bluuebunny/crossref_metadata_2025 huggingface dataset:
The segment polarity gene porcupine encodes a putative multitransmembrane protein involved in Wingless processing.
Authors: Kadowaki T , Wilder E , Klingensmith J , Zachary K , Perrimon N
Published: June 2007

The Wnt protein Wingless (Wg) functions as a signal in patterning of both the Drosophila embryo and imaginal discs. Lack of porcupine (porc) activity is associated with mutant phenotypes similar to those of wg mutations. In porc mutant embryos, Wg protein is confined to the cells that produce it, suggesting that Porc plays a role in processing or secretion of Wg. porc encodes a novel transmembrane protein that appears to be concentrated at the endoplasmic reticulum. We present both genetic and in vitro evidence demonstrating that porc is involved specifically in the processing of Wg. We identified a human sequence related to Porc suggesting the existence of a family of proteins involved in processing of Wnts.
Segmentation and specification of the Drosophila mesoderm.
Authors: Azpiazu N , Lawrence P A , Vincent J P , Frasch M
Published: June 2007

Patterning of the developing mesoderm establishes primordia of the visceral, somatic, and cardiac tissues at defined anteroposterior and dorsoventral positions in each segment. Here we examine the mechanisms that locate and determine these primordia. We focus on the regulation of two mesodermal genes: bagpipe (bap), which defines the anlagen of the visceral musculature of the midgut, and serpent (srp), which marks the anlagen of the fat body. These two genes are activated in specific groups of mesodermal cells in the anterior portions of each parasegment. Other genes mark the anlagen of the cardiac and somatic mesoderm and these are expressed mainly in cells derived from posterior portions of each parasegment. Thus the parasegments appear to be subdivided, at least with respect to these genes, a subdivision that depends on pair-rule genes such as even-skipped (eve). We show with genetic mosaics that eve acts autonomously within the mesoderm. We also show that hedgehog (hh) and wingless (wg) mediate pair-rule gene functions in the mesoderm, probably partly by acting within the mesoderm and partly by inductive signaling from the ectoderm. hh is required for the normal activation of bap and srp in anterior portions of each parasegment, whereas wg is required to suppress bap and srp expression in posterior portions. Hence, hh and wg play opposing roles in mesoderm segmentation.
Targeted in vivo expression of the cyclin-dependent kinase inhibitor p21 halts hepatocyte cell-cycle progression, postnatal liver development and regeneration.
Authors: Wu H , Wade M , Krall L , Grisham J , Xiong Y , Van Dyke T
Published: June 2007

The CDK inhibitor p21 (WAF-1/CIP-1/SDI-1) has been implicated in DNA damage-induced p53-mediated G1 arrest, as well as in physiological processes, such as cell differentiation and senescence, that do not involve p53 function. To determine the impact of p21 on normal development and cell-cycle regulation in vivo, we have generated transgenic mice that abundantly express p21 specifically in hepatocytes. During postnatal liver development, when transgenic p-21 protein becomes detectable, hepatocyte proliferation is inhibited dramatically. This disturbance causes a reduction in the overall number of adult hepatocytes, resulting in aberrant tissue organization, runted liver and body growth, and increased mortality. The transgenic p21 protein is associated with most, if not all, of the cyclin D1-CDK4 in liver but not significantly with other cyclin/CDK proteins, indicating the importance of cyclin D1-CDK4 function in normal liver development. The appearance of large polyploid nuclei in some hepatocytes indicates that p21 may also cause arrest during the G2 phase of the cell cycle. Significantly, partial hepatectomy failed to stimulate hepatocytes to proliferate in p21 transgenic animals. These results provide the first in vivo evidence that appropriate p21 levels are critical in normal development and further implicate p21 in the control of multiple cell-cycle phases.
Protein phosphatase 2A positively and negatively regulates Ras1-mediated photoreceptor development in Drosophila.
Authors: Wassarman D A , Solomon N M , Chang H C , Karim F D , Therrien M , Rubin G M
Published: June 2007

Protein phosphatase 2A (PP2A), a heterotrimeric serine/threonine phosphatase present in most tissues and cell types, has been implicated in the regulation of cell cycle progression, DNA replication, transcription, and translation. Here we present genetic evidence suggesting that PP2A functions downstream of Ras1 in the Sevenless receptor tyrosine kinase (RTK) signal transduction pathway that specifies R7 photoreceptor cell fate in the developing Drosophila eye. Ras1 and downstream cytoplasmic kinases, Raf, MEK, and MAPK, comprise an evolutionarily conserved cascade that mediates the transmission of signals from RTKs at the plasma membrane to specific factors in the nucleus. Using transgenic flies expressing constitutively activated Ras1 or Raf proteins that function independently of upstream signaling events, we show that a reduction in the dose of the gene encoding the catalytic subunit of PP2A stimulates signaling from Ras1 but impairs signaling from Raf. This suggests that PP2A both negatively and positively regulates the Ras1 cascade by dephosphorylating factors that function at different steps in the cascade.
Intron mobility in phage T4 occurs in the context of recombination-dependent DNA replication by way of multiple pathways.
Authors: Mueller J E , Clyman J , Huang Y J , Parker M M , Belfort M
Published: June 2007

Numerous group I introns in both prokaryotes and eukaryotes behave as mobile genetic elements. The functional requirements for intron mobility were determined in the T4 phage system using an in vivo assay to measure intron homing with wild-type and mutant derivatives. Thus, it was demonstrated that intron mobility occurs in the context of phage recombination-dependent replication, a pathway that uses overlapping subsets of replication and recombination functions. The functional requirements for intron homing and the nature of recombinant products are only partially consistent with the accepted double-strand-break repair (DSBR) model for intron inheritance, and implicate additional homing pathways. Whereas ambiguities in resolvase requirements and underrepresentation of crossover recombination products are difficult to rationalize strictly by DSBR, these properties are most readily consistent with a synthesis-dependent strand annealing (SDSA) pathway. These pathways share common features in the strand invasion steps, but differ in subsequent repair synthesis and resolution steps, influencing the genetic consequences of the intron transfer event.
A ceramide-activated protein phosphatase mediates ceramide-induced G1 arrest of Saccharomyces cerevisiae.
Authors: Nickels J T , Broach J R
Published: June 2007

Certain mammalian growth modulators, such as tumor necrosis factor alpha, interleukin-1beta, and gamma-interferon, induce an antiproliferative response-terminal differentiation, apoptosis, or cell cycle arrest-through a novel signal transduction pathway mediated by the lipid ceramide as a second messenger. Both a ceramide-activated protein phosphatase and a ceramide-activated protein kinase have been implicated in transmitting the signals elicited by ceramide. We have determined that ceramide addition to the yeast Saccharomyces causes a similar antiproliferative response, resulting in arrest of cells in the G1 phase of the cell cycle. We have also determined that yeast cells contain a ceramide-activated protein phosphatase composed of regulatory subunits encoded by TPD3 and CDC55 and a catalytic subunit encoded by SIT4. Because mutation of any one of these three genes renders strains resistant to ceramide inhibition, we conclude that the G1 effects of ceramide are mediated at least in part by the yeast ceramide-activated protein phosphatase. These results highlight the conservation of signaling systems in yeast and mammalian cells and provide a novel approach to dissecting this ubiquitous signal transduction pathway.
Spk1/Rad53 is regulated by Mec1-dependent protein phosphorylation in DNA replication and damage checkpoint pathways.
Authors: Sun Z , Fay D S , Marini F , Foiani M , Stern D F
Published: June 2007

SPK1/RAD53/MEC2/SAD1 of Saccharomyces cerevisiae encodes an essential protein kinase that is required for activation of replication-sensitive and DNA damage-sensitive checkpoint arrest. We have investigated the regulation of phosphorylation and kinase activity of Spk1p during the cell cycle and by conditions that activate checkpoint pathways. Phosphorylation of Spk1p is induced by treatment of cells with agents that damage DNA or interfere with DNA synthesis. Although only S- and G2-phase cdc mutants arrest with hyperphosphorylated Spk1p, damage-induced phosphorylation of Spk1p can occur in G1 and M as well. Hydroxyurea (HU) induces phosphorylation of kinase-defective forms of Spk1p, demonstrating that this regulated phosphorylation of Spk1p occurs in trans. HU-induced phosphorylation is associated with increased catalytic activity of Spk1p. Furthermore, overexpression of wild-type SPK1, but not checkpoint-defective alleles, delays progression through the G1/S boundary. Damage-dependent phosphorylation of Spk1p requires both MEC1 and MEC3, whereas MEC1 but not MEC3, is required for replication block-induced phosphorylation. These data support the model that Spk1p is an essential intermediate component in a signal transduction pathway coupling damage and checkpoint functions to cell cycle arrest. This regulation is mediated through a protein kinase cascade that potentially includes Mec1p and Tel1p as the upstream kinases.
Redundancy of Saccharomyces cerevisiae MSH3 and MSH6 in MSH2-dependent mismatch repair.
Authors: Marsischky G T , Filosi N , Kane M F , Kolodner R
Published: June 2007

Saccharomyces cerevisiae encodes six genes, MSH1-6, which encode proteins related to the bacterial MutS protein. In this study the role of MSH2, MSH3, and MSH6 in mismatch repair has been examined by measuring the rate of accumulating mutations and mutation spectrum in strains containing different combinations of msh2, msh3, and msh6 mutations and by studying the physical interaction between the MSH2 protein and the MSH3 and MSH6 proteins. The results indicate that S. cerevisiae has two pathways of MSH2-dependent mismatch repair: one that recognized single-base mispairs and requires MSH2 and MSH6, and a second that recognizes insertion/deletion mispairs and requires a combination of either MSH2 and MSH6 or MSH2 and MSH3. The redundancy of MSH3 and MSH6 explains the greater prevalence of hmsh2 mutations in HNPCC families and suggests how the role of hmsh3 and hmsh6 mutations in cancer susceptibility could be analyzed.
Transcription factor Spo0A switches the localization of the cell division protein FtsZ from a medial to a bipolar pattern in Bacillus subtilis.
Authors: Levin P A , Losick R
Published: June 2007

Entry into sporulation by the Gram-positive bacterium Bacillus subtilis is governed by two transcription factors, Spo0A and sigma H, and involves a switch in the site of division from a medial to a polar location. We report that at the onset of sporulation, assembly of the cell division protein FtsZ shifts from midcell to potential division sites near both poles. The switch to a bipolar pattern of FtsZ localization is dependent on Spo0A. Additionally, synthesis of an activated form of Spo0A during growth artificially activates the switch in FtsZ localization and results in the formation of polar septa. The sigma H factor, on the other hand, is dispensable for the switch in the position of the FtsZ assembly site, although it is required for formation of the polar septum. Our results suggest that during the transition from growth to sporulation, Spo0A induces the expression of genes that suppress FtsZ assembly at the midcell site and activate sites at both poles, whereas sigma H induces genes required for a subsequent step in cytokinesis.
The 3' end of yeast 5.8S rRNA is generated by an exonuclease processing mechanism.
Authors: Mitchell P , Petfalski E , Tollervey D
Published: June 2007

Eukaryotic rRNAs (with the exception of 5S rRNA) are synthesized from a contiguous pre-rRNA precursor by a complex series of processing reactions. Final maturation of yeast 5.8S rRNA involves processing of a 3'-extended, 7S precursor that contains approximately 140 nucleotides of the internal transcribed spacer 2 (ITS2) region. In yeast strains carrying the temperature-sensitive (ts) rrp4-1 mutation, 5.8S rRNA species were observed with 3' extensions of variable length extending up to the 3' end of the 7S pre-rRNA. These 3'-extended 5.8S rRNA species were observed at low levels in rrp4-1 strains under conditions permissive for growth and increased in abundance upon transfer to the nonpermissive temperature. The RRP4 gene was cloned by complementation of the ts growth phenotype of rrp4-1 strains. RRP4 encodes an essential protein of 39-kD predicted molecular mass. Immunoprecipitated Rrp4p exhibited a 3'-->5' exoribonuclease activity in vitro that required RNA with a 3'-terminal hydroxyl group and released nucleoside 5' monophosphates. We conclude that the 7S pre-rRNA is processed to 5.8S rRNA by a 3'-->5' exonuclease activity involving Rrp4p. Homologs of Rrp4p are found in both humans and fission yeast Schizosaccharomyces pombe (43% and 52% identity, respectively), suggesting that the mechanism of 5.8S rRNA 3' end formation has been conserved throughout eukaryotes.
Rum1 and Cdc18 link inhibition of cyclin-dependent kinase to the initiation of DNA replication in Schizosaccharomyces pombe.
Authors: Jallepalli P V , Kelly T J
Published: June 2007

Eukaryotic cells have evolved regulatory mechanisms to ensure the strict alternation of DNA replication and mitosis. Recent work has suggested that the mitotic form of cyclin-dependent kinase (Cdc2/cyclin B) has a role in preventing re-replication of the genome before mitosis, but the relevant targets of this inhibition are unknown. In this report we present evidence that the mitotic cyclin-dependent kinase affects DNA replication by inhibiting the accumulation and function of Cdc18, a critical regulator of S-phase entry. We found that the ruml+ gene efficiently suppresses the lethality of a conditional cdc18 mutant. Conversely, deletion of ruml+ increases the severity of the cdc18 mutant phenotype, resulting in inappropriate cell division and a rapid loss of viability. Biochemical experiments indicate that Ruml potently inhibits Cdc2 phosphorylation of histone H1 or a Cdc18 fusion protein by directly interacting with the Cdc2/cyclin B complex. Overexpression of Ruml under conditions that promote re-replication of the genome induces a striking accumulation of Cdc18 protein by a largely post-transcriptional mechanism. Overexpression of SIC1, an unrelated cyclin-dependent kinase inhibitor from budding yeast, causes a similar accumulation of Cdc18 and also leads to re-replication. Our data link a potent inhibitor of Cdc2 kinase to a key protein required for the initiation of DNA replication and strongly suggest that inhibition of Cdc18 by cyclin-dependent kinases has an important role in ensuring that the genome is duplicated precisely once each cell cycle.
Drosophila terminal structure development is regulated by the compensatory activities of positive and negative phosphotyrosine signaling sites on the Torso RTK.
Authors: Cleghon V , Gayko U , Copeland T D , Perkins L A , Perrimon N , Morrison D K
Published: June 2007

Specification of cell fates in the nonsegmented terminal regions of developing Drosophila embryos is under the control of a signal transduction pathway mediated by the receptor tyrosine kinase Torso (Tor). Here, we identify tyrosines (Y) 630 and 918 as the major sites of Tor autophosphorylation. We demonstrate that mutation of Y630, a site required for association with and tyrosine phosphorylation of the tyrosine phosphatase Corkscrew, decreases the efficiency of Tor signaling. In contrast, mutation of Y918, a site capable of binding mammalian rasGAP and PLC-gammal, increases Tor signaling. Interestingly, when receptors contain mutations in both the Y630 and Y918 sites, Tor signaling is restored to wild-type levels. These results identify a novel mechanism whereby Tor function is regulated using compensatory signals generated from distinct autophosphorylation sites and reveal an underlying signaling pathway for terminal development.
Developing Caenorhabditis elegans neurons may contain both cell-death protective and killer activities.
Authors: Shaham S , Horvitz H R
Published: June 2007

We developed a method for examining the effects of overexpressing cell-death-related genes in specific Caenorhabditis elegans neurons that normally live. Using this method, we demonstrated that the cell-death genes ced-3, ced-4, and ced-9 all can act cell autonomously to control programmed cell death. Our observations indicate further that not only the protective activity of ced-9 but also the killer activities of ced-3 and ced-4 are likely to be present in cells that normally live. We propose that both in C. elegans and in other organisms a competition between antagonistic protective and killer activities determines whether specific cells will live or die. Our results suggest a genetic pathway for programmed cell death in C. elegans in which ced-4 acts upstream of or in parallel to ced-3 and ced-9 negatively regulates the activity of ced-4.
The Saccharomyces retrotransposon Ty5 integrates preferentially into regions of silent chromatin at the telomeres and mating loci.
Authors: Zou S , Ke N , Kim J M , Voytas D F
Published: June 2007

The nonrandom integration of retrotransposons and retroviruses suggests that chromatin influences target choice. Targeted integration, in turn, likely affects genome organization. In Saccharomyces, native Ty5 retrotransposons are located near telomeres and the silent mating locus HMR. To determine whether this distribution is a consequence of targeted integration, we isolated a transposition-competent Ty5 element from S. paradoxus, a species closely related to S. cerevisiae. This Ty5 element was used to develop a transposition assay in S. cerevisiae to investigate target preference of de novo transposition events. Of 87 independent Ty5 insertions, approximately 30% were located on chromosome III, indicating this small chromosome (approximately 1/40 of the yeast genome) is a highly preferred target. Mapping of the exact location of 19 chromosome III insertions showed that 18 were within or adjacent to transcriptional silencers flanking HML and HMR or the type X subtelomeric repeat. We predict Ty5 target preference is attributable to interactions between transposition intermediates and constituents of silent chromatin assembled at these sites. Ty5 target preference extends the link between telomere structure and reverse transcription as carried out by telomerase and Drosophila retrotransposons.
Protein kinase A is a common negative regulator of Hedgehog signaling in the vertebrate embryo.
Authors: Hammerschmidt M , Bitgood M J , McMahon A P
Published: June 2007

Midline signaling by Hedgehog (Hh) family members has been implicated in patterning the vertebrate embryo. We have explored the potential regulatory role of cAMP-dependent protein kinase A (PKA) in these events. Zebrafish embryos injected with RNAs encoding Sonic hedgehog (Shh), Indian hedgehog (Ihh), or a dominant-negative regulatory subunit of PKA, PKI, have equivalent phenotypes. These include the expansion of proximal fates in the eye, ventral fates in the brain, and adaxial fates in somites and head mesenchyme. Moreover, ectopic expression of PKI partially rescues somite and optic stalk defects in no tail and cyclops mutants that lack midline structures that normally synthesize Shh. Conversely, all cell types promoted by ectopic expression of hhs and PKI are suppressed in embryos injected with RNA encoding a constitutively active catalytic subunit of PKA (PKA*). These results, together with epistasis studies on the block of ectopic Hh signaling by PKA*, indicate that PKA acts in target cells as a common negative regulator of Hedgehog signaling.
CIF, an essential cofactor for TFIID-dependent initiator function.
Authors: Kaufmann J , Verrijzer C P , Shao J , Smale S T
Published: June 2007

The core promoters for mammalian protein-coding genes often contain a TATA box, an initiator (Inr) element, or both of these control elements. The TFIID complex is essential both for TATA activity and for the activity of a common class of Inr elements characterized by an approximate consensus sequence PyPyA+1NT/APyPy. Although the complete set of proteins required for basal TATA-mediated transcription has been established, the requirements for TFIID-dependent Inr activity remain undefined. In this study we set out to reconstitute Inr activity with purified and recombinant general transcription factors. For this analysis, Inr activity was measured as the ability of an Inr to enhance the strength of a core promoter containing an upstream TATA box. Inr activity was not detected in reactions containing TFIIB, RAP30, RAP74, RNA polymerase II, and either TBP or TFIID, even though these factors were sufficient for TATA-mediated transcription from supercoiled templates. By use of a complementation assay, a factor that imparts Inr activity was identified. This factor, named CIF, stimulated Inr activity in reactions containing the TFIID complex, but activity was not detected with TBP. Further characterization of CIF suggested that it contains multiple components. Functional and immunological experiments demonstrated that one of the CIF components is the mammalian homolog of Drosophila TAF(II)150, which is not tightly associated with mammalian TFIID. These results reveal significant differences in the factor requirements for basal TATA and Inr activity. Further elucidation of these differences is likely to explain the need for the core promoter heterogeneity found within protein-coding genes.
A reversible, p53-dependent G0/G1 cell cycle arrest induced by ribonucleotide depletion in the absence of detectable DNA damage.
Authors: Linke S P , Clarkin K C , Di Leonardo A , Tsou A , Wahl G M
Published: June 2007

Cells with a functional p53 pathway undergo a G0/G1 arrest or apoptosis when treated with gamma radiation or many chemotherapeutic drugs. It has been proposed that DNA damage is the exclusive signal that triggers the arrest response. However, we found that certain ribonucleotide biosynthesis inhibitors caused a p53-dependent G0 or early G1 arrest in the absence of replicative DNA synthesis or detectable DNA damage in normal human fibroblasts. CTP, GTP, or UTP depletion alone was sufficient to induce arrest. In contrast to the p53-dependent response to DNA damage, characterized by long-term arrest and irregular cellular morphologies, the antimetabolite-induced arrest was highly reversible and cellular morphologies remained relatively normal. Both arrest responses correlated with prolonged induction of p53 and the Cdk inhibitor P21(WAF1/CIP1/SDI1) and with dephosphorylation of pRb. Thus, we propose that p53 can serve as a metabolite sensor activated by depletion of ribonucleotides or products or processes dependent on ribonucleotides. Accordingly, p53 may play a role in inducing a quiescence-like arrest state in response to nutrient challenge and a senescence-like arrest state in response to DNA damage. These results have important implications for the mechanisms by which p53 prevents the emergence of genetic variants and for developing more effective approaches to chemotherapy based on genotype.
Posterior patterning by the Caenorhabditis elegans even-skipped homolog vab-7.
Authors: Ahringer J
Published: June 2007

Patterning of the posterior end in animals is not well understood. Homologs of Drosophila even-skipped (eve) have a similar posterior expression pattern in many animals, and in vertebrates they are linked physically to the "posterior" ends of homeotic clusters (HOM-C), suggesting a conserved role in posterior development. However, the function of this posterior expression is not known. Here I show that the Caenorhabditis elegans gene vab-7 encodes an eve homolog that is required for posterior development and expressed in a pattern strikingly similar to that of vertebrate eve genes. Using a four-dimensional recording system, I found that posterior body muscles and the posterior epidermis are patterned abnormally in vab-7 mutants, but commitment to muscle and epidermal fates is normal. Furthermore, vab-7 activity is required for the complete expression of the most posterior HOM-C gene egl-5 in muscle cells, supporting the idea that eve homologs may act with the HOM-C to determine posterior cell fates. The conservation of sequence and expression pattern between vab-7 and eve homologs in other animals argues that most eve genes have posterior mesodermal and ectodermal patterning functions.
Meiotic cells monitor the status of the interhomolog recombination complex.
Authors: Xu L , Weiner B M , Kleckner N
Published: June 2007

During meiosis, mutations that cause defects at intermediate stages in the recombination process confer arrest at the end of prophase (e.g., pachytene). In yeast, mutations of this type include rad50S, dmc1, rad51, and zip1. Rad50 is likely part of a recombination initiation complex. DMC1, RAD51, and ZIP1 encode two RecA homologs and a synaptonemal complex protein, respectively. We report here the effects of mutations in two other (meiosis-specific) genes, RED1 and MEK1/MRE4, that encode a chromosome structure component and a protein kinase, respectively. A red1 or mek1/mre4 mutation alleviates completely rad50S, dmc1, rad51, and zip1 arrest. Furthermore, the red1 and mek1/mre4 mutations define a unique, previously unrecognized aspect of recombination imposed very early in the process, during DSB formation. Finally, the red1 and mek1/mre4 mutations appear to alleviate prophase arrest directly rather than by eliminating, or permitting bypass of, the rad50S, dmc1, rad51, or zip1 defects. These and other observations suggest that a meiosis-specific regulatory surveillance process monitors the status of the protein/DNA interhomolog recombination machinery as an integral entity, in its proper chromosomal context, and dependent upon its appropriate Red1 and Mek1/Mre4-promoted development. We speculate that a properly developed recombination complex emits an inhibitory signal to delay progression of meiotic cells out of prophase until or unless the recombination process has progressed, at least past certain critical steps, and perhaps to completion.
Endothelial PAS domain protein 1 (EPAS1), a transcription factor selectively expressed in endothelial cells.
Authors: Tian H , McKnight S L , Russell D W
Published: June 2007

Here we describe the cloning and characterization of a PAS domain transcription factor termed endothelial PAS-1 (EPAS1). This protein shares 48% sequence identity with hypoxia inducible factor (HIF-1alpha) and lesser similarity with other members of the basic helix-loop-helix/PAS domain family of transcription factors. Like HIF-1alpha, EPAS1 binds to and activates transcription from a DNA element originally isolated from the erythropoietin gene and containing the sequence 5'-GCCCTACGTGCTGTCTCA-3'. Activation by both HIF-1alpha and EPAS1 is stimulated by hypoxic conditions. EPAS1 forms a heterodimeric complex with the aryl hydrocarbon nuclear transporter prior to transcriptional activation of target genes. EPAS1 expression is limited to the endothelium of mouse embryos and, in agreement with its cell type-specific expression pattern, is capable of specifically activating the transcription of the endothelial tyrosine kinase gene Tie-2. These observations raise the possibility that EPAS1 may represent an important regulator of vascularization, perhaps involving the regulation of endothelial cell gene expression in response to hypoxia.
strawberry notch encodes a conserved nuclear protein that functions downstream of Notch and regulates gene expression along the developing wing margin of Drosophila.
Authors: Majumdar A , Nagaraj R , Banerjee U
Published: June 2007

The dorsal/ventral (D/V) boundary functions as an organizer in the growth and patterning of the Drosophila wing disc and gives rise to the wing margin in the adult fly. Here we show that strawberry notch (sno) is a downstream component of the Notch signaling pathway and is important for the specification of this organizer. sno encodes a novel nuclear protein conserved in C. elegans, mouse, and humans. Mutations in wing margin genes interact dominantly with sno and loss of sno function results in loss of expression of wingless, vestigial, cut, and E(spl)-m8 at the D/V boundary. In regulating these genes, sno functions in close cooperation with Suppressor of Hairless and Hairless. Finally, sno has no role in lateral inhibition suggesting that it may contribute to the specificity between lateral and inductive Notch signaling pathways.
A family of LIM domain-associated cofactors confer transcriptional synergism between LIM and Otx homeodomain proteins.
Authors: Bach I , Carrière C , Ostendorff H P , Andersen B , Rosenfeld M G
Published: June 2007

The essential roles of LIM homeodomain proteins in cell fate determination during development have been demonstrated in organisms as divergent as Drosophila and higher mammals. We have isolated murine cDNAs encoding two highly homologous proteins that specifically interact with the LIM domains of P-Lim/Lhx3 and several other LIM homeodomain factors. Transcripts encoding these factors can be detected as early as mouse E8.5, with maximal expression observed in regions of the embryo in which the LIM homeodomain factors P-Lim/Lhx3, Isl-1, and LH-2 are selectively expressed. These proteins can potentiate transactivation by P-Lim/Lhx-3 and are required for a synergistic activation of the glycoprotein hormone alpha-subunit promoter by P-Lim/Lhx3 and a pituitary Otx class homeodomain transcription factor, with which they also specifically associate. Our results link LIM homeodomain proteins and members of the Otx class of transcription factors in gene activation events during embryogenesis via the actions of specific cofactors.
Human TAF(II)135 potentiates transcriptional activation by the AF-2s of the retinoic acid, vitamin D3, and thyroid hormone receptors in mammalian cells.
Authors: Mengus G , May M , Carré L , Chambon P , Davidson I
Published: June 2007

We report for the first time the cloning of a complete cDNA encoding the human TFIID subunit hTAF(II)135 (hTAF(II)130). Full-length hTAF(II)135 comprises 1083 amino acids and contains two conserved domains present also in dTAF(II)110 and hTAF(II)105. We show that expression of hTAF(II)135 in mammalian cells strongly and selectively potentiates transcriptional stimulation by the activation function-2 (AF-2) of the retinoic acid, thyroid hormone, and vitamin D3 receptors (RAR, TR, and VDR), but does not affect the AF-2s of the estrogen (ER) or retinoid X (RXR) receptors. The coactivator activity requires an hTAF(II)135 region that is located between the conserved domains but is itself not conserved in dTAF(II)110 and hTAF(II)105. Expression of hTAF(II)135 also stimulates RAR AF-2 activity when a promoter with a low-affinity TATA element (TGTA) is used, indicating that hTAF(II)135 overexpression compensates for the low-affinity of TBP for this promoter and may facilitate the recruitment of TFIID by the RAR AF-2.
The mesenchymal winged helix transcription factor Fkh6 is required for the control of gastrointestinal proliferation and differentiation.
Authors: Kaestner K H , Silberg D G , Traber P G , Schütz G
Published: June 2007

The winged helix transcription factor Fkh6 is expressed in the mesoderm of the gastrointestinal tract directly adjacent to the endoderm-derived epithelium. Homozygous null mice for Fkh6 showed postnatal growth retardation secondary to severe structural abnormalities of the stomach, duodenum, and jejunum. Dysregulation of epithelial cell proliferation in these organs resulted in an approximately fourfold increase in the number of dividing intestinal epithelial cells and marked expansion of the proliferative zone. As a consequence, the tissue architecture of the stomach and small intestine was distorted, with abnormal crypt structure, formation of mucin filled cysts, and lengthening of villi. Changes in the cellular phenotype and composition of the gastric and intestinal epithelia also suggests that epithelial cell-lineage allocation or differentiation may be affected by loss of Fkh6. From the analysis of a number of potential signaling molecules, we found Bmp2 and Bmp4 expression reduced in the gastrointestinal tract of Fkh6 mutant mice, suggesting that Fkh6 directs a signaling cascade that mediates communication between the mesenchyme and endoderm of the gut to regulate cell proliferation.
Deletion of the H19 transcription unit reveals the existence of a putative imprinting control element.
Authors: Ripoche M A , Kress C , Poirier F , Dandolo L
Published: June 2007

The distal region of mouse chromosome 7 contains a cluster of imprinted genes that includes H19 and Igf2 (insulin-like growth factor 2). H19 is expressed as an untranslated RNA found at high levels in endodermal and mesodermal embryonic tissues. This gene is imprinted and exclusively expressed from the allele of maternal origin. The Igf2 gene shows a similar pattern of expression but is expressed from the paternal allele. We have generated a targeted deletion of the H19 transcription unit by insertion of a neo replacement cassette. The homozygous mutant animals are viable and fertile and display an overgrowth phenotype of 8% compared with wild-type littermates. This is associated with the disruption of Igf2 imprinting and the consequent biallelic expression of this gene. A striking feature of the recombinant H19 allele is the occurrence of a parental imprint set on the neo replacement cassette. Therefore imprinting of the H19 locus is independent of the H19 gene itself. Taken together with the results of a larger H19 mutation described previously, this indicates that an imprinting control element is located within the region 10 kb upstream of H19.
Genetic analysis reveals that PAX6 is required for normal transcription of pancreatic hormone genes and islet development.
Authors: Sander M , Neubüser A , Kalamaras J , Ee H C , Martin G R , German M S
Published: June 2007

We present genetic and biochemical evidence that PAX6 is a key regulator of pancreatic islet hormone gene transcription and is required for normal islet development. In embryos homozygous for a mutant allele of the Pax6 gene, Small eye (Sey(Neu)), the numbers of all four types of endocrine cells in the pancreas are decreased significantly, and islet morphology is abnormal. In the remaining islet cells, hormone production, particularly glucagon production, is markedly reduced because of decreased gene transcription. These effects appear to result from a lack of PAX6 protein in the mutant embryos. Biochemical studies identify wild-type PAX6 protein as the transcription factor that binds to a common element in the glucagon, insulin, and somatostatin promoters, and show that PAX6 transactivates the glucagon and insulin promoters.
Cdc37 is a molecular chaperone with specific functions in signal transduction.
Authors: Kimura Y , Rutherford S L , Miyata Y , Yahara I , Freeman B C , Yue L , Morimoto R I , Lindquist S
Published: June 2007

Cdc37 is required for cyclin-dependent kinase activation and is genetically linked with the activity of several other kinases, including oncogenic v-Src, casein kinase II, MPS-1 kinase, and sevenless. Strikingly, many pathways involving Cdc37 also involve the protein chaperone Hsp90. The identification of Cdc37 as the 50-kD protein in several Hsp90-kinase complexes, together with other data, led to the recent suggestion that Cdc37 is a kinase-targeting "subunit" of Hsp90. We directly examined the effect of Cdc37 on Hsp90 functions. Rather than simply acting as an accessory factor for Hsp90, Cdc37 is itself a protein chaperone with properties remarkably similar to those of Hsp90. In vitro, Cdc37 maintains denatured beta-galactosidase in an activation-competent state without reactivating it and stabilizes mature, but unstable, casein kinase II. In vivo, Cdc37 overexpression can compensate for decreased Hsp90 function, but the proteins are not interchangeable. Cdc37 can compensate for Hsp90 in maintaining the activity of v-Src kinase but does not maintain the activity of the glucocorticoid receptor. Thus, the very similar chaperone activities of the two proteins, uncovered through in vitro analysis, diverge in vivo in specific signal transduction pathways.
Xist-deficient mice are defective in dosage compensation but not spermatogenesis.
Authors: Marahrens Y , Panning B , Dausman J , Strauss W , Jaenisch R
Published: June 2007

The X-linked Xist gene encodes a large untranslated RNA that has been implicated in mammalian dosage compensation and in spermatogenesis. To investigate the function of the Xist gene product, we have generated male and female mice that carry a deletion in the structural gene but maintain a functional Xist promoter. Mutant males were healthy and fertile. Females that inherited the mutation from their mothers were also normal and had the wild-type paternal X chromosome inactive in every cell. In contrast to maternal transmission, females that carry the mutation on the paternal X chromosome were severely growth-retarded and died early in embryogenesis. The wild-type maternal X chromosome was inactive in every cell of the growth-retarded embryo proper, whereas both X chromosomes were expressed in the mutant female trophoblast where X inactivation is imprinted. However, an XO mouse with a paternally inherited Xist mutation was healthy and appeared normal. The imprinted lethal phenotype of the mutant females is therefore due to the inability of extraembryonic tissue with two active X chromosomes to sustain the embryo. Our results indicate that the Xist RNA is required for female dosage compensation but plays no role in spermatogenesis.
Immunological defects in mice with a targeted disruption in Bcl-3.
Authors: Schwarz E M , Krimpenfort P , Berns A , Verma I M
Published: June 2007

The proto-oncogene bcl-3 is a member of the IkappaB family. The Bcl-3 protein is known to interact specifically with the p50 and p52 subunits of NFkappaB. However, the function of this interaction is not well understood. To determine the in vivo role of Bcl-3, mice were generated that lack the bcl-3 gene, Bcl 3(-/-). Here we report that Bcl 3(-/-) mice appear developmentally normal, but exhibit severe defects in humoral immune responses and protection from in vivo pathogenic challenges. Relative to wild-type mice, Bcl 3(-/-) mice are unable to clear L. monocytogenes and are more susceptible to infection with S. pneumoniae. This phenotype is similar to that observed in the p50(-/-) mice and the cross between the Bcl-3(-/-) and p50(-/-) mice generates animals with an enhanced phenotype. In accordance with the observed defects in their immune response, the Bcl 3(-/-) mice have normal immunoglobulin levels before and after immunization, but fail to produce antigen-specific antibodies. Additionally, spleens from Bcl-3(-/-) mice are abnormal and void of germinal centers. In contrast, the p50(-/-) mice have normal germinal centers. We propose that in in vivo, Bcl-3 can function independently of p50.
An unusual form of transcriptional silencing in yeast ribosomal DNA.
Authors: Smith J S , Boeke J D
Published: June 2007

Generalized transcriptional repression of large chromosomal regions in Saccharomyces cerevisiae occurs at the silent mating loci and at telomeres and is mediated by the silent information regulator (SIR) genes. We have identified a novel form of transcriptional silencing in S. cerevisiae in the ribosomal DNA (rDNA) tandem array. Ty1 retrotransposons marked with a weakened URA3 gene (Ty1-mURA3) efficiently integrated into rDNA. The mURA3 marker in rDNA was transcriptionally silenced in a SIR2-dependent manner. MET15 and LEU2 were also partially silenced, indicating that rDNA silencing may be quite general. Deletion of SIR4 enhanced mURA3 and MET15 silencing, but deletion of SIR1 or SIR3 did not affect silencing, indicating that the mechanism of silencing differs from that at telomeres and silent mating loci. Deletion of SIR2 resulted in increased psoralen cross-linking of the rDNA in vivo, suggesting that a specific chromatin structure in rDNA down-regulates polymerase II promoters.
Transcriptional silencing of Ty1 elements in the RDN1 locus of yeast.
Authors: Bryk M , Banerjee M , Murphy M , Knudsen K E , Garfinkel D J , Curcio M J
Published: June 2007

We demonstrate that in Saccharomyces cerevisiae, the tandem array of ribosomal RNA genes (RDN1) is a target for integration of the Ty1 retrotransposon that results in silencing of Ty1 transcription and transposition. Ty1 elements transpose into random rDNA repeat units and are mitotically stable. In addition, we have found that mutation of several putative modifiers of RDN1 chromatin structure abolishes silencing of Ty1 elements in the rDNA array. Disruption of SIR2, which elevates recombination in RDN1, or TOP1, which increases psoralen accessibility in rDNA, or HTA1-HTB1, which reduces histone H2A-H2B levels and causes localized chromatin perturbations, abolishes transcriptional silencing of Ty1 elements in RDN1. Furthermore, deletion of the gene for the ubiquitin conjugating enzyme Ubc2p, which ubiquitinates histones in vitro, derepresses not only Ty1 transcription but also mitotic recombination in RDN1. On the basis of these results, we propose that a specialized chromatin structure exists in RDN1 that silences transcription of the Ty1 retrotransposon.
Ultraviolet radiation sensitivity and reduction of telomeric silencing in Saccharomyces cerevisiae cells lacking chromatin assembly factor-I.
Authors: Kaufman P D , Kobayashi R , Stillman B
Published: June 2007

In vivo, nucleosomes are formed rapidly on newly synthesized DNA after polymerase passage. Previously, a protein complex from human cells, termed chromatin assembly factor-I (CAF-I), was isolated that assembles nucleosomes preferentially onto SV40 DNA templates that undergo replication in vitro. Using a similar assay, we now report the purification of CAF-I from the budding yeast Saccharomyces cerevisiae. Amino acid sequence data from purified yeast CAF-I led to identification of the genes encoding each subunit in the yeast genome data base. The CAC1 and CAC2 (chromatin assembly complex) genes encode proteins similar to the p150 and p60 subunits of human CAF-I, respectively. The gene encoding the p50 subunit of yeast CAF-I (CAC3) is similar to the human p48 CAF-I subunit and was identified previously as MSI1, a member of a highly conserved subfamily of WD repeat proteins implicated in histone function in several organisms. Thus, CAF-I has been conserved functionally and structurally from yeast to human cells. Genes encoding the CAF-I subunits (collectively referred to as CAC genes) are not essential for cell viability. However, deletion of any CAC gene causes an increase in sensitivity to ultraviolet radiation, without significantly increasing sensitivity to gamma rays. This is consistent with previous biochemical data demonstrating the ability of CAF-I to assemble nucleosomes on templates undergoing nucleotide excision repair. Deletion of CAC genes also strongly reduces silencing of genes adjacent to telomeric DNA; the CAC1 gene is identical to RLF2 (Rap1p localization factor-2), a gene required for the normal distribution of the telomere-binding Rap1p protein within the nucleus. Together, these data suggest that CAF-I plays a role in generating chromatin structures in vivo.
Disruption of the terminal base pairs of retroviral DNA during integration.
Authors: Scottoline B P , Chow S , Ellison V , Brown P O
Published: June 2007

Integrase catalyzes two essential steps in the integration of the retroviral genome--end processing and strand transfer--both of which require the interaction of integrase with viral att sites located at the ends of viral genomic DNA. These two different polynucleotidyl transfer reactions are apparently carried out by a single active site. The end product of these reactions, the integrated provirus, does not undergo transposition and remains a stable part of the host cell genome. A central question in understanding the mechanism of integration is how a single active site accomplishes two distinct polynucleotidyl transfer reactions. We propose that integrase distorts DNA substrates to accommodate both reactions within the active site. Evidence is provided for disruption of base-pairing at the terminus of viral DNA during end processing. Furthermore, we show that this end fraying is a required step in end processing and that it appears to occur after initial binding of the viral DNA end. This requirement for base-pair disruption may account for the inability of integrase to use internal sites on DNA molecules as viral att sites. The specificity of integrase for DNA ends solves a problem posed by the long terminal repeat structure of the viral genome, and may help to prevent transposition of integrated proviruses.
Two domains of MyoD mediate transcriptional activation of genes in repressive chromatin: a mechanism for lineage determination in myogenesis.
Authors: Gerber A N , Klesert T R , Bergstrom D A , Tapscott S J
Published: June 2007

Genetic studies have demonstrated that MyoD and Myf5 establish the skeletal muscle lineage, whereas myogenin mediates terminal differentiation, yet the molecular basis for this distinction is not understood. We show that MyoD can remodel chromatin at binding sites in muscle gene enhancers and activate transcription at previously silent loci. TGF-beta, basic-FGF, and sodium butyrate blocked MyoD-mediated chromatin reorganization and the initiation of transcription. In contrast, TGF-beta and sodium butyrate did not block transcription when added after chromatin remodeling had occurred. MyoD and Myf-5 were 10-fold more efficient than myogenin at activating genes in regions of transcriptionally silent chromatin. Deletion mutagenesis of the MyoD protein demonstrated that the ability to activate endogenous genes depended on two regions: a region rich in cysteine and histidine residues between the acidic activation domain and the bHLH domain, and a second region in the carboxyl terminus of the protein. Neither region has been shown previously to regulate gene transcription and both have domains that are conserved in the Myf5 protein. Our results establish a mechanism for chromatin modeling in the skeletal muscle lineage and define domains of MyoD, independent of the activation domain, that participate in chromatin reorganization.
cash4, a novel achaete-scute homolog induced by Hensen's node during generation of the posterior nervous system.
Authors: Henrique D , Tyler D , Kintner C , Heath J K , Lewis J H , Ish-Horowicz D , Storey K G
Published: June 2007

In vertebrate embryos, the precursor cells of the central nervous system (CNS) are induced by signaling from the organizer region. Here we report the isolation of a novel vertebrate achaete-scute homolog, cash4, which is expressed in the presumptive posterior nervous system in response to such signaling. cash4 is first expressed in epiblast cells flanking the late-phase organizer (Hensen's node), which retains its ability to induce cash4 during regression to the caudal end of the embryo. We show that these node-derived signals can be mimicked in vivo by the activity of fibroblast growth factor (FGF). We demonstrate that cash4 can substitute for the achaete/scute genes in the fly and that it also has proneural activity in vertebrate embryos. Together these results suggest that cash4 functions as a proneural gene downstream of node-derived signals (including FGF) to promote the formation of the neural precursors that will give rise to the posterior CNS in the chick embryo.