bluuebunny/crossref_metadata_2025 huggingface dataset:
The tumor suppressor gene Smad4/Dpc4 is required for gastrulation and later for anterior development of the mouse embryo
Authors: Sirard Christian , de la Pompa José Luis , Elia Andrew , Itie Annick , Mirtsos Christine , Cheung Alison , Hahn Stephan , Wakeham Andrew , Schwartz Lois , Kern Scott E. , Rossant Janet , Mak Tak W.
Published: February 2008

Mutations in the SMAD4/DPC4 tumor suppressor gene, a key signal transducer in most TGFβ-related pathways, are involved in 50% of pancreatic cancers. Homozygous Smad4 mutant mice die before day 7.5 of embryogenesis. Mutant embryos have reduced size, fail to gastrulate or express a mesodermal marker, and show abnormal visceral endoderm development. Growth retardation of theSmad4-deficient embryos results from reduced cell proliferation rather than increased apoptosis. Aggregation of mutant Smad4 ES cells with wild-type tetraploid morulae rescues the gastrulation defect. These results indicate that Smad4 is initially required for the differentiation of the visceral endoderm and that the gastrulation defect in the epiblast is secondary and non-cell autonomous. Rescued embryos show severe anterior truncations, indicating a second important role for Smad4 in anterior patterning during embryogenesis.
Hoxc13 mutant mice lack external hair
Authors: Godwin Alan R. , Capecchi Mario R.
Published: February 2008

Hox genes are usually expressed temporally and spatially in a colinear manner with respect to their positions in the Hoxcomplex. Consistent with the expected pattern for a paralogous group 13 member, early embryonic Hoxc13 expression is found in the nails and tail. Hoxc13 is also expressed in vibrissae, in the filiform papillae of the tongue, and in hair follicles throughout the body; a pattern that apparently violates spatial colinearity. Mice carrying mutant alleles of Hoxc13 have been generated by gene targeting. Homozygotes have defects in every region in which gene expression is seen. The most striking defect is brittle hair resulting in alopecia (hairless mice). One explanation for this novel role is that Hoxc13 has been recruited for a function common to hair, nail, and filiform papilla development.
The Med proteins of yeast and their function through the RNA polymerase II carboxy-terminal domain
Authors: Myers Lawrence C. , Gustafsson Claes M. , Bushnell David A. , Lui Mary , Erdjument-Bromage Hediye , Tempst Paul , Kornberg Roger D.
Published: February 2008

Mediator was resolved from yeast as a multiprotein complex on the basis of its requirement for transcriptional activation in a fully defined system. Three groups of mediator polypeptides could be distinguished: the products of five SRB genes, identified as suppressors of carboxy-terminal domain (CTD)-truncation mutants; products of four genes identified as global repressors; and six members of a new protein family, termed Med, thought to be primarily responsible for transcriptional activation. Notably absent from the purified mediator were Srbs 8, 9, 10, and 11, as well as members of the SWI/SNF complex. The CTD was required for function of mediator in vitro, in keeping with previous indications of involvement of the CTD in transcriptional activation in vivo. Evidence for human homologs of several mediator proteins, including Med7, points to similar mechanisms in higher cells.
clr-1 encodes a receptor tyrosine phosphatase that negatively regulates an FGF receptor signaling pathway in Caenorhabditis elegans
Authors: Kokel Michelle , Borland Christina Z. , DeLong Leslie , Horvitz H. Robert , Stern Michael J.
Published: February 2008

Receptor tyrosine phosphatases have been implicated in playing important roles in cell signaling events by their ability to regulate the level of protein tyrosine phosphorylation. Although the catalytic activity of their phosphatase domains has been well established, the biological roles of these molecules are, for the most part, not well understood. Here we show that the Caenorhabditis elegansprotein CLR-1 (CLeaR) is a receptor tyrosine phosphatase (RTP) with a complex extracellular region and two intracellular phosphatase domains. Mutations in clr-1 result in a dramatic Clr phenotype that we have used to study the physiological requirements for the CLR-1 RTP. We show that the phosphatase activity of the membrane-proximal domain is essential for the in vivo function of CLR-1. By contrast, we present evidence that the membrane-distal domain is not required to prevent the Clr phenotype in vivo. The Clr phenotype of clr-1 mutants is mimicked by activation of the EGL-15 fibroblast growth factor receptor (FGFR) and is suppressed by mutations that reduce or eliminate the activity of egl-15. Our data strongly indicate that CLR-1 attenuates the action of an FGFR-mediated signaling pathway by dephosphorylation.
Noggin-mediated antagonism of BMP signaling is required for growth and patterning of the neural tube and somite
Authors: McMahon Jill A. , Takada Shinji , Zimmerman Lyle B. , Fan Chen-Ming , Harland Richard M. , McMahon Andrew P.
Published: February 2008

Embryonic patterning in vertebrates is dependent upon the balance of inductive signals and their specific antagonists. We show thatNoggin, which encodes a bone morphogenetic protein (BMP) antagonist expressed in the node, notochord, and dorsal somite, is required for normal mouse development. Although Noggin has been implicated in neural induction, examination of null mutants in the mouse indicates that Noggin is not essential for this process. However, Noggin is required for subsequent growth and patterning of the neural tube. Early BMP-dependent dorsal cell fates, the roof plate and neural crest, form in the absence of Noggin. However, there is a progressive loss of early, Sonic hedgehog(Shh)-dependent ventral cell fates despite the normal expression of Shh in the notochord. Further, somite differentiation is deficient in both muscle and sclerotomal precursors. Addition of BMP2 or BMP4 to paraxial mesoderm explants blocks Shh-mediated induction of Pax-1, a sclerotomal marker, whereas addition of Noggin is sufficient to induce Pax-1. Noggin and Shh induce Pax-1 synergistically. Use of protein kinase A stimulators blocks Shh-mediated induction of Pax-1, but not induction by Noggin, suggesting that induction is mediated by different pathways. Together these data demonstrate that inhibition of BMP signaling by axially secreted Noggin is an important requirement for normal patterning of the vertebrate neural tube and somite.
Phosphorylation and association with the transcription factor Atf1 regulate localization of Spc1/Sty1 stress-activated kinase in fission yeast
Authors: Gaits Frédérique , Degols Geneviève , Shiozaki Kazuhiro , Russell Paul
Published: February 2008

Control of gene expression by stress-activated protein kinase (SAPK) cascades is crucial for combating cytotoxic stress. Elements of these cascades have been investigated in detail, but regulation of stress signal transduction from the cytoplasm to the nucleus is poorly understood. Herein are reported subcellular localization studies of fission yeast Spc1, a homolog of human p38 and budding yeast Hog1p SAPKs. Stress induces transient nuclear localization of Spc1. Nuclear translocation of Spc1 is coupled with disassociation from its activator kinase Wis1. However, Spc1 does not concentrate in the nucleus ofΔwis1cells; therefore Wis1 does not tether Spc1 in the cytoplasm. Unphosphorylatable forms of Spc1 are dispersed in the cytoplasm and nucleus, even in cells that also produce wild-type Spc1. Thus, Spc1 must be phosphorylated by Wis1 to localize in the nucleus. Nuclear retention of Spc1 requires Atf1, a transcription factor that is the key nuclear substrate of Spc1. Nuclear localization of Atf1 requires Pcr1, a heterodimerization partner of Atf1. These studies show that phosphorylation and association with Atf1 are required for nuclear localization of Spc1.
Ultrabithorax regulates genes at several levels of the wing-patterning hierarchy to shape the development of theDrosophila haltere
Authors: Weatherbee Scott D. , Halder Georg , Kim Jaeseob , Hudson Angela , Carroll Sean
Published: February 2008

Arthropods and vertebrates are constructed of many serially homologous structures whose individual patterns are regulated byHoxgenes. The Hox-regulated target genes and developmental pathways that determine the morphological differences between any homologous structures are not known. The differentiation of theDrosophilahaltere from the wing through the action of theUltrabithorax(Ubx) gene is a classic example ofHoxregulation of serial homology, although no Ubx-regulated genes in the haltere have been identified previously. Here, we show that Ubx represses the expression of the Wingless (Wg) signaling protein and a subset of Wg- and Decapentaplegic-activated genes such asspalt-related, vestigial, Serum Response Factor,andachaete-scute,whose products regulate morphological features that differ between the wing and haltere. In addition, we found that some genes in the same developmental pathway are independently regulated by Ubx. Our results suggest that Ubx, andHoxgenes in general, independently and selectively regulate genes that act at many levels of regulatory hierarchies to shape the differential development of serially homologous structures.
Exit from mitosis inDrosophila syncytial embryos requires proteolysis and cyclin degradation, and is associated with localized dephosphorylation
Authors: Su Tin Tin , Sprenger Frank , DiGregorio Paul J. , Campbell Shelagh D. , O’Farrell Patrick H.
Published: February 2008

The cyclin proteolysis that accompanies the exit from mitosis in diverse systems appears to be essential for restoration of interphase. The early syncytial divisions of Drosophila embryos, however, occur without detectable oscillations in the total cyclin level or Cdk1 activity. Nonetheless, we found that injection of an established inhibitor of cyclin proteolysis, a cyclin B amino-terminal peptide, prevents exit from mitosis in syncytial embryos. Similarly, injection of a version of Drosophila cyclin B that is refractory to proteolysis results in mitotic arrest. We infer that proteolysis of cyclins is required for exit from syncytial mitoses. This inference can be reconciled with the failure to observe oscillations in total cyclin levels if only a small pool of cyclins is destroyed in each cycle. We find that antibody detection of histone H3 phosphorylation (PH3) acts as a reporter for Cdk1 activity. A gradient of PH3 along anaphase chromosomes suggests local Cdk1 inactivation near the spindle poles in syncytial embryos. This pattern of Cdk1 inactivation would be consistent with local cyclin destruction at centrosomes or kinetochores. The local loss of PH3 during anaphase is specific to the syncytial divisions and is not observed after cellularization. We suggest that exit from mitosis in syncytial cycles is modified to allow nuclear autonomy within a common cytoplasm.
RNA-binding protein conserved in both microtubule- and microfilament-based RNA localization
Authors: Havin Leora , Git Anna , Elisha Zichrini , Oberman Froma , Yaniv Karina , Schwartz Sigal Pressman , Standart Nancy , Yisraeli Joel K.
Published: February 2008

Vg1 mRNA translocation to the vegetal cortex ofXenopus oocytes requires intact microtubules, and a 3′ UTRcis-acting element (termed VLE), which also mediates sequence-specific binding of several proteins. One protein, the 69-kD Vg1 RBP, associates Vg1 RNA to microtubules in vitro. Here we show that Vg1 RBP-binding sites correlate with vegetal localization. Purification and cloning of Vg1 RBP revealed five RNA-binding motifs: four KH and one RRM domains. Surprisingly, Vg1 RBP is highly homologous to the zipcode binding protein implicated in the microfilament-mediated localization of β actin mRNA in fibroblasts. These data support Vg1 RBP’s direct role in vegetal localization and suggest the existence of a general, evolutionarily conserved mechanism for mRNA targeting.
Targeted disruption ofSHIPleads to hemopoietic perturbations, lung pathology, and a shortened life span
Authors: Helgason Cheryl D. , Damen Jacqueline E. , Rosten Patty , Grewal Rewa , Sorensen Poul , Chappel Suzanne M. , Borowski Anita , Jirik Frank , Krystal Gerald , Humphries R. Keith
Published: February 2008

SHIP is a 145-kDSH2-containinginositol-5-phosphatase widely expressed in hemopoietic cells. It was first identified as a tyrosine phosphoprotein associated with Shc in response to numerous cytokines. SHIP has been implicated in FcγRIIB receptor-mediated negative signaling in B cells and mast cells and is postulated to down-regulate cytokine signal transduction in myeloid cells. To define further its role in the proliferation and differentiation of hemopoietic progenitors, as well as its function in mature cells, we have generated embryonic stem cells and mice bearing a targeted disruption of bothSHIPalleles. Here we show that although SHIP null mice are viable and fertile, they fail to thrive and survival is only 40% by 14 weeks of age. Mortality is associated with extensive consolidation of the lungs resulting from infiltration by myeloid cells. Increased numbers of granulocyte–macrophage progenitors are observed in both the bone marrow and spleen ofSHIP−/−mice, perhaps as a consequence of hyper-responsiveness to stimulation by macrophage–colony stimulating factor, granulocyte–macrophage colony stimulating factor, interleukin-3, or Steel factor as observed in vitro. In contrast, numbers of bone marrow lymphoid and late erythroid progenitors (CFU-E) are reduced. Thus, homozygous disruption ofSHIPestablishes the crucial role of this molecule in modulating cytokine signaling within the hemopoietic system and provides a powerful model for further delineating its function.
Purification and characterization of the nuclear RNase P holoenzyme complex reveals extensive subunit overlap with RNase MRP
Authors: Chamberlain Joel R. , Lee Yoon , Lane William S. , Engelke David R.
Published: February 2008

Ribonuclease P (RNase P) is a ribonucleoprotein enzyme that cleaves precursor tRNA transcripts to give mature 5′ ends. RNase P in eubacteria has a large, catalytic RNA subunit and a small protein subunit that are required for precursor tRNA cleavage in vivo. Although the eukaryotic holoenzymes have similar, large RNA subunits, previous work in a number of systems has suggested that the eukaryotic enzymes require a greater protein content. We have purified theSaccharomyces cerevisiae nuclear RNase P to apparent homogeneity, allowing the first comprehensive analysis of an unexpectedly complex subunit composition. Peptide sequencing by ion trap mass spectrometry identifies nine proteins that copurify with the nuclear RNase P RNA subunit, totaling 20-fold more protein than in the bacterial enzyme. All of these proteins are encoded by genes essential for RNase P activity and for cell viability. Previous genetic studies suggested that four proteins might be subunits of both RNase P and RNase MRP, the related rRNA processing enzyme. We demonstrate that all four of these proteins, Pop1p, Pop3p, Pop4p, and Rpp1p, are integral subunits of RNase P. In addition, four of the five newly identified protein subunits, Pop5p, Pop6p, Pop7p, and Pop8p, also appear to be shared between RNase P and RNase MRP. Only one polypeptide, Rpr2p, is unique to the RNase P holoenzyme by genetic depletion and immunoprecipitation studies. The large increase in the number of protein subunits over eubacterial RNase P is consistent with an increase in functional complexity in eukaryotes. The degree of structural similarity between nuclear RNase P and RNase MRP suggests that some aspects of their functions in pre-tRNA and pre-rRNA processing pathways might overlap or be coordinated.
κ chain monoallelic demethylation and the establishment of allelic exclusion
Authors: Mostoslavsky Raul , Singh Nandita , Kirillov Andrei , Pelanda Roberta , Cedar Howard , Chess Andrew , Bergman Yehudit
Published: February 2008

Allelic exclusion in κ light-chain synthesis is thought to result from a feedback mechanism by which the expression of a functional κ light chain on the surface of the B cell leads to an intracellular signal that down-regulates the V(D)J recombinase, thus precluding rearrangement of the other allele. Whereas such a feedback mechanism clearly plays a role in the maintenance of allelic exclusion, here we provide evidence suggesting that the initial establishment of allelic exclusion involves differential availability of the two κ alleles for rearrangement. Analysis of κ+ B-cell populations and of individual κ+ B cells that have rearranged only one allele demonstrates that in these cells, critical sites on the rearranged allele are unmethylated, whereas the nonrearranged allele remains methylated. This pattern is apparently generated by demethylation that is initiated at the small pre-B cell stage, on a single allele, in a process that occurs prior to rearrangement and requires the presence in cis of both the intronic and 3′ κ enhancers. Taken together with data demonstrating that undermethylation is required for rearrangement, these results indicate that demethylation may actually underly the process of allelic exclusion by directing the initial choice of a single κ allele for rearrangement.
Miranda as a multidomain adapter linking apically localized Inscuteable and basally localized Staufen and Prospero during asymmetric cell division inDrosophila
Authors: Shen Chun-Pyn , Knoblich Juergen A. , Chan Yee-Ming , Jiang Ming-Ming , Jan Lily Yeh , Jan Yuh Nung
Published: February 2008

Neuroblasts in the developing Drosophila CNS asymmetrically localize the cell fate determinants Numb and Prospero as well as prospero RNA to the basal cortex during mitosis. The localization of Prospero requires the function of inscuteableand miranda, whereas prospero RNA localization requiresinscuteable and staufen function. We demonstrate that Miranda contains multiple functional domains: an amino-terminal asymmetric localization domain, which interacts with Inscuteable, a central Numb interaction domain, and a more carboxy-terminal Prospero interaction domain. We also show that Miranda and Staufen have similar subcellular localization patterns and interact in vitro. Furthermore,miranda function is required for the asymmetric localization of Staufen. Miranda localization is disrupted by the microfilament disrupting agent latrunculin A. Our results suggest that Miranda directs the basal cortical localization of multiple molecules, including Staufen and prospero RNA, in mitotic neuroblasts in an actin-dependent manner.
Proneural gene self-stimulation in neural precursors: an essential mechanism for sense organ development that is regulated byNotch signaling
Authors: Culí Joaquim , Modolell Juan
Published: February 2008

To learn about the acquisition of neural fate by ectodermal cells, we have analyzed a very early sign of neural commitment inDrosophila, namely the specific accumulation ofachaete–scute complex (AS-C) proneural proteins in the cell that becomes a sensory organ mother cell (SMC). We have characterized an AS-C enhancer that directs expression specifically in SMCs. This enhancer promotes Scute protein accumulation in these cells, an event essential for sensory organ development in the absence of other AS-C genes. Interspecific sequence comparisons and site-directed mutagenesis show the presence of several conserved motifs necessary for enhancer action, some of them binding sites for proneural proteins. These and other data indicate that the enhancer mediates scuteself-stimulation, although only in the presence of additional activating factors, which most likely interact with conserved motifs reminiscent of NF-κB-binding sites. Cells neighboring the SMC do not acquire the neural fate because the Notch signaling pathway effectors, the Enhancer of split bHLH proteins, block this proneural gene self-stimulatory loop, possibly by antagonizing the action on the enhancer of the NF-κB-like factors or the proneural proteins. These data suggest a mechanism for SMC committment.
Human papillomavirus 16 E6 oncoprotein binds to interferon regulatory factor-3 and inhibits its transcriptional activity
Authors: Ronco Lucienne V. , Karpova Alla Y. , Vidal Marc , Howley Peter M.
Published: February 2008

Interferon regulatory factor-3 (IRF-3) was found to specifically interact with HPV16 E6 in a yeast two-hybrid screen. IRF-3 is activated by the presence of double-stranded RNA or by virus infection to form a stable complex with other transcriptional regulators that bind to the regulatory elements of the IFNβ promoter. We show that IRF-3 is a potent transcriptional activator and demonstrate that HPV16 E6 can inhibit its transactivation function. The expression of HPV16 E6 in primary human keratinocytes inhibits the induction of IFNβ mRNA following Sendai virus infection. The binding of HPV16 E6 to IRF-3 does not result in its ubiquitination or degradation. We propose that the interaction of E6 with IRF-3 and the inhibition of IRF-3’s transcriptional activity may provide the virus a means to circumvent the normal antiviral response of an HPV16-infected cell.
A new NEDD8-ligating system for cullin-4A
Authors: Osaka Fumio , Kawasaki Hiroshi , Aida Noriko , Saeki Mihoro , Chiba Tomoki , Kawashima Seiichi , Tanaka Keiji , Kato Seishi
Published: February 2008

NEDD8 is a ubiquitin (Ub)-like protein. Here we report a novel ubiquitinylation-related pathway for modification by NEDD8. NEDD8 was activated by an E1 (Ub-activating enzyme)-like complex, consisting of APP–BP1 and hUba3 with high respective homologies to the amino- and carboxy-terminal regions of E1 and then linked to hUbc12 (a human homolog of yeast Ub-conjugating enzyme Ubc12p). The major target protein modified by NEDD8 was found to be Hs–cullin-4A (Cul-4A), a member of the family of human cullin/Cdc53 proteins functioning as an essential component of a multifunctional Ub–protein ligase E3 complex that has a critical role in Ub-mediated proteolysis.
Inhibition of DNA synthesis by RB: effects on G1/S transition and S-phase progression
Authors: Knudsen Erik S. , Buckmaster Carolan , Chen Tung-Ti , Feramisco James R. , Wang Jean Y.J.
Published: February 2008

The retinoblastoma tumor suppressor protein, RB, is a negative regulator of cell proliferation. Growth inhibitory activity of RB is attenuated by phosphorylation. Mutation of a combination of phosphorylation sites leads to a constitutively active RB. In Rat-1 cells, the phosphorylation-site-mutated (PSM)-RB, but not wild-type RB, can inhibit S-phase entry. In PSM-RB-arrested G1 cells, normal levels of cyclin E and cyclin E-associated kinase activity were detected, but the expression of cyclin A was inhibited. The ectopic expression of cyclin E restored cyclin A expression and drove the PSM-RB expressing cells into S phase. Interestingly, Rat-1 cells coexpressing cyclin E and PSM-RB could not complete DNA replication. Microinjection of cells that have passed through the G1restriction point with plasmids expressing PSM-RB also led to the inhibition of DNA synthesis. The S-phase inhibitory activity of PSM-RB could be attenuated by the coinjection of SV40 T-antigen, adenovirus E1A, or a high level of E2F-1 expression plasmids. However, the S-phase inhibitory activity of PSM-RB could not be overcome by the coinjection of cyclin E or cyclin A expression plasmids. These results reveal a novel role for RB in the inhibition of S-phase progression that is distinct from the inhibition of the G1/S transition, and suggest that continued phosphorylation of RB beyond G1/S is required for the completion of DNA replication.
Opposite phenotypes of hypomorphic and Y766 phosphorylation site mutations reveal a function for Fgfr1 in anteroposterior patterning of mouse embryos
Authors: Partanen Juha , Schwartz Lois , Rossant Janet
Published: February 2008

Intercellular communication is needed for both the generation of the mesodermal germ layer and its division into distinct subpopulations. To dissect the functions of fibroblast growth factor receptor-1 (FGFR1) during mouse gastrulation as well as to gain insights into its possible roles during later embryonic development, we have introduced specific mutations into the Fgfr1 locus by gene targeting. Our results show functional dominance of one of the receptor isoforms and suggest a function for the autophosphorylation of site Y766 in the negative regulation of FGFR1 activity. Y766F and hypomorphic mutations in Fgfr1 generate opposite phenotypes in terms of homeotic vertebral transformations, suggesting a role for FGFR1 in patterning the embryonic anteriorposterior axis by way of regulation of Hox gene activity.
A novel homeobox gene, dharma, can induce the organizer in a non-cell-autonomous manner
Authors: Yamanaka Yojiro , Mizuno Toshiro , Sasai Yoshiki , Kishi Masashi , Takeda Hiroyuki , Kim Cheol-Hee , Hibi Masahiko , Hirano Toshio
Published: February 2008

The formation of Spemann organizer is one of the most important steps in dorsoventral axis determination in vertebrate development. However, whether the organizer forms autonomously or is induced non-cell-autonomously is controversial. In this report we have isolated a novel zebrafish homeobox gene, dharma, capable of inducing the organizer ectopically. The expression of dharma was first detected in several blastomeres at one side of the margin soon after the mid-blastula transition and continued in the dorsal side of the yolk syncytial layer (YSL) under the embryonic shield, the zebrafish organizer, until the onset of gastrulation. Furthermore, dharmaexpressed in the YSL induced the organizer in a non-cell-autonomous manner. These results provided the first identification of a zygotic gene to be implicated in the formation of an organizer-inducing center.
The Drosophila Ste20-related kinase misshapen is required for embryonic dorsal closure and acts through a JNK MAPK module on an evolutionarily conserved signaling pathway
Authors: Su Yi-Chi , Treisman Jessica E. , Skolnik Edward Y.
Published: February 2008

Dorsal closure in the Drosophila embryo occurs during the later stages of embryogenesis and involves changes in cell shape leading to the juxtaposition and subsequent adherence of the lateral epidermal primordia over the amnioserosa. Dorsal closure requires the activation of a conserved c-jun amino-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) module, as it is blocked by null mutations in JNK kinase [hemipterous (hep)] and JNK [basket (bsk)]. Drosophila JNK (DJNK) functions by phosphorylating and activating DJun, which in turn induces the transcription of decapentaplegic (dpp). We provide biochemical and genetic evidence that a Ste20-related kinase,misshapen (msn), functions upstream of hep andbsk to stimulate dorsal closure in the Drosophilaembryo. Mammalian (NCK-interactingkinase [NIK]) and Caenorhabditis elegans(mig-15) homologs of msn have been identified;mig-15 is necessary for several developmental processes inC. elegans. These data suggest that msn, mig-15, and NIK are components of a signaling pathway that is conserved among flies, worms, and mammals to control developmentally regulated pathways.
PU.1 induces myeloid lineage commitment in multipotent hematopoietic progenitors
Authors: Nerlov Claus , Graf Thomas
Published: February 2008

Little is known about the transcription factors that mediate lineage commitment of multipotent hematopoietic precursors. One candidate is the Ets family transcription factor PU.1, which is expressed in myeloid and B cells and is required for the development of both these lineages. We show here that the factor specifically instructs transformed multipotent hematopoietic progenitors to differentiate along the myeloid lineage. This involves not only the up-regulation of myeloid-specific cell surface antigens and the acquisition of myeloid growth-factor dependence but also the down-regulation of progenitor/thrombocyte-specific cell-surface markers and GATA-1. Both effects require an intact PU.1 transactivation domain. Whereas sustained activation of an inducible form of the factor leads to myeloid lineage commitment, short-term activation leads to the formation of immature eosinophils, indicating the existence of a bilineage intermediate. Our results suggest that PU.1 induces myeloid lineage commitment by the suppression of a master regulator of nonmyeloid genes (such as GATA-1) and the concomitant activation of multiple myeloid genes.
Reduced skin tumor development in cyclin D1-deficient mice highlights the oncogenic ras pathway in vivo
Authors: Robles Ana I. , Rodriguez-Puebla Marcelo L. , Glick Adam B. , Trempus Carol , Hansen Laura , Sicinski Piotr , Tennant Raymond W. , Weinberg Robert A. , Yuspa Stuart H. , Conti Claudio J.
Published: February 2008

Cyclin D1 is part of a cell cycle control node consistently deregulated in most human cancers. However, studies with cyclin D1-null mice indicate that it is dispensable for normal mouse development as well as cell growth in culture. Here, we provide evidence thatras-mediated tumorigenesis depends on signaling pathways that act preferentially through cyclin D1. Cyclin D1 expression and the activity of its associated kinase are up-regulated in keratinocytes in response to oncogenic ras. Furthermore, cyclin D1 deficiency results in up to an 80% decrease in the development of squamous tumors generated through either grafting of retroviral ras-transduced keratinocytes, phorbol ester treatment of ras transgenic mice, or two-stage carcinogenesis.
A calcineurin-dependent transcriptional pathway controls skeletal muscle fiber type
Authors: Chin Eva R. , Olson Eric N. , Richardson James A. , Yang Quan , Humphries Caroline , Shelton John M. , Wu Hai , Zhu Weiguang , Bassel-Duby Rhonda , Williams R. Sanders
Published: February 2008

Slow- and fast-twitch myofibers of adult skeletal muscles express unique sets of muscle-specific genes, and these distinctive programs of gene expression are controlled by variations in motor neuron activity. It is well established that, as a consequence of more frequent neural stimulation, slow fibers maintain higher levels of intracellular free calcium than fast fibers, but the mechanisms by which calcium may function as a messenger linking nerve activity to changes in gene expression in skeletal muscle have been unknown. Here, fiber-type-specific gene expression in skeletal muscles is shown to be controlled by a signaling pathway that involves calcineurin, a cyclosporin-sensitive, calcium-regulated serine/threonine phosphatase. Activation of calcineurin in skeletal myocytes selectively up-regulates slow-fiber-specific gene promoters. Conversely, inhibition of calcineurin activity by administration of cyclosporin A to intact animals promotes slow-to-fast fiber transformation. Transcriptional activation of slow-fiber-specific transcription appears to be mediated by a combinatorial mechanism involving proteins of the NFAT and MEF2 families. These results identify a molecular mechanism by which different patterns of motor nerve activity promote selective changes in gene expression to establish the specialized characteristics of slow and fast myofibers.
Activated transcription independent of the RNA polymerase II holoenzyme in budding yeast
Authors: McNeil J. Bryan , Agah Helga , Bentley David
Published: February 2008

We investigated whether the multisubunit holoenzyme complex of RNA polymerase II (Pol II) and mediator is universally required for transcription in budding yeast. ΔCTD Pol II lacking the carboxy-terminal domain of the large subunit cannot assemble with mediator but can still transcribe the CUP1 gene. CUP1transcripts made by ΔCTD Pol II initiated correctly and some extended past the normal poly(A) site yielding a novel dicistronic mRNA. MostCUP1 transcripts made by ΔCTD Pol II were degraded but could be stabilized by deletion of the XRN1 gene. Unlike other genes, transcription of CUP1 and HSP82 also persisted after inactivation of the CTD kinase Kin28 or the mediator subunit Srb4. The upstream-activating sequence (UAS) of the CUP1 promoter was sufficient to drive Cu2+ inducible transcription without Srb4 and heat shock inducible transcription without the CTD. We conclude that the Pol II holoenzyme is not essential for all UAS-dependent activated transcription in yeast.
Masking, unmasking, and regulated polyadenylation cooperate in the translational control of a dormant mRNA in mouse oocytes
Authors: Stutz André , Conne Béatrice , Huarte Joachim , Gubler Pascale , Völkel Valérie , Flandin Pierre , Vassalli Jean-Dominique
Published: February 2008

The mechanisms responsible for translational silencing of certain mRNAs in growing oocytes, and for their awakening during meiotic maturation, are not completely elucidated. We show that binding of a ∼80-kD protein to a UA-rich element in the 3′ UTR of tissue-type plasminogen activator mRNA, a mouse oocyte mRNA that is translated during meiotic maturation, silences the mRNA in primary oocytes. Translation can be triggered by injecting a competitor transcript that displaces this silencing factor, without elongation of a pre-existing short poly(A) tail, the presence of which is mandatory. During meiotic maturation, cytoplasmic polyadenylation is necessary to maintain a poly(A) tail, but the determining event for translational activation appears to be the modification or displacement of the silencing factor.
Phosphorylation regulates association of the transcription factor Pho4 with its import receptor Pse1/Kap121
Authors: Kaffman Arie , Rank Nicole Miller , O’Shea Erin K.
Published: February 2008

The transcription factor Pho4 is phosphorylated and localized predominantly to the cytoplasm when budding yeast are grown in phosphate-rich medium and is unphosphorylated and localized to the nucleus upon phosphate starvation. We have investigated the requirements for nuclear import of Pho4 and find that Pho4 enters the nucleus via a nonclassical import pathway that utilizes the importin β family member Pse1/Kap121. Pse1 binds directly to Pho4 and is required for its import in vivo. We have defined the nuclear localization signal on Pho4 and demonstrate that it is required for Pse1 binding in vitro and is sufficient for PSE1-dependent import in vivo. Phosphorylation of Pho4 inhibits its interaction with Pse1, providing a mechanism by which phosphorylation may regulate import of Pho4 in vivo.
A novel homeobox gene mediates the Dpp signal to establish functional specificity within target cells
Authors: Nakagoshi Hideki , Hoshi Minako , Nabeshima Yo-ichi , Matsuzaki Fumio
Published: February 2008

Morphogen gradients of secreted molecules play critical roles in the establishment of the spatial pattern of gene expression. During midgut development in Drosophila, secreted molecules of Decapentaplegic (Dpp) and Wingless (Wg) establish unique transcriptional regulation within target cells to specify the resultant cell types. Here we report the identification of a novel homeobox gene,defective proventriculus (dve), which is required for the midgut specification under the control of Dpp and Wg. Indve mutants, two distinct parts of the midgut, the proventriculus and middle midgut, are abnormally organized. The Wg signal regulates dve expression during proventriculus development. On the other hand, dve is a downstream target of Dpp in the middle midgut and defines the functional specificity of copper cells along with another Dpp target gene, labial. Thus, the dve gene acts under the two distinct extracellular signals at distant parts of the midgut primordia.
Transcriptional termination in the Balbiani ring 1 gene is closely coupled to 3′-end formation and excision of the 3′-terminal intron
Authors: Baurén Göran , Belikov Sergey , Wieslander Lars
Published: February 2008

We have analyzed transcription termination, 3′-end formation, and excision of the 3′-terminal intron in vivo in the Balbiani ring 1 (BR1) gene and its pre-mRNA. We show that full-length RNA transcripts are evenly spaced on the gene from a position 300 bp upstream to a region 500–700 bp downstream of the polyadenylation sequence. Very few full-length transcripts and no short, cleaved, nascent transcripts could be observed downstream of this region. Pre-mRNA with 10–20 adenylate residues accumulates at the active gene and then rapidly leaves from the gene locus. Only polyadenylated pre-mRNAs could be detected in the nucleoplasm. Our results are consistent with the hypothesis that transcription termination occurs in a narrow region for the majority of transcripts, simultaneous with 3′-end formation. Excision of the 3′-terminal intron occurs before 3′-end formation in about 5% of the nascent transcripts. When transcription terminates, 3′ cleavage takes place and 10–20 adenylate residues are added, the 3′-terminal intron is excised from additionally about 75% of the pre-mRNA at the gene locus. Our data support a close temporal and spatial coupling of transcription termination and the cleavage and initial polyadenylation of 3′-end formation. Excision of the 3′-terminal intron is highly stimulated as the cleavage/polyadenylation complex assembles and 3′-end formation is initiated.
Repression of yeast Ste12 transcription factor by direct binding of unphosphorylated Kss1 MAPK and its regulation by the Ste7 MEK
Authors: Bardwell Lee , Cook Jeanette G. , Voora Deepak , Baggott Daniel M. , Martinez Anthony R. , Thorner Jeremy
Published: February 2008

The mitogen-activated protein kinase (MAPK) Kss1 has a dual role in regulating filamentous (invasive) growth of the yeast Saccharomyces cerevisiae. The stimulatory function of Kss1 requires both its catalytic activity and its activation by the MAPK/ERK kinase (MEK) Ste7; in contrast, the inhibitory function of Kss1 requires neither. This study examines the mechanism by which Kss1 inhibits invasive growth, and how Ste7 action overcomes this inhibition. We found that unphosphorylated Kss1 binds directly to the transcription factor Ste12, that this binding is necessary for Kss1-mediated repression of Ste12, and that Ste7-mediated phosphorylation of Kss1 weakens Kss1–Ste12 interaction and relieves Kss1-mediated repression. Relative to Kss1, the MAPK Fus3 binds less strongly to Ste12 and is correspondingly a weaker inhibitor of invasive growth. Analysis of Kss1 mutants indicated that the activation loop of Kss1 controls binding to Ste12. Potent repression of a transcription factor by its physical interaction with the unactivated isoform of a protein kinase, and relief of this repression by activation of the kinase, is a novel mechanism for signal-dependent regulation of gene expression.
Negative control of replication initiation by a novel chromosomal locus exhibiting exceptional affinity for Escherichia coli DnaA protein
Authors: Kitagawa Risa , Ozaki Toru , Moriya Shigeki , Ogawa Tohru
Published: February 2008

Replication of the Escherichia coli chromosome is initiated at a unique site, oriC. Concurrent initiation occurs at alloriC sites present in a cell once, and only once, per cell cycle. A mechanism to ensure cyclic initiation events was found operating through the chromosomal site, datA, a 1-kb segment located at 94.7 min on the genetic map that titrates exceptionally large amounts of the bacterial initiator protein, DnaA. A strain lacking datA grew normally but exhibited an asynchronous initiation phenotype as a result of extra initiation events. This mutant phenotype was suppressed by DnaA-titrating plasmids. Furthermore, mutations in a 9-bp DnaA-binding sequence (the DnaA box) in datA were enough to induce the mutant phenotype. Thus,datA is a novel chromosomal element that appears to adjust a balance between free and bound DnaA for a single initiation event at a fixed time in the bacterial cell cycle. Titration of DnaA to newly duplicated datA during oriC sequestration, which is mediated by hemimethylated GATC sequences in oriC and the SeqA protein, would contribute to prevention of reinitiations whenoriC is desequestered.
Eph signaling is required for segmentation and differentiation of the somites
Authors: Durbin Lindsey , Brennan Caroline , Shiomi Kensuke , Cooke Julie , Barrios Arantza , Shanmugalingam Shantha , Guthrie Brenda , Lindberg Rick , Holder Nigel
Published: February 2008

Somitogenesis involves the segmentation of the paraxial mesoderm into units along the anteroposterior axis. Here we show a role for Eph and ephrin signaling in the patterning of presomitic mesoderm and formation of the somites. Ephrin-A-L1 and ephrin-B2 are expressed in an iterative manner in the developing somites and presomitic mesoderm, as is the Eph receptor EphA4. We have examined the role of these proteins by injection of RNA, encoding dominant negative forms of Eph receptors and ephrins. Interruption of Eph signaling leads to abnormal somite boundary formation and reduced or disturbed myoD expression in the myotome. Disruption of Eph family signaling delays the normal down-regulation of her1 andDelta D expression in the anterior presomitic mesoderm and disrupts myogenic differentiation. We suggest that Eph signaling has a key role in the translation of the patterning of presomitic mesoderm into somites.
The flagellar anti-ς factor FlgM actively dissociates Salmonella typhimurium ς28 RNA polymerase holoenzyme
Authors: Chadsey Meggen S. , Karlinsey Joyce E. , Hughes Kelly T.
Published: February 2008

The anti-ς factor FlgM of Salmonella typhimurium inhibits transcription of class 3 flagellar genes through a direct interaction with the flagellar-specific ς factor, ς28. FlgM is believed to prevent RNA polymerase (RNAP) holoenzyme formation by sequestering free ς28. We have analyzed FlgM-mediated inhibition of ς28 activity in vitro. FlgM is able to inhibit ς28 activity even when ς28 is first allowed to associate with core RNAP. Surface plasmon resonance (SPR) was used to evaluate the interaction between FlgM and both ς28 and ς28 holoenzyme (Eς28). TheKd of the ς28–FlgM complex is ∼2 × 10−10m; missense mutations in FlgM that cause a defect in ς28 inhibition in vivo increase theKd of this interaction by 4- to 10-fold. SPR measurements of Eς28 dissociation in the presence of FlgM indicate that FlgM destabilizes Eς28, presumably via an interaction with the ς subunit. Our data provide the first direct evidence of an interaction between FlgM and Eς28. We propose that this secondary activity of FlgM, which we term holoenzyme destabilization, enhances the sensitivity of the cell to changes in FlgM levels during flagellar biogenesis.
Identification of APN2, the Saccharomyces cerevisiae homolog of the major human AP endonuclease HAP1, and its role in the repair of abasic sites
Authors: Johnson Robert E. , Torres-Ramos Carlos A. , Izumi Tadahide , Mitra Sankar , Prakash Satya , Prakash Louise
Published: February 2008

Abasic (AP) sites arise in DNA through spontaneous base loss and enzymatic removal of damaged bases. APN1 encodes the major AP-endonuclease of Saccharomyces cerevisiae. Human HAP1(REF1) encodes the major AP endonuclease which, in addition to its role in DNA repair, functions as a redox regulatory protein. We identify APN2, the yeast homolog of HAP1 and provide evidence that Apn1 and Apn2 represent alternate pathways for repairing AP sites. The apn1Δ apn2Δ strain displays a highly elevated level of MMS-induced mutagenesis, which is dependent on the REV3, REV7, and REV1 genes. Our findings indicate that AP sites are highly cytotoxic and mutagenic in eukaryotes, and that the REV3, REV7-encoded DNA polymerase ζ mediates the mutagenic bypass of AP sites.
Smad6 inhibits BMP/Smad1 signaling by specifically competing with the Smad4 tumor suppressor
Authors: Hata Akiko , Lagna Giorgio , Massagué Joan , Hemmati-Brivanlou Ali
Published: February 2008

Bone morphogenetic protein (BMP) receptors signal by phosphorylating Smad1, which then associates with Smad4; this complex moves into the nucleus and activates transcription. Here we report the existence of a natural inhibitor of this process, Smad6, a longer version of the previously reported JV15-1. In Xenopus embryos and in mammalian cells, Smad6 specifically blocks signaling by the BMP/Smad1 pathway. Smad6 inhibits BMP/Smad1 signaling without interfering with receptor-mediated phosphorylation of Smad1. Smad6 specifically competes with Smad4 for binding to receptor-activated Smad1, yielding an apparently inactive Smad1–Smad6 complex. Therefore, Smad6 selectively antagonizes BMP-activated Smad1 by acting as a Smad4 decoy.
Fgf-10 is required for both limb and lung development and exhibits striking functional similarity to Drosophila branchless
Authors: Min Hosung , Danilenko Dimitry M. , Scully Sheila A. , Bolon Brad , Ring Brian D. , Tarpley John E. , DeRose Margaret , Simonet W. Scott
Published: February 2008

Fgf-10-deficient mice (Fgf-10−/−) were generated to determine the role(s) of Fgf-10 in vertebrate development. Limb bud initiation was abolished inFgf-10−/− mice. Strikingly,Fgf-10−/− fetuses continued to develop until birth, despite the complete absence of both fore- and hindlimbs. Fgf-10 is necessary for apical ectodermal ridge (AER) formation and acts epistatically upstream of Fgf-8, the earliest known AER marker in mice.Fgf-10−/− mice exhibited perinatal lethality associated with complete absence of lungs. Although tracheal development was normal, main-stem bronchial formation, as well as all subsequent pulmonary branching morphogenesis, was completely disrupted. The pulmonary phenotype ofFgf-10−/− mice is strikingly similar to that of the Drosophila mutant branchless, anFgf homolog.
Cooperation between the Cdk inhibitors p27KIP1 and p57KIP2 in the control of tissue growth and development
Authors: Zhang Pumin , Wong Calvin , DePinho Ronald A. , Harper J. Wade , Elledge Stephen J.
Published: February 2008

Cell cycle exit is required for terminal differentiation of many cell types. The retinoblastoma protein Rb has been implicated both in cell cycle exit and differentiation in several tissues.Rb is negatively regulated by cyclin-dependent kinases (Cdks). The main effectors that down-regulate Cdkactivity to activate Rb are not known in the lens or other tissues. In this study, using multiple mutant mice, we show that theCdk inhibitors p27KIP1 andp57KIP2 function redundantly to control cell cycle exit and differentiation of lens fiber cells and placental trophoblasts. These studies demonstrate that p27KIP1and p57KIP2 are critical terminal effectors of signal transduction pathways that control cell differentiation.
Activation of Rac1 by a Crk SH3-binding protein, DOCK180
Authors: Kiyokawa Etsuko , Hashimoto Yuko , Kobayashi Shin , Sugimura Haruhiko , Kurata Takeshi , Matsuda Michiyuki
Published: February 2008

DOCK180 is involved in integrin signaling through CrkII-p130Cas complexes. We have studied the involvement of DOCK180 in Rac1 signaling cascades. DOCK180 activated JNK in a manner dependent on Rac1, Cdc42Hs, and SEK, and overexpression of DOCK180 increased the amount of GTP-bound Rac1 in 293T cells. Coexpression of CrkII and p130Cas enhanced this DOCK180-dependent activation of Rac1. Furthermore, we observed direct binding of DOCK180 to Rac1, but not to RhoA or Cdc42Hs. Dominant-negative Rac1 suppressed DOCK180-induced membrane spreading. These results strongly suggest that DOCK180 is a novel activator of Rac1 and involved in integrin signaling.