A pair of adjacent glucocorticoid response elements regulate expression of two mouse metallothionein genes

Synthesis of mouse metallothionein (MT)-I and MT-II is transcriptionally induced by the synthetic glucocorticoid, dexamethasone (DEX) or both in vivo as well as in numerous cell lines. However, the location(s) of a glucocorticoid response element (GRE) has not been described. The observation that a marked MT-I gene, as well as heterologous genes, when placed in the context of 17 kb of flanking sequence from the MT locus, are inducible by DEX and lipopolysaccharide in transgenic mice renewed the search for the GRE. Analysis of a series of deletion constructs from this 17-kb region in cultured cells identified a single 455-bp region that conferred DEX induction on a reporter gene. This 455-bp region contains two GREs that bind to the glucocorticoid receptor as assessed by gel mobility shift. Deletion of this fragment from the 17-kb flanking region eliminates the DEX responsiveness of reporter genes. The two GREs, which are located ≈1 kb upstream of the MT-II gene and ≈7 kb upstream of the MT-I gene, are necessary for induction of both genes and can function independently of elements within the proximal promoter region of either gene.

Association of CYP3A4 genotype with treatment-related leukemia

Epipodophyllotoxins are associated with leukemias characterized by translocations of the MLL gene at chromosome band 11q23 and other translocations. Cytochrome P450 (CYP) 3A metabolizes epipodophyllotoxins and other chemotherapeutic agents. CYP3A metabolism generates epipodophyllotoxin catechol and quinone metabolites, which could damage DNA. There is a polymorphism in the 5′ promoter region of the CYP3A4 gene (CYP3A4-V) that might alter the metabolism of anticancer drugs. We examined 99 de novo and 30 treatment-related leukemias with a conformation-sensitive gel electrophoresis assay for the presence of the CYP3A4-V. In all treatment-related cases, there was prior exposure to one or more anticancer drugs metabolized by CYP3A. Nineteen of 99 de novo (19%) and 1 of 30 treatment-related (3%) leukemias carried the CYP3A4-V (P = 0.026; Fisher’s Exact Test, FET). Nine of 42 de novo leukemias with MLL gene translocations (21%), and 0 of 22 treatment-related leukemias with MLL gene translocations carried the CYP3A4-V (P = 0.016, FET). This relationship remained significant when 19 treatment-related leukemias with MLL gene translocations that followed epipodophyllotoxin exposure were compared with the same 42 de novo cases (P = 0.026, FET). These data suggest that individuals with CYP3A4-W genotype may be at increased risk for treatment-related leukemia and that epipodophyllotoxin metabolism by CYP3A4 may contribute to the secondary cancer risk. The CYP3A4-W genotype may increase production of potentially DNA-damaging reactive intermediates. The variant may decrease production of the epipodophyllotoxin catechol metabolite, which is the precursor of the potentially DNA-damaging quinone.

The pir gene of Erwinia chrysanthemi EC16 regulates hyperinduction of pectate lyase virulence genes in response to plant signals

The plant pathogenic bacterium Erwinia chrysanthemi secretes pectate lyase proteins that are important virulence factors attacking the cell walls of plant hosts. Bacterial production of these enzymes is induced by the substrate polypectate-Na (NaPP) and further stimulated by the presence of plant extracts. The bacterial regulator responsible for induction by plant extracts was identified and purified by using a DNA-binding assay with the promoter region of pelE that encodes a major pectate lyase. A novel bacterial protein, called Pir, was isolated that produced a specific gel shift of the pelE promoter DNA, and the corresponding pir gene was cloned and sequenced. The Pir protein contains 272 amino acids with a molecular mass of 30 kDa and appears to function as a dimer. A homology search indicates that Pir belongs to the IclR family of transcriptional regulators. Pir bound to a 35-bp DNA sequence in the promoter region of pelE. This site overlaps that of a previously described negative regulator, KdgR. Gel shift experiments showed that the binding of either Pir or KdgR interfered with binding of the other protein.

Transcriptional coupling between the divergent promoters of a prototypic LysR-type regulatory system, the ilvYC operon of Escherichia coli

The twin-domain model [Liu, L. F. & Wang, J. C. (1987) Proc. Natl. Acad. Sci. USA 84, 7024–7027] suggests that closely spaced, divergent, superhelically sensitive promoters can affect the transcriptional activity of one another by transcriptionally induced negative DNA supercoiling generated in the divergent promoter region. This gene arrangement is observed for many LysR-type-regulated operons in bacteria. We have examined the effects of divergent transcription in the prototypic LysR-type system, the ilvYC operon of Escherichia coli. Double-reporter constructs with the lacZ gene under transcriptional control of the ilvC promoter and the galK gene under control of the divergent ilvY promoter were used to demonstrate that a down-promoter mutation in the ilvY promoter severely decreases in vivo transcription from the ilvC promoter. However, a down-promoter mutation in the ilvC promoter only slightly affects transcription from the ilvY promoter. In vitro transcription assays with DNA topoisomers showed that transcription from the ilvC promoter increases over the entire range of physiological superhelical densities, whereas transcription initiation from the ilvY promoter exhibits a broad optimum at a midphysiological superhelical density. Evidence that this promoter coupling is DNA supercoiling-dependent is provided by the observation that a novobiocin-induced decrease in global negative superhelicity results in an increase in ilvY promoter activity and a decrease in ilvC promoter activity predicted by the in vitro data. We suggest that this transcriptional coupling is important for coordinating basal level expression of the ilvYC operon with the nutritional and environmental conditions of cell growth.

Transcriptional Regulation of Fibroblast Growth Factor-2 Expression in Human Astrocytes: Implications for Cell Plasticity

Induction of the fibroblast growth factor-2 (FGF-2) gene and the consequent accumulation of FGF-2 in the nucleus are operative events in mitotic activation and hypertrophy of human astrocytes. In the brain, these events are associated with cellular degeneration and may reflect release of the FGF-2 gene from cell contact inhibition. We used cultures of human astrocytes to examine whether expression of FGF-2 is also controlled by soluble growth factors. Treatment of subconfluent astrocytes with interleukin-1β, epidermal or platelet-derived growth factors, 18-kDa FGF-2, or serum or direct stimulation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate or adenylate cyclase with forskolin increased the levels of 18-, 22-, and 24-kDa FGF-2 isoforms and FGF-2 mRNA. Transfection of FGF-2 promoter–luciferase constructs identified a unique −555/−513 bp growth factor-responsive element (GFRE) that confers high basal promoter activity and activation by growth factors to a downstream promoter region. It also identified a separate region (−624/−556 bp) essential for PKC and cAMP stimulation. DNA–protein binding assays indicated that novel cis-acting elements and trans-acting factors mediate activation of the FGF-2 gene. Southwestern analysis identified 40-, 50-, 60-, and 100-kDa GFRE-binding proteins and 165-, 112-, and 90-kDa proteins that interacted with the PKC/cAMP-responsive region. The GFRE and the element essential for PKC and cAMP stimulation overlap with the region that mediates cell contact inhibition of the FGF-2 promoter. The results show a two-stage regulation of the FGF-2 gene: 1) an initial induction by reduced cell contact, and 2) further activation by growth factors or the PKC-signaling pathway. The hierarchic regulation of the FGF-2 gene promoter by cell density and growth factors or PKC reflects a two-stage activation of protein binding to the GFRE and to the PKC/cAMP-responsive region, respectively.

Myeloblastin is a granulocyte colony-stimulating factor-responsive gene conferring factor-independent growth to hematopoietic cells

Hematopoiesis depends on a pool of quiescent hematopoietic stem/progenitor cells. When exposed to specific cytokines, a portion of these cells enters the cell cycle to generate an amplified progeny. Myeloblastin (MBN) initially was described as involved in proliferation of human leukemia cells. The granulocyte colony-stimulating factor (G-CSF), which stimulates the proliferation of granulocytic precursors, up-regulates MBN expression. Here we show that constitutive overexpression of MBN confers factor-independent growth to murine bone marrow-derived Ba/F3/G-CSFR cells. Our results point to MBN as a G-CSF responsive gene critical to factor-independent growth and indicate that expression of the G-CSF receptor is a prerequisite to this process. A 91-bp MBN promoter region containing PU.1, C/EBP, and c-Myb binding sites is responsive to G-CSF treatment. Although PU.1, C/EBP, and c-Myb transcription factors all were critical for expression of MBN, its up-regulation by G-CSF was associated mainly with PU.1. These findings suggest that MBN is an important target of PU.1 and a key protease for factor-independent growth of hematopoietic cells.

Overexpression of Mdm2 in mice reveals a p53-independent role for Mdm2 in tumorigenesis

The Mdm2 proto-oncogene is amplified to high copy numbers in human sarcomas and is overexpressed in a wide variety of other human cancers. Because Mdm2 protein forms a complex with the p53 tumor suppressor protein and down-regulates p53 function, the oncogenic potential of Mdm2 is presumed to be p53-dependent. To model these conditions in mice, we have used the entire Mdm2 gene, under transcriptional control of its native promoter region, as a transgene to create mice that overexpress Mdm2. The transgenic mice are predisposed to spontaneous tumor formation, and the incidence of sarcomas observed in the Mdm2-transgenic mice in the presence or absence of functional p53 demonstrates that, in addition to Mdm2-mediated inactivation of p53, there exists a p53-independent role for Mdm2 in tumorigenesis.

In vitro roles of invariant helix–turn–helix motif residue R383 in σ54 (σN)

In vitro DNA-binding and transcription properties of σ54 proteins with the invariant Arg383 in the putative helix–turn–helix motif of the DNA-binding domain substituted by lysine or alanine are described. We show that R383 contributes to maintaining stable holoenzyme–promoter complexes in which limited DNA opening downstream of the –12 GC element has occurred. Unlike wild-type σ54, holoenzymes assembled with the R383A or R383K mutants could not form activator-independent, heparin-stable complexes on heteroduplex Sinorhizobium meliloti nifH DNA mismatched next to the GC. Using longer sequences of heteroduplex DNA, heparin-stable complexes formed with the R383K and, to a lesser extent, R383A mutant holoenzymes, but only when the activator and a hydrolysable nucleotide was added and the DNA was opened to include the –1 site. Although R383 appears inessential for polymerase isomerisation, it makes a significant contribution to maintaining the holoenzyme in a stable complex when melting is initiating next to the GC element. Strikingly, Cys383-tethered FeBABE footprinting of promoter DNA strongly suggests that R383 is not proximal to promoter DNA in the closed complex. This indicates that R383 is not part of the regulatory centre in the σ54 holoenzyme, which includes the –12 promoter region elements. R383 contributes to several properties, including core RNA polymerase binding and to the in vivo stability of σ54.

PromEC: An updated database of Escherichia coli mRNA promoters with experimentally identified transcriptional start sites

PromEC is an updated compilation of Escherichia coli mRNA promoter sequences. It includes documentation on the location of experimentally identified mRNA transcriptional start sites on the E.coli chromosome, as well as the actual sequences in the promoter region. The database was updated as of July 2000 and includes 472 entries. PromEC is accessible at http://bioinfo.md.huji.ac.il/marg/promec

Establishment of medaka (Oryzias latipes) transgenic lines with the expression of green fluorescent protein fluorescence exclusively in germ cells: A useful model to monitor germ cells in a live vertebrate

We have generated transgenic medaka (teleost, Oryzias latipes), which allow us to monitor germ cells by green fluorescent protein (GFP) fluorescence in live specimens. Two medaka strains, himedaka (orange–red variety) and inbred QurtE, were used. The transgenic lines were achieved by microinjection of a construct containing the putative promoter region and 3′ region of the medaka vasa gene (olvas). The intensity of GFP fluorescence increases dramatically in primordial germ cells (PGCs) located in the ventrolateral region of the posterior intestine around stage 25 (the onset of blood circulation). Whole-mount in situ hybridization and monitoring of ectopically located cells by GFP fluorescence suggested that (i) the increase in zygotic olvas expression occurs after PGC specification and (ii) PGCs can maintain their cell characteristics ectopically after stages 20–25. Around the day of hatching, the QurtE strain clearly exhibits sexual dimorphisms in the number of GFP fluorescent germ cells, a finding consistent with the appearance of leucophores, a sex-specific marker of QurtE. The GFP expression persists throughout the later stages in the mature ovary and testis. Thus, these transgenic medaka represent a live vertebrate model to investigate how germ cells migrate to form sexually dimorphic gonads, as well as a potential assay system for environmental substances that may affect gonad development. The use of a transgenic construct as a selective marker to efficiently isolate germ-line-transmitting founders during embryogenesis is also discussed.

The nucleoporin Nup98 associates with the intranuclear filamentous protein network of TPR

The Nup98 gene codes for several alternatively spliced protein precursors. Two in vitro translated and autoproteolytically cleaved precursors yielded heterodimers of Nup98-6kDa peptide and Nup98-Nup96. TPR (translocated promoter region) is a protein that forms filamentous structures extending from nuclear pore complexes (NPCs) to intranuclear sites. We found that in vitro translated TPR bound to in vitro translated Nup98 and, via Nup98, to Nup96. Double-immunofluorescence microscopy with antibodies to TPR and Nup98 showed colocalization. In confocal sections the nucleolus itself was only weakly stained but there was intensive perinucleolar staining. Striking spike-like structures emanated from this perinucleolar ring and attenuated into thinner structures as they extended to the nuclear periphery. This characteristic staining pattern of the TPR network was considerably enhanced when a myc-tagged pyruvate kinase-6kDa fusion protein was overexpressed in HeLa cells. Double-immunoelectron microscopy of these cells using anti-myc and anti-TPR antibodies and secondary gold-coupled antibodies yielded row-like arrangements of gold particles. Taken together, the immunolocalization data support previous electron microscopical data, suggesting that TPR forms filaments that extend from the NPC to the nucleolus. We discuss the possible implications of the association of Nup98 with this intranuclear TPR network for an intranuclear phase of transport.

A Single Limit Dextrinase Gene Is Expressed Both in the Developing Endosperm and in Germinated Grains of Barley1

The single gene encoding limit dextrinase (pullulan 6-glucanohydrolase; EC 3.2.1.41) in barley (Hordeum vulgare) has 26 introns that range in size from 93 to 822 base pairs. The mature polypeptide encoded by the gene has 884 amino acid residues and a calculated molecular mass of 97,417 D. Limit dextrinase mRNA is abundant in gibberellic acid-treated aleurone layers and in germinated grain. Gibberellic acid response elements were found in the promoter region of the gene. These observations suggest that the enzyme participates in starch hydrolysis during endosperm mobilization in germinated grain. The mRNA encoding the enzyme is present at lower levels in the developing endosperm of immature grain, a location consistent with a role for limit dextrinase in starch synthesis. Enzyme activity was also detected in developing grain. The limit dextrinase has a presequence typical of transit peptides that target nascent polypeptides to amyloplasts, but this would not be expected to direct secretion of the mature enzyme from aleurone cells in germinated grain. It remains to be discovered how the enzyme is released from the aleurone and whether another enzyme, possibly of the isoamylase group, might be equally important for starch hydrolysis in germinated grain.

Oncolytic herpes simplex virus vector with enhanced MHC class I presentation and tumor cell killing

Oncolytic herpes simplex virus type 1 (HSV-1) vectors are promising therapeutic agents for cancer. Their efficacy depends on the extent of both intratumoral viral replication and induction of a host antitumor immune response. To enhance these properties while employing ample safeguards, two conditionally replicating HSV-1 vectors, termed G47Δ and R47Δ, have been constructed by deleting the α47 gene and the promoter region of US11 from γ34.5-deficient HSV-1 vectors, G207 and R3616, respectively. Because the α47 gene product is responsible for inhibiting the transporter associated with antigen presentation (TAP), its absence led to increased MHC class I expression in infected human cells. Moreover, some G47Δ-infected human melanoma cells exhibited enhanced stimulation of matched antitumor T cell activity. The deletion also places the late US11 gene under control of the immediate-early α47 promoter, which suppresses the reduced growth properties of γ34.5-deficient mutants. G47Δ and R47Δ showed enhanced viral growth in a variety of cell lines, leading to higher virus yields and enhanced cytopathic effect in tumor cells. G47Δ was significantly more efficacious in vivo than its parent G207 at inhibiting tumor growth in both immune-competent and immune-deficient animal models. Yet, when inoculated into the brains of HSV-1-sensitive A/J mice at 2 × 106 plaque forming units, G47Δ was as safe as G207. These results suggest that G47Δ may have enhanced antitumor activity in humans.

Crystal structure of the SarR protein from Staphylococcus aureus

The expression of virulence determinants in Staphylococcus aureus is controlled by global regulatory loci (e.g., sarA and agr). The sar (Staphylococcus accessory regulator) locus is composed of three overlapping transcripts (sarA P1, P3, and P2, transcripts initiated from the P1, P3, and P2 promoters, respectively), all encoding the 124-aa SarA protein. The level of SarA, the major regulatory protein, is partially controlled by the differential activation of the sarA promoters. We previously partially purified a 13.6-kDa protein, designated SarR, that binds to the sarA promoter region to down-modulate sarA transcription from the P1 promoter and subsequently SarA expression. SarR shares sequence similarity to SarA, and another SarA homolog, SarS. Here we report the 2.3 Å-resolution x-ray crystal structure of the dimeric SarR-MBP (maltose binding protein) fusion protein. The structure reveals that the SarR protein not only has a classic helix–turn–helix module for DNA binding at the major grooves, but also has an additional loop region involved in DNA recognition at the minor grooves. This interaction mode could represent a new functional class of the “winged helix” family. The dimeric SarR structure could accommodate an unusually long stretch of ≈27 nucleotides with two or four bending points along the course, which could lead to the bending of DNA by 90° or more, similar to that seen in the catabolite activator protein (CAP)–DNA complex. The structure also demonstrates the molecular basis for the stable dimerization of the SarR monomers and possible motifs for interaction with other proteins.

The candidate tumor suppressor gene, RASSF1A, from human chromosome 3p21.3 is involved in kidney tumorigenesis

Clear cell-type renal cell carcinomas (clear RCC) are characterized almost universally by loss of heterozygosity on chromosome 3p, which usually involves any combination of three regions: 3p25-p26 (harboring the VHL gene), 3p12-p14.2 (containing the FHIT gene), and 3p21-p22, implying inactivation of the resident tumor-suppressor genes (TSGs). For the 3p21-p22 region, the affected TSGs remain, at present, unknown. Recently, the RAS association family 1 gene (isoform RASSF1A), located at 3p21.3, has been identified as a candidate lung and breast TSG. In this report, we demonstrate aberrant silencing by hypermethylation of RASSF1A in both VHL-caused clear RCC tumors and clear RCC without VHL inactivation. We found hypermethylation of RASSF1A's GC-rich putative promoter region in most of analyzed samples, including 39 of 43 primary tumors (91%). The promoter was methylated partially or completely in all 18 RCC cell lines analyzed. Methylation of the GC-rich putative RASSF1A promoter region and loss of transcription of the corresponding mRNA were related causally. RASSF1A expression was reactivated after treatment with 5-aza-2′-deoxycytidine. Forced expression of RASSF1A transcripts in KRC/Y, a renal carcinoma cell line containing a normal and expressed VHL gene, suppressed growth on plastic dishes and anchorage-independent colony formation in soft agar. Mutant RASSF1A had reduced growth suppression activity significantly. These data suggest that RASSF1A is the candidate renal TSG gene for the 3p21.3 region.