bluuebunny/crossref_metadata_2025 huggingface dataset:
CLB5 and CLB6 are required for premeiotic DNA replication and activation of the meiotic S/M checkpoint
Authors: Stuart David , Wittenberg Curt
Published: February 2008

Initiation of DNA replication during the mitotic cell cycle requires the activation of a cyclin-dependent protein kinase (CDK). The B-type cyclins Clb5 and Clb6 are the primary activators of the S phase function of the budding yeast CDK Cdc28. However, in mitotically growing cells this role can be fulfilled by the other B-type cyclins Clb1–Clb4. We report here that cells undergoing meiotic development also require Clb dependent CDK activity for DNA replication. Diploidclb5/clb5 clb6/clb6 mutants are unable to perform premeiotic DNA replication. Despite this defect, the mutant cells progress into the meiotic program and undergo lethal segregation of unreplicated DNA suggesting that they fail to activate a checkpoint that restrains meiotic M phase until DNA replication is complete. We have found that a DNA replication checkpoint dependent on the ATM homolog MEC1 operates in wild-type cells during meiosis and can be invoked in response to inhibition of DNA synthesis. Although cells that lack clb5 and clb6 are unable to activate the meiotic DNA replication checkpoint, they do possess an intact DNA damage checkpoint which can restrain chromosome segregation in the face of DNA damage. We conclude that CLB5 and CLB6 are essential for premeiotic DNA replication and, consequently, for activation of a meiotic DNA replication checkpoint.
Transcription occurs in pulses in muscle fibers
Authors: Newlands Sarah , Levitt Linda K. , Robinson C. Stephen , Karpf A.B. Carmen , Hodgson Vanessa R.M. , Wade Robert P. , Hardeman Edna C.
Published: February 2008

We report a novel mechanism of gene regulation in skeletal muscle fibers. Within an individual myofiber nucleus, not all muscle loci are transcriptionally active at a given time and loci are regulated independently. This phenomenon is particularly remarkable because the nuclei within a myofiber share a common cytoplasm. Both endogenous muscle-specific and housekeeping genes and transgenes are regulated in this manner. Therefore, despite the uniform protein composition of the contractile apparatus along the length of the fiber, the loci that encode this structure are not transcribed continuously. The total number of active loci for a particular gene is dynamic, changing during fetal development, regeneration, and in the adult, and potentially reflects the growth status of the fiber. The data reveal that transcription in particular stages of muscle fiber maturation occurs in pulses and is defined by a stochastic mechanism.
Ribonuclease E organizes the protein interactions in the Escherichia coli RNA degradosome
Authors: Vanzo Nathalie F. , Li Yeun Shan , Py Béatrice , Blum Erwin , Higgins Christopher F. , Raynal Lelia C. , Krisch Henry M. , Carpousis Agamemnon J.
Published: February 2008

The Escherichia coli RNA degradosome is the prototype of a recently discovered family of multiprotein machines involved in the processing and degradation of RNA. The interactions between the various protein components of the RNA degradosome were investigated by Far Western blotting, the yeast two-hybrid assay, and coimmunopurification experiments. Our results demonstrate that the carboxy-terminal half (CTH) of ribonuclease E (RNase E) contains the binding sites for the three other major degradosomal components, the DEAD-box RNA helicase RhlB, enolase, and polynucleotide phosphorylase (PNPase). The CTH of RNase E acts as the scaffold of the complex upon which the other degradosomal components are assembled. Regions for oligomerization were detected in the amino-terminal and central regions of RNase E. Furthermore, polypeptides derived from the highly charged region of RNase E, containing the RhlB binding site, stimulate RhlB activity at least 15-fold, saturating at one polypeptide per RhlB molecule. A model for the regulation of the RhlB RNA helicase activity is presented. The description of RNase E now emerging is that of a remarkably complex multidomain protein containing an amino-terminal catalytic domain, a central RNA-binding domain, and carboxy-terminal binding sites for the other major components of the RNA degradosome.
Sequence and DNA structural determinants of N4 virion RNA polymerase–promoter recognition
Authors: Dai Xing , Rothman-Denes Lucia B.
Published: February 2008

Coliphage N4-coded, virion-encapsidated RNA polymerase (vRNAP) is able to bind to and transcribe promoter-containing double-stranded DNAs when the template is supercoiled and Escherichia colisingle-stranded DNA-binding protein (Eco SSB) is present. We report that vRNAP–promoter recognition and activity on these templates require specific sequences and a hairpin structure on the template strand. Hairpin extrusion, induced by Mg(II) and physiological superhelical density, is essential to provide the correct DNA structure for polymerase recognition, as mutant promoters that do not form hairpins show reduced in vitro activity. Therefore, a supercoil-induced DNA structural transition regulates N4 vRNAP transcription. EcoSSB activates transcription at physiological superhelical densities by stabilizing the template-strand hairpin. Specific sequences at the promoters are conserved to provide proper contacts for vRNAP, to support hairpin extrusion, or both. We propose a model for in vivo utilization of the vRNAP promoters, and discuss the roles of DNA supercoiling and Eco SSB in promoter activation.
Communication between Hin recombinase and Fis regulatory subunits during coordinate activation of Hin-catalyzed site-specific DNA inversion
Authors: Merickel Stacy K. , Haykinson Michael J. , Johnson Reid C.
Published: February 2008

The Hin DNA invertase becomes catalytically activated when assembled in an invertasome complex containing two Fis dimers bound to an enhancer segment. The region of Fis responsible for transactivation of Hin contains a mobile β-hairpin arm that extends from each dimer subunit. We show here that whereas both Fis dimers must be capable of activating Hin, Fis heterodimers that have only one functional activating β-arm are sufficient to form catalytically competent invertasomes. Analysis of homodimer and heterodimer mixes of different Hin mutants suggests that Fis must activate each subunit of the two Hin dimers that participate in catalysis. These experiments also indicate that all four Hin subunits must be coordinately activated prior to initiation of the first chemical step of the reaction and that the process of activation is independent of the catalytic steps of recombination. We propose a molecular model for the invertasome structure that is consistent with current information on protein–DNA structures and the topology of the DNA strands within the recombination complex. In this model, a single Fis activation arm could contact amino acids from both Hin subunits at the dimer interface to induce a conformational change that coordinately positions the active sites close to the scissile phosphodiester bonds.
Altered DNA-binding specificity mutants of EKLF and Sp1 show that EKLF is an activator of the β-globin locus control region in vivo
Authors: Gillemans Nynke , Tewari Rita , Lindeboom Fokke , Rottier Robbert , de Wit Ton , Wijgerde Mark , Grosveld Frank , Philipsen Sjaak
Published: February 2008

The locus control region of the β-globincluster contains five DNase I hypersensitive sites (5′HS1–5) required for locus activation. 5′HS3 contains six G-rich motifs that are essential for its activity. Members of a protein family, characterized by three zinc fingers highly homologous to those found in transcription factor Sp1, interact with these motifs. Because point mutagenesis cannot distinguish between family members, it is not known which protein activates 5′HS3. We show that the function of such closely related proteins can be distinguished in vivo by matching point mutations in 5′HS3 with amino acid changes in the zinc fingers of Sp1 and EKLF. Testing their activity in transgenic mice shows that EKLF is a direct activator of 5′HS3.
The Hog1 MAPK prevents cross talk between the HOG and pheromone response MAPK pathways in Saccharomyces cerevisiae
Authors: O’Rourke Sean M. , Herskowitz Ira
Published: February 2008

The MAPKKK Ste11p functions in three Saccharomyces cerevisiae MAPK cascades [the high osmolarity glycerol (HOG), pheromone response, and pseudohyphal/invasive growth pathways], but its activation in response to high osmolarity stimulates only the HOG pathway. To determine what restricts cross-activation of MAPK cascades (cross talk), we have studied mutants in which the pheromone response pathway is activated by high osmolarity (1 m sorbitol). We found that mutations in the HOG1gene, encoding the p38-type MAPK of the HOG pathway, and in thePBS2 gene, encoding the activating kinase for Hog1p, allowed osmolarity-induced activation of the pheromone response pathway. This cross talk required the osmosensor Sho1p, as well as Ste20p, Ste50p, the pheromone response MAPK cascade (Ste11p, Ste7p, and Fus3p or Kss1p), and Ste12p but not Ste4p or the MAPK scaffold protein, Ste5p. The cross talk in hog1 mutants induced multiple responses of the pheromone response pathway: induction of a FUS1::lacZreporter, morphological changes, and mating in ste4 andste5 mutants. We suggest that Hog1p may prevent osmolarity-induced cross talk by inhibiting Sho1p, perhaps as part of a feedback control on the HOG pathway. We have also shown that Ste20p and Ste50p function in the Sho1p branch of the HOG pathway and that a second osmosensor in addition to Sho1p may activate Ste11p. Finally, we have found that pseudohyphal growth exhibited by wild-type (HOG1) strains depends on SHO1, suggesting that Sho1p may be a receptor that feeds into the pseudohyphal growth pathway.
CDK inhibitors p18INK4c and p27Kip1 mediate two separate pathways to collaboratively suppress pituitary tumorigenesis
Authors: Franklin David S. , Godfrey Virginia L. , Lee Hayyoung , Kovalev Grigoriy I. , Schoonhoven Robert , Chen-Kiang Selina , Su Lishan , Xiong Yue
Published: February 2008

INK4 and CIP/KIP are two distinct families of cyclin-dependent kinase (CDK) inhibitors implicated in mediating a wide range of cell growth control signals. We have created p18INK4c-deficient mice. These mice develop gigantism and widespread organomegaly. The pituitary gland, spleen, and thymus are disproportionately enlarged and hyperplastic. T and B lymphocytes develop normally in p18-deficient mice, but both exhibit increased cellularity and a higher proliferative rate upon mitogenic stimulation. Loss of p18, like that of p27, but not other CDK inhibitor genes, leads to a gradual progression from intermediate lobe pituitary hyperplasia in young mice to an adenoma by 10 months of age with a nearly complete penetrance. Mice lacking both p18 and p27, like mice chimeric for Rb deficiency, invariably died from pituitary adenomas by 3 months. Hence, p18 and p27 mediate two separate pathways to collaboratively suppress pituitary tumorigenesis, likely by controlling the function of Rb.
Premature senescence involving p53 and p16 is activated in response to constitutive MEK/MAPK mitogenic signaling
Authors: Lin Athena W. , Barradas Marta , Stone James C. , van Aelst Linda , Serrano Manuel , Lowe Scott W.
Published: February 2008

Oncogenic Ras transforms immortal rodent cells to a tumorigenic state, in part, by constitutively transmitting mitogenic signals through the mitogen-activated protein kinase (MAPK) cascade. In primary cells, Ras is initially mitogenic but eventually induces premature senescence involving the p53 and p16INK4a tumor suppressors. Constitutive activation of MEK (a component of the MAPK cascade) induces both p53 and p16, and is required for Ras-induced senescence of normal human fibroblasts. Furthermore, activated MEK permanently arrests primary murine fibroblasts but forces uncontrolled mitogenesis and transformation in cells lacking either p53 orINK4a. The precisely opposite response of normal and immortalized cells to constitutive activation of the MAPK cascade implies that premature senescence acts as a fail-safe mechanism to limit the transforming potential of excessive Ras mitogenic signaling. Consequently, constitutive MAPK signaling activates p53 and p16 as tumor suppressors.
Pleiotropic control of glucose and hormone responses by PRL1, a nuclear WD protein, in Arabidopsis
Authors: Németh Kinga , Salchert Klaus , Putnoky Péter , Bhalerao Rishikesh , Koncz-Kálmán Zsuzsanna , Stankovic-Stangeland Biljana , Bakó László , Mathur Jaideep , Ökrész László , Stabel Sylvia , Geigenberger Peter , Stitt Mark , Rédei George P. , Schell Jeff , Koncz Csaba
Published: February 2008

The prl1 mutation localized by T-DNA tagging onArabidopsis chromosome 4-44 confers hypersensitivity to glucose and sucrose. The prl1 mutation results in transcriptional derepression of glucose responsive genes defining a novel suppressor function in glucose signaling. The prl1 mutation also augments the sensitivity of plants to growth hormones including cytokinin, ethylene, abscisic acid, and auxin; stimulates the accumulation of sugars and starch in leaves; and inhibits root elongation. PRL1encodes a regulatory WD protein that interacts with ATHKAP2, an α-importin nuclear import receptor, and is imported into the nucleus in Arabidopsis. Potential functional conservation of PRL1 homologs found in other eukaryotes is indicated by nuclear localization of PRL1 in monkey COS-1 cells and selective interaction of PRL1 with a nuclear protein kinase C–βII isoenzyme involved in human insulin signaling.
Synergistic cooperation of TFE3 and Smad proteins in TGF-β-induced transcription of the plasminogen activator inhibitor-1 gene
Authors: Hua Xianxin , Liu Xuedong , Ansari Dominic O. , Lodish Harvey F.
Published: February 2008

Members of the TGF-β superfamily influence a broad range of biological activities including stimulation of wound healing and inhibition of cell growth. TGF-β signals through type I and II receptor serine/ threonine kinases and induces transcription of many genes including plasminogen activator inhibitor-1 (PAI-1). To identify proteins that participate in TGF-β-induced gene expression, we developed a novel retrovirus-mediated expression cloning strategy; and using this approach, we established that transcription factor μE3 (TFE3) is involved in TGF-β-induced activation of the PAI-1 promoter. We showed that TFE3 binds to an E-box sequence in PE2, a 56-bp promoter fragment of the PAI-1 promoter, and that mutation of this sequence abolishes both TFE3 binding as well as TGF-β-dependent activation. TFE3 and Smad3 synergistically activate the PE2 promoter and phosphorylated Smad3 and Smad4 bind to a sequence adjacent to the TFE3-binding site in this promoter. Binding of both TFE3 and the Smad proteins to their cognate sequences is indispensable for TGF-β-inducible activation of the PE2 promoter. Hence, TFE3 is an important transcription factor in at least one TGF-β-activated signal transduction pathway.
Interaction of a nascent RNA structure with RNA polymerase is required for hairpin-dependent transcriptional pausing but not for transcript release
Authors: Artsimovitch Irina , Landick Robert
Published: February 2008

Nascent RNA structures may regulate RNA chain elongation either directly through interaction with RNA polymerase or indirectly by disrupting nascent RNA contacts with polymerase or DNA. To distinguish these mechanisms we tested whether the effects of the hisleader pause RNA hairpin could be mimicked by pairing of antisense DNA or RNA oligonucleotides to the nascent transcript. The hispause hairpin inhibits nucleotide addition when it forms 11 nucleotides from the transcript 3′ end. It also can terminate transcription when base changes extend its stem to ≤8 nucleotides from the 3′ end. All oligonucleotides that disrupted the pause hairpin reduced the dwell time of RNA polymerase at the pause site dramatically, even when they mimicked the 11-nucleotide 3′-proximal RNA spacing or created a suitably positioned RNA loop. Oligonucleotides that paired ≤8 nucleotides from the pause RNA 3′ end could trigger transcript release, but only when added to an already paused complex. These results argue that direct interaction of a nascent RNA hairpin with RNA polymerase delays escape from a pause, but that indirect effects of a hairpin may trigger transcript release from a paused complex. Resistance of the paused complex to pyrophosphorolysis and its reversal by antisense oligonucleotides further suggest that interaction of the pause hairpin with RNA polymerase disengages the RNA 3′ end from the active site.
Cellular and developmental control of O2 homeostasis by hypoxia-inducible factor 1α
Authors: Iyer Narayan V. , Kotch Lori E. , Agani Faton , Leung Sandra W. , Laughner Erik , Wenger Roland H. , Gassmann Max , Gearhart John D. , Lawler Ann M. , Yu Aimee Y. , Semenza Gregg L.
Published: February 2008

Hypoxia is an essential developmental and physiological stimulus that plays a key role in the pathophysiology of cancer, heart attack, stroke, and other major causes of mortality. Hypoxia-inducible factor 1 (HIF-1) is the only known mammalian transcription factor expressed uniquely in response to physiologically relevant levels of hypoxia. We now report that in Hif1a−/−embryonic stem cells that did not express the O2-regulated HIF-1α subunit, levels of mRNAs encoding glucose transporters and glycolytic enzymes were reduced, and cellular proliferation was impaired. Vascular endothelial growth factor mRNA expression was also markedly decreased in hypoxicHif1a−/− embryonic stem cells and cystic embryoid bodies. Complete deficiency of HIF-1α resulted in developmental arrest and lethality by E11 ofHif1a−/− embryos that manifested neural tube defects, cardiovascular malformations, and marked cell death within the cephalic mesenchyme. InHif1a+/+ embryos, HIF-1α expression increased between E8.5 and E9.5, coincident with the onset of developmental defects and cell death inHif1a−/− embryos. These results demonstrate that HIF-1α is a master regulator of cellular and developmental O2 homeostasis.
Overcoming cellular senescence in human cancer pathogenesis
Authors: Yeager Thomas R. , DeVries Sandy , Jarrard David F. , Kao Chinghai , Nakada Stephen Y. , Moon Timothy D. , Bruskewitz Reginald , Stadler Walter M. , Meisner Lorraine F. , Gilchrist Kennedy W. , Newton Michael A. , Waldman Frederic M. , Reznikoff Catherine A.
Published: February 2008

Elevation of p16, the CDKN2/p16 tumor suppressor gene (TSG) product, occurs at senescence in normal human uroepithelial cells (HUC). Immortal HUCs and bladder cancer cell lines show either alteration of p16 or pRb, the product of the retinoblastoma (RB) TSG. In addition, many human cancers show p16 or pRb alteration along with other genetic alterations that we associated with immortalization, including +20q and −3p. These observations led us to hypothesize that p16 elevation plays a critical role in senescence cell cycle arrest and that overcoming this block is an important step in tumorigenesis in vivo, as well as immortalization in vitro. Using a novel approach, we tested these hypotheses in the present study by examining p16 and pRb status in primary culture (P0) and after passage in vitro of transitional cell carcinoma (TCC) biopsies that represented both superficial bladder tumors and invasive bladder cancers. We demonstrated that all superficial TCCs showed elevated p16 after limited passage in vitro and then senesced, like normal HUCs. In contrast, all muscle invasive TCCs contained either a p16 or a pRb alteration at P0 and all spontaneously bypassed senescence (P = 0.001). Comparative genomic hybridization (CGH) was used to identify regions of chromosome loss or gain in all TCC samples. The application of a statistical model to the CGH data showed a high probability of elevated alteration rates of +20q11−q12 (0.99) and +8p22−pter (0.94) in the immortal muscle invasive TCCs, and of −9q (0.99) in the superficial TCCs. Three myoinvasive TCCs lost 3p13−p14. In this study, four of six myoinvasive TCCs also showedTP53 mutation that associated well with genome instability (P = 0.001), as previously hypothesized. Notably,TP53 mutation, which has been used as a marker of tumor progression in many human cancers, was less significant in associating with progression in this study (P = 0.04) than was p16 or pRb alteration (P = 0.001). Thus, these data support a new model in which overcoming senescence plays a critical role in human cancer pathogenesis and requires at least two genetic changes that occur in several combinations that can include either p16 or pRb loss and at least one additional alteration, such as +20q11−q12, −3p13−p14, or −8p21−pter.
The maize geneliguleless2 encodes a basic leucine zipper protein involved in the establishment of the leaf blade–sheath boundary
Authors: Walsh Justine , Waters Cynthia A. , Freeling Michael
Published: February 2008

The blade and sheath of a maize leaf are separated by a linear epidermal fringe, the ligule, and two wedge-like structures, the auricles. In plants homozygous for the null mutation,liguleless2-reference (lg2-R), the ligule and auricles are often absent or positioned incorrectly and the blade–sheath boundary is diffuse. This phenotype is in contrast to that ofliguleless1-reference (lg1-R) mutant plants, which have a more defined boundary even in the absence of the ligule and auricles. Additionally, mosaic analysis indicates the lg2-R phenotype is cell-nonautonomous and the lg1-R phenotype is cell-autonomous. Using scanning electron microscopy we show that lg2-R mutant plants are affected before the first visible sign of ligule and auricle formation. We have cloned the Lg2+ gene through aMutator8 transposon insertion allele, and verified it with five independently derived alleles. The comparison of genomic DNA and cDNA sequences reveals an open reading frame encoding a protein of 531 amino acids with partial homology to a subclass of plant basic leucine zipper (bZIP) transcription factors. Although a large body of molecular and biochemical characterization exists on this subclass of bZIP proteins, our work represents the first report of a mutant phenotype within this group. A specific reverse transcriptase (RT)–PCR assay shows LG2 mRNA expression in meristem/developing ligule regions. RT–PCR also shows that LG2 mRNA accumulation precedes that of LG1 mRNA. The mutant phenotype and expression analysis of lg2 suggest an early role in initiating an exact blade–sheath boundary within the young leaf primordia.
p300 and ATF-2 are components of the DRF complex, which regulates retinoic acid- and E1A-mediated transcription of the c-jun gene in F9 cells
Authors: Kawasaki Hiroaki , Song Jun , Eckner Richard , Ugai Hideyo , Chiu Robert , Taira Kazunari , Shi Yang , Jones Nic , Yokoyama Kazunari K.
Published: February 2008

Transcriptional activation of the c-jun gene is a critical event in the differentiation of F9 cells. In our previous studies we characterized an element [differentiation response element (DRE)] in the c-jun promoter that is both necessary and sufficient to confer the capacity for differentiation-dependent up-regulation. This element binds the differentiation regulatory factor (DRF) complex, of which one component is the adenovirus E1A-associated protein p300. We have now identified activation transcription factor-2 (ATF-2) as a DNA-binding subunit of the DRF complex. p300 and ATF-2 interact with each other in vivo and in vitro. The bromodomain and the C/H2 domain of p300 mediate the binding to ATF-2, which in turn requires a proline-rich region between amino acids 112 and 350 for its interaction with p300. The phosphorylation of the serine residue at position 121 of ATF-2 appears to be induced by protein kinase Cα (PKCα) after treatment of cells with retinoic acid (RA) or induction with E1A. In cotransfection assays, wild-type ATF-2 enhanced the transcription of an E2/tk–luciferase construct, in conjunction with p300–E2. However, a mutant form of ATF-2 with a mutation at position 121 (pCMVATF–2Ser121–Ala) did not. These results suggest that ATF-2 and p300 cooperate in the control of transcription by forming a protein complex that is responsive to differentiation-inducing signals, such as RA or E1A, and moreover, that the phosphorylation of ATF-2 by PKCα is probably a signaling event in the pathway that leads to the transactivation of the c-jun gene in F9 cells.
Regulation of POU genes by castor andhunchback establishes layered compartments in theDrosophila CNS
Authors: Kambadur Ravi , Koizumi Keita , Stivers Chad , Nagle James , Poole Stephen J. , Odenwald Ward F.
Published: February 2008

POU transcription factors participate in cell-identity decisions during nervous system development, yet little is known about the regulatory networks controlling their expression. We report all knownDrosophila POU genes require castor (cas) for correct CNS expression. drifter and I-POU depend oncas for full expression, whereas pdm-1 andpdm-2 are negatively regulated. cas encodes a zinc finger protein that shares DNA-binding specificity with anotherpdm repressor: the gap segmentation gene regulator Hunchback (Hb). Our studies reveal that the embryonic CNS contains sequentially generated neuroblast sublineages that can be distinguished by their expression of either Hb, Pdm-1, or Cas. Hb and Cas may directly silencepdm expression in early and late developing sublineages, given that pdm-1 cis-regulatory DNA contains ⩾32 Hb/Cas-binding sites and its enhancer(s) are ectopically activated in cas− neuroblasts. In addition, the targeted misexpression of Cas in all neuroblast lineages reduces Pdm-1 expression without altering Hb expression. By ensuring correct POU gene expression boundaries, hb and cas maintain temporal subdivisions in the cell-identity circuitry controlling CNS development.
Life without white fat: a transgenic mouse
Authors: Moitra Jaideep , Mason Mark M. , Olive Michelle , Krylov Dmitry , Gavrilova Oksana , Marcus-Samuels Bernice , Feigenbaum Lionel , Lee Eric , Aoyama Toshifumi , Eckhaus Michael , Reitman Marc L. , Vinson Charles
Published: February 2008

We have generated a transgenic mouse with no white fat tissue throughout life. These mice express a dominant-negative protein, termed A-ZIP/F, under the control of the adipose-specific aP2 enhancer/promoter. This protein prevents the DNA binding of B-ZIP transcription factors of both the C/EBP and Jun families. The transgenic mice (named A-ZIP/F-1) have no white adipose tissue and dramatically reduced amounts of brown adipose tissue, which is inactive. They are initially growth delayed, but by week 12, surpass their littermates in weight. The mice eat, drink, and urinate copiously, have decreased fecundity, premature death, and frequently die after anesthesia. The physiological consequences of having no white fat tissue are profound. The liver is engorged with lipid, and the internal organs are enlarged. The mice are diabetic, with reduced leptin (20-fold) and elevated serum glucose (3-fold), insulin (50- to 400-fold), free fatty acids (2-fold), and triglycerides (3- to 5-fold). The A-ZIP/F-1 phenotype suggests a mouse model for the human disease lipoatrophic diabetes (Seip-Berardinelli syndrome), indicating that the lack of fat can cause diabetes. The myriad of consequences of having no fat throughout development can be addressed with this model.
SXR, a novel steroid and xenobioticsensing nuclear receptor
Authors: Blumberg Bruce , Sabbagh Walid , Juguilon Henry , Bolado Jack , van Meter Casey M. , Ong Estelita S. , Evans Ronald M.
Published: February 2008

An important requirement for physiologic homeostasis is the detoxification and removal of endogenous hormones and xenobiotic compounds with biological activity. Much of the detoxification is performed by cytochrome P-450 enzymes, many of which have broad substrate specificity and are inducible by hundreds of different compounds, including steroids. The ingestion of dietary steroids and lipids induces the same enzymes; therefore, they would appear to be integrated into a coordinated metabolic pathway. Instead of possessing hundreds of receptors, one for each inducing compound, we propose the existence of a few broad specificity, low-affinity sensing receptors that would monitor aggregate levels of inducers to trigger production of metabolizing enzymes. In support of this model, we have isolated a novel nuclear receptor, termed the steroid and xenobiotic receptor (SXR), which activates transcription in response to a diversity of natural and synthetic compounds. SXR forms a heterodimer with RXR that can bind to and induce transcription from response elements present in steroid-inducible cytochrome P-450 genes and is expressed in tissues in which these catabolic enzymes are expressed. These results strongly support the steroid sensor hypothesis and suggest that broad specificity sensing receptors may represent a novel branch of the nuclear receptor superfamily.
Inorganic pyrophosphatase is a component of theDrosophilanucleosome remodeling factor complex
Authors: Gdula David A. , Sandaltzopoulos Raphael , Tsukiyama Toshio , Ossipow Vincent , Wu Carl
Published: February 2008

TheDrosophilanucleosome remodeling factor (NURF) is a protein complex consisting of four polypeptides that facilitates the perturbation of chromatin structure in vitro in an ATP-dependent manner. The 140-kD NURF subunit, imitation switch (ISWI), is related to the SWI2/SNF2 ATPase. Another subunit, NURF-55, is a 55-kD WD repeat protein homologous to the human retinoblastoma-associated protein RbAp48. Here, we report the cloning and characterization of the smallest (38 kD) component of NURF. NURF-38 is strikingly homologous to known inorganic pyrophosphatases. Both recombinant NURF-38 alone and the purified NURF complex are shown to have inorganic pyrophosphatase activity. Inhibition of the pyrophosphatase activity of NURF with sodium fluoride has no significant effect on chromatin remodeling, indicating that these two activities may be biochemically uncoupled. Our results suggest that NURF-38 may serve a structural or regulatory role in the complex. Alternatively, because accumulation of unhydrolyzed pyrophosphate during nucleotide incorporation inhibits polymerization, NURF may also have been adapted to deliver pyrophosphatase to chromatin to assist in replication or transcription by efficient removal of the inhibitory metabolite.
HSP101 functions as a specific translational regulatory protein whose activity is regulated by nutrient status
Authors: Wells Douglas R. , Tanguay Robert L. , Le Hanh , Gallie Daniel R.
Published: February 2008

The 5′ leader (Ω) of tobacco mosaic viral RNA functions as a translational enhancer. Sequence analysis of a 102-kD protein, identified previously as a specific Ω RNA-binding protein, revealed homology to the HSP101/HSP104/ClpB family of heat shock proteins and its expression in yeast complemented a thermotolerance defect caused by a deletion of the HSP104 gene. Up to a 50-fold increase in the translation of Ω-luc, but notluc mRNA was observed in yeast expressing the tobacco HSP101 whereas Ω failed to enhance translation in the absence of HSP101. Therefore, HSP101 and Ω comprise a two-component translational regulatory mechanism that can be recapitulated in yeast. Analysis of HSP101 function in yeast translation mutants suggested that the initiation factor (eIF) 3 and specifically one (TIF4632) of the two eIF4G proteins were required for the HSP101-mediated enhancement. The RNA-binding and translational regulatory activities of HSP101 were inactive in respiring cells or in cells subject to nutrient limitation, but its thermotolerance function remained unaffected. This is the first identification of a protein required for specific translational enhancement of capped mRNAs, the first report of a translational regulatory function for any heat-shock protein, and the first functional distinction between the two eIF4G proteins present in eukaryotes.
Development of neuroendocrine lineages requires the bHLH–PAS transcription factor SIM1
Authors: Michaud Jacques L. , Rosenquist Thomas , May Noah R. , Fan Chen-Ming
Published: February 2008

The bHLH–PAS transcription factor SIM1 is expressed during the development of the hypothalamic–pituitary axis in three hypothalamic nuclei: the paraventricular nucleus (PVN), the anterior periventricular nucleus (aPV), and the supraoptic nucleus (SON). To investigateSim1 function in the hypothalamus, we produced mice carrying a null allele of Sim1 by gene targeting. Homozygous mutant mice die shortly after birth. Histological analysis shows that the PVN and the SON of these mice are hypocellular. At least five distinct types of secretory neurons, identified by the expression of oxytocin, vasopressin, thyrotropin-releasing hormone, corticotropin-releasing hormone, and somatostatin, are absent in the mutant PVN, aPV, and SON. Moreover, we show that SIM1 controls the development of these secretory neurons at the final stages of their differentiation. A subset of these neuronal lineages in the PVN/SON are also missing in mice bearing a mutation in the POU transcription factor BRN2. We provide evidence that, during development of the Sim1 mutant hypothalamus, the prospective PVN/SON region fails to express Brn2. Our results strongly indicate that SIM1 functions upstream to maintain Brn2 expression, which in turn directs the terminal differentiation of specific neuroendocrine lineages within the PVN/SON.
A surface of Escherichia coli ς70 required for promoter function and antitermination by phage λ Q protein
Authors: Ko Dennis C. , Marr Michael T. , Guo Jingshu , Roberts Jeffrey W.
Published: February 2008

The ς initiation factor ς70 of Escherichia coli acts not only in promoter recognition and DNA strand opening, but also to mediate the transformation of RNA polymerase (RNAP) to an antiterminating form by the phage λ gene Q protein. Q is able to bind and modify RNAP when α70, still present in the initially elongating enzyme, recognizes a repeat of the −10 promoter element and induces a transcription pause. We have isolated mutations in the rpoD gene for ς70 that impair Q function because they reduce the ability of ς70 to recognize the downstream pause site. These mutations identify a locus of ς70 that is important for the formation and stability of open promoter complex, likely because it mediates protein interactions with RNAP core.
Specific telomerase RNA residues distant from the template are essential for telomerase function
Authors: Roy Jagoree , Fulton Tracy B. , Blackburn Elizabeth H.
Published: February 2008

The reverse transcriptase telomerase is a ribonucleoprotein complex that adds telomeric repeats to chromosome ends, using a sequence within its endogenous RNA component as a template. Although templating domains of telomerase RNA have been studied in detail, little is known about the roles of the remaining residues, particularly in yeast. We examined the functions of nontemplate telomerase residues in the telomerase RNA of budding yeast Kluyveromyces lactis. Although approximately half of the RNA residues were dispensable for function, four specific regions were essential for telomerase action in vivo. We analyzed the effects of mutating these regions on in vivo function, in vitro telomerase activity, and telomerase RNP assembly. Deletion of two regions resulted in synthesis of stable RNAs that appeared unable to assemble into a stable RNP. Mutating a region near the 5′ end of the RNA allowed RNP assembly but abolished enzymatic activity. Mutations in another specific small region of the RNA led to an inactive telomerase RNP with an altered RNA conformation.
GAGA mediates the enhancer blocking activity of the eve promoter in the Drosophila embryo
Authors: Ohtsuki Sumio , Levine Michael
Published: February 2008

Insulator DNAs and promoter competition regulate enhancer–promoter interactions within complex genetic loci. A transgenic embryo assay was used to obtain evidence that theDrosophila eve promoter possesses an insulator activity that can be uncoupled from the core elements that mediate competition. Theeve promoter contains an optimal TATA element and a GAGA sequence. The analysis of various chimeric promoters provides evidence that TATA is essential for promoter competition, whereas GAGA mediates enhancer blocking. The Trithorax-like (Trl) protein interacts with GAGA, and mutations in trl attenuate eve promoter insulator activity. We suggest that Trl–GAGA increases the stability of enhancer–promoter interactions by creating an open chromatin configuration at the core promoter.
Stage-specific apoptosis, developmental delay, and embryonic lethality in mice homozygous for a targeted disruption in the murine Bloom’s syndrome gene
Authors: Chester Nicholas , Kuo Frank , Kozak Christine , O’Hara Cathie D. , Leder Philip
Published: February 2008

Bloom’s syndrome is a human autosomal genetic disorder characterized at the cellular level by genome instability and increased sister chomatid exchanges (SCEs). Clinical features of the disease include proportional dwarfism and a predisposition to develop a wide variety of malignancies. The human BLM gene has been cloned recently and encodes a DNA helicase. Mouse embryos homozygous for a targeted mutation in the murine Bloom’s syndrome gene (Blm) are developmentally delayed and die by embryonic day 13.5. The fact that the interrupted gene is the homolog of the human BLM gene was confirmed by its homologous sequence, its chromosomal location, and by demonstrating high numbers of SCEs in cultured murineBlm−/− fibroblasts. The proportional dwarfism seen in the human is consistent with the small size and developmental delay (12–24 hr) seen during mid-gestation in murine Blm−/− embryos. Interestingly, the growth retardation in mutant embryos can be accounted for by a wave of increased apoptosis in the epiblast restricted to early post-implantation embryogenesis. Mutant embryos do not survive past day 13.5, and at this time exhibit severe anemia. Red blood cells and their precursors fromBlm−/− embryos are heterogeneous in appearance and have increased numbers of macrocytes and micronuclei. Both the apoptotic wave and the appearance of micronuclei in red blood cells are likely cellular consequences of damaged DNA caused by effects on replicating or segregating chromosomes.
Neuronal patterning by BMPs: a requirement for GDF7 in the generation of a discrete class of commissural interneurons in the mouse spinal cord
Authors: Lee Kevin J. , Mendelsohn Monica , Jessell Thomas M.
Published: February 2008

Inductive factors are known to direct the regional differentiation of the vertebrate central nervous system (CNS) but their role in the specification of individual neuronal cell types is less clear. We have examined the function of GDF7, a BMP family member expressed selectively by roof plate cells, in the generation of neuronal cell types in the dorsal spinal cord. We find that GDF7 can promote the differentiation in vitro of two dorsal sensory interneuron classes, D1A and D1B neurons. In Gdf7-null mutant embryos, the generation of D1A neurons is eliminated but D1B neurons and other identified dorsal interneurons are unaffected. These findings show that GDF7 is an inductive signal from the roof plate required for the specification of neuronal identity in the dorsal spinal cord and that GDF7 and other BMP family members expressed by the roof plate have non-redundant functions in vivo. More generally, these results suggest that BMP signaling may have a prominent role in the assignment of neuronal identity within the mammalian CNS.
The ovo gene required for cuticle formation and oogenesis in flies is involved in hair formation and spermatogenesis in mice
Authors: Dai Xing , Schonbaum Christopher , Degenstein Linda , Bai Wenyu , Mahowald Anthony , Fuchs Elaine
Published: February 2008

The Drosophila svb/ovo gene gives rise to differentially expressed transcripts encoding a zinc finger protein.svb/ovo has two distinct genetic functions:shavenbaby (svb) is required for proper formation of extracellular projections that are produced by certain epidermal cells in late-stage differentiation; ovo is required for survival and differentiation of female germ cells. We cloned a mouse gene,movo1 encoding a nuclear transcription factor that is highly similar to its fly counterpart in its zinc-finger sequences. In mice, the gene is expressed in skin, where it localizes to the differentiating cells of epidermis and hair follicles, and in testes, where it is present in spermatocytes and spermatids. Using gene targeting, we show that movo1 is required for proper development of both hair and sperm.movo1−/− mice are small, produce aberrant hairs, and display hypogenitalism, with a reduced ability to reproduce. These mice also develop abnormalities in kidney, wheremovo1 is also expressed. Our findings reveal remarkable parallels between mice and flies in epidermal appendage formation and in germ-cell maturation. Furthermore, they uncover a phenotype similar to that of Bardet–Biedl syndrome, a human disorder that maps to the same locus as human ovo1.
The molecular mechanism of mitotic inhibition of TFIIH is mediated by phosphorylation of CDK7
Authors: Akoulitchev Sasha , Reinberg Danny
Published: February 2008

TFIIH is a multisubunit complex, containing ATPase, helicases, and kinase activities. Functionally, TFIIH has been implicated in transcription by RNA polymerase II (RNAPII) and in nucleotide excision repair. A member of the cyclin-dependent kinase family, CDK7, is the kinase subunit of TFIIH. Genetically, CDK7 homologues have been implicated in transcription inSaccharomyces cerevisiae, and in mitotic regulation inSchizosaccharomyces pombe. Here we show that in mitosis the CDK7 subunit of TFIIH and the largest subunit of RNAPII become hyperphosphorylated. MPF-induced phosphorylation of CDK7 results in inhibition of the TFIIH-associated kinase and transcription activities. Negative and positive regulation of TFIIH requires phosphorylation within the T-loop of CDK7. Our data establishes TFIIH and its subunit CDK7 as a direct link between the regulation of transcription and the cell cycle.
GATA6 regulates HNF4 and is required for differentiation of visceral endoderm in the mouse embryo
Authors: Morrisey Edward E. , Tang Zhihua , Sigrist Kirsten , Lu Min Min , Jiang Fang , Ip Hon S. , Parmacek Michael S.
Published: February 2008

GATA6 belongs to a family of zinc finger transcription factors that play important roles in transducing nuclear events that regulate cellular differentiation and embryonic morphogenesis in vertebrate species. To examine the function of GATA6 during embryonic development, gene targeting was used to generate GATA6-deficient (GATA6−/−) ES cells and mice harboring a null mutation in GATA6. Differentiated embryoid bodies derived from GATA6−/− ES cells lack a covering layer of visceral endoderm and severely attenuate, or fail to express, genes encoding early and late endodermal markers, including HNF4, GATA4, α-fetoprotein (AFP), and HNF3β. Homozygous GATA6−/− mice died between embryonic day (E) 6.5 and E7.5 and exhibited a specific defect in endoderm differentiation including severely down-regulated expression of GATA4 and absence of HNF4 gene expression. Moreover, widespread programmed cell death was observed within the embryonic ectoderm of GATA6-deficient embryos, a finding also observed in HNF4-deficient embryos. Consistent with these data, forced expression of GATA6 activated the HNF4 promoter in nonendodermal cells. Finally, to examine the function of GATA6 during later embryonic development,GATA6−/−-C57BL/6 chimeric mice were generated. lacZ-taggedGATA6−/− ES cells contributed to all embryonic tissues with the exception of the endodermally derived bronchial epithelium. Taken together, these data suggest a model in which GATA6 lies upstream of HNF4 in a transcriptional cascade that regulates differentiation of the visceral endoderm. In addition, these data demonstrate that GATA6 is required for establishment of the endodermally derived bronchial epithelium.
Dorsoventral patterning in the Drosophila central nervous system: the vnd homeobox gene specifies ventral column identity
Authors: McDonald Jocelyn A. , Holbrook Scott , Isshiki Takako , Weiss Joseph , Doe Chris Q. , Mellerick Dervla M.
Published: February 2008

The Drosophila CNS develops from three columns of neuroectodermal cells along the dorsoventral (DV) axis: ventral, intermediate, and dorsal. In this and the accompanying paper, we investigate the role of two homeobox genes, vnd andind, in establishing ventral and intermediate cell fates within the Drosophila CNS. During early neurogenesis, Vnd protein is restricted to ventral column neuroectoderm and neuroblasts; later it is detected in a complex pattern of neurons. We use molecular markers that distinguish ventral, intermediate, and dorsal column neuroectoderm and neuroblasts, and a cell lineage marker for selected neuroblasts, to show that loss of vnd transforms ventral into intermediate column identity and that specific ventral neuroblasts fail to form. Conversely, ectopic vnd produces an intermediate to ventral column transformation. Thus, vnd is necessary and sufficient to induce ventral fates and repress intermediate fates within theDrosophila CNS. Vertebrate homologs of vnd(Nkx2.1 and 2.2) are similarly expressed in the ventral CNS, raising the possibility that DV patterning within the CNS is evolutionarily conserved.
Transcription factor CREM coordinates the timing of hepatocyte proliferation in the regenerating liver
Authors: Servillo Giuseppe , Della Fazia Maria Agnese , Sassone-Corsi Paolo
Published: February 2008

The liver regenerates upon partial hepatectomy (PH) as terminally differentiated hepatocytes undergo a tremendous proliferative process. CREM gene expression is powerfully induced during liver regeneration. We show that cell proliferation is significantly reduced upon PH in CREM−/− mice. There is a reduction in DNA synthesis, in the number of mitosis and of phosphorylated histone H3-positive cells. The post-PH proliferation peak is delayed by 10 hr, indicating an altered hepatocyte cell cycle. Expression of cyclins A, B, D1, E, and cdc2, of c-fos and tyrosine aminotransferase is deregulated. CREM mutation results in delayed S-phase entry, impairing the synchronization of proliferation.
Defective potassium currents in ataxia telangiectasia fibroblasts
Authors: Rhodes Nelson , D’Souza Theresa , Foster Christine D. , Ziv Yael , Kirsch David G. , Shiloh Yosef , Kastan Michael B. , Reinhart Peter H. , Gilmer Tona M.
Published: February 2008

Similarities exist between the progressive cerebellar ataxia in ataxia telangiectasia (AT) patients and a number of neurodegenerative diseases in both mouse and man involving specific mutations in ion channels and/or ion channel activity. These relationships led us to investigate the possibility of defective ion channel activity in AT cells. We examined changes in the membrane potential of AT fibroblasts in response to extracellular cation addition and found that the ability of AT fibroblasts to depolarize in response to increasing concentrations of extracellular K+ is significantly reduced when compared with control fibroblasts. Electrophysiological measurements performed with a number of AT cell lines, as well as two matched sets of primary AT fibroblast cultures, reveal that outward rectifier K+currents are largely absent in AT fibroblasts in comparison with control cells. These K+ current defects can be corrected in AT fibroblasts transfected with the full-length ATM cDNA. These data implicate, for the first time, a role for ATM in the regulation of K+ channel activity and membrane potential.