bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of tissue-specific P-element pre-mRNA splicing requires the RNA-binding protein PSI.
Authors: Siebel C W , Kanaar R , Rio D C
Published: June 2007

Binding of a multiprotein complex to a 5' exon inhibitory element appears to repress splicing of the Drosophila P-element third intron (IVS3) in the soma. We have purified 97- and 50-kD proteins that interact specifically with the inhibitory element using RNA affinity chromatography. Antibodies specific for the 97-kD protein relieve inhibition of IVS3 splicing in somatic extracts, providing direct evidence that inhibition requires this protein, P-element somatic inhibitor (PSI). We identify the 50-kD protein as hrp48, a protein similar to the mammalian splicing factor hnRNP A1, and show that hrp48 recognizes specific nucleotides in a pseudo-5' splice site within the inhibitory element. The results indicate that PSI is an alternative splicing factor that regulates tissue-specific splicing, probably through interactions with generally expressed factors such as hrp48.
p21-containing cyclin kinases exist in both active and inactive states.
Authors: Zhang H , Hannon G J , Beach D
Published: June 2007

In normal fibroblasts CDKs exist predominantly in p21/PCNA/cyclin/CDK quaternary complexes, whereas in p53-deficient cells, p21 expression is depressed and the kinases are reduced to a cyclin/CDK binary state. p21 is a universal cyclin kinase inhibitor, but we show here that p21-containing complexes exist in both catalytically active and inactive forms. This finding challenges the current view that active cyclin kinases function only in the binary state and reveals the subtlety with which tumor-suppressor proteins modulate the cell cycle.
Dominant-negative and targeted null mutations in the endothelial receptor tyrosine kinase, tek, reveal a critical role in vasculogenesis of the embryo.
Authors: Dumont D J , Gradwohl G , Fong G H , Puri M C , Gertsenstein M , Auerbach A , Breitman M L
Published: June 2007

The receptor tyrosine kinases (RTKs) expressed on the surface of endothelial cells are likely to play key roles in initiating the program of endothelial cell growth during development and subsequent vascularization during wound healing and tumorigenesis. Expression of the Tek RTK during mouse development is restricted primarily to endothelial cells and their progenitors, the angioblasts, suggesting that Tek is a key participant in vasculogenesis. To investigate the role that Tek plays within the endothelial cell lineage, we have disrupted the Tek signaling pathway using two different genetic approaches. First, we constructed transgenic mice expressing a dominant-negative form of the Tek receptor. Second, we created a null allele of the tek gene by homologous recombination in embryonic stem (ES) cells. Transgenic mice expressing dominant-negative alleles of Tek or homozygous for a null allele of the tek locus both died in utero with similar defects in the integrity of their endothelium. By crossing transgenic mice that express the lacZ reporter gene under the transcriptional control of the endothelial cell-specific tek promoter, we found that the extraembryonic and embryonic vasculature was patterned correctly. However, homozygous tek embryos had approximately 30% and 75% fewer endothelial cells at day 8.5 and 9.0, respectively. Homozygous null embryos also displayed abnormalities in heart development, consistent with the conclusion that Tek is necessary for endocardial/myocardial interactions during development. On the basis of the analysis of mice carrying either dominant-negative or null mutations of the tek gene, these observations demonstrate that the Tek signaling pathway plays a critical role in the differentiation, proliferation, and survival of endothelial cells in the mouse embryo.
Mot1, a global repressor of RNA polymerase II transcription, inhibits TBP binding to DNA by an ATP-dependent mechanism.
Authors: Auble D T , Hansen K E , Mueller C G , Lane W S , Thorner J , Hahn S
Published: June 2007

Basal transcription of many genes in yeast is repressed by Mot1, an essential protein which is a member of the Snf2/Swi2 family of conserved nuclear factors. ADI is an ATP-dependent inhibitor of TATA-binding protein (TBP) binding to DNA that inhibits transcription in vitro. Here we demonstrate that ADI is encoded by the MOT1 gene. Mutation of MOT1 abolishes ADI activity and derepresses basal transcription in vitro and in vivo. Recombinant Mot1 removes TBP from DNA and Mot1 contains an ATPase activity which is essential for its function. Genetic interactions between Mot1 and TBP indicate that their functions are interlinked in vivo. These results provide a general model for understanding the mechanism of action of a large family of nuclear factors involved in processes such as transcription and DNA repair.
The yeast activator HAP1--a GAL4 family member--binds DNA in a directly repeated orientation.
Authors: Zhang L , Guarente L
Published: June 2007

The yeast transcriptional activator HAP1 contains a DNA-binding domain homologous to GAL4, PPR1, and related factors. By selecting random HAP1-binding sites, we found that HAP1, like GAL4, binds to two CGG triplets. Unlike GAL4, the CGGs in the HAP1 consensus are in a direct and not inverted orientation. Sites with inverted CGGs were not recovered, and mutations converting the direct repeat of CGGs to an inverted repeat greatly reduce HAP1-binding affinity. Also, the 6-bp spacer between the CGGs contains a consensus TA that is positioned asymmetrically. Dimethylsulfate protection patterns on six of these sites show protections and enhancements that also lie in a directly repeated orientation, suggesting that the two HAP1 DNA recognition domains of a HAP1 homodimer are oriented in a directly repeated configuration on the DNA. Moreover, substitution of the HAP1 dimerization domain with that of PPR1, which forms coiled-coils and dimerizes symmetrically, did not diminish the ability of the protein to bind selectively to a direct repeat. This result suggests that one DNA-binding domain of the HAP1 homodimer must be able to swivel 180 degrees relative to the dimerization domain to make specific contacts with the second CGG triplet. Our results present a novel example of domain swiveling in one of the two identical subunits of a homodimer to accommodate specific DNA contacts to both CGG triplets of a direct repeat.
Homologs of the mouse Brachyury gene are involved in the specification of posterior terminal structures in Drosophila, Tribolium, and Locusta.
Authors: Kispert A , Herrmann B G , Leptin M , Reuter R
Published: June 2007

The Brachyury (T) gene is required for notochord differentiation in vertebrates. We have identified a Drosophila gene, the T-related gene (Trg), with high similarity to T within a stretch of approximately 200 amino acids, the DNA-binding domain of T. Trg is expressed throughout embryogenesis, first at the blastoderm stage in the hindgut primordium under the control of the terminal gap genes tll and hkb, and then until the end of embryogenesis in the differentiating hindgut. Drosophila embryos deficient for Trg do not form the hindgut, a phenotype that can be rescued by a Trg transgene. Thus, a common feature of T and Trg is their requirement in specifying the development of a single embryonic structure. Homologs of Trg are also expressed in the developing hindgut of Tribolium and Locusta embryos suggesting a highly conserved function of Trg in insects. This conservation and the high similarity of T and Trg raise the question of a common evolutionary origin of the hindgut of insects and the notochord of chordates.
Tissue-specific gene activation by MyoD: determination of specificity by cis-acting repression elements.
Authors: Weintraub H , Genetta T , Kadesch T
Published: June 2007

MyoD is a muscle-specific transcriptional activator; E12 is a B-cell activator. An IgH enhancer is activated almost 100-fold by E12 but not at all by Myo; an MCK enhancer is activated almost 1000-fold by MyoD and not at all by E12. MyoD and E12 are both basic helix-loop-helix proteins that bind to similar E-box sequences (CANNTG); the IgH enhancer contains the same E boxes as the MCK enhancer, yet each retains exclusive specificity for either E12 or MyoD, respectively. We show that the IgH enhancer contains a cis-acting negative element that is directed at MyoD, but not at E12. This repression requires the mu E5 E box within the IgH enhancer; however, the specificity for repression, as opposed to activation, is associated with 2 bp flanking each side of the mu E5 E box. The target for repression of MyoD in the IgH enhancer is the bHLH region of MyoD. Our results suggest that MyoD only activates myogenic genes because nonmuscle enhancers that contain E boxes also contain negative elements that prevent MyoD activity.
Transcriptional enhancer factor 1 disruption by a retroviral gene trap leads to heart defects and embryonic lethality in mice.
Authors: Chen Z , Friedrich G A , Soriano P
Published: June 2007

We have used a retroviral gene trap in embryonic stem (ES) cells to derive a recessive embryonic lethal mouse strain, ROSA beta-geo5. Mutant embryos display an enlarged pericardial cavity, bradycardia, a dilated fourth ventricle in the brain, and die between embryonic days 11 and 12. Whereas heart development in the mutant embryos is extensive, the ventricular wall is abnormally thin with a reduced number of trabeculae. Cloning of the trapped gene indicates that proviral insertion creates a null mutation in the transcriptional enhancer factor 1 (TEF-1) gene. Although transcription of a number of muscle-specific genes believed to be TEF-1 targets appears normal, the defect in cardiogenesis is likely attributable to diminished transcription of one or several cardiac-specific genes.
The nuclear receptor steroidogenic factor 1 acts at multiple levels of the reproductive axis.
Authors: Ingraham H A , Lala D S , Ikeda Y , Luo X , Shen W H , Nachtigal M W , Abbud R , Nilson J H , Parker K L
Published: June 2007

Steroidogenic factor 1 (SF-1), an orphan nuclear receptor, regulates the enzymes that produce sex steroids, and disruption of the Ftz-F1 gene encoding SF-1 precludes adrenal and gonadal development. We now study the role of SF-1 at other levels of the hypothalamic/pituitary/gonadal axis. In Ftz-F1-disrupted mice, immunohistochemical analyses with antibodies against pituitary trophic hormones showed a selective loss of gonadotrope-specific markers, supporting the role of SF-1 in gonadotrope function. In situ hybridization analyses confirmed these results; pituitaries from Ftz-F1-disrupted mice lacked transcripts for three gonadotrope-specific markers (LH beta, FSH beta, and the receptor for gonadotropin-releasing hormone), whereas they exhibited decreased but detectable expression of the alpha-subunit of glycoprotein hormones. SF-1 transcripts in the developing mouse pituitary, which first became detectable at embryonic day 13.5-14.5, preceded the appearance of FSH beta and LH beta transcripts. In adult rat pituitary cells, SF-1 transcripts colocalized with immunoreactivity for the gonadotrope-specific LH. Finally, SF-1 interacted with a previously defined promoter element in the glycoprotein hormone alpha-subunit gene, providing a possible mechanism for the impaired gonadotropin expression in Ftz-F1-disrupted mice. These studies establish novel roles of this orphan nuclear receptor in reproductive function.
Suppression of activated Let-60 ras protein defines a role of Caenorhabditis elegans Sur-1 MAP kinase in vulval differentiation.
Authors: Wu Y , Han M
Published: June 2007

The let-60 ras gene of Caenorhabditis elegans is one of the key players in a signal transduction pathway that controls the choice between vulval and epidermal differentiation in response to extracellular signals. To identify components acting downstream of let-60 ras in the vulval signaling pathway, we have identified a reduction-of-function mutation in the sur-1 gene that completely suppresses the multivulva phenotype of a hyperactive let-60 ras mutation. About 10% of animals homozygous for the sur-1 mutation also display a specific and intriguing vulval cell lineage defect. In addition, the sur-1 mutation results in a cold-sensitive egg-laying defective phenotype and a partial larval lethal phenotype. We have cloned the sur-1 gene by DNA-mediated transformation and have shown that it encodes a protein similar in overall structure to mammalian MAP kinases (ERKs). The functional homology between Sur-1 MAP kinase and mammalian MAP kinases was also demonstrated by the ability of a rat ERK2 kinase to rescue the sur-1 mutant phenotypes. Genetic double-mutant analyses place sur-1 downstream of let-60 ras but upstream of lin-1 in the vulval signaling pathway. Our results provide further evidence for the extreme conservation of Ras-mediated signaling pathway between worms and humans and for the function of MAP kinases in cell signaling processes that control cell differentiation and animal development.
Wnt-3a regulates somite and tailbud formation in the mouse embryo.
Authors: Takada S , Stark K L , Shea M J , Vassileva G , McMahon J A , McMahon A P
Published: June 2007

Amphibian studies have implicated Wnt signaling in the regulation of mesoderm formation, although direct evidence is lacking. We have characterized the expression of 12 mammalian Wnt-genes, identifying three that are expressed during gastrulation. Only one of these, Wnt-3a, is expressed extensively in cells fated to give rise to embryonic mesoderm, at egg cylinder stages. A likely null allele of Wnt-3a was generated by gene targeting. All Wnt-3a-/Wnt-3a- embryos lack caudal somites, have a disrupted notochord, and fail to form a tailbud. Thus, Wnt-3a may regulate dorsal (somitic) mesoderm fate and is required, by late primitive steak stages, for generation of all new embryonic mesoderm. Wnt-3a is also expressed in the dorsal CNS. Mutant embryos show CNS dysmorphology and ectopic expression of a dorsal CNS marker. We suggest that dysmorphology is secondary to the mesodermal and axial defects and that dorsal patterning of the CNS may be regulated by inductive signals arising from surface ectoderm.
A new bipartite DNA-binding domain: cooperative interaction between the cut repeat and homeo domain of the cut homeo proteins.
Authors: Andrés V , Chiara M D , Mahdavi V
Published: June 2007

The recently cloned Clox (Cut-like homeo box) and CDP (CCAAT displacement protein), two mammalian counterparts of the Drosophila Cut homeo protein, correspond to alternatively spliced products of the same gene (mClox, for mammalian Cut-like homeo box). Although these proteins reportedly bind to apparently unrelated DNA sequences, we show by in vitro selection of optimal binding sites that both Clox and CDP have the same preferred DNA-binding specificity. The palindromic consensus target sequence, 5'-(t/a)(a/t)tATCGATTAt(t/c)(t/g)(t/a)-3', contains a bona fide homeo domain binding motif (ATTA). In addition, 37% of the in vitro-selected sequences have a CCAAT box, the canonical target for members of the family of CCAAT-binding factors. A characteristic feature of the cut homeo proteins is the presence of three evolutionarily conserved 73-amino-acid repeats of unknown function, the so-called cut repeats. We present evidence that the cut repeat II binds to mClox consensus targets independently of the DNA-binding activity of the homeo domain. In vitro selection of binding sites shows that the optimal targets for the cut repeat II contain one or more CCAAT boxes and, like the homeo domain, an ATTA core. These results indicate that the DNA-binding activity of the second cut repeat can account for the suggested role of CDP mClox as CCAAT displacement protein, a putative repressor of gene expression. We also report that the mClox homeo domain and cut repeat II interact in vitro in the absence of DNA. This interaction, which greatly enhances the DNA-binding activity of the binary complex, is specific to the cut homeo proteins. No cooperativity was observed between the cut repeat II and the homeo domains of Oct-1 and Gtx. Furthermore, the Drosophila cut repeat II, which does not appear to bind to DNA, also enhances the DNA-binding activity of the mClox homeo domain. Thus, the bifunctional cut repeat II, which defines a new family of bipartite DNA-binding proteins, is likely to play an important role in the function of the cut homeo proteins.
The screw gene encodes a ubiquitously expressed member of the TGF-beta family required for specification of dorsal cell fates in the Drosophila embryo.
Authors: Arora K , Levine M S , O'Connor M B
Published: June 2007

The decapentaplegic (dpp) gene product, a TGF-beta related ligand, acts as an extracellular morphogen to establish at least two cellular response thresholds within the dorsal half of the Drosophila embryo. Null mutations in the screw (scw) gene are phenotypically similar to moderate dpp mutants and cause dorsal cells to adopt ventral fates. We show that scw encodes a novel TGF-beta protein and is an integral part of the signal that specifies dorsal pattern. Although scw is expressed uniformly during blastoderm stages, its effect on development appears graded and is restricted to the dorsal side of the embryo. Our results indicate that DPP activity alone is insufficient to specify different dorsal cell fates. We propose that SCW and DPP act together to establish distinct response boundaries within the dorsal half of the embryo, perhaps by forming heterodimers that have higher activity than homodimers of either molecule alone.
Direct control of transcription of the Drosophila morphogen bicoid by the serendipity delta zinc finger protein, as revealed by in vivo analysis of a finger swap.
Authors: Payre F , Crozatier M , Vincent A
Published: June 2007

Determination of the anterior structures of the Drosophila embryo is under control of the maternal gene product Bicoid (bcd), which specifies distinct domains of embryonic gene expression in a concentration-dependent manner. We show here that bcd transcription is controlled by serendipity delta (sry delta), a zygotic-lethal zinc finger protein gene. This sry delta germ-line function was revealed by transgenic expression of a modified Sry delta protein, Sry DB56, carrying a two-finger swap. Although it almost fully rescues sry delta lethality, Sry DB56 does not substitute for the wild-type protein in activating bcd transcription. Two overlapping sites binding the Sry delta protein were identified in the bcd promoter region, a few base pairs upstream of the putative TATA box. Mutating one site impairs bcd transcription in vivo, indicating that Sry delta acts directly upstream of bcd. The specific requirement of sry delta for bcd transcription in the female germ line constitutes an unexpected link between a zygotic gene with pleiotropic functions and the establishment of coordinates of the Drosophila egg. It highlights the fundamental role of ubiquitous transcription factors in bringing about a specific developmental program.
Novel mechanism of C/EBP beta (NF-M) transcriptional control: activation through derepression.
Authors: Kowenz-Leutz E , Twamley G , Ansieau S , Leutz A
Published: June 2007

Phosphorylation of transcription factors is regarded as a major mechanism to control their activity in regulation of gene expression. C/EBP beta is a transcription factor that becomes activated after phosphorylation to induce genes involved in inflammation, acute-phase response, cytokine expression, cell growth, and differentiation. The chicken homolog NF-M collaborates with Myb and various kinase oncogenes in normal myeloid differentiation as well as in the leukemic transformation of myelomonocytic cells. Here, we examined the structure of NF-M and its mechanism of activation. We show that NF-M is a repressed transcription factor with concealed activation potential. Derepressed NF-M exhibits enhanced transcriptional efficacy in reporter assays. More importantly, NF-M activates resident chromatin-embedded, myelomonocyte-specific target genes, even in heterologous cell types such as fibroblasts or erythroblasts. We identified two regions within NF-M that act to repress trans-activation. Repression is abolished by deletion of these regions, activation of signal transduction kinases including v-erbB, polyoma middle T, ras and mil/raf, or point mutation of a critical phosphorylation site for MAP kinases. We provide evidence that phosphorylation plays a unique role to derepress rather than to enhance the trans-activation domain as a novel mechanism to regulate gene expression by NF-M/C/EBP beta.
Intercellular C-signaling and the traveling waves of Myxococcus.
Authors: Sager B , Kaiser D
Published: June 2007

Early in their development into fruiting bodies, Myxococcus xanthus cells organize themselves into dense bands that move as trains of traveling waves. C-factor, a 20-kD cell-surface bound protein, is a short-range developmental signal molecule required for these waves. What is the role of C-factor in the wave pattern? It is proposed that oriented collisions between cells initiate C-signaling, which, in turn, causes individual cells to reverse their direction of gliding. Cells would move about one wavelength and then reverse. Several lines of experimental evidence support these proposals: (1) Cells that suffered a mutation in the signal transduction pathway that controls the spontaneous reversal frequency lost the ability to form waves; (2) presentation of developing cells with detergent-solubilized C-factor increased the mean frequency of single cell reversal by three-fold; and (3) fluorescently labeled cells in the waves were tracked, and it was found that they moved and reversed on linear paths along the axis of wave propagation. Similar numbers of cells were found moving in the direction of ripple propagation, and in the reverse direction, as expected. (4) Dilution of C-signaling-competent cells with C-factor-deficient cells increased the wavelength as the probability of productive collision decreased. The waves exemplify a way that a multicellular pattern of stripes can be produced de novo, one that maintains a uniform 50-microns separation between stripes over a distance as large as 1 cm.
Myc-mediated apoptosis requires wild-type p53 in a manner independent of cell cycle arrest and the ability of p53 to induce p21waf1/cip1.
Authors: Wagner A J , Kokontis J M , Hay N
Published: June 2007

Deregulated expression of the c-myc proto-oncogene can lead to apoptosis under certain physiological conditions. By introducing a conditionally active Myc allele into primary embryo fibroblasts null for p53, and into fibroblasts without endogenous p53 expression but ectopically expressing a temperature-sensitive p53 allele, we show that expression of wild-type p53 is required for susceptibility to Myc-mediated apoptosis. Although ectopic expression of wild-type p53 blocked cells in the G1 phase of the cell cycle, G1 arrest by isoleucine starvation, in a manner independent of p53, did not confer susceptibility to apoptosis. Thus, growth arrest per se is not sufficient to induce Myc-mediated apoptosis; instead, a property intrinsic to p53 is specifically required. Moreover, apoptosis did not require induction of p53 target proteins, including the cyclin-dependent kinase inhibitor p21waf1/cip1. Therefore, the role of p53 in apoptosis may be distinct from its role in cell cycle arrest.
Arabidopsis mutants define downstream branches in the phototransduction pathway.
Authors: Li H M , Altschmied L , Chory J
Published: June 2007

Light regulates the development of Arabidopsis seedlings in a variety of ways, including inhibition of hypocotyl growth and promotion of leaf development, chloroplast differentiation, and light-responsive gene expression. Mutations that uncouple most or all of these responses from light control have been described, for example, det1, det2, and cop1. To identify regulatory components that define downstream branches in the light-regulated signal transduction pathway, mutants specifically affected in only one light-regulated response were isolated. A screen was designed to isolate mutants that overexpressed the CAB (photosystem II type I chlorophyll a/b-binding proteins) genes in the dark, by use of transgenic line containing a T-DNA construct with two CAB3 promoter-reporter fusions. Eight mutants that showed aberrant expression of both CAB3 promoters were isolated and were designated doc mutants (for dark overepression of CAB). All of the mutants have normal etiolated morphology in the dark. Genetic and phenotypic analyses indicate that most of the mutations are recessive and define at least three loci (doc1, doc2, doc3). Unlike det1 and det2 mutants, which affect the expression of CAB and RBCS (the small subunit of RuBP carboxylase) to approximately the same extent, all three doc mutations are much more specific in derepressing the expression of CAB. The phenotypes of doc mutants suggest that morphological changes can be genetically separated from changes in CAB gene expression. Moreover, the regulation of CAB gene expression can be separated further from the regulation of RBCS gene expression. Epistasis studies suggest that DOC1 and DET3 act downstream from DET1 on two separate branches in the phototransduction pathway. In contrast, DOC2 appears to act on a distinct pathway from DET1. Mutations in doc1, doc2, or doc3 also impair plant growth under short-day conditions.
Three levels of functional interaction determine the activity of CCAAT/enhancer binding protein-alpha on the serum albumin promoter.
Authors: Nerlov C , Ziff E B
Published: June 2007

We have studied the activation of the serum albumin promoter by transcription factor CCAAT/enhancer binding protein-alpha (C/EBP alpha) in the HepG2 hepatoma cell line. We find that three distinct mechanisms determine the ability of C/EBP alpha to activate this promoter in a cell-type-specific and cooperative manner. First, the trans-activating function of C/EBP alpha is generated through cooperation between three separate domains of the protein that we have named trans-activation elements (TE-I through TE-III). The TEs have little or no ability to activate transcription by themselves, but any two can cooperate to do so, both in the C/EBP alpha protein and when linked to the GAL4 DNA-binding domain. Second, TE-III was found to contain a negative regulatory subdomain, the function of which was alleviated when C/EBP alpha was bound in the environment of the albumin promoter. This formed the basis for cooperative activation of this promoter by C/EBP alpha. Finally, we demonstrate that the leucine zipper of C/EBP alpha participates in determining the cell type specificity of albumin promoter activation, as it exerts a strong negative effect on albumin promoter activation in the nonhepatic HeLa cell line but not in HepG2 cells. These findings shed new light on the mode of action of C/EBP alpha and show a novel function for leucine zipper in cell-type-specific gene expression.
Cloning and characterization of the G-box binding factor, an essential component of the developmental switch between early and late development in Dictyostelium.
Authors: Schnitzler G R , Fischer W H , Firtel R A
Published: June 2007

During Dictyostelium development, the cAMP-regulated induction of cell-type-specific late genes marks a developmental switch from the initial formation of the multicellular organism to the differentiation of the various cell types that mediate morphogenesis and eventually give rise to the mature fruting body. The G-box binding factor (GBF) is a developmentally regulated Dictyostelium transcription factor whose affinity for a DNA sequence correlates with the ability of that sequence to confer inducibility to late gene promoters in response to high, continuous levels of extracellular cAMP. We report the purification of GBF and cloning of the gene that encodes it, as confirmed by in vitro production of GBF activity. The predicted protein is highly basic and contains two putative zinc fingers. Disruption of the GBF gene by homologous recombination results in the loss of all GBF DNA-binding activity, developmental arrest at the loose aggregate stage, and the loss of late gene induction during development or in response to extracellular cAMP. Constitutive expression of GBF complements the null phenotype and allows for the rapid activation of a class of late genes in response to cAMP. Our results indicate that GBF acts as an extracellular cAMP-responsive transcriptional activator regulating late gene expression and is an essential component of a developmental switch between aggregation and cellular morphogenesis.
NOT1(CDC39), NOT2(CDC36), NOT3, and NOT4 encode a global-negative regulator of transcription that differentially affects TATA-element utilization.
Authors: Collart M A , Struhl K
Published: June 2007

The yeast HIS3 TR and TC TATA elements support basal transcription, but only TR can respond to transcriptional activators. Four genes, NOT1(CDC39), NOT2(CDC36), NOT3, NOT4, act as general negative regulators and preferentially affect TC-dependent transcription. Allele-specific suppression, a two-hybrid interaction, and biochemical confractionation suggest that NOT1 and NOT2 are nuclear proteins associated in a discrete, 500-kD complex. NOT4 interacts with NOT1 and NOT3 in the two-hybrid assay, and overexpression of NOT3 or NOT4 suppresses not1 and not2 mutations. Repression by the NOT proteins is not attributable to inhibition of transcriptional activators, does not involve the CYC8/TUP1 negative regulatory complex, and is distinct from repression by nucleosomes or by the SPT4, 5, 6 proteins that affect chromatin structure. We propose that the NOT protein inhibit the basic RNA polymerase II transcription machinery, possibly by affecting TFIID function.
Isoform-specific amino-terminal domains dictate DNA-binding properties of ROR alpha, a novel family of orphan hormone nuclear receptors.
Authors: Giguère V , Tini M , Flock G , Ong E , Evans R M , Otulakowski G
Published: June 2007

Three isoforms of a novel member of the steroid hormone nuclear receptor superfamily related to the retinoic acid receptors have been identified. The three isoforms, referred to as ROR alpha 1, ROR alpha 2, and ROR alpha 3, share common DNA- and putative ligand-binding domains but are characterized by distinct amino-terminal domains generated by alternative RNA processing. An exon encoding a functionally important subregion of the amino-terminal domain of the ROR alpha 2 isoform resides on the opposite strand of a cytochrome c-processed pseudogene. Binding site selection using in vitro-synthesized proteins reveals that the ROR alpha 1 and ROR alpha 2 isoforms bind DNA as monomers to hormone response elements composed of a 6-bp AT-rich sequence preceding a half-site core motif PuGGTCA (RORE). However, ROR alpha 1 and ROR alpha 2 display different binding specificities: ROR alpha 1 binds to and constitutively activates transcription from a large subset of ROREs, whereas ROR alpha 2 recognizes ROREs with strict specificity and displays weaker transcriptional activity. The differential DNA-binding activity of each isoform maps to their respective amino-terminal domains. Whereas truncation of the amino-terminal domain diminishes the ability of ROR alpha 1 to bind DNA, a similar deletion relaxes ROR alpha 2-binding specificity to that displayed by ROR alpha 1. Remarkably, transfer of the entire amino-terminal region of ROR alpha 1 or amino-terminal deletion of ROR alpha 2 confers RORE-binding specificities to heterologous receptors. These results demonstrate that the amino-terminal domain and the zinc finger region work in concert to confer high affinity and specific DNA-binding properties to the ROR isoforms and suggest a novel strategy to control DNA-binding activity of nuclear receptors.
orb is required for anteroposterior and dorsoventral patterning during Drosophila oogenesis.
Authors: Christerson L B , McKearin D M
Published: June 2007

We describe mutations in the orb gene, identified previously as an ovarian-specific member of a large family of RNA-binding proteins. Strong orb alleles arrest oogenesis prior to egg chamber formation, an early step of oogenesis, whereas females mutant for a maternal-effect lethal orb allele lay eggs with ventralized eggshell structures. Embryos that develop within these mutant eggs display posterior patterning defects and abnormal dorsoventral axis formation. Consistent with such embryonic phenotypes, orb is required for the asymmetric distribution of oskar and gurken mRNAs within the oocyte during the later stages of oogenesis. In addition, double heterozygous combinations of orb and grk or orb and top/DER alleles reveal that mutations in these genes interact genetically, suggesting that they participate in a common pathway. Orb protein, which is localized within the oocyte in wild-type females, is distributed ubiquitously in stage 8-10 orb mutant oocytes. These data will be discussed in the context of a model proposing that Orb is a component of the cellular machinery that delivers mRNA molecules to specific locations within the oocyte and that this function contributes to both D/V and A/P axis specification during oogenesis.
LagC is required for cell-cell interactions that are essential for cell-type differentiation in Dictyostelium.
Authors: Dynes J L , Clark A M , Shaulsky G , Kuspa A , Loomis W F , Firtel R A
Published: June 2007

Strain AK127 is a developmental mutant of Dictyostelium discoideum that was isolated by restriction enzyme-mediated integration (REMI). Mutant cells aggregate normally but are unable to proceed past the loose aggregate stage. The cloned gene, lagC (loose aggregate C), encodes a novel protein of 98 kD that contains an amino-terminal signal sequence and a putative carboxy-terminal transmembrane domain. The mutant strain AK127 shows no detectable lagC transcript upon Northern analysis, indicating that the observed phenotype is that of a null allele. Expression of the lagC cDNA in AK127 cells complements the arrest at the loose aggregate stage, indicating that the mutant phenotype results from disruption of the lagC gene. In wild-type cells, lagC mRNA is induced at the loose aggregate stage and is expressed through the remainder of development. lagC- null cells aggregate but then disaggregate and reaggregate to form small granular mounds. Mature spores are produced at an extremely low efficiency (< 0.1% of wild type), appearing only after approximately 72 hr, whereas wild-type strains produce mature spores by 26 hr. lagC- null cells accumulate reduced levels of transcripts for the prestalk-enriched genes rasD and CP2 and do not express the DIF-induced prestalk-specific gene ecmA or the cAMP-induced prespore-specific gene SP60 to significant levels. In chimeric organisms resulting from the coaggregation of lagC- null and wild-type cells, cell-type-specific gene expression is rescued in the lagC- null cells; however, lagC- prespore cells are localized to the posterior of the prespore region and do not form mature spores, suggesting that LagC protein has both no cell-autonomous and cell-autonomous functions. Overexpression of lagC from an actin promoter in both wild-type and lagC- cells causes a delay at the tight aggregate stage, the first stage requiring LagC activity. These results suggest that the LagC protein functions as a nondiffusible cell-cell signaling molecule that is required for multicellular development.
Cloning of yeast HAP5: a novel subunit of a heterotrimeric complex required for CCAAT binding.
Authors: McNabb D S , Xing Y , Guarente L
Published: June 2007

The CCAAT-binding factor is a conserved heteromeric transcription factor that binds to CCAAT box-containing upstream activation sites (UASs) within the promoters of numerous eukaryotic genes. The CCAAT-binding factor of Saccharomyces cerevisiae activates the transcription of these genes in response to growth in a nonfermentable carbon source. Previous studies have demonstrated that the HAP2, HAP3, and HAP4 subunits of the yeast CCAAT-binding factor are required for the transcriptional activation of genes containing a CCAAT box. Using the two-hybrid screening method, we have identified an additional component of the CCAAT-binding factor. We present the identification and characterization of a novel gene, HAP5, that encodes an additional subunit of the CCAAT-binding factor required for the assembly and DNA-binding activity of the complex. In a hap5 mutant, we show that CCAAT-binding activity is abolished in vitro. Furthermore, we demonstrate that purified recombinant HAP2, HAP3, and HAP5 are able to reconstitute CCAAT-binding activity in mobility shift analysis. These data suggest that the HAP2/3/5 heterotrimer represents a unique DNA-binding factor in which all three subunits of the complex are absolutely required for DNA-binding activity.
Exonucleolytic processing of small nucleolar RNAs from pre-mRNA introns.
Authors: Kiss T , Filipowicz W
Published: June 2007

Many small nucleolar RNAs (snoRNAs) in vertebrates are encoded within introns of protein genes. We have reported previously that two isoforms of human U17 snoRNA are encoded in introns of the cell-cycle regulatory gene, RCC1. We have now investigated the mechanism of processing of U17 RNAs and of another intron-encoded snoRNA, U19. Experiments in which the processing of intronic RNA substrates was tested in HeLa cell extracts suggest that exonucleases rather than endonucleases are involved in the excision of U17 and U19 RNAs: (1) Cutoff products that would be expected from endonucleolytic cleavages were not detected; (2) capping or circularization of substrates inhibited formation of snoRNAs; and (3) U17 RNA was faithfully processed from a substrate carrying unrelated flanking sequences. To study in vivo processing the coding regions of snoRNAs were inserted into intron 2 of the human beta-globin gene. Expression of resulting pre-mRNAs in simian COS cells resulted in formation of correctly processed snoRNAs and of the spliced globin mRNA, demonstrating that snoRNAs can be excised from a nonhost intron and that their sequences contain all the signals essential for accurate processing. When the U17 sequence was placed in a beta-globin exon, no formation of U17 RNA took place, and when two U17 RNA-coding regions were placed in a single intron, doublet U17 RNA molecules accumulated. The results support a model according to which 5'-->3' and 3'-->5' exonucleases are involved in maturation of U17 and U19 RNAs and that excised and debranched introns are the substrates of the processing reaction.
The transcription factor E2F is required for S phase during Drosophila embryogenesis.
Authors: Duronio R J , O'Farrell P H , Xie J E , Brook A , Dyson N
Published: June 2007

Overexpression of the E2F-1 cDNA in mammalian cells disrupts normal control of the cell cycle and drives cells into S phase. Whereas eliminating E2F activity would test its inferred involvement in the G1-S transition, elimination is complicated by the existence of gene families encoding mammalian E2F. Here we identify mutations in a single essential Drosophila gene, dE2F, that encodes a homolog of the mammalian E2F gene family. Embryos homozygous for null mutations of dE2F complete early cell cycles, presumably using maternal contributions of gene products, but DNA synthesis falls to virtually undetectable levels in cycle 17. Mutant embryos also lack the pulses of coordinate transcription of genes encoding replication functions that usually accompany each transition from quiescence to S phase. We conclude that in most cells dE2F is essential for a G1-S transcriptional program and for G1-S progression.
A developmentally regulated chromosomal origin of replication uses essential transcription elements.
Authors: Marczynski G T , Lentine K , Shapiro L
Published: June 2007

Only one of the two chromosomes in the asymmetric Caulobacter predivisional cell initiates replication in the progeny cells. Transcription from a strong promoter within the origin occurs uniquely from the replication-competent chromosome at the stalked pole of the predivisional cell. This regulated promoter has an unusual sequence organization, and transcription from this promoter is essential for regulated (cell type-specific) replication. Our analysis defines a new class of bacterial origins and suggests a coupling between transcription and replication that is consistent with the phylogenetic relationship of Caulobacter to the ancestral mitochondrion.
The protein kinase C-activated MAP kinase pathway of Saccharomyces cerevisiae mediates a novel aspect of the heat shock response.
Authors: Kamada Y , Jung U S , Piotrowski J , Levin D E
Published: June 2007

The PKC1 gene of budding yeast encodes a homolog of the alpha, beta, and gamma isoforms of mammalian PKC that is proposed to regulate a MAPK-activation pathway. Mutants in this pathway undergo cell lysis resulting from a deficiency in cell wall construction when they attempt to grow at elevated temperatures. We show that the PKC1-regulated pathway is important for induced thermotolerance and that the MPK1 protein kinase (the MAPK of this pathway) is strongly activated by mild heat shock. This activation is sustained during growth at high temperature and is dependent on the function of pathway components proposed to function upstream of MPK1, including PKC1. Expression of genes under the control of known heat shock-inducible promoter elements (HSEs and STREs) was not compromised in PKC1 pathway mutants, indicating that this pathway mediates a novel aspect of the yeast heat shock response. We propose that the heat-induced signal for pathway activation is generated in response to weakness in the cell wall created during growth under thermal stress, perhaps as a result of increased membrane fluidity. Evidence is presented that the mechanism by which the cell detects this weakness is by measuring stretch of the plasma membrane.
The head involution defective gene of Drosophila melanogaster functions in programmed cell death.
Authors: Grether M E , Abrams J M , Agapite J , White K , Steller H
Published: June 2007

Deletions of chromosomal region, 75C1,2 block virtually all programmed cell death (PCD) in the Drosophila embryo. We have identified a gene previously in this interval, reaper (rpr), which encodes an important regulator of PCD. Here we report the isolation of a second gene in this region, head involution defective (hid), which plays a similar role in PCD. hid mutant embryos have decreased levels of cell death and contain extra cells in the head. We have cloned the hid gene and find that its expression is sufficient to induce PCD in cell death defective mutants. The hid gene appears to encode a novel 410-amino-acid protein, and its mRNA is expressed in regions of the embryo where cell death occurs. Ectopic expression of hid in the Drosophila retina results in eye ablation. This phenotype can be suppressed completely by expression of the anti-apoptotic p35 protein from baculovirus, indicating that p35 may act genetically downstream from hid.
Ste20-like protein kinases are required for normal localization of cell growth and for cytokinesis in budding yeast.
Authors: Cvrcková F , De Virgilio C , Manser E , Pringle J R , Nasmyth K
Published: June 2007

The yeast Ste20 protein kinase is involved in pheromone response. Mammalian homologs of Ste20 exist, but their function remains unknown. We identified a novel yeast STE20 homolog, CLA4, in a screen for mutations lethal in the absence of the G1 cyclins Cln1 and Cln2. Cla4 is involved in budding and cytokinesis and interacts with Cdc42, a GTPase required for polarized cell growth. Despite a cytokinesis defect, cla4 mutants are viable. However, double cla4 ste20 mutants cannot maintain septin rings at the bud neck and cannot undergo cytokinesis. Mutations in CDC12, which encodes one of the septins, were found in the same screen. Cla4 and Ste20 kinases apparently share a function in localizing cell growth with respect to the septin ring.
Cis-regulation of achaete and scute: shared enhancer-like elements drive their coexpression in proneural clusters of the imaginal discs.
Authors: Gómez-Skarmeta J L , Rodríguez I , Martínez C , Culí J , Ferrés-Marcó D , Beamonte D , Modolell J
Published: June 2007

The pattern of bristles and other sensory organs on the adult cuticle of Drosophila is prefigured in the imaginal discs by the pattern of expression of the proneural achaete (ac) and scute (sc) genes, two members of the ac-sc complex (AS-C). These genes are simultaneously expressed by groups of cells (the proneural clusters) located at constant positions in discs. Their products (transcription factors of the basic-helix-loop-helix family) allow cells to become sensory organ mother cells (SMCs), a fate normally realized by only one or a few cells per cluster. Here we show that the highly complex pattern of proneural clusters is constructed piecemeal, by the action on ac and sc of site-specific, enhancer-like elements distributed along most of the AS-C (approximately 90 kb). Fragments of AS-C DNA containing these enhancers drive reporter lacZ genes in only one or a few proneural clusters. This expression is independent of the ac and sc endogenous genes, indicating that the enhancers respond to local combinations of factors (prepattern). We show further that the cross-activation between ac and sc, discovered by means of transgenes containing either ac or sc promoter fragments linked to lacZ and thought to explain the almost identical patterns of ac and sc expression, does not occur detectably between the endogenous ac and sc genes in most proneural clusters. Our data indicate that coexpression is accomplished by activation of both ac and sc by the same set of position-specific enhancers.
Deletion of beta 1 integrins in mice results in inner cell mass failure and peri-implantation lethality.
Authors: Stephens L E , Sutherland A E , Klimanskaya I V , Andrieux A , Meneses J , Pedersen R A , Damsky C H
Published: June 2007

Integrin receptors for extracellular matrix receptors are important effectors of cell adhesion, differentiation, and migration in cultured cells and are believed to be critical effectors of these processes during development. To determine when beta 1 integrins become critical during embryonic development, we generated mutant mice with a targeted disruption of the beta 1 integrin subunit gene. Heterozygous mutant mice were normal. Homozygous loss of beta 1 integrin expression was lethal during early postimplantation development. Homozygous embryos lacking beta 1 integrins formed normal-looking blastocysts and initiated implantation at E4.5. However, the E4.5 beta 1-null embryos in situ had collapsed blastocoeles, and whereas the trophoblast penetrated the uterine epithelium, extensive invasion of the decidua was not observed. Laminin-positive endoderm cells were detected in the inner cell mass area, but endoderm morphogenesis and migration were defective. By E5.5 beta 1-null embryos had degenerated extensively. In vitro analysis showed that trophoblast function in beta 1-null peri-implantation embryos was largely normal, including expression of tissue-specific markers, and outgrowth on fibronectin- and vitronectin-coated, although not on laminin-coated substrates. In contrast, the inner cell mass region of beta 1-null blastocyst outgrowths, and inner cell masses isolated from beta 1-null blastocysts, showed highly retarded growth and defective extraembryonic endoderm morphogenesis and migration. These data suggest that beta 1 integrins are required for normal morphogenesis of the inner cell mass and are essential mediators of growth and survival of cells of the inner cell mass. Failure of continued trophoblast development in beta 1-null embryos after inner cell mass failure could be attributable to either an intrinsic requirement for beta 1 integrins for later stages of trophoblast development, or to the lack of trophic signals from the beta 1-null inner cell mass.