bluuebunny/crossref_metadata_2025 huggingface dataset:
Tcn1p/Crz1p, a calcineurin-dependent transcription factor that differentially regulates gene expression inSaccharomyces cerevisiae
Authors: Matheos Dina P. , Kingsbury Tami J. , Ahsan U. Salma , Cunningham Kyle W.
Published: February 2008

Ca2+signals regulate gene expression in animal and yeast cells through mechanisms involving calcineurin, a protein phosphatase activated by binding Ca2+and calmodulin. Tcn1p, also named Crz1p, was identified as a transcription factor in yeast required for the calcineurin-dependent induction ofPMC1, PMR1, PMR2A,andFKS2which confer tolerance to high Ca2+, Mn2+, Na+, and cell wall damage, respectively. Tcn1p was not required for other calcineurin-dependent processes, such as inhibition of a vacuolar H+/Ca2+exchanger and inhibition of a pheromone-stimulated Ca2+uptake system, suggesting that Tcn1p functions downstream of calcineurin on a branch of the calcium signaling pathway leading to gene expression. Tcn1p contains three zinc finger motifs at its carboxyl terminus resembling the DNA-binding domains of Zif268, Swi5p, and other transcription factors. When fused to the transcription activation domain of Gal4p, the carboxy terminal domain of Tcn1p directed strong calcineurin-independent expression ofPMC1–lacZand other target genes. The amino-terminal domain of Tcn1p was found to function as a calcineurin-dependent transcription activation domain when fused to the DNA-binding domain of Gal4p. This amino-terminal domain also formed Ca2+-dependent and FK506-sensitive interactions with calcineurin in the yeast two-hybrid assay. These findings suggest that Tcn1p functions as a calcineurin-dependent transcription factor. Interestingly, induction of Tcn1p-dependent genes was found to be differentially controlled in response to physiological Ca2+signals generated by treatment with mating pheromone and high salt. We propose that different promoters are sensitive to variations in the strength of Ca2+signals generated by these stimuli and to effects of other signaling pathways.
Cloning and characterization of developmental endothelial locus-1: An embryonic endothelial cell protein that binds the αvβ3 integrin receptor
Authors: Hidai Chiaki , Zupancic Thomas , Penta Kalyani , Mikhail Adel , Kawana Masatoshi , Quertermous Elena E. , Aoka Yoshikazu , Fukagawa Masafumi , Matsui Yasuhisa , Platika Doros , Auerbach Robert , Hogan Brigid L.M. , Snodgrass Ralph , Quertermous Thomas
Published: February 2008

We have taken advantage of an enhancer trap event in a line of transgenic mice to identify a unique developmentally regulated endothelial cell locus (Del1). The protein encoded in this locus contains three EGF-like repeats homologous to those in Notch and related proteins, including an EGF-like repeat that contains an RGD motif, and two discoidin I-like domains. Del1 is shown to be a matrix protein and to promote adhesion of endothelial cells through interaction with the αvβ3 integrin receptor. Embryonic endothelial-like yolk sac cells expressing recombinant Del1 protein, or grown on an extracellular matrix containing Del1 protein, are inhibited from forming vascular-like structures. Expression of Del1 protein in the chick chorioallantoic membrane leads to loss of vascular integrity and promotes vessel remodeling. Del1 is thus a new ligand for the αvβ3 integrin receptor and may function to regulate vascular morphogenesis or remodeling in embryonic development.
New core promoter element in RNA polymerase II-dependent transcription: sequence-specific DNA binding by transcription factor IIB
Authors: Lagrange Thierry , Kapanidis Achillefs N. , Tang Hong , Reinberg Danny , Ebright Richard H.
Published: February 2008

A sequence element located immediately upstream of the TATA element, and having the consensus sequence 5′-G/C-G/C-G/A-C-G-C-C-3′, affects the ability of transcription factor IIB to enter transcription complexes and support transcription initiation. The sequence element is recognized directly by the transcription factor IIB. Recognition involves α-helices 4′ and 5′ of IIB, which comprise a helix–turn–helix DNA-binding motif. These observations establish that transcription initiation involves a fourth core promoter element, the IIB recognition element (BRE), in addition to the TATA element, the initiator element, and the downstream promoter element, and involves a second sequence-specific general transcription factor, IIB, in addition to transcription factor IID.
Nucleosome positioning by the winged helix transcription factor HNF3
Authors: Shim Eun Yong , Woodcock Christopher , Zaret Kenneth S.
Published: February 2008

Nucleosome positioning at genetic regulatory sequences is not well understood. The transcriptional enhancer of the mouse serum albumin gene is active in liver, where regulatory factors occupy their target sites on three nucleosome-like particles designated N1, N2, and N3. The winged helix transcription factor HNF3 binds to two sites near the center of the N1 particle. We created dinucleosome templates using the albumin enhancer sequence and found that site-specific binding of HNF3 protein resulted in nucleosome positioning in vitro similar to that seen in liver nuclei. Thus, binding of a transcription factor can position an underlying nucleosome core.
A specific subset of SR proteins shuttles continuously between the nucleus and the cytoplasm
Authors: Cáceres Javier F. , Screaton Gavin R. , Krainer Adrian R.
Published: February 2008

The SR proteins constitute a large family of nuclear phosphoproteins required for constitutive pre-mRNA splicing. These factors also have global, concentration-dependent effects on alternative splicing regulation and this activity is antagonized by members of the hnRNP A/B family of proteins. We show here that whereas some human SR proteins are confined to the nucleus, three of them—SF2/ASF, SRp20, and 9G8—shuttle rapidly and continuously between the nucleus and the cytoplasm. By swapping the corresponding domains between shuttling and nonshuttling SR proteins, we show that the carboxy-terminal arginine/serine-rich (RS) domain is required for shuttling. This domain, however, is not sufficient to promote shuttling of an unrelated protein reporter, suggesting that stable RNA binding mediated by the RNA-recognition motifs may be required for shuttling. Consistent with such a requirement, a double point-mutation in RRM1 of SF2/ASF that impairs RNA binding prevents the protein from shuttling. In addition, we show that phosphorylation of the RS domain affects the shuttling properties of SR proteins. These findings show that different SR proteins have unique intracellular transport properties and suggest that the family members that shuttle may have roles not only in nuclear pre-mRNA splicing but also in mRNA transport, cytoplasmic events, and/or processes that involve communication between the nucleus and the cytoplasm.
Stable binding to E2F is not required for the retinoblastoma protein to activate transcription, promote differentiation, and suppress tumor cell growth
Authors: Sellers William R. , Novitch Bennett G. , Miyake Satoshi , Heith Agnieszka , Otterson Gregory A. , Kaye Frederic J. , Lassar Andrew B. , Kaelin William G.
Published: February 2008

The retinoblastoma tumor suppressor protein (pRB) can inhibit cell cycle progression and promote differentiation. pRB interacts with a variety of transcription factors, including members of the E2F and C-EBP protein families and MyoD, and can either repress or activate transcription depending on the promoter under study. These biological and biochemical activities of pRB have been mapped previously to a core domain, referred to as the pRB pocket. Using a panel of synthetic pRB pocket mutants, we found that the acute induction of a G1/S block by pRB is linked to its ability to both bind to E2F and to repress transcription. In contrast, these functions were not required for pRB to promote differentiation, which correlated with its ability to activate transcription in concert with fate-determining proteins such as MyoD. All tumor-derived pRB mutants tested to date failed to bind to E2F and did not repress transcription. Despite an inability to bind to E2F, pRB mutants associated with a low risk of retinoblastoma, unlike high-risk mutants, retained the ability to activate transcription and promote differentiation. Thus, the pRB pocket participates in dual tumor suppressor functions, one linked to cell cycle progression and the other to differentiation control, and these functions can be genetically and mechanistically dissociated.
Oncogenic Abl and Src tyrosine kinases elicit the ubiquitin-dependent degradation of target proteins through a Ras-independent pathway
Authors: Dai Zonghan , Quackenbush Robert C. , Courtney Kevin D. , Grove Matthew , Cortez David , Reuther Gary W. , Pendergast Ann Marie
Published: February 2008

Oncogenic forms of the Abl and Src tyrosine kinases trigger the destruction of the Abi proteins, a family of Abl-interacting proteins that antagonize the oncogenic potential of Abl after overexpression in fibroblasts. The destruction of the Abi proteins requires tyrosine kinase activity and is dependent on the ubiquitin–proteasome pathway. We show that degradation of the Abi proteins occurs through a Ras-independent pathway. Significantly, expression of the Abi proteins is lost in cell lines and bone marrow cells isolated from patients with aggressive Bcr–Abl-positive leukemias. These findings suggest that loss of Abi proteins may be a component in the progression of Bcr–Abl-positive leukemias and identify a novel pathway linking activated nonreceptor protein tyrosine kinases to the destruction of specific target proteins through the ubiquitin–proteasome pathway.
pavarotti encodes a kinesin-like protein required to organize the central spindle and contractile ring for cytokinesis
Authors: Adams Richard R. , Tavares Alvaro A.M. , Salzberg Adi , Bellen Hugo J. , Glover David M.
Published: February 2008

Mutations in the Drosophila gene pavarotti result in the formation of abnormally large cells in the embryonic nervous system. In mitotic cycle 16, cells of pav mutant embryos undergo normal anaphase but then develop an abnormal telophase spindle and fail to undertake cytokinesis. We show that the septin Peanut, actin, and the actin-associated protein Anillin, do not become correctly localized in pav mutants. pav encodes a kinesin-like protein, PAV–KLP, related to the mammalian MKLP-1. In cellularized embryos, the protein is localized to centrosomes early in mitosis, and to the midbody region of the spindle in late anaphase and telophase. We show that Polo kinase associates with PAV–KLP with which it shows an overlapping pattern of subcellular localization during the mitotic cycle and this distribution is disrupted in pavmutants. We suggest that PAV–KLP is required both to establish the structure of the telophase spindle to provide a framework for the assembly of the contractile ring, and to mobilize mitotic regulator proteins.
Modulation of telomerase activity by telomere DNA-binding proteins in Oxytricha
Authors: Froelich-Ammon Stacie J. , Dickinson Brent A. , Bevilacqua Joanne M. , Schultz Steve C. , Cech Thomas R.
Published: February 2008

Telomere proteins protect the chromosomal terminus from nucleolytic degradation and end-to-end fusion, and they may contribute to telomere length control and the regulation of telomerase. The current studies investigate the effect of Oxytrichasingle-stranded telomere DNA-binding protein subunits α and β on telomerase elongation of telomeric DNA. A native agarose gel system was used to evaluate telomere DNA-binding protein complex composition, and the ability of telomerase to use these complexes as substrates was characterized. Efficient elongation occurred in the presence of the α subunit. Moreover, the α–DNA cross-linked complex was a substrate for telomerase. At higher α concentrations, two α subunits bound to the 16-nucleotide single-stranded DNA substrate and rendered it inaccessible to telomerase. The formation of this α · DNA · α complex may contribute to regulation of telomere length. The α · β · DNA ternary complex was not a substrate for telomerase. Even when telomerase was prebound to telomeric DNA, the addition of α and β inhibited elongation, suggesting that these telomere protein subunits have a greater affinity for the DNA and are able to displace telomerase. In addition, the ternary complex was not a substrate for terminal deoxynucleotidyltransferase. We conclude that the telomere protein inhibits telomerase by rendering the telomeric DNA inaccessible, thereby helping to maintain telomere length.
The mammalian transcriptional repressor RBP (CBF1) targets TFIID and TFIIA to prevent activated transcription
Authors: Olave Ivan , Reinberg Danny , Vales Lynne D.
Published: February 2008

RBP is a cellular protein that functions as a transcriptional repressor in mammalian cells. RBP has elicited great interest lately because of its established roles in regulating gene expression, inDrosophila and mouse development, and as a component of the Notch signal transduction pathway. This report focuses on the mechanism by which RBP represses transcription and thereby regulates expression of a relatively simple, but natural, promoter. The results show that, irrespective of the close proximity between RBP and other transcription factors bound to the promoter, RBP does not occlude binding by these other transcription factors. Instead, RBP interacts with two transcriptional coactivators: dTAFII110, a subunit of TFIID, and TFIIA to repress transcription. The domain of dTAFII110 targeted by RBP is the same domain that interacts with TFIIA, but is disparate from the domain that interacts with Sp1. Repression can be thwarted when stable transcription preinitiation complexes are formed before RBP addition, suggesting that RBP interaction with TFIIA and TFIID perturbs optimal interactions between these coactivators. Consistent with this, interaction between RBP and TFIIA precludes interaction with dTAFII110. This is the first report of a repressor specifically targeting these two coactivators to subvert activated transcription.
Multistep signaling requirements for pituitary organogenesis in vivo
Authors: Treier Mathias , Gleiberman Anatoli S. , O’Connell Shawn M. , Szeto Daniel P. , McMahon Jill A. , McMahon Andrew P. , Rosenfeld Michael G.
Published: February 2008

During development of the mammalian pituitary gland specific hormone-producing cell types, critical in maintaining homeostasis, emerge in a spatially and temporally specific fashion from an ectodermal primordium. We have investigated the molecular basis of generating diverse pituitary cell phenotypes from a common precursor, providing in vivo and in vitro evidence that their development involves three sequential phases of signaling events and the action of a gradient at an ectodermal boundary. In the first phase, the BMP4 signal from the ventral diencephalon, expressing BMP4, Wnt5a, andFGF8, represents a critical dorsal neuroepithelial signal for pituitary organ commitment in vivo. Subsequently, a BMP2 signal emanates from a ventral pituitary organizing center that forms at the boundary of a region of oral ectoderm in which Shh expression is selectively excluded. This BMP2 signal together with a dorsal FGF8 signal, appears to create opposing activity gradients that are suggested to generate overlapping patterns of specific transcription factors underlying cell lineage specification events, whereas Wnt4 is needed for the expansion of ventral pituitary cell phenotypes. In the third phase, temporally specific loss of the BMP2 signal is required to allow terminal differentiation. The consequence of these sequential organ and cellular determination events is that each of the hormone-producing pituitary cell types—gonadotropes, thyrotropes, somatotropes, lactotropes, corticotropes, and melanotropes—appear to be determined, in a ventral-to-dorsal gradient, respectively.
Mcm1 regulates donor preference controlled by the recombination enhancer in Saccharomyces mating-type switching
Authors: Wu Cherry , Weiss Kerstin , Yang Chen , Harris Midori A. , Tye Bik-Kwoon , Newlon Carol S. , Simpson Robert T. , Haber James E.
Published: February 2008

Switching of Saccharomyces mating type by replacement of sequences at the MAT locus involves a choice between two donors, HML and HMR. MATα cells inhibit recombination along the entire left arm of chromosome III, including HML,whereas MATa cells activate this same region.MATa -dependent activation of HML depends on a small, cis-acting DNA sequence designated the recombination enhancer (RE), located 17 kb centromere-proximal to HML. A comparison of RE sequences interchangeable between Saccharomyces cerevisiae and Saccharomyces carlsbergensis defines a minimum RE of 244 bp. RE activity is repressed in MATα cells by binding of the Matα2–Mcm1 corepressor to a site within the RE. Mutation of the two Matα2 binding sites removes most, but not all, of this repression, and RE chromatin structure in MATα cells becomes indistinguishable from that seen in MATa. Surprisingly, a 2-bp mutation in the Mcm1 binding site completely abolishes RE activity in MATa cells; moreover, RE chromatin structure in the MATa mutant becomes very similar to that seen in MATα cells with a normal RE, displaying highly ordered nucleosomes despite the absence of Matα2. Further, a mutation that alters the ability of Mcm1 to act with Matα2 in repressing a -specific genes also alters donor preference in either mating type. Thus, Mcm1 is critically responsible for the activation as well as the Matα2-Mcm1-mediated repression of RE activity.
A novel Myb homolog initiates Dictyostelium development by induction of adenylyl cyclase expression
Authors: Otsuka Hideshi , Van Haastert Peter J.M.
Published: February 2008

Dictyostelium development is induced by starvation. The adenylyl cyclase gene ACA is one of the first genes expressed upon starvation. ACA produces extracellular cAMP that induces chemotaxis, aggregation, and differentiation in neighboring cells. Using insertional mutagenesis we have isolated a mutant that does not aggregate upon starvation but is rescued by adding extracellular cAMP. Sequencing of the mutated locus revealed a new gene, DdMYB2,whose product contains three Myb repeats, the DNA-binding motif of Myb-related transcription factors. Ddmyb2–null cells show undetectable levels of ACA transcript and no cAMP production. Ectopic expression of ACA from a constitutive promotor rescues differentiation and morphogenesis of Ddmyb2–null mutants. The results suggest that development in Dictyostelium starts by starvation-mediated DdMyb2 activation, which induces adenylyl cyclase activity producing the differentiation-inducing signal cAMP.
β-Cell-specific inactivation of the mouseIpf1/Pdx1 gene results in loss of the β-cell phenotype and maturity onset diabetes
Authors: Ahlgren Ulf , Jonsson Jörgen , Jonsson Lena , Simu Karin , Edlund Helena
Published: February 2008

To study the late β-cell-specific function of the homeodomain protein IPF1/PDX1 we have generated mice in which theIpf1/Pdx1 gene has been disrupted specifically in β cells. These mice develop diabetes with age, and we show that IPF1/PDX1 is required for maintaining the β cell identity by positively regulating insulin and islet amyloid polypeptide expression and by repressing glucagon expression. We also provide evidence that IPF1/PDX1 regulates the expression of Glut2 in a dosage-dependent manner suggesting that lowered IPF1/PDX1 activity may contribute to the development of type II diabetes by causing impaired expression of both Glut2 and insulin.
Proteasomal regulation of nuclear receptor corepressor-mediated repression
Authors: Zhang Jinsong , Guenther Matthew G. , Carthew Richard W. , Lazar Mitchell A.
Published: February 2008

Repression of gene transcription is a fundamental property of nuclear hormone receptors. We report here that cell-specific repression by nuclear receptors correlates with levels of nuclear receptor corepressor (N-CoR) protein. N-CoR protein levels are regulated by mSiah2, a mammalian homolog of Drosophila Seven in absentiathat targets N-CoR for proteasomal degradation. mSiah2 expression is cell-type specific and differentially regulates the repressive activities of nuclear receptors. These findings establish targeted proteolysis of transcriptional coregulators as a mechanism for cell-specific regulation of gene transcription.
A novel protein complex that interacts with the vitamin D3 receptor in a ligand-dependent manner and enhances VDR transactivation in a cell-free system
Authors: Rachez Christophe , Suldan Zalman , Ward Jeremy , Chang Chao-Pei Betty , Burakov Darya , Erdjument-Bromage Heidye , Tempst Paul , Freedman Leonard P.
Published: February 2008

Nuclear receptors transduce hormonal signals by binding directly to DNA target sites in promoters and modulating the transcription of linked genes. Receptor-mediated transactivation appears to be potentiated in response to ligand by a number of coactivators that may provide key interactions with components of the transcription preinitiation complex and/or alter chromatin structure. Here, we use the vitamin D3 receptor ligand-binding domain (VDR LBD) as an affinity matrix to identify components of a transcriptionally active nuclear extract that interact with VDR in response to ligand. We describe the purification of a complex of at least 10 VDR interacting proteins (DRIPs) ranging from 65 to 250 kD that associate with the receptor in a strictly 1,25-dihydroxyvitamin D3-dependent manner. These proteins also appear to interact with other, but not all, nuclear receptors, such as the thyroid hormone receptor. The DRIPs are distinct from known nuclear receptor coactivators, although like these coactivators, their interaction also requires the AF-2 transactivation motif of VDR. In addition, the DRIP complex contains histone acetyltransferase activity, indicating that at least one or more of the DRIPs may function at the level of nucleosomal modification. However, we show that the DRIPs selectively enhance the transcriptional activity of VDR on a naked DNA template utilizing a cell-free, ligand-dependent transcription assay. Moreover, this activity can be specifically depleted from the extract by liganded, but not unliganded, VDR-LBD. Overexpression of DRIP100 in vivo resulted in a strong squelching of VDR transactivation, suggesting the sequestration of other limiting factors, including components of the DRIP complex. These results demonstrate the existence of a new complex of novel functional nuclear receptor coactivators.
The ErbB2 and ErbB3 receptors and their ligand, neuregulin-1, are essential for development of the sympathetic nervous system
Authors: Britsch Stefan , Li Li , Kirchhoff Susanne , Theuring Franz , Brinkmann Volker , Birchmeier Carmen , Riethmacher Dieter
Published: February 2008

Neuregulins (NDF, heregulin, GGF ARIA, or SMDF) are EGF-like growth and differentiation factors that signal through tyrosine kinase receptors of the ErbB family. Here, we report a novel phenotype in mice with targeted mutations in the erbB2, erbB3, orneuregulin-1 genes. These three mutations cause a severe hypoplasia of the primary sympathetic ganglion chain. We provide evidence that migration of neural crest cells to the mesenchyme lateral of the dorsal aorta, in which they differentiate into sympathetic neurons, depends on neuregulin-1 and its receptors. Neuregulin-1 is expressed at the origin of neural crest cells. Moreover, a tight link between neuregulin-1 expression, the migratory path, and the target site of sympathogenic neural crest cells is observed. Sympathetic ganglia synthesize catecholamines in the embryo and the adult. Accordingly, catecholamine levels in mutant embryos are severely decreased, and we suggest that the lack of catecholamines contributes to the embryonal lethality of the erbB3 mutant mice. Thus,neuregulin-1, erbB2, and erbB3 are required for the formation of the sympathetic nervous system; the block in development observed in mutant mice is caused by a lack of neural crest precursor cells in the anlage of the primary sympathetic ganglion chain. Together with previous observations, these findings establish the neuregulin signaling system as a key regulator in the development of neural crest cells.
The C/EBPβ transcription factor regulates epithelial cell proliferation and differentiation in the mammary gland
Authors: Robinson Gertraud W. , Johnson Peter F. , Hennighausen Lothar , Sterneck Esta
Published: February 2008

Studies of C/EBPβ-deficient mice have demonstrated a pivotal role for this transcription factor in hematopoiesis, adipogenesis, and ovarian function. Here we show that C/EBPβ is also essential for normal development and function of the mammary gland. Ductal morphogenesis in virgin C/EBPβ-deficient mice was disrupted, with ducts displaying reduced growth and branching. To distinguish whether the effect of C/EBPβ deficiency on mammary epithelium is indirect or cell autonomous, we performed ovarian and mammary gland transplants. Transplants of wild-type ovaries into mutant females partially restored ductal morphogenesis during puberty but failed to support mammopoiesis during pregnancy. At term, mutant mice harboring wild-type ovaries exhibited reduced alveolar proliferation and impaired epithelial cell differentiation, including a complete absence of milk protein expression. Mammary gland transplant experiments demonstrated that development of C/EBPβ-deficient epithelium was defective within a wild-type stroma and host background. Cell proliferation during pregnancy was reduced and differentiation, as measured by the activity of milk protein genes, was inhibited. However, wild-type epithelium developed in a C/EBPβ-deficient stroma. Thus, C/EBPβ plays an essential, cell autonomous role in the proliferation and differentiation of mammary secretory epithelial cells and is required for the activation of milk protein genes.
Inhibition of the anti-apoptotic PI(3)K/Akt/Bad pathway by stress
Authors: Zundel Wayne , Giaccia Amato
Published: February 2008

The initiation of apoptosis often transpires in the presence of agents that regulate cell survival. This study evaluated the effects of stress-induced ceramide on the anti-apoptotic activity of the phosphoinositide-3 kinase [PI(3)K] pathway. PI(3)K activity is directly down-regulated by stress-induced ceramide in a dose-dependent manner with rapid kinetics and high specificity. Ceramide inhibition of PI(3)K is dependent on acid–sphingomyelinase. Down-regulation of PI(3)K by ceramide results in inhibition of the kinase Akt and decreased phosphorylation of the death effector Bad. Thus, ceramide levels could act as a general apoptotic rheostat controlling cell survival by regulating PI(3)K anti-apoptotic effector mechanisms.
Negative regulation of the heat shock transcriptional response by HSBP1
Authors: Satyal Sanjeev H. , Chen Dayue , Fox Susan G. , Kramer James M. , Morimoto Richard I.
Published: February 2008

In response to stress, heat shock factor 1 (HSF1) acquires rapid DNA binding and transient transcriptional activity while undergoing conformational transition from an inert non-DNA-binding monomer to active functional trimers. Attenuation of the inducible transcriptional response occurs during heat shock or upon recovery at non-stress conditions and involves dissociation of the HSF1 trimer and loss of activity. We have used the hydrophobic repeats of the HSF1 trimerization domain in the yeast two-hybrid protein interaction assay to identify heat shock factor binding protein 1 (HSBP1), a novel, conserved, 76-amino-acid protein that contains two extended arrays of hydrophobic repeats that interact with the HSF1 heptad repeats. HSBP1 is nuclear-localized and interacts in vivo with the active trimeric state of HSF1 that appears during heat shock. During attenuation of HSF1 to the inert monomer, HSBP1 associates with Hsp70. HSBP1 negatively affects HSF1 DNA-binding activity, and overexpression of HSBP1 in mammalian cells represses the transactivation activity of HSF1. To establish a biological role for HSBP1, the homologousCaenorhabditis elegans protein was overexpressed in body wall muscle cells and was shown to block activation of the heat shock response from a heat shock promoter–reporter construct. Alteration in the level of HSBP1 expression in C. elegans has severe effects on survival of the animals after thermal and chemical stress, consistent with a role for HSBP1 as a negative regulator of the heat shock response.
Suppression of the p300-dependent mdm2negative-feedback loop induces the p53 apoptotic function
Authors: Thomas Anju , White Eileen
Published: February 2008

The p53 tumor suppressor gene product interacts with the p300 transcriptional coactivator that regulates the transactivation of p53-inducible genes. The adenovirus E1A protein has been shown to bind to p300 and inhibit its function. E1A inhibits p53 transactivation and also promotes p53 accumulation by a p300-dependent mechanism. Murine double minute 2 (Mdm2) is a transcriptional target of p53 that binds to p53 and inhibits its transcriptional activity. E1A inhibitedmdm2 transactivation without affecting the expression ofp21WAF1 or Bax, which resulted in high levels of p53 accumulation and apoptosis. Ectopic expression of p300 restored Mdm2 levels and inhibited p53-dependent apoptosis, as did ectopic expression of Mdm2. Thus, p300 is required for mdm2 induction by p53 and the subsequent inhibition of p53 stabilization. Inhibition of p300 by E1A results in stabilization of p53 and causes apoptosis. Moreover, E1B 19K or Bcl-2 expression in E1A-transformed cells abrogated p53-dependent apoptosis by restoring mdm2transactivation by p53. Hence, p300 regulation of mdm2expression controls apoptotic activity of p53, and 19K or Bcl-2 bypass E1A inhibition of p300 transactivation of Mdm2.
TheROOT HAIRLESS 1gene encodes a nuclear protein required for root hair initiation in Arabidopsis
Authors: Schneider Katharina , Mathur Jaideep , Boudonck Kurt , Wells Brian , Dolan Liam , Roberts Keith
Published: February 2008

The epidermis ofArabidopsiswild-type primary roots, in which some cells grow hairs and others remain hairless in a position-dependent manner, has become an established model system to study cell differentiation. Here we present a molecular analysis of theRHL1(ROOTHAIRLESS1) gene that, if mutated, prevents the formation of hairs on primary roots and causes a seedling lethal phenotype. We have cloned theRHL1gene by use of a T-DNA-tagged mutant and found that it encodes a protein that appears to be plant specific. The predictedRHL1gene product is a small hydrophilic protein (38.9 kD) containing putative nuclear localization signals and shows no significant homology to any known amino acid sequence. We demonstrate that a 78-amino-acid sequence at its amino terminus is capable of directing an RHL1–GFP fusion protein to the nucleus. TheRHL1transcript is present throughout the wild-type plant and in suspension culture cells, but in very low amounts, suggesting a regulatory function for theRHL1protein. Structural evidence suggests a role for theRHL1gene product in the nucleolus. We have examined the genetic relationship betweenRHL1andGL2,an inhibitor of root hair initiation in non-hair cells. Our molecular and genetic data with double mutants, together with the expression analysis of aGL2promoter–GUS reporter gene construct, indicate that theRHL1gene acts independently ofGL2.
Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3
Authors: Niwa Hitoshi , Burdon Tom , Chambers Ian , Smith Austin
Published: February 2008

The propagation of embryonic stem (ES) cells in an undifferentiated pluripotent state is dependent on leukemia inhibitory factor (LIF) or related cytokines. These factors act through receptor complexes containing the signal transducer gp130. The downstream mechanisms that lead to ES cell self-renewal have not been delineated, however. In this study, chimeric receptors were introduced into ES cells. Biochemical and functional studies of transfected cells demonstrated a requirement for engagement and activation of the latent trancription factor STAT3. Detailed mutational analyses unexpectedly revealed that the four STAT3 docking sites in gp130 are not functionally equivalent. The role of STAT3 was then investigated using the dominant interfering mutant, STAT3F. ES cells that expressed this molecule constitutively could not be isolated. An episomal supertransfection strategy was therefore used to enable the consequences of STAT3F expression to be examined. In addition, an inducible STAT3F transgene was generated. In both cases, expression of STAT3F in ES cells growing in the presence of LIF specifically abrogated self-renewal and promoted differentiation. These complementary approaches establish that STAT3 plays a central role in the maintenance of the pluripotential stem cell phenotype. This contrasts with the involvement of STAT3 in the induction of differentiation in somatic cell types. Cell type-specific interpretation of STAT3 activation thus appears to be pivotal to the diverse developmental effects of the LIF family of cytokines. Identification of STAT3 as a key transcriptional determinant of ES cell self-renewal represents a first step in the molecular characterization of pluripotency.
The tumor suppressor Smad4/DPC4 and transcriptional adaptor CBP/p300 are coactivators for Smad3 in TGF-β-induced transcriptional activation
Authors: Feng Xin-Hua , Zhang Ying , Wu Rui-Yun , Derynck Rik
Published: February 2008

Smads regulate transcription of defined genes in response to TGF-β receptor activation, although the mechanisms of Smad-mediated transcription are not well understood. We demonstrate that the TGF-β-inducible Smad3 uses the tumor suppressor Smad4/DPC4 and CBP/p300 as transcriptional coactivators, which associate with Smad3 in response to TGF-β. The association of CBP with Smad3 was localized to the carboxyl terminus of Smad3, which is required for transcriptional activation, and a defined segment in CBP. Furthermore, CBP/p300 stimulated both TGF-β- and Smad-induced transcription in a Smad4/DPC4-dependent fashion. Smad3 transactivation and TGF-β-induced transcription were inhibited by expressing E1A, which interferes with CBP functions. The coactivator functions and physical interactions of Smad4 and CBP/p300 with Smad3 allow a model for the induction of gene expression in response to TGF-β.
EIR1, a root-specific protein involved in auxin transport, is required for gravitropism inArabidopsis thaliana
Authors: Luschnig Christian , Gaxiola Roberto A. , Grisafi Paula , Fink Gerald R.
Published: February 2008

The EIR1 gene of Arabidopsis is a member of a family of plant genes with similarities to bacterial membrane transporters. This gene is expressed only in the root, which is consistent with the phenotypes of the eir1 mutants—the roots are agravitropic and have a reduced sensitivity to ethylene. The roots of eir1 mutants are also insensitive to the excess auxin produced by alf1-1 and fail to induce an auxin-inducible gene in the expansion zone. Although they fail to respond to internally generated auxin, they respond normally to externally applied auxin. Expression of the EIR1 gene in Saccharomyces cerevisiaeconfers resistance to fluorinated indolic compounds. Taken together, these data suggest that the EIR1 protein has a root-specific role in the transport of auxin.
DNA end-independent activation of DNA-PK mediated via association with the DNA-binding protein C1D
Authors: Yavuzer Ugur , Smith Graeme C.M. , Bliss Tonya , Werner Dieter , Jackson Stephen P.
Published: February 2008

DNA-dependent protein kinase (DNA-PK), which is involved in DNA double-strand break repair and V(D)J recombination, is comprised of a DNA-targeting component termed Ku and an ∼465-kD catalytic subunit, DNA-PKcs. Although DNA-PK phosphorylates proteins in the presence of DSBs or other discontinuities in the DNA double helix in vitro, the possibility exists that it is also activated in other circumstances via its association with additional proteins. Here, through use of the yeast two-hybrid screen, we discover that the recently identified high affinity DNA binding protein C1D interacts with the putative leucine zipper region of DNA-PKcs. Furthermore, we show that C1D can interact with DNA-PK in mammalian cells and that C1D is a very effective DNA-PK substrate in vitro. Finally, we establish that C1D directs the activation of DNA-PK in a manner that does not require DNA termini. Therefore, these studies provide a function for C1D and suggest novel mechanisms for DNA-PK activation in vivo.
Genetic analysis of the SR protein ASF/SF2: interchangeability of RS domains and negative control of splicing
Authors: Wang Jin , Xiao Shou-Hua , Manley James L.
Published: February 2008

The SR proteins constitute a family of splicing factors, highly conserved in metazoans, that contain one or two amino-terminal RNA-binding domains (RBDs) and a region enriched in arginine/serine repeats (RS domain) at the carboxyl terminus. Previous studies have shown that SR proteins possess distinct RNA-binding specificities that likely contribute to their unique functions, but it is unclear whether RS domains have specific roles in vivo. Here, we used a genetic system developed in the chicken B cell line DT40 to address this question. Expression of chimeric proteins generated by fusion of the RS domains of heterologous SR proteins, or a human TRA-2 protein, with the RBDs of ASF/SF2 allowed cell growth following genetic inactivation of endogenous ASF/SF2, indicating that RS domains are interchangeable for all functions required to maintain cell viability. However, a chimera containing the RS domain from a related splicing factor, U2AF65, could not rescue viability and was inactive in in vitro splicing assays, suggesting that this domain performs a distinct function. We also used the DT40 system to show that depletion of ASF/SF2 affects splicing of specific transcripts in vivo. Although splicing of several simple constitutive introns was not significantly affected, the alternative splicing patterns of two model pre-mRNAs switched in a manner consistent with predictions from previous studies. Unexpectedly, ASF/SF2 depletion resulted in a substantial increase in splicing of an HIV-1 tat pre-mRNA substrate, indicating that ASF/SF2 can repress tat splicing in vivo. These results provide the first demonstration that an SR protein can influence splicing of specific pre-mRNAs in vivo.
Functional analysis of the secretory precursor processing machinery of Bacillus subtilis: identification of a eubacterial homolog of archaeal and eukaryotic signal peptidases
Authors: Tjalsma Harold , Bolhuis Albert , van Roosmalen Maarten L. , Wiegert Thomas , Schumann Wolfgang , Broekhuizen Cees P. , Quax Wim J. , Venema Gerard , Bron Sierd , van Dijl Jan Maarten
Published: February 2008

Approximately 47% of the genes of the Gram-positive bacteriumBacillus subtilis belong to paralogous gene families. The present studies were aimed at the functional analysis of thesip gene family of B. subtilis, consisting of five chromosomal genes, denoted sipS, sipT, sipU, sipV, andsipW. All five sip genes specify type I signal peptidases (SPases), which are actively involved in the processing of secretory preproteins. Interestingly, strains lacking as many as four of these SPases could be obtained. As shown with a temperature-sensitive SipS variant, only cells lacking both SipS and SipT were not viable, which may be caused by jamming of the secretion machinery with secretory preproteins. Thus, SipS and SipT are of major importance for protein secretion. This conclusion is underscored by the observation that only the transcription of the sipS andsipT genes is temporally controlled via the DegS–DegU regulatory system, in concert with the transcription of most genes for secretory preproteins. Notably, the newly identified SPase SipW is highly similar to SPases from archaea and the ER membrane of eukaryotes, suggesting that these enzymes form a subfamily of the type I SPases, which is conserved in the three domains of life.
Smad proteins act in combination with synergistic and antagonistic regulators to target Dpp responses to theDrosophila mesoderm
Authors: Xu Xiaolei , Yin Zhizhang , Hudson John B. , Ferguson Edwin L. , Frasch Manfred
Published: February 2008

Dorsal mesoderm induction in arthropods and ventral mesoderm induction in vertebrates are closely related processes that involve signals of the BMP family. In Drosophila, induction of visceral mesoderm, dorsal muscles, and the heart by Dpp is, at least in part, effected through the transcriptional activation and function of the homeobox gene tinman in dorsal mesodermal cells during early embryogenesis. Here we present a functional dissection of atinman enhancer that mediates the Dpp response. We provide evidence that mesoderm-specific induction of tinman requires the binding of both activators and repressors. Screens for binding factors yielded Tinman itself and the Smad4 homolog Medea. We show that the binding and synergistic activities of Smad and Tinman proteins are critical for mesodermal tinman induction, whereas repressor binding sites prevent induction in the dorsal ectoderm and amnioserosa. Thus, integration of positive and negative regulators on enhancers of target genes appears to be an important mechanism in tissue-specific induction by TGF-β molecules.
Myc signaling via the ARF tumor suppressor regulates p53-dependent apoptosis and immortalization
Authors: Zindy Frederique , Eischen Christine M. , Randle David H. , Kamijo Takehiko , Cleveland John L. , Sherr Charles J. , Roussel Martine F.
Published: February 2008

Establishment of primary mouse embryo fibroblasts (MEFs) as continuously growing cell lines is normally accompanied by loss of the p53 or p19ARF tumor suppressors, which act in a common biochemical pathway. myc rapidly activates ARF andp53 gene expression in primary MEFs and triggers replicative crisis by inducing apoptosis. MEFs that survive mycoverexpression sustain p53 mutation or ARF loss during the process of establishment and become immortal. MEFs lackingARF or p53 exhibit an attenuated apoptotic response tomyc ab initio and rapidly give rise to cell lines that proliferate in chemically defined medium lacking serum. Therefore,ARF regulates a p53-dependent checkpoint that safeguards cells against hyperproliferative, oncogenic signals.
Nucleolar localization of early tRNA processing
Authors: Bertrand Edouard , Houser-Scott Felicia , Kendall Ann , Singer Robert H. , Engelke David R.
Published: February 2008

There is little information as to the location of early tRNA biosynthesis. Using fluorescent in situ hybridization in the budding yeast, Saccharomyces cerevisiae, examples of nuclear pre-tRNAs are shown to reside primarily in the nucleoli. We also probed the RNA subunit of RNase P. The majority of the signal from RNase P probes was nucleolar, with less intense signals in the nucleoplasm. These results demonstrate that a major portion of the tRNA processing pathway is compartmentalized in nucleoli with rRNA synthesis and ribosomal assembly. The spatial juxtaposition suggests the possibility of direct coordination between tRNA and ribosome biosynthesis.
Selective regulation ofBcl-XLby a Jak kinase-dependent pathway is bypassed in murine hematopoietic malignancies
Authors: Packham Graham , White Elsie L. , Eischen Christine M. , Yang Hui , Parganas Evan , Ihle James N. , Grillot Didier A.M. , Zambetti Gerard P. , Nuñez Gabriel , Cleveland John L.
Published: February 2008

Bcl-2 family proteins are key regulators of apoptosis and function as cell death antagonists (e.g., Bcl-2, Bcl-XL, and Mcl-1) or agonists (e.g., Bax, Bad, and Bak). Here we report that among the Bcl-2 family of proteins tested (Bcl-2, Bcl-XL, Mcl-1, Bax, Bad, and Bak), Bcl-XLwas unique in that its protein levels were tightly regulated by hemopoietins in both immortal and primary myeloid progenitors. Investigating signaling pathways utilized by cytokine receptors established that the regulation of Bcl-XLprotein levels is mediated by the Jak kinase pathway and is independent of other signaling effectors including STATs, PI-3′ kinase, and Ras. Moreover, we provide the first direct evidence thatBcl-Xis altered in cancer, becausebcl-Xexpression was activated selectively by retroviral insertions in murine myeloid and T-cell hemopoietic malignancies. Tumors harboringbcl-Xinsertions had alteredbcl-XRNAs, expressed elevated levels of Bcl-XLprotein, and lacked the requirements for cytokines normally essential for cell survival. Finally, overexpression of Bcl-XLeffectively protected IL-3-dependent myeloid cells from apoptosis following removal of trophic factors. Therefore, Bcl-XLfunctions as a key cytokine regulated anti-apoptotic protein in myelopoiesis and contributes to leukemia cell survival.
Caenorhabditis elegans Akt/PKB transduces insulin receptor-like signals from AGE-1 PI3 kinase to the DAF-16 transcription factor
Authors: Paradis Suzanne , Ruvkun Gary
Published: February 2008

A neurosecretory pathway regulates a reversible developmental arrest and metabolic shift at the Caenorhabditis elegans dauer larval stage. Defects in an insulin-like signaling pathway cause arrest at the dauer stage. We show here that two C. elegansAkt/PKB homologs, akt-1 and akt-2,transduce insulin receptor-like signals that inhibit dauer arrest and that AKT-1 and AKT-2 signaling are indispensable for insulin receptor-like signaling in C. elegans. A loss-of-function mutation in the Fork head transcription factor DAF-16 relieves the requirement for Akt/PKB signaling, which indicates that AKT-1 and AKT-2 function primarily to antagonize DAF-16. This is the first evidence that the major target of Akt/PKB signaling is a transcription factor. An activating mutation in akt-1,revealed by a genetic screen, as well as increased dosage of wild-typeakt-1 relieves the requirement for signaling from AGE-1 PI3K, which acts downstream of the DAF-2 insulin/IGF-1 receptor homolog. This demonstrates that Akt/PKB activity is not necessarily dependent on AGE-1 PI3K activity. akt-1 andakt-2 are expressed in overlapping patterns in the nervous system and in tissues that are remodeled during dauer formation.
The upstream sequence element of the C2 complement poly(A) signal activates mRNA 3′ end formation by two distinct mechanisms
Authors: Moreira Alexandra , Takagaki Yoshio , Brackenridge Simon , Wollerton Matthew , Manley James L. , Proudfoot Nicholas J.
Published: February 2008

The poly(A) signal of the C2 complement gene is unusual in that it possesses an upstream sequence element (USE) required for full activity in vivo. We describe here in vitro experiments demonstrating that this USE enhances both the cleavage and poly(A) addition reactions. We also show that the C2 USE can be cross-linked efficiently to a 55-kD protein that we identify as the polypyrimidine tract-binding protein (PTB), implicated previously in modulation of pre-mRNA splicing. Mutation of the PTB-binding site significantly reduces the efficiency of the C2 poly(A) site both in vivo and in vitro. Furthermore, addition of PTB to reconstituted processing reactions enhances cleavage at the C2 poly(A) site, indicating that PTB has a direct role in recognition of this signal. The C2 USE, however, also increases the affinity of general polyadenylation factors independently for the C2 poly(A) signal as detected by enhanced binding of cleavage-stimulaton factor (CstF). Strikingly, this leads to a novel CstF-dependant enhancement of the poly(A) synthesis phase of the reaction. These studies both emphasize the interconnection between splicing and polyadenylation and indicate an unexpected flexibility in the organization of mammalian poly(A) sites.