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At least 631 records · Page 35Linked to original sources
PubMed2003

Differential regulation of E2F1 apoptotic target genes in response to DNA damage.

E2F1, a member of the E2F family of transcription factors, in addition to its established proliferative effect, has also been implicated in the induction of apoptosis through p53-dependent and p53-independent pathways. Several genes involved in the activation or execution of the apoptotic programme have recently been shown to be upregulated at the transcriptional level by E2F1 overexpression, including the genes encoding INK4a/ARF, Apaf-1, caspase 7 and p73 (refs 3-5). E2F1 is stabilized in response to DNA damage but it has not been established how this translates into the activation of specific subsets of E2F target genes. Here, we applied a chromatin immunoprecipitation approach to show that, in response to DNA damage, E2F1 is directed from cell cycle progression to apoptotic E2F target genes. We identify p73 as an important E2F1 apoptotic target gene in DNA damage response and we show that acetylation is required for E2F1 recruitment on the P1p73 promoter and for its transcriptional activation.

Acetylation↗
PubMed1999

Construction of gene targeting vectors from lambda KOS genomic libraries.

We describe a highly redundant murine genomic library in a new lambda phage, lambda knockout shuttle (lambda KOS) that facilitates the very rapid construction of replacement-type gene targeting vectors. The library consists of 94 individually amplified subpools, each containing an average of 40,000 independent genomic clones. The subpools are arrayed into a 96-well format that allows a PCR-based efficient recovery of independent genomic clones. The lambda KOS vector backbone permits the CRE-mediated conversion into high-copy number pKOS plasmids, wherein the genomic inserts are automatically flanked by negative-selection cassettes. The lambda KOS vector system exploits the yeast homologous recombination machinery to simplify the construction of replacement-type gene targeting vectors independent of restriction sites within the genomic insert. We outline procedures that allow the generation of simple and more sophisticated conditional gene targeting vectors within 3-4 weeks, beginning with the screening of the lambda KOS genomic library.

Animals↗
PubMed2006

Identification of novel candidate target genes in amplicons of Glioblastoma multiforme tumors detected by expression and CGH microarray profiling.

BACKGROUND: Conventional cytogenetic and comparative genomic hybridization (CGH) studies in brain malignancies have shown that glioblastoma multiforme (GBM) is characterized by complex structural and numerical alterations. However, the limited resolution of these techniques has precluded the precise identification of detailed specific gene copy number alterations. RESULTS: We performed a genome-wide survey of gene copy number changes in 20 primary GBMs by CGH on cDNA microarrays. A novel amplicon at 4p15, and previously uncharacterized amplicons at 13q32-34 and 1q32 were detected and are analyzed here. These amplicons contained amplified genes not previously reported. Other amplified regions containing well-known oncogenes in GBMs were also detected at 7p12 (EGFR), 7q21 (CDK6), 4q12 (PDGFRA), and 12q13-15 (MDM2 and CDK4). In order to identify the putative target genes of the amplifications, and to determine the changes in gene expression levels associated with copy number change events, we carried out parallel gene expression profiling analyses using the same cDNA microarrays. We detected overexpression of the novel amplified genes SLA/LP and STIM2 (4p15), and TNFSF13B and COL4A2 (13q32-34). Some of the candidate target genes of amplification (EGFR, CDK6, MDM2, CDK4, and TNFSF13B) were tested in an independent set of 111 primary GBMs by using FISH and immunohistological assays. The novel candidate 13q-amplification target TNFSF13B was amplified in 8% of the tumors, and showed protein expression in 20% of the GBMs. CONCLUSION: This high-resolution analysis allowed us to propose novel candidate target genes such as STIM2 at 4p15, and TNFSF13B or COL4A2 at 13q32-34 that could potentially contribute to the pathogenesis of these tumors and which would require futher investigations. We showed that overexpression of the amplified genes could be attributable to gene dosage and speculate that deregulation of those genes could be important in the development and progression of GBM. Our findings highlight the important influence in GBM of signaling pathways such as the PI3K/AKT, consistent with the invasive features of this tumor.

Adult↗
PubMed2001

Towards genetic genome projects: genomic library screening and gene-targeting vector construction in a single step.

We have developed technologies that simplify genomic library construction and screening, substantially reducing both the time and the cost associated with traditional library screening methods and facilitating the generation of gene-targeting constructs. By taking advantage of homologous recombination in Escherichia coli, we were able to use as little as 80 bp of total sequence homology to screen for a specific gene from a genomic library in plasmid or phage form. This method, called recombination cloning (REC), takes only a few days instead of the several weeks required for traditional plaque-lift methods. In addition, because every clone in the mouse genomic library we have constructed has a negative selection marker adjacent to the genomic insert, REC screening can generate gene-targeting vectors in one step, from library screening to finished construct. Conditional targeting constructs can be generated easily with minimal additional manipulation.

Animals↗
PubMed2003

Insulin and hypoxia share common target genes but not the hypoxia-inducible factor-1alpha.

Both hypoxia and insulin induce common target genes, including vascular endothelial growth factors and several glycolytic enzymes. However, these two signals eventually trigger quite different metabolic pathways. Hypoxia induces glycolysis, resulting in anaerobic ATP production, while insulin increases glycolysis for energy storage. Hypoxia-induced gene expression is mediated by the hypoxia-inducible factor-1 (HIF-1) that consists of HIF-1alpha and the aromatic hydrocarbon nuclear translocator (Arnt). Hypoxia-induced gene expression is initiated by the stabilization of the HIF-1alpha subunit. Here we investigated whether insulin-induced gene expression also requires stabilization of HIF-1alpha. Our results indicate that hypoxia but not insulin stabilizes HIF-1alpha protein levels, whereas both insulin- and hypoxia-induced gene expression require the presence of the Arnt protein. Insulin treatment fails to inactivate proline hydroxylation of HIF-1alpha, which triggers recruitment of the von Hippel-Lindau protein and oxygen-dependent degradation of HIF-1alpha. Insulin-induced gene expression is inhibited by the presence of the phosphoinositide (PI) 3-kinase inhibitor LY294002 and the dominant negative mutant of the p85 subunit of PI 3-kinase, whereas hypoxia-induced gene expression is not. Pyrrolidine dithiocarbamate, a scavenger of H2O2, reduces insulin-induced gene expression but not hypoxia-induced gene expression. Although both hypoxia and insulin induce the expression of common target genes through a hypoxia-responsive element- and Arnt-dependent mechanism, insulin cannot stabilize the HIF-1alpha protein. We believe that insulin activates other putative partner proteins for Arnt in PI 3-kinase- and H2O2-dependent pathways.

3T3 Cells↗
PubMed2005

ERE-independent ERalpha target genes differentially expressed in human breast tumors.

The classical pathway for estrogen receptor (ER) signaling is mediated by ER binding to an estrogen response element (ERE) in DNA. ERalpha can also act via a nonclassical pathway by altering the activities of other transcription factors (e.g., Sp1, AP-1, or NF-kappaB) at their cognate sites on DNA. We previously generated a mutant form of ERalpha (E207A/G208A) that does not bind to EREs, and therefore lacks signaling via the classical pathway but retains signaling via the nonclassical pathway. In the current study, we introduce this mutant ERalpha into MDA-MB231 ERalpha-negative breast carcinoma cells to identify nonclassical pathway genes that respond to 17beta-estradiol (E2), selective estrogen receptor modulators (SERMs) tamoxifen (TAM) or raloxifene (RAL), or the estrogen antagonist ICI 182,780 (ICI). Consistent with a role for nonclassical signaling in SERM action, microarray analyses identify 268 responsive nonclassical ERalpha pathway target genes. ICI elicits the largest number of nonclassical genes, followed by RAL, TAM, and E2. Custom microarrays containing identified nonclassical ERalpha responsive genes are used to compare gene expression in human breast tumor (n = 34) and normal mammary epithelial cell (n = 9) samples. A subset of nonclassical genes (n = 32) are differentially expressed in breast tumors. In summary, we show that nonclassical ERalpha pathway target genes exhibit a range of transcriptional responses to SERMs and identify targets of this pathway as potentially relevant to breast cancer. The identification of nonclassical ERalpha target genes offers new insight into estrogen receptor signaling and cross talk with pathways that mediate breast tumor response to SERM therapy.

Breast Neoplasms↗
PubMed1999

Overexpression of ptc1 inhibits induction of Shh target genes and prevents normal patterning in the neural tube.

Patched (Ptc) is a human tumor suppressor protein and a candidate receptor for Hedgehog (Hh) proteins, which regulate growth and patterning in embryos. Ptc represses expression of Hh target genes such as Gli1 and ptc1 itself. Localized secretion of Hh appears to induce transcription of target genes in specific patterns by binding to Ptc and preventing it from functioning in recipient cells. People who are heterozygous for PTC1 exhibit a range of developmental defects, suggesting that some genes are inappropriately expressed when there is not enough Ptc protein. To test the idea that a balance between Hh and Ptc activities is essential for normal development, we overexpressed Ptc in the neural tube. We find that excess Ptc is sufficient to inhibit expression of Gli1 and ptc1, suggesting that Sonic hedgehog (Shh) cannot signal effectively. This leads to partial dorsalization of the neural tube and a wide spectrum of neural defects, ranging from embryonic lethality to hydrocephaly.

Animals↗
PubMed2003

Microarray analysis of embryonic retinoic acid target genes in the ascidian Ciona intestinalis.

Many chordate- and vertebrate-specific characteristics develop depending on retinoic acid (RA). Because the gene encoding the RA receptor exists only in chordates, RA function seems to be involved in chordate evolution. A cDNA microarray analysis of 9287 non-redundant cDNA clones was used to screen for RA target genes in the ascidian Ciona intestinalis. In addition, the spatial expression pattern of 94 candidate RA target genes was examined by in situ hybridization in RA-treated and control embryos. Strong RA-induced upregulation of Hox-1 and Cyp26 was observed, as is the case in vertebrates. In addition, a number of novel candidate target genes was identified. These included transcription factors and signaling molecules, suggesting that various differentiation and/or morphogenetic pathways are modulated by RA. The expression of cell adhesion molecules, cytoskeletal proteins and extracellular matrix components was affected by RA. Changes in the expression pattern of these genes may be a direct cause of abnormal morphogenesis of the anterior neural tissues. RA also affected the expression of genes that seemed to be involved in neuronal functions. Although obvious homeotic transformation has not been observed, the function of various neural cell types seemed to be impaired by RA. The microarray data are reliable and will contribute to comprehensive understanding of RA action in the development and evolution of chordates.

Animals↗
PubMed2005

Ontogeny of mRNA abundance of nuclear receptors and nuclear receptor target genes in young cattle.

After birth the development of appropriate detoxification mechanisms is important. Nuclear receptors (NR), such as constitutive androstane receptor (CAR), pregnane X receptor (PXR), peroxisome proliferator-activated receptor-alpha (PPARalpha), retinoid receptors (RAR, RXR), and NR target genes are involved in the detoxification of exogenous and endogenous substances. We quantified abundances of hepatic mRNA of NR and several NR target genes (cytochromes, CYP; cytochrome P450 reductase, CPR; UDP-glucuronosyl transferase, UDP) in calves at different ages. Gene expression was quantified by real-time RT-PCR. Abundance of mRNA of CAR and PXR increased from low levels at birth in pre-term calves (P0) and full-term calves (F0) to higher levels in 5-day-old calves (F5) and in 159-day-old veal calves (F159), whereas mRNA levels of PPARalpha did not exhibit significant ontogenetic changes. RARbeta mRNA levels were higher in F5 and F159 than in F0, whereas no age differences were observed for RARalpha levels. Levels of RXRalpha and RXRbeta mRNA were lower in F5 than in P0 and F0. Abundance of CYP2C8 and CYP3A4 increased from low levels in P0 and F0 to higher levels in F5 and to highest levels in F159. Abundance of CPR was transiently decreased in F0 and F5 calves. Levels of UGT1A1 mRNA increased from low levels in P0 and F0 to maximal level in F5 and F159. In conclusion, mRNA levels of NR and NR target genes exhibited ontogenetic changes that are likely of importance for handling of xeno- and endobiotics with increasing age.

Age Factors↗
PubMed2000

Targeting gene expression to tumor cells with loss of wild-type p53 function.

The tumor suppressor protein p53 is a transcription factor that can positively regulate the expression of critical target genes involved in negative control of cell growth or induction of apoptosis; p53 is also able to suppress the transcription of other genes by virtue of its ability to bind components of the basal transcription machinery. Over 50% of human tumors are characterized by p53 mutations that result in a loss of wild-type p53 (wtp53) function in the transcriptional control of these target genes. We have exploited this loss of p53 function in the regulation of gene transcription to develop a novel gene therapy strategy that maximizes expression of the potential therapeutic gene in tumors while simultaneously down-regulating the same gene in normal cells. In one construct (unit I), the potential therapeutic gene (in this case represented by a luciferase reporter) is placed under the control of a promoter such as the heat shock protein 70 gene promoter, which is repressed by wtp53 but overexpressed in many tumor cells with defective p53 function. Residual expression of the reporter in normal cells is repressed by cotransfection of another construct (unit II) consisting of a repressor of unit I under the control of a promoter that is activated by wtp53 expression. Unit II contains a promoter with a consensus wtp53 binding site driving a transcriptional repressor or an antisense construct for the gene in unit I. Our results suggest that this dual control approach may represent a strategy with wide applications in the field of cancer gene therapy.

Feasibility Studies↗
PubMed1992

An animal model for cystic fibrosis made by gene targeting.

Cystic fibrosis results from defects in the gene encoding a cyclic adenosine monophosphate-dependent chloride ion channel known as the cystic fibrosis transmembrane conductance regulator (CFTR). To create an animal model for cystic fibrosis, mice were generated from embryonic stem cells in which the CFTR gene was disrupted by gene targeting. Mice homozygous for the disrupted gene display many features common to young human cystic fibrosis patients, including failure to thrive, meconium ileus, alteration of mucous and serous glands, and obstruction of glandlike structures with inspissated eosinophilic material. Death resulting from intestinal obstruction usually occurs before 40 days of age.

Animals↗
PubMed2006

Rapid loss of intestinal crypts upon conditional deletion of the Wnt/Tcf-4 target gene c-Myc.

Inhibition of the mutationally activated Wnt cascade in colorectal cancer cell lines induces a rapid G1 arrest and subsequent differentiation. This arrest can be overcome by maintaining expression of a single Tcf4 target gene, the proto-oncogene c-Myc. Since colorectal cancer cells share many molecular characteristics with proliferative crypt progenitors, we have assessed the physiological role of c-Myc in adult crypts by conditional gene deletion. c-Myc-deficient crypts are lost within weeks and replaced by c-Myc-proficient crypts through a fission process of crypts that have escaped gene deletion. Although c-Myc(-/-) crypt cells remain in the cell cycle, they are on average much smaller than wild-type cells, cycle slower, and divide at a smaller cell size. c-Myc appears essential for crypt progenitor cells to provide the necessary biosynthetic capacity to successfully progress through the cell cycle.

Animals↗
PubMed2005

Multiple expression control mechanisms of peroxisome proliferator-activated receptors and their target genes.

The peroxisome proliferator-activated receptors (PPAR) alpha, beta/delta and gamma belong to the nuclear hormone receptor superfamily. As ligand-activated receptors, they form a functional transcriptional unit upon heterodimerization with retinoid X receptors (RXRs). PPARs are activated by fatty acids and their derivatives, whereas RXR is activated by 9-cis retinoic acid. This heterodimer binds to peroxisome proliferator response elements (PPRE) residing in target genes and stimulates their expression. Recent reports now indicate that PPARs and RXRs can function independently, in the absence of a hetero-partner, to modulate gene expression. Of importance, these non-canonical mechanisms underscore the impact of both cofactors and DNA on gene expression. Furthermore, these different mechanisms reveal the increasing repertoire of PPAR 'target' genes that now encompasses non-PPREs containing genes. It is also becoming apparent that understanding the regulation of PPAR expression and activity, can itself have a significant influence on how the expression of subgroups of target genes is studied and integrated in current knowledge.

Animals↗
PubMed2003

[In vivo functional analysis of the neuropeptide PACAP using gene-targeted mice].

Mutant strains of mice with precise genetic mutations generated by gene-targeting technology have proved to be useful tools for linking specific genes with biological processes in vivo and serve as models for human diseases. Pituitary adenylate cyclase-activating polypeptide (PACAP) is a highly conserved neuropeptide that is widely expressed in the mammalian brain, and it has been implicated in a broad variety of physiological and pathophysiological processes. To assess the function of PACAP in vivo, recently, we have generated PAC1 receptor- and PACAP-targeted mice and transgenic mice overexpressing PACAP in the pancreatic beta-cells. The phenotypes of these mutant mice revealed both expected and unexpected roles of PACAP in the brain and pancreatic functions. A significant contribution of genetic background as well as environmental factors to the knockout phenotypes was also observed. In this article, we briefly describe the technique of gene targeting and discuss how this method was used to generate PACAP and its receptor deficient mice. We also analyze how these mutants can contribute to our understanding of the molecular mechanism underlying higher nervous functions.

Animals↗
PubMed1999

[Molecular dissection by gene targeting in the chicken B cell line DT40].

The gene-targeting experiment is a powerful tool for clarifying the functions of each gene product. For instance, establishment of a gene-knockout mouse, undoubtedly, contributes to our understanding of the functions of each gene in vivo. However, assuming that the gene product is expressed in various lineage cells, the phenotype in this knockout mouse exhibits a mixture of defects in various lineage cells. Thus, to compensate for this weak point, cell-based gene-knock experiments are required. Since the DT40 chicken B cell line shows a high frequency of homologous recombination, this line can be used for rapid dissection of each gene product at the cellular level.

Animals↗
PubMed2003

Gene targeting in livestock.

The development of nuclear transfer from tissue culture cells in livestock made it possible in principle to produce animals with subtle, directed genetic changes by in vitro modification of nuclear donor cells. In the short period since nuclear transfer was first performed, gene targeting in livestock has become a reality. Although gene targeting has immediate potential in biotechnology, it is unclear whether there are practical agricultural applications, at present. The first livestock targeting experiments have been directed at engineering animals either to render their organs immunologically compatible for human transplantation, or for improving the commercial production of recombinant proteins in the transgenic mammary gland. All successful examples of targeting have involved target loci that are expressed in the nuclear donor cell line. Two important barriers to the further development of this technology are adapting protocols for non-expressed genes and modifying procedures to enhance the lifespan of targeted cells in vitro. This review provides data that illustrate the difficulty in targeting non-expressed genes and discusses some of the practical issues associated with providing targeted nuclear donor cells that are competent for nuclear transfer.

Animals↗
PubMed2000

E-cadherin is a WT1 target gene.

The WT1 tumor suppressor gene encodes a transcription factor that can activate and repress gene expression. Transcriptional targets relevant for the growth suppression functions of WT1 are poorly understood. We found that mesenchymal NIH 3T3 fibroblasts stably expressing WT1 exhibit growth suppression and features of epithelial differentiation including up-regulation of E-cadherin mRNA. Acute expression of WT1 in NIH 3T3 fibroblasts after retroviral infection induced murine E-cadherin expression. In transient transfection experiments, the human and murine E-cadherin promoters were activated by co-expression of WT1. E-cadherin promoter activity was increased in cells overexpressing WT1 and was blocked by a dominant negative form of WT1. WT1 activated the murine E-cadherin promoter through a conserved GC-rich sequence similar to an EGR-1 binding site as well as through a CAAT box sequence. WT1 produced in vitro or derived from nuclear extracts bound to the WT1-response element within the murine E-cadherin promoter, but not the CAAT box. E-cadherin, a gene important in epithelial differentiation and neoplastic transformation, represents a downstream target gene that links the roles of the WT1 in differentiation and growth control.

3T3 Cells↗
PubMed2006

A functional hierarchy for c-Myc target genes? Lessons from MT-MC1.

Deregulation of CMYC is a hallmark of many human cancers. As a general bHLH-ZIP transcription factor, the c-Myc oncoprotein directly regulates the expression of >1500 genes controlled by RNA polymerases I, II and III. This represents a formidable challenge to determining which are the most critical for transformation. Previous work from our laboratory has indicated that MT-MC1, a direct c-Myc target gene, mimics many c-Myc phenotypes. More recently, genome-wide microarray experiments indicate that MT-MC1 itself regulates fewer than 50 downstream target genes, many of which, if not all, are also c-Myc targets. These observations suggest that this small, overlapping subset of target genes plays a particularly important role in c-Myc-mediated transformation. This is supported by the finding that nearly half of MT-MC1-regulated genes have been previously implicated in cancer.

Animals↗