Creation of genome-wide protein expression libraries using random activation of gene expression

Harrington, John; Sherf, Bruce A.; Rundlett, Stephen E.; Jackson, P. David; Perry, Rob; Cain, Scott; Leventhal, Christina; Thornton, Mark; Rakesh, Ramachandran; Whittington, Jessica; Lerner, Laura; Costanzo, Dana; McElligott, Karen; Boozer, Sherry; Mays, Robert W.; Smith, Emery; Veloso, Neil; Klika, Alison; Hess, J. Fred; Cothren, Kevin; Lo, Kalok; Offenbacher, Jason; Danzig, Joel; Ducar, Matt

doi:10.1038/88107

Cited by 65 publications

(30 citation statements)

References 37 publications

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“…Whether the regulation of expression of these genes is aected in this tumour by the viral integration is unclear. Interestingly, the cellular sequence identi®ed in tumour number 3 is part of an expressed gene (Harrington et al, 2001). It is very likely that the integration event in this case has disrupted a gene.…”

Section: Discussionmentioning

confidence: 95%

Involvement of intact HPV16 E6/E7 gene expression in head and neck cancers with unaltered p53 status and perturbed pRb cell cycle control

Wiest¹,

Schwarz

Enders³

et al. 2002

Oncogene

336

296

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We have identi®ed parameters which de®ne a causal role of HPV16 in head and neck cancer. Twenty-eight tumours which were typed positive for HPV16 DNA, were comprehensively analysed for expression of the viral oncogenes E6 and E7, the status of the p53 gene, and the protein status of pRb and p16 INK4a. In a subset of cases, we have searched for integrated viral DNA, and have determined the genomic status of the E6 gene. Expression of E6/E7 was found in 12 tumours most of which were derived from the oropharynx, whereas p53 mutations were present in 13 tumours from various sites. The tumours either carried p53 mutations but did not express E6/E7, or they did express E6/E7 but were p53-wild-type. Coexistence of E6/E7 expression with a mutated p53 was found in only one case. Strikingly, in most p53-mutated tumours without E6/E7 expression, we found the E6 gene to be disrupted. E6/E7 expression was associated with reduced pRb and overexpressed p16INK4a . Viral-cellular fusion transcripts were found in two cases. Our data demonstrate that HPV16 DNA-positivity in head and neck cancers is not indicative of a causal role. A causal role of HPV16 in head and neck cancer is de®ned by: E6/E7 expression, viral integration with an intact E6 gene, and perturbation of pRb cell cycle control. Mostly, the p53 gene is wild-type.

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Section: Discussionmentioning

confidence: 95%

Involvement of intact HPV16 E6/E7 gene expression in head and neck cancers with unaltered p53 status and perturbed pRb cell cycle control

Wiest¹,

Schwarz

Enders³

et al. 2002

Oncogene

336

296

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“…To our knowledge, PNPLA1 has yet to be assessed for enzymatic activities, and the only evidence we are aware of regarding transcript expression of PNPLA1 is via in silico assessments of EST clones. PNPLA1 is present in only one cDNA library, HT1080 cells treated with agents to induce random gene activation (17). In regard to adipose tissue expression, murine ATGL and adiponutrin show marked cell type specificity to adipocytes (4,22,28,73,81).…”

Section: Functional Analysis Of the Atgl Promoter Region Identifies Amentioning

confidence: 99%

The adipose tissue triglyceride lipase ATGL/PNPLA2 is downregulated by insulin and TNF-α in 3T3-L1 adipocytes and is a target for transactivation by PPARγ

Kim¹,

Tillison²,

Lee³

et al. 2006

American Journal of Physiology-Endocrinology and Metabolism

177

183

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The minimal adipose phenotype of hormone-sensitive lipase (HSL)-null mice suggested that other hormonally responsive lipase(s) were present in adipocytes. Recent studies have characterized a new adipose tissue triglyceride lipase, ATGL/PNPLA2/destnutrin/iPLA2/ TTS2.2 (ATGL). We had previously cloned a novel adipose-enriched transcript by differential screening and recently determined its identity with murine ATGL. We report here on the regulation of ATGL by TNF-␣ and insulin in 3T3-L1 adipocytes and identify ATGL as a target for transcriptional activation by the key adipogenic transcription factor PPAR␥. Insulin at 100 nM resulted in a marked decrease in ATGL transcript that was effectively blocked by inhibitors for PI 3-kinase and p70 ribosomal protein S6 kinase. TNF-␣ treatment decreased ATGL transcript in a time-dependent manner that paralleled TNF-␣ downregulation of PPAR␥ with a maximal decrease noted by 6 h. TNF-␣ effects on ATGL were attenuated by pretreatment with PD-98059, LY-294002, or rapamycin, suggesting involvement of the p44/42 MAP kinase, PI 3-kinase, and p70 ribosomal protein S6 kinase signals. To study transcriptional regulation of ATGL, we cloned 2,979 bp of the murine ATGL 5Ј-flanking region. Compared with promoterless pGL2-Basic, the Ϫ2979/ϩ21 ATGL luciferase construct demonstrated 120-and 40-fold increases in activity in white and brown adipocytes, respectively. Luciferase reporter activities for a series of eight ATGL promoter deletions revealed that the Ϫ928/ϩ21, Ϫ1738/ϩ21, Ϫ1979/ϩ21, and Ϫ2979/ϩ21 constructs were transactivated by PPAR␥. Our findings identify the novel lipase ATGL to be a target gene for TNF-␣ and insulin action in adipocytes and reveal that it is subject to transcriptional control by PPAR␥-mediated signals.

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“…Although many of the trapped sequences were not mapped to exons, it cannot be ruled out that many of them might be located at the sites of unknown genes. In fact, it has been demonstrated that the poly(A) trap scheme is useful for the identification of novel genes (14,35).…”

Section: Vol 23 2003mentioning

confidence: 99%

Characterization ofSleeping BeautyTransposition and Its Application to Genetic Screening in Mice

Horie¹,

Yusa²,

Yae

et al. 2003

Molecular and Cellular Biology

143

151

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The use of mutant mice plays a pivotal role in determining the function of genes, and the recently reported germ line transposition of the Sleeping Beauty (SB) transposon would provide a novel system to facilitate this approach. In this study, we characterized SB transposition in the mouse germ line and assessed its potential for generating mutant mice. Transposition sites not only were clustered within 3 Mb near the donor site but also were widely distributed outside this cluster, indicating that the SB transposon can be utilized for both region-specific and genome-wide mutagenesis. The complexity of transposition sites in the germ line was high enough for large-scale generation of mutant mice. Based on these initial results, we conducted germ line mutagenesis by using a gene trap scheme, and the use of a green fluorescent protein reporter made it possible to select for mutant mice rapidly and noninvasively. Interestingly, mice with mutations in the same gene, each with a different insertion site, were obtained by local transposition events, demonstrating the feasibility of the SB transposon system for region-specific mutagenesis. Our results indicate that the SB transposon system has unique features that complement other mutagenesis approaches.The analysis of mutant mice plays a key role in the understanding of gene functions, and the importance of this approach is expected to increase (2) with the recent availability of the mouse genome sequence (25). However, large-scale genetic screening for mice has been lagging far behind that for other model organisms, such as Drosophila melanogaster and Caenorhabditis elegans, because of the lack of a system allowing for both mutagenesis and subsequent rapid identification of the mutation. Large-scale generation of mutant mice has been conducted recently by using N-ethyl-N-nitrosourea (ENU), and a number of mutant mice with various phenotypes have been generated successfully (6, 26). The drawback of this approach is that identification of the causative point mutations is time-consuming. The embryonic stem (ES) cell-based gene trap is another effective approach (11,12,31). However, largescale generation of mutant mice, which is a prerequisite for genetic screening, is not easy because the ES cell-based methods involve labor-intensive processes such as tissue culture or embryo manipulation.Transposon-tagged mutagenesis has been used in a wide range of organisms, such as D. melanogaster (1, 32), C. elegans (13, 24), and plants (27). Although the mutation rate resulting from transposition is not as high as that in ENU mutagenesis, transposon-tagged mutagenesis has been used as an alternative genetic screening method for the following reasons. First, the genes responsible for the phenotypes can be identified rapidly by using the transposon sequence as a tag. Second, desired elements can be introduced into the transposon sequence to expand the application range of the mutant lines. This principle has been demonstrated in the P element of D. melanogaster, where various GAL4 en...

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Creation of genome-wide protein expression libraries using random activation of gene expression

Cited by 65 publications

References 37 publications

Involvement of intact HPV16 E6/E7 gene expression in head and neck cancers with unaltered p53 status and perturbed pRb cell cycle control

Involvement of intact HPV16 E6/E7 gene expression in head and neck cancers with unaltered p53 status and perturbed pRb cell cycle control

The adipose tissue triglyceride lipase ATGL/PNPLA2 is downregulated by insulin and TNF-α in 3T3-L1 adipocytes and is a target for transactivation by PPARγ

Characterization ofSleeping BeautyTransposition and Its Application to Genetic Screening in Mice

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