Mammalian Trithorax and Polycomb-group homologues are antagonistic regulators of homeotic development

Control of cell identity during development is specified in large part by the unique expression patterns of multiple homeobox-containing (Hox) genes in specific segments of an embryo. Trithorax and Polycomb-group (Trx-G and Pc-G) proteins in Drosophila maintain Hox expression or repression, respectively. Mixed lineage leukemia (MLL) is frequently involved in chromosomal translocations associated with acute leukemia and is the one established mammalian homologue of Trx. Bmi-1 was first identified as a collaborator in c-myc-induced murine lymphomagenesis and is homologous to the Drosophila Pc-G member Posterior sex combs. Here, we note the axial-skeletal transformations and altered Hox expression patterns of Mll-deficient and Bmi-1-deficient mice were normalized when both Mll and Bmi-1 were deleted, demonstrating their antagonistic role in determining segmental identity. Embryonic fibroblasts from Mll-deficient compared with Bmi-1-deficient mice demonstrate reciprocal regulation of Hox genes as well as an integrated Hoxc8-lacZ reporter construct. Reexpression of MLL was able to overcome repression, rescuing expression of Hoxc8-lacZ in Mll-deficient cells. Consistent with this, MLL and BMI-I display discrete subnuclear colocalization. Although Drosophila Pc-G and Trx-G members have been shown to maintain a previously established transcriptional pattern, we demonstrate that MLL can also dynamically regulate a target Hox gene.

Beryllofluoride mimics phosphorylation of NtrC and other bacterial response regulators

Two-component systems, sensor kinase-response regulator pairs, dominate bacterial signal transduction. Regulation is exerted by phosphorylation of an Asp in receiver domains of response regulators. Lability of the acyl phosphate linkage has limited structure determination for the active, phosphorylated forms of receiver domains. As assessed by both functional and structural criteria, beryllofluoride yields an excellent analogue of aspartyl phosphate in response regulator NtrC, a bacterial enhancer-binding protein. Beryllofluoride also appears to activate the chemotaxis, sporulation, osmosensing, and nitrate/nitrite response regulators CheY, Spo0F, OmpR, and NarL, respectively. NMR spectroscopic studies indicate that beryllofluoride will facilitate both biochemical and structural characterization of the active forms of receiver domains.

14-3-3 Proteins Act as Negative Regulators of the Mitotic Inducer Cdc25 in Xenopus Egg Extracts

Cdc25, the dual-specificity phosphatase that dephosphorylates the Cdc2–cyclin B complex at mitosis, is highly regulated during the cell cycle. In Xenopus egg extracts, Cdc25 is associated with two isoforms of the 14-3-3 protein. Cdc25 is complexed primarily with 14-3-3ε and to a lesser extent with 14-3-3ζ. The association of these 14-3-3 proteins with Cdc25 varies dramatically during the cell cycle: binding is high during interphase but virtually absent at mitosis. Interaction with 14-3-3 is mediated by phosphorylation of Xenopus Cdc25 at Ser-287, which resides in a consensus 14-3-3 binding site. Recombinant Cdc25 with a point mutation at this residue (Cdc25-S287A) is incapable of binding to 14-3-3. Addition of the Cdc25-S287A mutant to Xenopus egg extracts accelerates mitosis and overrides checkpoint-mediated arrests of mitotic entry due to the presence of unreplicated and damaged DNA. These findings indicate that 14-3-3 proteins act as negative regulators of Cdc25 in controlling the G2–M transition.

Identification of Regulators for Ypt1 GTPase Nucleotide Cycling

Small GTPases of the Ypt/Rab family are involved in the regulation of vesicular transport. Cycling between the GDP- and GTP-bound forms and the accessory proteins that regulate this cycling are thought to be crucial for Ypt/Rab function. Guanine nucleotide exchange factors (GEFs) stimulate both GDP loss and GTP uptake, and GTPase-activating proteins (GAPs) stimulate GTP hydrolysis. Little is known about GEFs and GAPs for Ypt/Rab proteins. In this article we report the identification and initial characterization of two factors that regulate nucleotide cycling by Ypt1p, which is essential for the first two steps of the yeast secretory pathway. The Ypt1p-GEF stimulates GDP release and GTP uptake at least 10-fold and is specific for Ypt1p. Partially purified Ypt1p-GEF can rescue the inhibition caused by the dominant-negative Ypt1p-D124N mutant of in vitro endoplasmic reticulum-to-Golgi transport. This mutant probably blocks transport by inhibiting the GEF, suggesting that we have identified the physiological GEF for Ypt1p. The Ypt1p-GAP stimulates GTP hydrolysis by Ypt1p up to 54-fold, has a higher affinity for the GTP-bound form of Ypt1p than for the GDP-bound form, and is specific to a subgroup of exocytic Ypt proteins. The Ypt1p-GAP activity is not affected by deletion of two genes that encode known Ypt GAPs, GYP7 and GYP1, nor is it influenced by mutations in SEC18, SEC17, or SEC22, genes whose products are involved in vesicle fusion. The GEF and GAP activities for Ypt1p localize to particulate cellular fractions. However, contrary to the predictions of current models, the GEF activity localizes to the fraction that functions as the acceptor in an endoplasmic reticulum-to-Golgi transport assay, whereas the GAP activity cofractionates with markers for the donor. On the basis of our current and previous results, we propose a new model for the role of Ypt/Rab nucleotide cycling and the factors that regulate this process.

A T42A Ran Mutation: Differential Interactions with Effectors and Regulators, and Defect in Nuclear Protein Import

Ran, the small, predominantly nuclear GTPase, has been implicated in the regulation of a variety of cellular processes including cell cycle progression, nuclear-cytoplasmic trafficking of RNA and protein, nuclear structure, and DNA synthesis. It is not known whether Ran functions directly in each process or whether many of its roles may be secondary to a direct role in only one, for example, nuclear protein import. To identify biochemical links between Ran and its functional target(s), we have generated and examined the properties of a putative Ran effector mutation, T42A-Ran. T42A-Ran binds guanine nucleotides as well as wild-type Ran and responds as well as wild-type Ran to GTP or GDP exchange stimulated by the Ran-specific guanine nucleotide exchange factor, RCC1. T42A-Ran·GDP also retains the ability to bind p10/NTF2, a component of the nuclear import pathway. In contrast to wild-type Ran, T42A-Ran·GTP binds very weakly or not detectably to three proposed Ran effectors, Ran-binding protein 1 (RanBP1), Ran-binding protein 2 (RanBP2, a nucleoporin), and karyopherin β (a component of the nuclear protein import pathway), and is not stimulated to hydrolyze bound GTP by Ran GTPase-activating protein, RanGAP1. Also in contrast to wild-type Ran, T42A-Ran does not stimulate nuclear protein import in a digitonin permeabilized cell assay and also inhibits wild-type Ran function in this system. However, the T42A mutation does not block the docking of karyophilic substrates at the nuclear pore. These properties of T42A-Ran are consistent with its classification as an effector mutant and define the exposed region of Ran containing the mutation as a probable effector loop.

The tomato ethylene receptors NR and LeETR4 are negative regulators of ethylene response and exhibit functional compensation within a multigene family

The plant hormone ethylene is involved in many developmental processes, including fruit ripening, abscission, senescence, and leaf epinasty. Tomato contains a family of ethylene receptors, designated LeETR1, LeETR2, NR, LeETR4, and LeETR5, with homology to the Arabidopsis ETR1 ethylene receptor. Transgenic plants with reduced LeETR4 gene expression display multiple symptoms of extreme ethylene sensitivity, including severe epinasty, enhanced flower senescence, and accelerated fruit ripening. Therefore, LeETR4 is a negative regulator of ethylene responses. Reduced expression of this single gene affects multiple developmental processes in tomato, whereas in Arabidopsis multiple ethylene receptors must be inactivated to increase ethylene response. Transgenic lines with reduced NR mRNA levels exhibit normal ethylene sensitivity but elevated levels of LeETR4 mRNA, indicating a functional compensation of LeETR4 for reduced NR expression. Overexpression of NR in lines with lowered LeETR4 gene expression eliminates the ethylene-sensitive phenotype, indicating that despite marked differences in structure these ethylene receptors are functionally redundant.

The LIM-domain binding protein Ldb1 and its partner LMO2 act as negative regulators of erythroid differentiation

The nuclear LIM domain protein LMO2, a T cell oncoprotein, is essential for embryonic erythropoiesis. LIM-only proteins are presumed to act primarily through protein-protein interactions. We, and others, have identified a widely expressed protein, Ldb1, whose C-terminal 76-residues are sufficient to mediate interaction with LMO2. In murine erythroleukemia cells, the endogenous Lbd1 and LMO2 proteins exist in a stable complex, whose binding affinity appears greater than that between LMO2 and the bHLH transcription factor SCL. However, Ldb1, LMO2, and SCL/E12 can assemble as a multiprotein complex on a consensus SCL binding site. Like LMO2, the Ldb1 gene is expressed in fetal liver and erythroid cell lines. Forced expression of Ldb1 in G1ER proerythroblast cells inhibited cellular maturation, a finding compatible with the decrease in Ldb1 gene expression that normally occurs during erythroid differentiation. Overexpression of the LMO2 gene also inhibited erythroid differentiation. Our studies demonstrate a function for Ldb1 in hemopoietic cells and suggest that one role of the Ldb1/LMO2 complex is to maintain erythroid precursors in an immature state.

Plastid redox state and sugars: Interactive regulators of nuclear-encoded photosynthetic gene expression

Feedback regulation of photosynthesis by carbon metabolites has long been recognized, but the underlying cellular mechanisms that control this process remain unclear. By using an Arabidopsis cell culture, we show that a block in photosynthetic electron flux prevents the increase in transcript levels of chlorophyll a/b-binding protein and the small subunit of Rubisco that typically occurs when intracellular sugar levels are depleted. In contrast, the expression of the nitrate reductase gene, which is induced by sugars, is not affected. These findings were confirmed in planta by using Arabidopsis carrying the firefly luciferase reporter gene fused to the plastocyanin and chlorophyll a/b-binding protein 2 gene promoters. Transcription from both promoters increases on carbohydrate depletion. Blocking photosynthetic electron transport with 3-(3′, 4′-dichlorophenyl)-1,1′-dimethylurea prevents this increase in transcription. We conclude that plastid-derived redox signaling can override the sugar-regulated expression of nuclear-encoded photosynthetic genes. In the sugar-response mutant, sucrose uncoupled 6 (sun6), plastocyanin-firefly luciferase transcription actually increases in response to exogenous sucrose rather than decreasing as in the wild type. Interestingly, plastid-derived redox signals do not influence this defective pattern of sugar-regulated gene expression in the sun6 mutant. A model, which invokes a positive inducer originating from the photosynthetic electron transport chain, is proposed to explain the nature of the plastid-derived signal.

Selection for in vivo regulators of bacterial virulence

We devised a noninvasive genetic selection strategy to identify positive regulators of bacterial virulence genes during actual infection of an intact animal host. This strategy combines random mutagenesis with a switch-like reporter of transcription that confers antibiotic resistance in the off state and sensitivity in the on state. Application of this technology to the human intestinal pathogen Vibrio cholerae identified several regulators of cholera toxin and a central virulence gene regulator that are operative during infection. These regulators function in chemotaxis, signaling pathways, transport across the cell envelope, biosynthesis, and adherence. We show that phenotypes that appear genetically independent in cell culture become interrelated in the host milieu.

The Friend of GATA proteins U-shaped, FOG-1, and FOG-2 function as negative regulators of blood, heart, and eye development in Drosophila

Friend of GATA (FOG) proteins regulate GATA factor-activated gene transcription. During vertebrate hematopoiesis, FOG and GATA proteins cooperate to promote erythrocyte and megakaryocyte differentiation. The Drosophila FOG homologue U-shaped (Ush) is expressed similarly in the blood cell anlage during embryogenesis. During hematopoiesis, the acute myeloid leukemia 1 homologue Lozenge and Glial cells missing are required for the production of crystal cells and plasmatocytes, respectively. However, additional factors have been predicted to control crystal cell proliferation. In this report, we show that Ush is expressed in hemocyte precursors and plasmatocytes throughout embryogenesis and larval development, and the GATA factor Serpent is essential for Ush embryonic expression. Furthermore, loss of ush function results in an overproduction of crystal cells, whereas forced expression of Ush reduces this cell population. Murine FOG-1 and FOG-2 also can repress crystal cell production, but a mutant version of FOG-2 lacking a conserved motif that binds the corepressor C-terminal binding protein fails to affect the cell lineage. The GATA factor Pannier (Pnr) is required for eye and heart development in Drosophila. When Ush, FOG-1, FOG-2, or mutant FOG-2 is coexpressed with Pnr during these developmental processes, severe eye and heart phenotypes result, consistent with a conserved negative regulation of Pnr function. These results indicate that the fly and mouse FOG proteins function similarly in three distinct cellular contexts in Drosophila, but may use different mechanisms to regulate genetic events in blood vs. cardial or eye cell lineages.

Dual-function regulators: the cAMP receptor protein and the CytR regulator can act either to repress or to activate transcription depending on the context.

Studies of gene regulation have revealed that several transcriptional regulators can switch between activator and repressor depending upon both the promoter and the cellular context. A relatively simple prokaryotic example is illustrated by the Escherichia coli CytR regulon. In this system, the cAMP receptor protein (CRP) assists the binding of RNA polymerase as well as a specific negative regulator, CytR. Thus, CRP functions either as an activator or as a corepressor. Here we show that, depending on promoter architecture, the CRP/CytR nucleoprotein complex has opposite effects on transcription. When acting from a site close to the DNA target for RNA polymerase, CytR interacts with CRP to repress transcription, whereas an interaction with CRP from appropriately positioned upstream binding sites can result in formation of a huge preinitiation complex and transcriptional activation. Based on recent results about CRP-mediated regulation of transcription initiation and the finding that CRP possesses discrete surface-exposed patches for protein-protein interaction with RNA polymerase and CytR, a molecular model for this dual regulation is discussed.

Arachidonate lipoxygenases as essential regulators of cell survival and apoptosis.

Arachidonic acid (AA) metabolites derived from both cyclooxygenase (COX) and lipoxygenase (LOX) pathways transduce a variety of signals related to cell growth. Here, we report that the AA LOX pathway also functions as a critical regulator of cell survival and apoptosis. Rat Walker 256 (W256) carcinosarcoma cells express 12-LOX and synthesize 12(S)- and 15(S)-hydroxyeicosatetraenoic acids as their major LOX metabolites. W256 cells transfected with 12-LOX-specific antisense oligonucleotide or antisense oligonucleotides directed to conserved regions of LOXs underwent time- and dose-dependent apoptosis. Likewise, treatment of W256 cells with various LOX but not COX inhibitors induced apoptotic cell death, which could be partially inhibited by exogenous 12(S)- or 15(S)-hydroxyeicosatetraenoic acids. The W256 cell apoptosis induced by antisense oligos and LOX inhibitors was followed by a rapid downregulation of bcl-2 protein, a dramatic decrease in the bcl-2/bax ratio, and could be suppressed by bcl-2 overexpression. In contrast, p53, which is wild type in W256 cells, did not undergo alterations during apoptosis induction. The results suggest that the LOX pathway plays an important physiological role in regulating apoptosis.

Transposon tools for recombinant DNA manipulation: characterization of transcriptional regulators from yeast, Xenopus, and mouse.

Transposon Tn1000 has been adapted to deliver novel DNA sequences for manipulating recombinant DNA. The transposition procedure for these "tagged" Tn1000s is simple and applicable to most plasmids in current use. For yeast molecular biology, tagged Tn1000s introduce a variety of yeast selective markers and replication origins into plasmids and cosmids. In addition, the beta-globin minimal promoter and lacZ gene of Tn(beta)lac serve as a mobile reporter of eukaryotic enhancer activity. In this paper, Tn(beta)lac was used to localize a mouse HoxB-complex enhancer in transgenic mice. Other tagged transposons create Gal4 DNA-binding-domain fusions, in either Escherichia coli or yeast plasmids, for use in one- and two-hybrid tests of transcriptional activation and protein-protein interaction, respectively. With such fusions, the Saccharomyces cerevisiae Swi6 G1/S-phase transcription factor and the Xenopus laevis Pintallavis developmental regulator are shown to activate transcription. Furthermore, the same transposon insertions also facilitated mapping of the Swi6 and Pintallavis domains responsible for transcriptional activation. Thus, as well as introducing novel sequences, tagged transposons share the numerous other applications of transposition such as producing insertional mutations, creating deletion series, or serving as mobile primer sites for DNA sequencing.

Orp1, a member of the Cdc18/Cdc6 family of S-phase regulators, is homologous to a component of the origin recognition complex.

cdc18+ of Schizosaccharomyces pombe is a periodically expressed gene that is required for entry into S phase and for the coordination of S phase with mitosis. cdc18+ is related to the Saccharomyces cerevisiae gene CDC6, which has also been implicated in the control of DNA replication. We have identified a new Sch. pombe gene, orp1+, that encodes an 80-kDa protein with amino acid sequence motifs conserved in the Cdc18 and Cdc6 proteins. Genetic analysis indicates that orp1+ is essential for viability. Germinating spores lacking the orp1+ gene are capable of undergoing one or more rounds of DNA replication but fail to progress further, arresting as long cells with a variety of deranged nuclear structures. Unlike cdc18+, orp1+ is expressed constitutively during the cell cycle. cdc18+, CDC6, and orp1+ belong to a family of related genes that also includes the gene ORC1, which encodes a subunit of the origin recognition complex (ORC) of S. cerevisiae. The products of this gene family share a 250-amino acid domain that is highly conserved in evolution and contains several characteristic motifs, including a consensus purine nucleotide-binding motif. Among the members of this gene family, orp1+ is most closely related to S. cerevisiae ORC1. Thus, the protein encoded by orp1+ may represent a component of an Sch. pombe ORC. The orp1+ gene is also closely related to an uncharacterized putative human homologue. It is likely that the members of the cdc18/CDC6 family play key roles in the regulation of DNA replication during the cell cycle of diverse species from archaebacteria to man.

Yeast global transcriptional regulators Sin4 and Rgr1 are components of mediator complex/RNA polymerase II holoenzyme.

Sin4 and Rgr1 proteins, previously shown by genetic studies to play both positive and negative roles in the transcriptional regulation of many genes, are identified here as components of mediator and RNA polymerase II holoenzyme complexes. Results with Sin4 deletion and Rgr1 truncation strains indicate the association of these proteins in a subcomplex comprising Sin4, Rgr1, Gal11, and a 50-kDa polypeptide. Taken together with the previous genetic evidence, our findings point to a role of the mediator in repression as well as in transcriptional activation.