bluuebunny/crossref_metadata_2025 huggingface dataset:
A stress response pathway from the endoplasmic reticulum to the nucleus requires a novel bifunctional protein kinase/endoribonuclease (Ire1p) in mammalian cells
Authors: Tirasophon Witoon , Welihinda Ajith A. , Kaufman Randal J.
Published: February 2008

Eukaryotes respond to the presence of unfolded protein in the endoplasmic reticulum (ER) by up-regulating the transcription of genes encoding ER protein chaperones, such as BiP. We have isolated a novel human cDNA encoding a homolog to Saccharomyces cerevisiaeIre1p, a proximal sensor for this signal transduction pathway in yeast. The gene product hIre1p is a type 1 transmembrane protein containing a cytoplasmic domain that is highly conserved to the yeast counterpart having a Ser/Thr protein kinase domain and a domain homologous to RNase L. However, the luminal domain has extensively diverged from the yeast gene product. hIre1p expressed in mammalian cells displayed intrinsic autophosphorylation activity and an endoribonuclease activity that cleaved the 5′ splice site of yeastHAC1 mRNA, a substrate for the endoribonuclease activity of yeast Ire1p. Overexpressed hIre1p was localized to the ER with particular concentration around the nuclear envelope and some colocalization with the nuclear pore complex. Expression of Ire1p mRNA was autoregulated through a process that required a functional hIre1p kinase activity. Finally, overexpression of wild-type hIre1p constitutively activated a reporter gene under transcriptional control of the rat BiP promoter, whereas expression of a catalytically inactive hIre1p acted in a trans-dominant-negative manner to prevent transcriptional activation of the BiP promoter in response to ER stress induced by inhibition of N-linked glycosylation. These results demonstrate that hIre1p is an essential proximal sensor of the unfolded protein response pathway in mammalian cells.
Binary sibling neuronal cell fate decisions in the Drosophila embryonic central nervous system are nonstochastic and require inscuteable-mediated asymmetry of ganglion mother cells
Authors: Buescher Marita , Yeo Su Ling , Udolph Gerald , Zavortink Michael , Yang Xiaohang , Tear Guy , Chia William
Published: February 2008

Asymmetric cell division is a widespread mechanism in developing tissues that leads to the generation of cell diversity. In the embryonic central nervous system of Drosophila melanogaster,secondary precursor cells—ganglion mother cells (GMCs)—divide and produce postmitotic neurons that take on different cell fates. In this study, we show that binary fate decision of two pairs of sibling neurons is accomplished through the interplay of Notch (N) signaling and the intrinsic fate determinant Numb. We show that GMCs have apical–basal polarity and Numb localization and the orientation of division are coordinated to segregate Numb to only one sibling cell. The correct positioning of Numb and the proper orientation of division require Inscuteable (Insc). Loss of insc results in the generation of equivalent sibling cells. Our results provide evidence that sibling neuron fate decision is nonstochastic and normally depends on the presence of Numb in one of the two siblings. Moreover, our data suggest that the fate of some sibling neurons may be regulated by signals that do not require lateral interaction between the sibling cells.
The checkpoint protein MAD2 and the mitotic regulator CDC20 form a ternary complex with the anaphase-promoting complex to control anaphase initiation
Authors: Fang Guowei , Yu Hongtao , Kirschner Marc W.
Published: February 2008

The spindle assembly checkpoint mechanism delays anaphase initiation until all chromosomes are aligned at the metaphase plate. Activation of the anaphase-promoting complex (APC) by binding of CDC20 and CDH1 is required for exit from mitosis, and APC has been implicated as a target for the checkpoint intervention. We show that the human checkpoint protein hMAD2 prevents activation of APC by forming a hMAD2–CDC20–APC complex. When injected into Xenopus embryos, hMAD2 arrests cells at mitosis with an inactive APC. The recombinant hMAD2 protein exists in two-folded states: a tetramer and a monomer. Both the tetramer and the monomer bind to CDC20, but only the tetramer inhibits activation of APC and blocks cell cycle progression. Thus, hMAD2 binding is not sufficient for inhibition, and a change in hMAD2 structure may play a role in transducing the checkpoint signal. There are at least three different forms of mitotic APC that can be detected in vivo: an inactive hMAD2–CDC20–APC ternary complex present at metaphase, a CDC20–APC binary complex active in degrading specific substrates at anaphase, and a CDH1–APC complex active later in mitosis and in G1. We conclude that the checkpoint-mediated cell cycle arrest involves hMAD2 receiving an upstream signal to inhibit activation of APC.
Antigen receptor signaling induces MAP kinase-mediated phosphorylation and degradation of the BCL-6 transcription factor
Authors: Niu Huifeng , Ye Bihui H. , Dalla-Favera Riccardo
Published: February 2008

Thebcl-6proto-oncogene encodes a POZ/zinc finger transcriptional repressor expressed in germinal center (GC) B and T cells and required for GC formation and antibody affinity maturation. Deregulation ofbcl-6expression by chromosomal rearrangements and point mutations of thebcl-6promoter region are implicated in the pathogenesis of B-cell lymphoma. The signals regulatingbcl-6expression are not known. Here we show that antigen receptor activation leads to BCL-6 phosphorylation by mitogen-activated protein kinase (MAPK). Phosphorylation, in turn, targets BCL-6 for rapid degradation by the ubiquitin/proteasome pathway. These findings indicate that BCL-6 expression is directly controlled by the antigen receptor via MAPK activation. This signaling pathway may be crucial for the control of B-cell differentiation and antibody response and has implications for the regulation of other POZ/zinc finger transcription factors in other tissues.
Identification of Xenopus SMC protein complexes required for sister chromatid cohesion
Authors: Losada Ana , Hirano Michiko , Hirano Tatsuya
Published: February 2008

The structural maintenance of chromosomes (SMC) family is a growing family of chromosomal ATPases. The founding class of SMC protein complexes, condensins, plays a central role in mitotic chromosome condensation. We report here a new class of SMC protein complexes containing XSMC1 and XSMC3, Xenopus homologs of yeast Smc1p and Smc3p, respectively. The protein complexes (termed cohesins) exist as two major forms with sedimentation coefficients of 9S and 14S. 9S cohesin is a heterodimer of XSMC1 and XSMC3, whereas 14S cohesin contains three additional subunits. One of them has been identified as a Xenopus homolog of the Schizosaccharomyces pombeRad21p implicated in DNA repair and the Saccharomyces cerevisiae Scc1p/Mcd1p implicated in sister chromatid cohesion. 14S cohesin binds to interphase chromatin independently of DNA replication and dissociates from it at the onset of mitosis. Immunodepletion of cohesins during interphase causes defects in sister chromatid cohesion in subsequent mitosis, whereas condensation is unaffected. These results suggest that proper assembly of mitotic chromosomes is regulated by two distinct classes of SMC protein complexes, cohesins and condensins.
Chromosome methylation patterns during mammalian preimplantation development
Authors: Rougier Nathalie , Bourc’his Déborah , Gomes Denise Molina , Niveleau Alain , Plachot Michelle , Pàldi Andràs , Viegas-Péquignot Evani
Published: February 2008

DNA methylation patterns were evaluated during preimplantation mouse development by analyzing the binding of monoclonal antibody to 5-methylcytosine (5-MeC) on metaphase chromosomes. Specific chromosome patterns were observed in each cell stage. A banding pattern predominated in chromosomes at the one-cell stage. Banding was replaced at the two-cell stage by an asymmetrical labeling of the sister chromatids. Then, the proportion of asymmetrical chromosomes decreased by one-half at each cell division until the blastocyst stage, and chromosomes became progressively symmetrical and weakly labeled. Our results indicate that chromosome demethylation is associated with each DNA replication and suggest that a passive mechanism predominates during early development.
Determinants of specificity in TGF-β signal transduction
Authors: Chen Ye-Guang , Hata Akiko , Lo Roger S. , Wotton David , Shi Yigong , Pavletich Nikola , Massagué Joan
Published: February 2008

Signal transduction by the TGF-β family involves sets of receptor serine/threonine kinases, Smad proteins that act as receptor substrates, and Smad-associated transcription factors that target specific genes. We have identified discrete structural elements that dictate the selective interactions between receptors and Smads and between Smads and transcription factors in the TGF-β and BMP pathways. A cluster of four residues in the L45 loop of the type I receptor kinase domain, and a matching set of two residues in the L3 loop of the Smad carboxy-terminal domain establish the specificity of receptor–Smad interactions. A cluster of residues in the highly exposed α-helix 2 of the Smad carboxy-terminal domain specify the interaction with the DNA-binding factor Fast1 and, as a result, the gene responses mediated by the pathway. By establishing specific interactions, these determinants keep the TGF-β and BMP pathways segregated from each other.
A histone deacetylase corepressor complex regulates the Notch signal transduction pathway
Authors: Kao Hung-Ying , Ordentlich Peter , Koyano-Nakagawa Naoko , Tang Zhenyu , Downes Michael , Kintner Chris R. , Evans Ronald M. , Kadesch Tom
Published: February 2008

The Delta–Notch signal transduction pathway has widespread roles in animal development in which it appears to control cell fate. CBF1/RBP-Jκ, the mammalian homolog ofDrosophila Suppressor of Hairless [Su(H)], switches from a transcriptional repressor to an activator upon Notch activation. The mechanism whereby Notch regulates this switch is not clear. In this report we show that prior to induction CBF1/RBP-Jκ interacts with a corepressor complex containing SMRT (silencing mediator ofretinoid and thyroid hormone receptors) and the histone deacetylase HDAC-1. This complex binds via the CBF1 repression domain, and mutants defective in repression fail to interact with the complex. Activation by Notch disrupts the formation of the repressor complex, thus establishing a molecular basis for the Notch switch. Finally, ESR-1, a Xenopus gene activated by Notch and X-Su(H), is induced in animal caps treated with TSA, an inhibitor of HDAC-1. The functional role for the SMRT/HDAC-1 complex in CBF1/RBP-Jκ regulation reveals a novel genetic switch in which extracellular ligands control the status of critical nuclear cofactor complexes.
The TEL/ETV6 gene is required specifically for hematopoiesis in the bone marrow
Authors: Wang Li Chun , Swat Wojciech , Fujiwara Yuko , Davidson Laurie , Visvader Jane , Kuo Frank , Alt Fred W. , Gilliland D. Gary , Golub Todd R. , Orkin Stuart H.
Published: February 2008

The TEL (translocation–Ets–leukemia orETV6) locus, which encodes an Ets family transcription factor, is frequently rearranged in human leukemias of myeloid or lymphoid origins. By gene targeting in mice, we previously showed thatTEL−/− mice are embryonic lethal because of a yolk sac angiogenic defect. TEL also appears essential for the survival of selected neural and mesenchymal populations within the embryo proper. Here, we have generated mouse chimeras withTEL−/− ES cells to examine a possible requirement in adult hematopoiesis. Although not required for the intrinsic proliferation and/or differentiation of adult-type hematopoietic lineages in the yolk sac and fetal liver, TEL function is essential for the establishment of hematopoiesis of all lineages in the bone marrow. This defect is manifest within the first week of postnatal life. Our data pinpoint a critical role for TEL in the normal transition of hematopoietic activity from fetal liver to bone marrow. This might reflect an inability ofTEL−/− hematopoietic stem cells or progenitors to migrate or home to the bone marrow or, more likely, the failure of these cells to respond appropriately and/or survive within the bone marrow microenvironment. These data establish TEL as the first transcription factor required specifically for hematopoiesis within the bone marrow, as opposed to other sites of hematopoietic activity during development.
DNA-binding polarity of human replication protein A positions nucleases in nucleotide excision repair
Authors: de Laat Wouter L. , Appeldoorn Esther , Sugasawa Kaoru , Weterings Eric , Jaspers Nicolaas G.J. , Hoeijmakers Jan H.J.
Published: February 2008

The human single-stranded DNA-binding replication A protein (RPA) is involved in various DNA-processing events. By comparing the affinity of hRPA for artificial DNA hairpin structures with 3′- or 5′-protruding single-stranded arms, we found that hRPA binds ssDNA with a defined polarity; a strong ssDNA interaction domain of hRPA is positioned at the 5′ side of its binding region, a weak ssDNA-binding domain resides at the 3′ side. Polarity appears crucial for positioning of the excision repair nucleases XPG and ERCC1–XPF on the DNA. With the 3′-oriented side of hRPA facing a duplex ssDNA junction, hRPA interacts with and stimulates ERCC1–XPF, whereas the 5′-oriented side of hRPA at a DNA junction allows stable binding of XPG to hRPA. Our data pinpoint hRPA to the undamaged strand during nucleotide excision repair. Polarity of hRPA on ssDNA is likely to contribute to the directionality of other hRPA-dependent processes as well.
JNK targets p53 ubiquitination and degradation in nonstressed cells
Authors: Fuchs Serge Y. , Adler Victor , Buschmann Thomas , Yin Zhimin , Wu Xiangwei , Jones Stephen N. , Ronai Ze’ev
Published: February 2008

In this study we elucidated the role of nonactive JNK in regulating p53 stability. The amount of p53–JNK complex was inversely correlated with p53 level. A peptide corresponding to the JNK binding site on p53 efficiently blocked ubiquitination of p53. Similarly, p53 lacking the JNK binding site exhibits a longer half-life than p53wt. Outcompeting JNK association with p53 increased the level of p53, whereas overexpression of a phosphorylation mutant form of JNK inhibited p53 accumulation. JNK–p53 and Mdm2–p53 complexes were preferentially found in G0/G1and S/G2M phases of the cell cycle, respectively. Altogether, these data indicate that JNK is an Mdm2-independent regulator of p53 stability in nonstressed cells.
SNAP19 mediates the assembly of a functional core promoter complex (SNAPc) shared by RNA polymerases II and III
Authors: Henry R. William , Mittal Vivek , Ma Beicong , Kobayashi Ryuji , Hernandez Nouria
Published: February 2008

The basal transcription factor SNAPc binds to the PSE, a core element in the RNA polymerase II and III human snRNA promoters. SNAPc contains at least four subunits, but it has not been possible to assemble a fully defined recombinant SNAPc. Here we reconstitute SNAPc from five recombinant subunits, SNAP43, SNAP45, SNAP50, SNAP190, and a newly identified subunit, SNAP19. This recombinant complex binds specifically to the PSE and directs both RNA polymerase II and III snRNA gene transcription. Thus, the same core SNAPc nucleates the assembly of two classes of initiation complexes.
Pax9-deficient mice lack pharyngeal pouch derivatives and teeth and exhibit craniofacial and limb abnormalities
Authors: Peters Heiko , Neubüser Annette , Kratochwil Klaus , Balling Rudi
Published: February 2008

Pax genes have been shown to play important roles in mammalian development and organogenesis. Pax9, a member of this transcription factor family, is expressed in somites, pharyngeal pouches, mesenchyme involved in craniofacial, tooth, and limb development, as well as other sites during mouse embryogenesis. To analyze its function in vivo, we generated Pax9 deficient mice and show that Pax9 is essential for the development of a variety of organs and skeletal elements. Homozygous Pax9-mutant mice die shortly after birth, most likely as a consequence of a cleft secondary palate. They lack a thymus, parathyroid glands, and ultimobranchial bodies, organs which are derived from the pharyngeal pouches. In all limbs, a supernumerary preaxial digit is formed, but the flexor of the hindlimb toes is missing. Furthermore, craniofacial and visceral skeletogenesis is disturbed, and all teeth are absent. InPax9-deficient embryos tooth development is arrested at the bud stage. At this stage, Pax9 is required for the mesenchymal expression of Bmp4, Msx1, and Lef1, suggesting a role for Pax9 in the establishment of the inductive capacity of the tooth mesenchyme. In summary, our analysis shows that Pax9 is a key regulator during the development of a wide range of organ primordia.
DNA damage activates p53 through a phosphorylation–acetylation cascade
Authors: Sakaguchi Kazuyasu , Herrera Julio E. , Saito Shin’ichi , Miki Toru , Bustin Michael , Vassilev Alex , Anderson Carl W. , Appella Ettore
Published: February 2008

Activation of p53-mediated transcription is a critical cellular response to DNA damage. p53 stability and site-specific DNA-binding activity and, therefore, transcriptional activity, are modulated by post-translational modifications including phosphorylation and acetylation. Here we show that p53 is acetylated in vitro at separate sites by two different histone acetyltransferases (HATs), the coactivators p300 and PCAF. p300 acetylates Lys-382 in the carboxy-terminal region of p53, whereas PCAF acetylates Lys-320 in the nuclear localization signal. Acetylations at either site enhance sequence-specific DNA binding. Using a polyclonal antisera specific for p53 that is phosphorylated or acetylated at specific residues, we show that Lys-382 of human p53 becomes acetylated and Ser-33 and Ser-37 become phosphorylated in vivo after exposing cells to UV light or ionizing radiation. In vitro, amino-terminal p53 peptides phosphorylated at Ser-33 and/or at Ser-37 differentially inhibited p53 acetylation by each HAT. These results suggest that DNA damage enhances p53 activity as a transcription factor in part through carboxy-terminal acetylation that, in turn, is directed by amino-terminal phosphorylation.
Loss of transcriptional activity of a transgene is accompanied by DNA methylation and histone deacetylation and is prevented by insulators
Authors: Pikaart Michael J. , Recillas-Targa Félix , Felsenfeld Gary
Published: February 2008

The constitutive DNase I hypersensitive site at the 5′ end of the chicken β-globin locus marks the boundary of the active chromatin domain in erythroid cells. The DNA sequence containing this site has the properties of an insulator, as shown by its ability in stable transformation experiments to block enhancer–promoter interaction when it lies between the two, but not when it lies outside, and to protect against position effects in Drosophila. We now show that the chicken insulator can protect a stably integrated gene, which is otherwise subject to great variability of expression, from chromatin-mediated repression in cell culture. When the integrated reporter gene is surrounded by insulator elements, stably transformed cell lines display consistent enhancer-dependent expression levels, in accord with the strength of the enhancer. In the absence of insulators, long-term nonselective propagation of cells carrying the integrated reporter gene results in gradual extinction of the reporter’s expression, with expression patterns from tandemly repeated inserted genes suggesting that the extinction of adjacent genes is coupled. We show that the uninsulated reporter genes, in addition to becoming transcriptionally inactive, lose several epigenetic hallmarks of active chromatin, including nuclease accessibility, DNA hypomethylation, and histone hyperacetylation during time in culture. Treatment with inhibitors of histone deacetylase or DNA methylation reverses the extinction of the uninsulated genes. Extinction is completely prevented by flanking the reporter construct with insulators. Furthermore, in contrast to the uninsulated reporter genes, chromatin over the insulated genes retains nuclease accessibility and histone hyperacetylation. However, there is no clear correlation between the presence of the insulators and the level of DNA methylation. This leads us to propose a model for the insulator’s ability to protect against extinction in the transformed cell lines and to function as a chromatin boundary for the chicken β-globin locus in normal erythroid cells.
Beadex encodes an LMO protein that regulates Apterous LIM–homeodomain activity in Drosophila wing development: a model for LMO oncogene function
Authors: Milán Marco , Diaz-Benjumea Fernando J. , Cohen Stephen M.
Published: February 2008

Formation of the dorsal–ventral axis of the Drosophila wing depends on activity of the LIM–homeodomain protein Apterous (Ap). Here we report that Ap activity levels are modulated by dLMO, the protein encoded by the Beadex (Bx) gene. Overexpression of dLMO in Bx mutants interferes with Apterous function. Conversely,Bx loss-of-function mutants fail to down-regulate Apterous activity at late stages of wing development. Biochemical analysis shows that dLMO protein competes for binding of Apterous to its cofactor Chip. These data suggest that Apterous activity depends on formation of a functional complex with Chip and that the relative levels of dLMO, Apterous, and Chip determine the level of Apterous activity. The dominant interference mechanism of dLMO action may serve as a model for the mechanism by which LMO oncogenes cause cancer when misexpressed in T cells.
Recovery from DNA replicational stress is the essential function of the S-phase checkpoint pathway
Authors: Desany Brian A. , Alcasabas Annette A. , Bachant Jeffrey B. , Elledge Stephen J.
Published: February 2008

RAD53 and MEC1 are essential genes required for the transcriptional and cell cycle responses to DNA damage and DNA replication blocks. We have examined the essential function of these genes and found that their lethality but not their checkpoint defects can be suppressed by increased expression of genes encoding ribonucleotide reductase. Analysis of viable null alleles revealed that Mec1 plays a greater role in response to inhibition of DNA synthesis than Rad53. The loss of survival in mec1 and rad53 null or point mutants in response to transient inhibition of DNA synthesis is not a result of inappropriate anaphase entry but primarily to an inability to complete chromosome replication. We propose that this checkpoint pathway plays an important role in the maintenance of DNA synthetic capabilities when DNA replication is stressed.
The TIR1 protein of Arabidopsis functions in auxin response and is related to human SKP2 and yeast Grr1p
Authors: Ruegger Max , Dewey Elizabeth , Gray William M. , Hobbie Lawrence , Turner Jocelyn , Estelle Mark
Published: February 2008

Genetic analysis in Arabidopsis has led to the identification of several genes that are required for auxin response. One of these genes, AXR1, encodes a protein related to yeast Aos1p, a protein that functions to activate the ubiquitin-related protein Smt3p. Here we report the identification of a new gene calledTRANSPORT INHIBITOR RESPONSE 1 (TIR1). The tir1mutants are deficient in a variety of auxin-regulated growth processes including hypocotyl elongation and lateral root formation. These results indicate that TIR1 is also required for normal response to auxin. Further, mutations in TIR1 display a synergistic interaction with mutations in AXR1, suggesting that the two genes function in overlapping pathways. The TIR1 protein contains a series of leucine-rich repeats and a recently identified motif called an F box. Sequence comparisons indicate that TIR1 is related to the yeast protein Grr1p and the human protein SKP2. Because Grr1p and other F-box proteins have been implicated in ubiquitin-mediated processes, we speculate that auxin response depends on the modification of a key regulatory protein(s) by ubiquitin or a ubiquitin-related protein.
WT1 interacts with the splicing factor U2AF65 in an isoform-dependent manner and can be incorporated into spliceosomes
Authors: Davies Rachel C. , Calvio Cinzia , Bratt Eva , Larsson Stefan H. , Lamond Angus I. , Hastie Nicholas D.
Published: February 2008

WT1 is essential for normal kidney development, and genetic alterations are associated with Wilms’ tumor, Denys Drash (DDS), and Frasier syndromes. Although generally considered a transcription factor this study has revealed that WT1 interacts with an essential splicing factor, U2AF65, and associates with the splicing machinery. WT1 is alternatively spliced and isoforms that include three amino acids, KTS, show stronger interaction with U2AF65 in vitro and better colocalization with splicing factors in vivo. Interestingly a mutation associated with DDS enhanced both −KTS WT1 binding to U2AF65 and splicing-factor colocalization. These data illustrate the functional importance of WT1 isoforms and suggest that WT1 plays a role in pre-mRNA splicing.
mRNA stabilization by poly(A) binding protein is independent of poly(A) and requires translation
Authors: Coller Jeffery M. , Gray Nicola K. , Wickens Marvin P.
Published: February 2008

Translation and mRNA stability are enhanced by the presence of a poly(A) tail. In vivo, the tail interacts with a conserved polypeptide, poly(A) binding protein (Pab1p). To examine Pab1p function in vivo, we have tethered Pab1p to the 3′ UTR of reporter mRNAs by fusing it to MS2 coat protein and placing MS2 binding sites in the 3′ UTR of the reporter. This strategy allows us to uncouple Pab1p function from its RNA binding activity. We show that mRNAs that lack a poly(A) tail in vivo are stabilized by Pab1p, and that the portions of Pab1p required for stabilization are genetically distinct from those required for poly(A) binding. In addition, stabilization by Pab1p requires ongoing translation of the mRNA. We conclude that the primary, or sole, function of poly(A) with respect to mRNA stability is simply to bring Pab1p to the mRNA, and that mRNA stabilization is an intrinsic property of Pab1p. The approach we describe may be useful in identifying and assaying 3′ UTR regulatory proteins, as it uncouples analysis of function from RNA binding.
The hypoxia-responsive transcription factor EPAS1 is essential for catecholamine homeostasis and protection against heart failure during embryonic development
Authors: Tian Hui , Hammer Robert E. , Matsumoto Alvin M. , Russell David W. , McKnight Steven L.
Published: February 2008

Mice lacking the hypoxia-inducible transcription factor EPAS1 die at mid-gestation. Despite normal morphological development of the circulatory system, EPAS1-deficient mice display pronounced bradycardia. In addition to the vascular endothelium, EPAS1 is expressed intensively in the organ of Zuckerkandl (OZ), the principle source of catecholamine production in mammalian embryos. EPAS1-deficient embryos contained substantially reduced catecholamine levels. Mid-gestational lethality was rescued by administration of the catecholamine precursor DOPS to pregnant females. We hypothesize that EPAS1 expressed in the OZ senses hypoxia during mid-gestational development and translates this signal into an altered pattern of gene expression, leading to increases in circulating catecholamine levels and proper cardiac function.
Structure and specificity of nuclear receptor–coactivator interactions
Authors: Darimont Beatrice D. , Wagner Richard L. , Apriletti James W. , Stallcup Michael R. , Kushner Peter J. , Baxter John D. , Fletterick Robert J. , Yamamoto Keith R.
Published: February 2008

Combinatorial regulation of transcription implies flexible yet precise assembly of multiprotein regulatory complexes in response to signals. Biochemical and crystallographic analyses revealed that hormone binding leads to the formation of a hydrophobic groove within the ligand binding domain (LBD) of the thyroid hormone receptor that interacts with an LxxLL motif-containing α-helix from GRIP1, a coactivator. Residues immediately adjacent to the motif modulate the affinity of the interaction; the motif and the adjacent sequences are employed to different extents in binding to different receptors. Such interactions of amphipathic α-helices with hydrophobic grooves define protein interfaces in other regulatory complexes as well. We suggest that these common structural elements impart flexibility to combinatorial regulation, whereas side chains at the interface impart specificity.
Determinants of coactivator LXXLL motif specificity in nuclear receptor transcriptional activation
Authors: McInerney Eileen M. , Rose David W. , Flynn Sarah E. , Westin Stefan , Mullen Tina-Marie , Krones Anna , Inostroza Juan , Torchia Joseph , Nolte Robert T. , Assa-Munt Nuria , Milburn Michael V. , Glass Christopher K. , Rosenfeld Michael G.
Published: February 2008

Ligand-dependent activation of gene transcription by nuclear receptors is dependent on the recruitment of coactivators, including a family of related NCoA/SRC factors, via a region containing three helical domains sharing an LXXLL core consensus sequence, referred to as LXDs. In this manuscript, we report receptor-specific differential utilization of LXXLL-containing motifs of the NCoA-1/SRC-1 coactivator. Whereas a single LXD is sufficient for activation by the estrogen receptor, different combinations of two, appropriately spaced, LXDs are required for actions of the thyroid hormone, retinoic acid, peroxisome proliferator-activated, or progesterone receptors. The specificity of LXD usage in the cell appears to be dictated, at least in part, by specific amino acids carboxy-terminal to the core LXXLL motif that may make differential contacts with helices 1 and 3 (or 3′) in receptor ligand-binding domains. Intriguingly, distinct carboxy-terminal amino acids are required for PPARγ activation in response to different ligands. Related LXXLL-containing motifs in NCoA-1/SRC-1 are also required for a functional interaction with CBP, potentially interacting with a hydrophobic binding pocket. Together, these data suggest that the LXXLL-containing motifs have evolved to serve overlapping roles that are likely to permit both receptor-specific and ligand-specific assembly of a coactivator complex, and that these recognition motifs underlie the recruitment of coactivator complexes required for nuclear receptor function.
Polar localization of the MinD protein of Bacillus subtilis and its role in selection of the mid-cell division site
Authors: Marston Adele L. , Thomaides Helena B. , Edwards David H. , Sharpe Michaela E. , Errington Jeffery
Published: February 2008

Cell division in rod-shaped bacteria is initiated by formation of a ring of the tubulin-like protein FtsZ at mid-cell. Division site selection is controlled by a conserved division inhibitor MinCD, which prevents aberrant division at the cell poles. The Bacillus subtilis DivIVA protein controls the topological specificity of MinCD action. Here we show that DivIVA is targeted to division sites late in their assembly, after some MinCD-sensitive step requiring FtsZ and other division proteins has been passed. DivIVA then recruits MinD to the division sites preventing another division from taking place near the newly formed cell poles. Sequestration of MinD to the poles also releases the next mid-cell sites for division. Remarkably, this mechanism of DivIVA action is completely different from that of the equivalent protein MinE of Escherichia coli, even though both systems operate via the same division inhibitor MinCD.
Bacterial senescence: stasis results in increased and differential oxidation of cytoplasmic proteins leading to developmental induction of the heat shock regulon
Authors: Dukan Sam , Nyström Thomas
Published: February 2008

Aging, or senescence, is the progressive deterioration of every bodily function over time. A fundamental question that applies to all life forms, including growth-arrested bacteria, is why growing older by necessity causes organisms to grow more fragile. In this work, we demonstrate that the levels of oxidized proteins is correlated to the age of a stationary-phase Escherichia coli culture; both disulfide bridge formation of a cytoplasmic leader-less alkaline phosphatase and protein carbonyl levels increase during stasis. The stasis-induced increase in protein oxidation is enhanced in cells lacking the global regulators OxyR and ςs. Some proteins were found to be specifically susceptible to stasis-induced oxidation; notably several TCA cycle enzymes, glutamine synthetase, glutamate synthase, pyruvate kinase, DnaK, and H-NS. Evidence that oxidation of target proteins during stasis serves as the signal for stationary-phase, developmental, induction of the heat shock regulon is presented by demonstrating that this induction is mitigated by overproducing the superoxide dismutase SodA. In addition, cells lacking cytoplasmic superoxide dismutase activity exhibit superinduction of heat shock proteins. The possibility that oxidative sensitivity of TCA cycle enzymes serves as a feedback mechanism down-regulating toxic respiration is discussed.
Not just scaffolding: plectin regulates actin dynamics in cultured cells
Authors: Andrä Kerstin , Nikolic Branislav , Stöcher Markus , Drenckhahn Detlev , Wiche Gerhard
Published: February 2008

Plectin, a major linker and scaffolding protein of the cytoskeleton, has been shown to be essential for the mechanical integrity of skin, skeletal muscle, and heart. Studying fibroblast and astroglial cell cultures derived from plectin (−/−) mice, we found that their actin cytoskeleton, including focal adhesion contacts, was developed more extensively than in wild-type cells. Also it failed to show characteristic short-term rearrangments in response to extracellular stimuli activating the Rho/Rac/Cdc42 signaling cascades. As a consequence, cell motility, adherence, and shear stress resistance were altered, and morphogenic processes were delayed. Furthermore, we show that plectin interacts with G-actin in vitro in a phosphatidylinositol-4,5-biphosphate-dependent manner and associates with actin stress fibers in living cells. The actin stress fiber phenotype of plectin-deficient fibroblasts could be reversed to a large degree by transient transfection of full-length plectin or plectin fragments containing the amino-terminal actin-binding domain (ABD). These results reveal a novel role of plectin as regulator of cellular processes involving actin filament dynamics that goes beyond its proposed role in scaffolding and mechanical stabilization of cells.
The interaction between HIV-1 Tat and human cyclin T1 requires zinc and a critical cysteine residue that is not conserved in the murine CycT1 protein
Authors: Garber Mitchell E. , Wei Ping , KewalRamani Vineet N. , Mayall Timothy P. , Herrmann Christine H. , Rice Andrew P. , Littman Dan R. , Jones Katherine A.
Published: February 2008

HIV-1 Tat activates transcription through binding to human cyclin T1, a regulatory subunit of the TAK/P-TEFb CTD kinase complex. Here we show that the cyclin domain of hCycT1 is necessary and sufficient to interact with Tat and promote cooperative binding to TAR RNA in vitro, as well as mediate Tat transactivation in vivo. A Tat:TAR recognition motif (TRM) was identified at the carboxy-terminal edge of the cyclin domain, and we show that hCycT1 can interact simultaneously with Tat and CDK9 on TAR RNA in vitro. Alanine-scanning mutagenesis of the hCycT1 TRM identified residues that are critical for the interaction with Tat and others that are required specifically for binding of the complex to TAR RNA. Interestingly, we find that the interaction between Tat and hCycT1 requires zinc as well as essential cysteine residues in both proteins. Cloning and characterization of the murine CycT1 protein revealed that it lacks a critical cysteine residue (C261) and forms a weak, zinc-independent complex with HIV-1 Tat that greatly reduces binding to TAR RNA. A point mutation in mCycT1 (Y261C) restores high-affinity, zinc-dependent binding to Tat and TAR in vitro, and rescues Tat transactivation in vivo. Although overexpression of hCycT1 in NIH3T3 cells strongly enhances transcription from an integrated proviral promoter, we find that this fails to overcome all blocks to productive HIV-1 infection in murine cells.
The Oct-1 POU domain activates snRNA gene transcription by contacting a region in the SNAPc largest subunit that bears sequence similarities to the Oct-1 coactivator OBF-1
Authors: Ford Ethan , Strubin Michel , Hernandez Nouria
Published: February 2008

The RNA polymerases II and III snRNA gene promoters contain an octamer sequence as part of the enhancer and a proximal sequence element (PSE) as part of the core promoter. The octamer and the PSE bind the POU domain activator Oct-1 and the basal transcription factor SNAPc, respectively. Oct-1, but not Oct-1 with a single E7R mutation within the POU domain, binds cooperatively with SNAPc and, in effect, recruits SNAPc to the PSE. Here, we show that SNAPc recruitment is mediated by an interaction between the Oct-1 POU domain and a small region of the largest subunit of SNAPc, SNAP190. This SNAP190 region is strikingly similar to a region in the B-cell-specific Oct-1 coactivator, OBF-1, that is required for interaction with octamer-bound Oct-1 POU domain. The Oct-1 POU domain–SNAP190 interaction is a direct protein–protein contact as determined by the isolation of a switched specificity SNAP190 mutant that interacts with Oct-1 POU E7R but not with wild-type Oct-1 POU. We also show that this direct protein–protein contact results in activation of transcription. Thus, we have identified an activation target of a human activator, Oct-1, within its cognate basal transcription complex.
The essential Gcd10p–Gcd14p nuclear complex is required for 1-methyladenosine modification and maturation of initiator methionyl-tRNA
Authors: Anderson James , Phan Lon , Cuesta Rafael , Carlson Bradley A. , Pak Marie , Asano Katsura , Björk Glenn R. , Tamame Mercedes , Hinnebusch Alan G.
Published: February 2008

Gcd10p and Gcd14p are essential proteins required for the initiation of protein synthesis and translational repression ofGCN4mRNA. The phenotypes ofgcd10mutants were suppressed by high-copy-numberIMTgenes, encoding initiator methionyl tRNA (tRNAiMet), orLHP1, encoding the yeast homolog of the human La autoantigen. Thegcd10-504mutation led to a reduction in steady-state levels of mature tRNAiMet, attributable to increased turnover rather than decreased synthesis of pre-tRNAiMet. Remarkably, the lethality of aGCD10deletion was suppressed by high-copy-numberIMT4, indicating that its role in expression of mature tRNAiMetis the essential function of Gcd10p. Agcd14-2mutant also showed reduced amounts of mature tRNAiMet, but in addition, displayed a defect in pre-tRNAiMetprocessing. Gcd10p and Gcd14p were found to be subunits of a protein complex with prominent nuclear localization, suggesting a direct role in tRNAiMetmaturation. The chromatographic behavior of elongator and initiator tRNAMeton a RPC-5 column indicated that both species are altered structurally ingcd10Δcells, and analysis of base modifications revealed that 1-methyladenosine (m1A) is undetectable ingcd10ΔtRNA. Interestingly,gcd10andgcd14mutations had no effect on processing or accumulation of elongator tRNAMet, which also contains m1A at position 58, suggesting a unique requirement for this base modification in initiator maturation.
Deletion of the H19 differentially methylated domain results in loss of imprinted expression of H19 and Igf2
Authors: Thorvaldsen Joanne L. , Duran Kristen L. , Bartolomei Marisa S.
Published: February 2008

Differentially methylated sequences associated with imprinted genes are proposed to control genomic imprinting. A 2-kb region located 5′ to the imprinted mouse H19 gene is hypermethylated on the inactive paternal allele throughout development. To determine whether this differentially methylated domain (DMD) is required for imprinted expression at the endogenous locus, we have generated mice harboring a 1.6-kb targeted deletion of the DMD and assayed for allelic expression of H19 and the linked, oppositely imprintedIgf2 gene. H19 is activated and Igf2 expression is reduced when the DMD deletion is paternally inherited; conversely, upon maternal transmission of the mutation, H19 expression is reduced and Igf2 is activated. Consistent with the DMD’s hypothesized role of setting up the methylation imprint, the mutation also perturbs allele-specific methylation of the remaining H19sequences. In conclusion, these experiments show that the H19hypermethylated 5′ flanking sequences are required to silence paternally derived H19. Additionally, these experiments demonstrate a novel role for the DMD on the maternal chromosome where it is required for the maximal expression of H19 and the silencing of Igf2. Thus, the H19 differentially methylated sequences are required for both H19 and Igf2imprinting.
PRDI-BF1/Blimp-1 repression is mediated by corepressors of the Groucho family of proteins
Authors: Ren Bing , Chee Kerlen J. , Kim Tae Hoon , Maniatis Tom
Published: February 2008

The PRDI-BF1/Blimp-1 protein is a transcriptional repressor required for normal B-cell differentiation, and it has been implicated in the repression of β-interferon(IFN-β) and c-myc gene expression. Here, we show that PRDI-BF1 represses transcription of theIFN-β promoter and of an artificial promoter through an active repression mechanism. We also identified a minimal repression domain in PRDI-BF1 that is sufficient for transcriptional repression when tethered to DNA as a Gal4 fusion protein. Remarkably, this repression domain interacts specifically with hGrg, TLE1, and TLE2 proteins, all of which are members of the Groucho family of transcriptional corepressors. In addition, the hGrg protein itself can function as a potent repressor when tethered to DNA through the Gal4 DNA-binding domain. We also find that the amino-terminal glutamine-rich domains of hGrg and TLE1 are sufficient to mediate dimerization of the two Groucho family proteins. Proteins containing only this domain can function as a dominant-negative inhibitor of PRDI-BF1 repression, and can significantly increase the IFN-β promoter activity after virus induction. We conclude that PRDI-BF1/Blimp-1 represses transcription by recruiting a complex of Groucho family proteins to DNA, and suggest that such corepressor complexes are required for the postinduction repression of the IFN-β promoter.
Localization of matrix metalloproteinase 9 to the cell surface provides a mechanism for CD44-mediated tumor invasion
Authors: Yu Qin , Stamenkovic Ivan
Published: February 2008

The cell surface hyaluronan receptor CD44 promotes tumor growth and metastasis by mechanisms that remain poorly understood. We show here that CD44 associates with a proteolytic form of the matrix metalloproteinase-9 (MMP-9) on the surface of mouse mammary carcinoma and human melanoma cells. CD44-associated cell surface MMP-9 promotes cell-mediated collagen IV degradation in vitro and mediates tumor cell invasion of G8 myoblast monolayers. Several distinct CD44 isoforms coprecipitate with MMP-9 and CD44/MMP-9 coclustering is observed to be dependent on the ability of CD44 to form hyaluronan-induced aggregates. Disruption of CD44/MMP-9 cluster formation, by overexpression of soluble or truncated cell surface CD44, is shown to inhibit tumor invasiveness in vivo. Our observations indicate that CD44 serves to anchor MMP-9 on the cell surface and define a mechanism for CD44-mediated tumor invasion.
Functional role of p35srj, a novel p300/CBP binding protein, during transactivation by HIF-1
Authors: Bhattacharya Shoumo , Michels Catherine L. , Leung Man-Kit , Arany Zoltàn P. , Kung Andrew L. , Livingston David M.
Published: February 2008

Recruitment of p300/CBP by the hypoxia-inducible factor, HIF-1, is essential for the transcriptional response to hypoxia and requires an interaction between the p300/CBP CH1 region and HIF-1α. A new p300-CH1 interacting protein, p35srj, has been identified and cloned. p35srj is an alternatively spliced isoform of MRG1, a human protein of unknown function. Virtually all endogenous p35srj is bound to p300/CBP in vivo, and it inhibits HIF-1 transactivation by blocking the HIF-1α/p300 CH1 interaction. p35srj did not affect transactivation by transcription factors that bind p300/CBP outside the CH1 region. Endogenous p35srj is up-regulated markedly by the HIF-1 activators hypoxia or deferoxamine, suggesting that it could operate in a negative-feedback loop. In keeping with this notion, a p300 CH1 mutant domain, defective in HIF-1 but not p35srj binding, enhanced endogenous HIF-1 function. In hypoxic cells, p35srj may regulate HIF-1 transactivation by controlling access of HIF-1α to p300/CBP, and may keep a significant portion of p300/CBP available for interaction with other transcription factors by partially sequestering and functionally compartmentalizing cellular p300/CBP.
The Drosophila Activin receptor Baboon signals through dSmad2 and controls cell proliferation but not patterning during larval development
Authors: Brummel Ted , Abdollah Shirin , Haerry Theodor E. , Shimell Mary Jane , Merriam John , Raftery Laurel , Wrana Jeffrey L. , O’Connor Michael B.
Published: February 2008

The TGF-β superfamily of growth and differentiation factors, including TGF-β, Activins and bone morphogenetic proteins (BMPs) play critical roles in regulating the development of many organisms. These factors signal through a heteromeric complex of type I and II serine/threonine kinase receptors that phosphorylate members of the Smad family of transcription factors, thereby promoting their nuclear localization. Although components of TGF-β/Activin signaling pathways are well defined in vertebrates, no such pathway has been clearly defined in invertebrates. In this study we describe the role of Baboon (Babo), a type I Activin receptor previously called Atr-I, in Drosophila development and characterize aspects of the Babo intracellular signal-transduction pathway. Genetic analysis of babo loss-of-function mutants and ectopic activation studies indicate that Babo signaling plays a role in regulating cell proliferation. In mammalian cells, activated Babo specifically stimulates Smad2-dependent pathways to induce TGF-β/Activin-responsive promoters but not BMP-responsive elements. Furthermore, we identify a newDrosophila Smad, termed dSmad2, that is most closely related to vertebrate Smads 2 and 3. Activated Babo associates with dSmad2 but not Mad, phosphorylates the carboxy-terminal SSXS motif and induces heteromeric complex formation with Medea, the Drosophila Smad4 homolog. Our results define a novel DrosophilaActivin/TGF-β pathway that is analogous to its vertebrate counterpart and show that this pathway functions to promote cellular growth with minimal effects on patterning.