon , Maurer Matthew , Zhao Kathy , Manfredi James J.
Published: February 2008

There are two response elements for p53 in the promoter of the gene for the cyclin-dependent kinase inhibitor p21. The binding of p53 to the 5′ site was enhanced by incubation with monoclonal antibody 421, whereas the binding of p53 to the 3′ site was inhibited. Mutational analysis showed that a single-base change caused one element to behave like the other. A response element in the humancdc25C promoter is bound by p53 with properties similar to the 3′ site. These results identify two classes of p53-binding sites and suggest a mechanism for target gene selectivity by p53.
TGF-β-stimulated cooperation of Smad proteins with the coactivators CBP/p300
Authors: Janknecht Ralf , Wells Nicholas J. , Hunter Tony
Published: February 2008

TGF-β and activin induce the phosphorylation and activation of Smad2 and Smad3, but how these proteins stimulate gene transcription is poorly understood. We report that TGF-β receptor phosphorylation of Smad3 promotes its interaction with the paralogous coactivators CBP and p300, whereas CBP/p300 binding to nonphosphorylated Smad3 or its oligomerization partner Smad4 is negatively regulated by Smad–intramolecular interactions. Furthermore, p300 and TGF-β receptor-phosphorylated Smad3 synergistically augment transcriptional activation. Thus, CBP/p300 are important components of activin/TGF-β signaling and may mediate the antioncogenic functions of Smad2 and Smad4.
E2F3 activity is regulated during the cell cycle and is required for the induction of S phase
Authors: Leone Gustavo , DeGregori James , Yan Zhen , Jakoi Laszlo , Ishida Seiichi , Williams R. Sanders , Nevins Joseph R.
Published: February 2008

Previous work has demonstrated the important role of E2F transcription activity in the induction of S phase during the transition from quiescence to proliferation. In addition to the E2F-dependent activation of a number of genes encoding DNA replication activities such as DNA Pol α, we now show that the majority of genes encoding initiation proteins, including Cdc6 and the Mcm proteins, are activated following the stimulation of cell growth and are regulated by E2F. The transcription of a subset of these genes, which includesCdc6, cyclin E, and cdk2, is also regulated during the cell cycle. Moreover, whereas overall E2F DNA-binding activity accumulates during the initial G1 following a growth stimulus, only E2F3-binding activity reaccumulates at subsequent G1/S transitions, coincident with the expression of the cell-cycle-regulated subset of E2F-target genes. Finally, we show that immunodepletion of E2F3 activity inhibits the induction of S phase in proliferating cells. We propose that E2F3 activity plays an important role during the cell cycle of proliferating cells, controlling the expression of genes whose products are rate limiting for initiation of DNA replication, thereby imparting a more dramatic control of entry into S phase than would otherwise be achieved by post-transcriptional control alone.
Control of Cyclin B1 localization through regulated binding of the nuclear export factor CRM1
Authors: Yang Jing , Bardes Elaine S.G. , Moore Jonathan D. , Brennan Jennifer , Powers Maureen A. , Kornbluth Sally
Published: February 2008

Activation of the Cyclin B/Cdc2 kinase complex triggers entry into mitosis in all eukaryotic cells. Cyclin B1 localization changes dramatically during the cell cycle, precipitously transiting from the cytoplasm to the nucleus at the beginning of mitosis. Presumably, this relocalization promotes the phosphorylation of nuclear targets critical for chromatin condensation and nuclear envelope breakdown. We show here that the previously characterized cytoplasmic retention sequence of Cyclin B1, responsible for its interphase cytoplasmic localization, is actually an autonomous nuclear export sequence, capable of directing nuclear export of a heterologous protein, and able to bind specifically to the recently identified export mediator, CRM1. We propose that the observed cytoplasmic localization of Cyclin B1 during interphase reflects the equilibrium between ongoing nuclear import and rapid CRM1-mediated export. In support of this hypothesis, we found that treatment of cells with leptomycin B, which disrupted Cyclin B1–CRM1 interactions, led to a marked nuclear accumulation of Cyclin B1. In mitosis, Cyclin B1 undergoes phosphorylation at several sites, a subset of which have been proposed to play a role in Cyclin B1 accumulation in the nucleus. Both CRM1 binding and the ability to direct nuclear export were affected by mutation of these phosphorylation sites; thus, we propose that Cyclin B1 phosphorylation at the G2/M transition prevents its interaction with CRM1, thereby reducing nuclear export and facilitating nuclear accumulation.
Impaired megakaryopoiesis and behavioral defects inmafG-null mutant mice
Authors: Shavit Jordan A. , Motohashi Hozumi , Onodera Ko , Akasaka Jun-etsu , Yamamoto Masayuki , Engel James Douglas
Published: February 2008

The small Maf proteins (MafG, MafK, and MafF), which serve as heterodimeric partner molecules of CNC family proteins for binding in vitro to MARE sites, have been implicated in the regulation of both transcription and chromatin structure, but there is no current evidence that the proteins fulfill these functions in vivo. To elucidate possible contributions of the small Maf proteins to gene regulation, we have ablated the mafG and mafK genes in mice by replacing their entire coding sequences with the Escherichia coli lacZ gene. mafG homozygous mutant animals exhibit impaired platelet formation accompanied by megakaryocyte proliferation, as well as behavioral abnormalities, whereas mafK-null mutant mice are phenotypically normal. Characterization of the mafG andmafK embryonic expression patterns show that their developmental programs are distinct and intersecting, but not entirely overlapping. These results provide direct evidence that the small Maf transcription factors are vital participants in embryonic development and cellular differentiation.
Igf2 imprinting does not require its own DNA methylation or H19 RNA
Authors: Jones Beverly K. , Levorse John M. , Tilghman Shirley M.
Published: February 2008

Three models have been proposed to explain the imprinting of the mouse Igf2 gene on the maternal chromosome. We ruled out the importance of DNA methylation at Igf2 by showing that silencing of Igf2 accompanying the loss of DNA methylation could be overcome by a mutation at the neighboring H19 gene that activates Igf2. By replacing the H19 structural gene with a protein-coding gene, we have ruled out a role for H19RNA in the imprinting of Igf2. This replacement resulted in sporadic activation of the H19 promoter on the paternal chromosome without affecting the level of expression of Igf2, a finding that is inconsistent with strict promoter competition between the genes. We conclude that a transcriptional model involving access to a common set of enhancers shared between Igf2 and H19is the most likely explanation for Igf2 imprinting.
Rad52 associates with RPA and functions with Rad55 and Rad57 to assemble meiotic recombination complexes
Authors: Gasior Stephen L. , Wong Anthony K. , Kora Yoshiteru , Shinohara Akira , Bishop Douglas K.
Published: February 2008

We show that the Saccharomyces cerevisiae recombination protein Rad52 and the single-strand DNA-binding protein RPA assemble into cytologically detectable subnuclear complexes (foci) during meiotic recombination. Immunostaining shows extensive colocalization of Rad52 and RPA and more limited colocalization of Rad52 with the strand exchange protein Rad51. Rad52 and RPA foci are distinct from those formed by Rad51, and its meiosis-specific relative Dmc1, in that they are also detected in meiosis during replication. In addition, RPA foci are observed during mitotic S phase. Double-strand breaks (DSBs) promote formation of RPA, Rad52, and Rad51 foci. Mutants that lack Spo11, a protein required for DSB formation, are defective in focus formation, and this defect is suppressed by ionizing radiation in a dose-dependent manner. DSBs are not sufficient for the appearance of Rad51 foci; Rad52, Rad55, and Rad57 are also required supporting a model in which these three proteins promote meiotic recombination by promoting the assembly of strand exchange complexes.
Maternal IL-11Rα function is required for normal decidua and fetoplacental development in mice
Authors: Bilinski Petra , Roopenian Derry , Gossler Achim
Published: February 2008

In eutherian mammals, implantation and establishment of the chorioallantoic placenta are essential for embryo development and survival. As a maternal response to implantation, uterine stromal cells proliferate, differentiate, and generate the decidua, which encapsulates the conceptus and forms the maternal part of the placenta. Little is known about decidual functions and the molecular interactions that regulate its development and maintenance. Here we show that the receptor for the cytokine interleukin-11 (IL-11Rα) is required specifically for normal establishment of the decidua. Females homozygous for a hypomorphicIL-11Rαallele are fertile and their blastocysts implant and elicit the decidual response. Because of reduced cell proliferation, however, only small deciduae form. Mutant deciduae degenerate progressively, and consequently embryo-derived trophoblast cells generate a network of trophoblast giant cells but fail to form a chorioallantoic placenta, indicating that the decidua is essential for normal fetoplacentation.IL-11Rαis expressed in the decidua as well as in numerous other tissues and cell types, including the ovary and lymphocytes. The differentiation state and proliferative responses of B and T-lymphocytes in mutant females were normal, and wild-type females carryingIL-11Rαmutant ovaries had normal deciduae, suggesting that the decidualization defects do not arise secondarily as a consequence of perturbedIL-11Rαsignaling defects in lymphoid organs or in the ovary. Therefore,IL-11Rαsignaling at the implantation site appears to be required for decidua development.
Switch from translation to RNA replication in a positive-stranded RNA virus
Authors: Gamarnik Andrea V. , Andino Raul
Published: February 2008

In positive-stranded viruses, the genomic RNA serves as a template for both translation and RNA replication. Using poliovirus as a model, we examined the interaction between these two processes. We show that the RNA polymerase is unable to replicate RNA templates undergoing translation. We discovered that an RNA structure at the 5′ end of the viral genome, next to the internal ribosomal entry site, carries signals that control both viral translation and RNA synthesis. The interaction of this RNA structure with the cellular factor PCBP up-regulates viral translation, while the binding of the viral protein 3CD represses translation and promotes negative-strand RNA synthesis. We propose that the interaction of 3CD with this RNA structure controls whether the genomic RNA is used for translation or RNA replication.
Enhancer control ofV(D)Jrecombination at the TCRβ locus: differential effects on DNA cleavage and joining
Authors: Hempel William M. , Stanhope-Baker Patricia , Mathieu Noëlle , Huang Fang , Schlissel Mark S. , Ferrier Pierre
Published: February 2008

Deletion of the TCRβ transcriptional enhancer (Eβ) results in nearly complete inhibition ofV(D)Jrecombination at the TCRβ locus and a block in αβ T cell development. This result, along with previous work from many laboratories, has led to the hypothesis that transcriptional enhancers affectV(D)Jrecombination by regulating the accessibility of the locus to the recombinase. Here we test this hypothesis by performing a detailed analysis of the recombination defect in Eβ-deleted (Eβ−/−) mice using assays that detect various reaction intermediates and products. We found double-strand DNA breaks at recombination signal sequences flankingDβ andJβ gene segments in Eβ−/−thymuses at about one-third to one-thirtieth the level found in thymuses with an unaltered TCRβ locus. These sites are also subject to in vitro cleavage by theV(D)Jrecombinase in both Eβ−/−and Eβ+/+thymocyte nuclei. However, the correspondingDβ-to-Jβ coding joints are further reduced (by 100- to 300-fold) in Eβ−/−thymuses. Formation of extrachromosomalDβ-to-Jβ signal joints appears to be intermediately affected and nonstandardDβ-to-Dβ joining occurs at the Eβ-deleted alleles. These data indicate that, unexpectedly, loss of accessibility alone cannot explain the loss of TCRβ recombination in the absence of the Eβ element and suggest an additional function for Eβ in the process of DNA repair at specific TCRβ sites during the late phase of the recombination reaction.
The ROTUNDIFOLIA3 gene of Arabidopsis thalianaencodes a new member of the cytochrome P-450 family that is required for the regulated polar elongation of leaf cells
Authors: Kim Gyung-Tae , Tsukaya Hirokazu , Uchimiya Hirofumi
Published: February 2008

The polarized processes of cell elongation play a crucial role in morphogenesis of higher plants. We reported previously that therotundifolia3 (rot3) mutant of Arabidopsis has a defect in the polar elongation of leaf cells. In the present study, we isolated two additional alleles with mutations in the ROT3gene. The ROT3 gene was cloned by a T-DNA-tagging method and isolation of the gene was confirmed by a molecular analysis of threerot3 mutant alleles obtained from different mutagenesis. TheROT3 gene encodes a cytochrome P-450 (CYP90C1) with domains homologous to regions of steroid hydroxylases of animals and plants. Expression of the ROT3 gene was detected in all major plant organs. Especially, higher expression was detected in the tissues that had high activity of cell division. We confirmed that the ROT3gene controls polar elongation specifically in leaf cells by an analysis of three rot3 mutants obtained from different mutagenesis experiments. Our results imply that the ROT3protein is a member of a new class of cytochrome P-450 encoding putative steroid hydroxylases, which is required for the regulated polar elongation of cells in leaves of Arabidopsis.
Distinct C/EBP functions are required for eosinophil lineage commitment and maturation
Authors: Nerlov Claus , McNagny Kelly M. , Döderlein Gabriele , Kowenz-Leutz Elisabeth , Graf Thomas
Published: February 2008

Hematopoietic differentiation involves the commitment of multipotent progenitors to a given lineage, followed by the maturation of the committed cells. To study the transcriptional events controlling these processes, we have investigated the role of C/EBP proteins in lineage choice of multipotent hematopoietic progenitors (MEPs) transformed by the E26 virus. We found that forced expression of either the α or β isoforms of C/EBP in MEPs induced eosinophil differentiation and that in addition, C/EBPβ could induce myeloid differentiation. Conversely, dominant-negative versions of C/EBPβ inhibited myeloid differentiation. C/EBP-induced eosinophil differentiation could be separated into two distinct events, lineage commitment and maturation. Thus, eosinophils induced by transactivation-deficient C/EBPβ alleles were found to be blocked in their maturation, whereas those expressing wild-type C/EBP proteins were not. Likewise, a 1-day activation of a conditional C/EBPβ allele in multipotent progenitors led to the formation of immature eosinophils, whereas sustained activation produced mature eosinophils. These results show that C/EBP can induce both myeloid and eosinophil lineage commitment and that transactivation independent and dependent C/EBP functions are required during eosinophil lineage commitment and maturation, respectively.
E1A signaling to p53 involves the p19ARFtumor suppressor
Authors: de Stanchina Elisa , McCurrach Mila E. , Zindy Frederique , Shieh Sheau-Yann , Ferbeyre Gerardo , Samuelson Andrew V. , Prives Carol , Roussel Martine F. , Sherr Charles J. , Lowe Scott W.
Published: February 2008

The adenovirus E1A oncogene activates p53 through a signaling pathway involving the retinoblastoma protein and the tumor suppressor p19ARF. The ability of E1A to induce p53 and its transcriptional targets is severely compromised in ARF-null cells, which remain resistant to apoptosis following serum depletion or adriamycin treatment. Reintroduction of p19ARF restores p53 accumulation and resensitizes ARF-null cells to apoptotic signals. Therefore, p19ARF functions as part of a p53-dependent failsafe mechanism to counter uncontrolled proliferation. Synergistic effects between the p19ARF and DNA damage pathways in inducing p53 may contribute to E1A’s ability to enhance radio- and chemosensitivity.
Telomere-mediated chromosome pairing during meiosis in budding yeast
Authors: Rockmill Beth , Roeder G. Shirleen
Published: February 2008

Certain haploid strains of Saccharomyces cerevisiae can undergo meiosis, but meiotic prophase progression and subsequent nuclear division are delayed if these haploids carry an extra chromosome (i.e., are disomic). Observations indicate that interactions between homologous chromosomes cause a delay in meiotic prophase, perhaps to allow time for interhomolog interactions to be completed. Analysis of meiotic mutants demonstrates that the relevant aspect of homolog recognition is independent of meiotic recombination and synaptonemal complex formation. A disome in which the extra chromosome is circular sporulates without a delay, indicating that telomeres are important for homolog recognition. Consistent with this hypothesis, fluorescent in situ hybridization demonstrates that a circular chromosome has a reduced capacity to pair with its homolog, and a telomere-associated meiotic protein (Ndj1) is required to delay sporulation in disomes. A circular dimer containing two copies of the same chromosome delays meiosis to the same extent as two linear homologs, implying that physical proximity bypasses the requirement for telomeres in homolog pairing. Analysis of a disome carrying two linear permuted chromosomes suggests that even nonhomologous chromosome ends can promote homolog pairing to a limited extent. We speculate that telomere-mediated chromosome movement and/or telomere clustering promote homolog pairing.
Differential recruitment of Dishevelled provides signaling specificity in the planar cell polarity and Wingless signaling pathways
Authors: Axelrod Jeffrey D. , Miller Jeffrey R. , Shulman Joshua M. , Moon Randall T. , Perrimon Norbert
Published: February 2008

In Drosophila, planar cell polarity (PCP) signaling is mediated by the receptor Frizzled (Fz) and transduced by Dishevelled (Dsh). Wingless (Wg) signaling also requires Dsh and may utilize DFz2 as a receptor. Using a heterologous system, we show that Dsh is recruited selectively to the membrane by Fz but not DFz2, and this recruitment depends on the DEP domain but not the PDZ domain in Dsh. A mutation in the DEP domain impairs both membrane localization and the function of Dsh in PCP signaling, indicating that translocation is important for function. Further genetic and molecular analyses suggest that conserved domains in Dsh function differently during PCP and Wg signaling, and that divergent intracellular pathways are activated. We propose that Dsh has distinct roles in PCP and Wg signaling. The PCP signal may selectively result in focal Fz activation and asymmetric relocalization of Dsh to the membrane, where Dsh effects cytoskeletal reorganization to orient prehair initiation.
Membrane recruitment of the kinase cascade scaffold protein Ste5 by the Gβγ complex underlies activation of the yeast pheromone response pathway
Authors: Pryciak Peter M. , Huntress Frederick A.
Published: February 2008

In the Saccharomyces cerevisiae pheromone response pathway, the Gβγ complex activates downstream responses by an unknown mechanism involving a MAP kinase cascade, the PAK-like kinase Ste20, and a Rho family GTPase, Cdc42. Here we show that Gβγ must remain membrane-associated after release from Gα to activate the downstream pathway. We also show that pheromone stimulates translocation of the kinase cascade scaffold protein Ste5 to the cell surface. This recruitment requires Gβγ function and the Gβγ-binding domain of Ste5, but not the kinases downstream of Gβγ, suggesting that it is mediated by Gβγ itself. Furthermore, this event has functional significance, as artificial targeting of Ste5 to the plasma membrane, but not intracellular membranes, activates the pathway in the absence of pheromone or Gβγ. Remarkably, although independent of Gβγ, activation by membrane-targeted Ste5 requires Ste20, Cdc42, and Cdc24, indicating that their participation in this pathway does not require them to be activated by Gβγ. Thus, membrane recruitment of Ste5 defines a molecular activity for Gβγ. Moreover, our results suggest that this event promotes kinase cascade activation by delivering the Ste5-associated kinases to the cell surface kinase Ste20, whose function may depend on Cdc42 and Cdc24.