Overexpression of MYC causes p53-dependent G2 arrest of normal fibroblasts

Overexpression of the proto-oncogene MYC has been implicated in the genesis of diverse human cancers. One explanation for the role of MYC in tumorigenesis has been that this gene might drive cells inappropriately through the division cycle, leading to the relentless proliferation characteristic of the neoplastic phenotype. Herein, we report that the overexpression of MYC alone cannot sustain the division cycle of normal cells but instead leads to their arrest in G2. We used an inducible form of the MYC protein to stimulate normal human and rodent fibroblasts. The stimulated cells passed through G1 and S but arrested in G2 and frequently became aneuploid, presumably as a result of inappropriate reinitiation of DNA synthesis. Absence of the tumor suppressor gene p53 or its downstream effector p21 reduced the frequency of both G2 arrest and aneuploidy, apparently by compromising the G2 checkpoint control. Thus, relaxation of the G2 checkpoint may be an essential early event in tumorigenesis by MYC. The loss of p53 function seems to be one mechanism by which this relaxation commonly occurs. These findings dramatize how multiple genetic events can collaborate to produce neoplastic cells.

The essential protein encoded by the UL31 gene of herpes simplex virus 1 depends for its stability on the presence of UL34 protein

To pursue an earlier observation that the protein encoded by the UL34 gene binds to intermediate chain of dynein, we constructed a series of mutants from which sequences encoding the entire protein (ΔUL34) or amino-terminal [UL34Δ(3–119)] or carboxyl-terminal [UL34Δ(245–275)] domains were deleted. The mutant lacking the sequence encoding the carboxyl-terminal domain grew in all cell lines tested. The two other mutants replicated only in cell type-dependent manner and poorly. Rescue of ΔUL34 mutant with a fragment that does not encompass the UL31 ORF restored wild-type phenotype. UL34 protein interacts physically with UL31, and the UL31 deletion mutant appears to have a phenotype similar to that of UL34 deletion mutant. Experiments designed to determine whether the phenotypes of the deletion mutants have a common base revealed that cells infected with the ΔUL34 mutant accumulate UL31 RNA but not the corresponding protein. The UL31 protein accumulated, however, to near wild-type virus-infected cell levels in cells infected with ΔUL34 mutant and treated with the MG132 proteosomal inhibitor at 6 h after infection. This is evidence that the stability of an essential viral protein requires the presence of another protein. The observation raises the bar for identification of gene function on the basis of analyses of the phenotype of mutants in which the gene has been deleted or rendered inoperative.

Regional and strain-specific gene expression mapping in the adult mouse brain

To determine the genetic causes and molecular mechanisms responsible for neurobehavioral differences in mice, we used highly parallel gene expression profiling to detect genes that are differentially expressed between the 129SvEv and C57BL/6 mouse strains at baseline and in response to seizure. In addition, we identified genes that are differentially expressed in specific brain regions. We found that approximately 1% of expressed genes are differentially expressed between strains in at least one region of the brain and that the gene expression response to seizure is significantly different between the two inbred strains. The results lead to the identification of differences in gene expression that may account for distinct phenotypes in inbred strains and the unique functions of specific brain regions.

Requirement for the lpA1 lysophosphatidic acid receptor gene in normal suckling behavior

Although extracellular application of lysophosphatidic acid (LPA) has been extensively documented to produce a variety of cellular responses through a family of specific G protein-coupled receptors, the in vivo organismal role of LPA signaling remains largely unknown. The first identified LPA receptor gene, lpA1/vzg-1/edg-2, was previously shown to have remarkably enriched embryonic expression in the cerebral cortex and dorsal olfactory bulb and postnatal expression in myelinating glia including Schwann cells. Here, we show that targeted deletion of lpA1 results in approximately 50% neonatal lethality, impaired suckling in neonatal pups, and loss of LPA responsivity in embryonic cerebral cortical neuroblasts with survivors showing reduced size, craniofacial dysmorphism, and increased apoptosis in sciatic nerve Schwann cells. The suckling defect was responsible for the death among lpA1(−/−) neonates and the stunted growth of survivors. Impaired suckling behavior was attributable to defective olfaction, which is likely related to developmental abnormalities in olfactory bulb and/or cerebral cortex. Our results provide evidence that endogenous lysophospholipid signaling requires an lp receptor gene and indicate that LPA signaling through the LPA1 receptor is required for normal development of an inborn, neonatal behavior.

Mutations in the chloride-bicarbonate exchanger gene AE1 cause autosomal dominant but not autosomal recessive distal renal tubular acidosis

Primary distal renal tubular acidosis (dRTA) is characterized by reduced ability to acidify urine, variable hyperchloremic hypokalemic metabolic acidosis, nephrocalcinosis, and nephrolithiasis. Kindreds showing either autosomal dominant or recessive transmission are described. Mutations in the chloride-bicarbonate exchanger AE1 have recently been reported in four autosomal dominant dRTA kindreds, three of these altering codon Arg589. We have screened 26 kindreds with primary dRTA for mutations in AE1. Inheritance was autosomal recessive in seventeen kindreds, autosomal dominant in one, and uncertain due to unknown parental phenotype or sporadic disease in eight kindreds. No mutations in AE1 were detected in any of the autosomal recessive kindreds, and analysis of linkage showed no evidence of linkage of recessive dRTA to AE1. In contrast, heterozygous mutations in AE1 were identified in the one known dominant dRTA kindred, in one sporadic case, and one kindred with two affected brothers. In the dominant kindred, the mutation Arg-589/Ser cosegregated with dRTA in the extended pedigree. An Arg-589/His mutation in the sporadic case proved to be a de novo mutation. In the third kindred, affected brothers both have an intragenic 13-bp duplication resulting in deletion of the last 11 amino acids of AE1. These mutations were not detected in 80 alleles from unrelated normal individuals. These findings underscore the key role of Arg-589 and the C terminus in normal AE1 function, and indicate that while mutations in AE1 cause autosomal dominant dRTA, defects in this gene are not responsible for recessive disease.

The Drosophila LIM-only gene, dLMO, is mutated in Beadex alleles and might represent an evolutionarily conserved function in appendage development

The process of wing patterning involves precise molecular mechanisms to establish an organizing center at the dorsal–ventral boundary, which functions to direct the development of the Drosophila wing. We report that misexpression of dLMO, a Drosophila LIM-only protein, in specific patterns in the developing wing imaginal disc, disrupts the dorsal–ventral (D-V) boundary and causes errors in wing patterning. When dLMO is misexpressed along the anterior–posterior boundary, extra wing outgrowth occurs, similar to the phenotype seen when mutant clones lacking Apterous, a LIM homeodomain protein known to be essential for normal D-V patterning of the wing, are made in the wing disc. When dLMO is misexpressed along the D-V boundary in third instar larvae, loss of the wing margin is observed. This phenotype is very similar to the phenotype of Beadex, a long-studied dominant mutation that we show disrupts the dLMO transcript in the 3′ untranslated region. dLMO normally is expressed in the wing pouch of the third instar wing imaginal disc during patterning. A mammalian homolog of dLMO is expressed in the developing limb bud of the mouse. This indicates that LMO proteins might function in an evolutionarily conserved mechanism involved in patterning the appendages.

β-Adrenergic receptor-induced activation of nerve growth factor gene transcription in rat cerebral cortex involves CCAAT/enhancer-binding protein δ

Stimulation of β-adrenergic receptors (BAR) by clenbuterol (CLE) increases nerve growth factor (NGF) biosynthesis in the rat cerebral cortex but not in other regions of the brain. We have explored the transcription mechanisms that may account for the cortex-specific activation of the NGF gene. Although the NGF promoter contains an AP-1 element, AP-1-binding activity in the cerebral cortex was not induced by CLE, suggesting that other transcription factors govern the brain area-specific induction of NGF. Because BAR activation increases cAMP levels, we examined the role of CCAAT/enhancer-binding proteins (C/EBP), some of which are known to be cAMP-inducible. In C6–2B glioma cells, whose NGF expression is induced by BAR agonists, (i) CLE increased C/EBPδ-binding activity, (ii) NGF mRNA levels were increased by overexpressing C/EBPδ, and (iii) C/EBPδ increased the activity of an NGF promoter–reporter construct. Moreover, DNase footprinting and deletion analyses identified a C/EBPδ site in the proximal region of the NGF promoter. C/EBPδ appears to be responsible for the BAR-mediated activation of the NGF gene in vivo, since CLE elicited a time-dependent increase in C/EBPδ-binding activity in the cerebral cortex only. Our data suggest that, while AP-1 may regulate basal levels of NGF expression, C/EBPδ is a critical component determining the area-specific expression of NGF in response to BAR stimulation.

Comparative studies on mammalian Hoxc8 early enhancer sequence reveal a baleen whale-specific deletion of a cis-acting element

Variations in regulatory regions of developmental control genes have been implicated in the divergence of axial morphologies. To find potentially significant changes in cis-regulatory regions, we compared nucleotide sequences and activities of mammalian Hoxc8 early enhancers. The nucleotide sequence of the early enhancer region is extremely conserved among mammalian clades, with five previously described cis-acting elements, A–E, being invariant. However, a 4-bp deletion within element C of the Hoxc8 early enhancer sequence is observed in baleen whales. When assayed in transgenic mouse embryos, a baleen whale enhancer (unlike other mammalian enhancers) directs expression of the reporter gene to more posterior regions of the neural tube but fails to direct expression to posterior mesoderm. We suggest that regulation of Hoxc8 in baleen whales differs from other mammalian species and may be associated with variation in axial morphology.

Repression of transcription mediated by dual elements in the CCAAT/enhancer binding protein α gene

During adipocyte differentiation, the expression of C/EBPα is activated, which in turn serves to transcriptionally activate numerous adipocyte genes. A previous search for cis elements that regulate transcription of the C/EBPα gene led to the identification of a potential repressive element within the proximal 5′ flanking region of the gene. Nuclear extracts from 3T3-L1 preadipocytes, but not adipocytes, were found to contain a factor, CUP (C/EBPα undifferentiated protein), that binds to this site (the CUP-1 site). In the present investigation, we show that C/EBPα promoter-luciferase constructs containing both the proximal 5′ flanking and the entire 5′ untranslated regions of the gene exhibit an expression pattern during adipocyte differentiation comparable to that of the endogenous C/EBPα gene. Mutation of the CUP-1 site in these constructs had little effect on reporter gene expression; however, when this mutation was combined with deletion of the 5′ untranslated region, reporter gene expression by preadipocytes was dramatically up-regulated. Consistent with this finding, a second CUP binding site (the CUP-2 site) was identified in the 5′ untranslated region. Although mutation of either CUP element in constructs containing both the 5′ flanking and 5′ untranslated region had little effect on reporter gene transcription, mutation of both CUP elements markedly activated transcription. Thus, it appears that dual CUP regulatory elements repress transcription of the C/EBPα gene prior to induction of the adipocyte differentiation program.

A ribozyme-mediated, gene “knockdown” strategy for the identification of gene function in zebrafish

The zebrafish system offers many unique opportunities for the study of molecular biology. To date, only random mutagenesis, and not directed gene knockouts, have been demonstrated in this system. To more fully develop the potential of the zebrafish system, an approach to effectively inhibit the expression of any targeted gene in the developing zebrafish embryo has been developed. This approach uses a transient, cytoplasmic, T7 expression system, injected into the fertilized zebrafish egg to rapidly produce high levels of a ribozyme directed against the mRNA encoded by the targeted gene to inhibit its expression. In a demonstration of this strategy, expression of the recessive dominant zebrafish no tail gene was effectively inhibited by using this strategy to yield a phenotype identical to that resulting from a known defective mutation in this same gene. This, ribozyme-mediated, message deletion strategy may have use in determining the function of genetic coding sequences of unknown function.

Both hypertrophic and dilated cardiomyopathies are caused by mutation of the same gene, δ-sarcoglycan, in hamster: An animal model of disrupted dystrophin-associated glycoprotein complex

Cardiomyopathy (CM) is a primary degenerative disease of myocardium and is traditionally categorized into hypertrophic and dilated CMs (HCM and DCM) according to its gross appearance. Cardiomyopathic hamster (CM hamster), a representative model of human hereditary CM, has HCM and DCM inbred sublines, both of which descend from the same ancestor. Herein we show that both HCM and DCM hamsters share a common defect in a gene for δ-sarcoglycan (δ-SG), the functional role of which is yet to be characterized. A breakpoint causing genomic deletion was found to be located at 6.1 kb 5′ upstream of the second exon of δ-SG gene, and its 5′ upstream region of more than 27.4 kb, including the authentic first exon of δ-SG gene, was deleted. This deletion included the major transcription initiation site, resulting in a deficiency of δ-SG transcripts with the consequent loss of δ-SG protein in all the CM hamsters, despite the fact that the protein coding region of δ-SG starting from the second exon was conserved in all the CM hamsters. We elucidated the molecular interaction of dystrophin-associated glycoproteins including δ-SG, by using an in vitro pull-down study and ligand overlay assay, which indicates the functional role of δ-SG in stabilizing sarcolemma. The present study not only identifies CM hamster as a valuable animal model for studying the function of δ-SG in vivo but also provides a genetic target for diagnosis and treatment of human CM.

Analysis of the Xenopus laevis CCAAT-enhancer binding protein α gene promoter demonstrates species-specific differences in the mechanisms for both auto-activation and regulation by Sp1

Transcription factors belonging to the CCAAT-enhancer binding protein (C/EBP) family have been implicated in the regulation of gene expression during differentiation, development and disease. Autoregulation is relatively common in the modulation of C/EBP gene expression and the murine and human C/EBPα genes have been shown to be auto-activated by different mechanisms. In the light of this finding, it is essential that autoregulation of C/EBPα genes from a wider range of different species be investigated in order to gauge the degree of commonality, or otherwise, that may exist. We report here studies that investigate the regulation of the Xenopus laevis C/EBPα gene (xC/EBPα). The –1131/+41 promoter region was capable of directing high levels of expression in both the human hepatoma Hep3B and the Xenopus kidney epithelial A6 cell lines, and was auto-activated by expression vectors specifying for xC/EBPα or xC/EBPβ. Deletion analysis showed that the –321/+41 sequence was sufficient for both the constitutive promoter activity and auto-activation and electrophoretic mobility shift assays identified the interaction of C/EBPs and Sp1 to this region. Although deletion of either the C/EBP or the Sp1 site drastically reduced the xC/EBPα promoter activity, multimers of only the C/EBP site could confer autoregulation to a heterologous SV40 promoter. These results indicate that, in contrast to the human promoter and in common with the murine gene, the xC/EBPα promoter was subject to direct autoregulation. In addition, we demonstrate a novel species-specific action of Sp1 in the regulation of C/EBPα expression, with the factor able to repress the murine promoter but activate the Xenopus gene.

Cis-acting requirements in flanking DNA for the programmed elimination of mse2.9: a common mechanism for deletion of internal eliminated sequences from the developing macronucleus of Tetrahymena thermophila

During macronuclear development in the ciliated protozoan Tetrahymena thermophila, extensive DNA deletions occur, eliminating thousands of internal eliminated sequences (IESs). Using an rDNA-based transformation assay we have analyzed the role during DNA deletion of DNA flanking mse2.9, an IES within the second intron of a gene encoding an as yet incompletely characterized protein. We establish that a cis-acting sequence for mse2.9 deletion acts at a distance to specify deletion boundaries. A complex sequence element necessary for efficient and accurate mse2.9 deletion is located in the region 47–81 bp from the right side of mse2.9. The ability of a variety of IES flanking sequences to rescue a processing deficient mse2.9 construct indicates that some cis-acting signal is shared among different IESs. In addition, the short intronic sequence that flanks mse2.9 is able to direct efficient and accurate processing. Despite no obvious sequence similarity between mse2.9 and other IESs, we suggest that a common mechanism is used to delete different families of IESs in Tetrahymena.

Inactivation of Saccharomyces cerevisiae OGG1 DNA repair gene leads to an increased frequency of mitochondrial mutants

The OGG1 gene encodes a highly conserved DNA glycosylase that repairs oxidized guanines in DNA. We have investigated the in vivo function of the Ogg1 protein in yeast mitochondria. We demonstrate that inactivation of ogg1 leads to at least a 2-fold increase in production of spontaneous mitochondrial mutants compared with wild-type. Using green fluorescent protein (GFP) we show that a GFP–Ogg1 fusion protein is transported to mitochondria. However, deletion of the first 11 amino acids from the N-terminus abolishes the transport of the GFP–Ogg1 fusion protein into the mitochondria. This analysis indicates that the N-terminus of Ogg1 contains the mitochondrial localization signal. We provide evidence that both yeast and human Ogg1 proteins protect the mitochondrial genome from spontaneous, as well as induced, oxidative damage. Genetic analyses revealed that the combined inactivation of OGG1 and OGG2 [encoding an isoform of the Ogg1 protein, also known as endonuclease three-like glycosylase I (Ntg1)] leads to suppression of spontaneously arising mutations in the mitochondrial genome when compared with the ogg1 single mutant or the wild-type. Together, these studies provide in vivo evidence for the repair of oxidative lesions in the mitochondrial genome by human and yeast Ogg1 proteins. Our study also identifies Ogg2 as a suppressor of oxidative mutagenesis in mitochondria.

Transcriptional activation of the small GTPase gene rhoB by genotoxic stress is regulated via a CCAAT element

The gene encoding the Ras-related GTPase RhoB-specific is immediate-early inducible by genotoxic treatments. Regulation of transcriptional activation of rhoB is still unclear. Here we show that cells lacking either p53 or c-Fos are not different from wild-type cells with respect to the level of rhoB induction upon UV irradiation, indicating that these transcription factors are not crucial for stimulation of rhoB mRNA expression. Extracts from UV-irradiated and non-irradiated cells revealed similar DNA-binding activities to a 0.17 kb rhoB promoter fragment harboring the functional element(s) necessary for stimulation of rhoB by UV light. By means of immunoprecipitation we found that an ATF-2-specific antibody co-precipitates the 32P-labeled 0.17 kb rhoB fragment, whereas an anti-AP1 antibody did not. Since no consensus sequence for binding of ATF-2 is present within the rhoB promoter, ATF-2 is likely to be associated with another factor that binds to the minimal promoter. Deletion analysis and site-directed mutagenesis of the 0.17 kb rhoB fragment revealed a CCAAT box to be an essential requirement for stimulation of rhoB by UV light and methyl methanesulfonate. Moreover, immunoprecipitation experiments showed that the CCAAT-binding factor NF-YA is complexed with ATF-2. Overall, the data strongly indicate that transcriptional activation of the rhoB gene by genotoxic stress is regulated via a CCAAT box and that interaction of CCAAT-binding factor and ATF-2 triggers the stress-inducible expression of rhoB.