Active transcriptomic and proteomic reprogramming in the C. elegans nucleotide excision repair mutant xpa-1.

Transcription-blocking oxidative DNA damage is believed to contribute to aging and to underlie activation of oxidative stress responses and down-regulation of insulin-like signaling (ILS) in Nucleotide Excision Repair (NER) deficient mice. Here, we present the first quantitative proteomic description of the Caenorhabditis elegans NER-defective xpa-1 mutant and compare the proteome and transcriptome signatures. Both methods indicated activation of oxidative stress responses, which was substantiated biochemically by a bioenergetic shift involving increased steady-state reactive oxygen species (ROS) and Adenosine triphosphate (ATP) levels. We identify the lesion-detection enzymes of Base Excision Repair (NTH-1) and global genome NER (XPC-1 and DDB-1) as upstream requirements for transcriptomic reprogramming as RNA-interference mediated depletion of these enzymes prevented up-regulation of genes over-expressed in the xpa-1 mutant. The transcription factors SKN-1 and SLR-2, but not DAF-16, were identified as effectors of reprogramming. As shown in human XPA cells, the levels of transcription-blocking 8,5'-cyclo-2'-deoxyadenosine lesions were reduced in the xpa-1 mutant compared to the wild type. Hence, accumulation of cyclopurines is unlikely to be sufficient for reprogramming. Instead, our data support a model where the lesion-detection enzymes NTH-1, XPC-1 and DDB-1 play active roles to generate a genomic stress signal sufficiently strong to result in transcriptomic reprogramming in the xpa-1 mutant.

Ex vivo and in vivo delivery of anti-tissue factor short interfering RNA inhibits mouse pulmonary metastasis of B16 melanoma cells.

PURPOSE: The coagulation trigger tissue factor has been implicated in tumor growth, angiogenesis, and metastasis. In this study, we explore the effects of ex vivo and in vivo delivery of short interfering RNA (siRNA) targeting tissue factor on B16 melanoma colonization of the lung in a murine model for metastasis. The purposes of this work are to establish a noncytotoxic in vivo model for investigation of tissue factor function and provide preclinical assessment of the therapeutic potential of tissue factor siRNA for prevention of metastasis. EXPERIMENTAL DESIGN AND RESULTS: C57BL/6 mice were evaluated for pulmonary metastases following tail vein injection of B16 cells transfected with either active or inactive siRNA. Mice receiving cells transfected with active siRNA had significantly lower numbers of pulmonary tumors compared with mice injected with control cells (transfected with inactive siRNA). The average time point at which the mice started to exhibit tumor-associated stress was also increased significantly from 22 days for the control group to 27 days for the experimental group (P = 0.01). In a therapeutically more relevant model, where the siRNA was delivered i.p. and the cells (untransfected) by tail vein injection, an inhibitory effect on metastasis was observed when the siRNA treatment was initiated either before or at the time of cell injection. CONCLUSIONS: The results suggest that tissue factor has a crucial function in promoting lung tumor metastasis of blood-borne tumor cells in the early stages of the tumor take process and further suggest that treatment with tissue factor siRNA may become a viable clinical strategy for prevention of tumor metastasis.

LXRα Regulates Hepatic ChREBPα Activity and Lipogenesis upon Glucose, but Not Fructose Feeding in Mice.

Liver X receptors (LXRα/ß) and carbohydrate response element-binding proteins (ChREBPα/ß) are key players in the transcriptional control of hepatic de novo lipogenesis. LXRα/ß double knockout (LXRα-/-/ß-/-) mice have reduced feeding-induced nuclear O-linked N-acetylglucosamine (O-GlcNAc) signaling, ChREBPα activity, and lipogenic gene expression in livers, suggesting important roles for LXRs in linking hepatic glucose utilization to lipid synthesis. However, the role of LXRs in fructose-induced ChREBP activation and lipogenesis is currently unknown. In this study, we studied the effects of high fructose or high glucose feeding on hepatic carbohydrate metabolism and lipogenic gene expression in livers from fasted (24 h) and fasted-refed (12 h) wild type and LXRα knockout (LXRα-/-) mice. Hepatic lipogenic gene expression was reduced in glucose fed, but not fructose fed LXRα-/- mice. This was associated with lower expression of liver pyruvate-kinase (L-pk) and Chrebpß, indicating reduced ChREBPα activity in glucose fed, but not fructose fed mice. Interestingly, ChREBP binding to the L-pk promoter was increased in fructose fed LXRα-/- mice, concomitant with increased glucose-6-phosphatase (G6pc) expression and O-GlcNAc modified LXRß, suggesting a role for LXRß in regulating ChREBPα activity upon fructose feeding. In conclusion, we propose that LXRα is an important regulator of hepatic lipogenesis and ChREBPα activity upon glucose, but not fructose feeding in mice.

Similar behaviour of single-strand and double-strand siRNAs suggests they act through a common RNAi pathway.

RNA interference (RNAi), mediated by either long double-stranded RNA (dsRNA) or short interfering RNA (siRNA), has become a routine tool for transient knockdown of gene expression in a wide range of organisms. The antisense strand of the siRNA duplex (antisense siRNA) was recently shown to have substantial mRNA depleting activity of its own. Here, targeting human Tissue Factor mRNA in HaCaT cells, we perform a systematic comparison of the activity of antisense siRNA and double-strand siRNA, and find almost identical target position effects, appearance of mRNA cleavage fragments and tolerance for mutational and chemical backbone modifications. These observations, together with the demonstration that excess inactive double-strand siRNA blocks antisense siRNA activity, i.e. shows sequence-independent competition, indicate that the two types of effector molecules share the same RNAi pathway. Interest ingly, both FITC-tagged and 3'-deoxy antisense siRNA display severely limited activity, despite having practically wild-type activity in a siRNA duplex. Finally, we find that maximum depletion of target mRNA expression occurs significantly faster with antisense siRNA than with double-strand siRNA, suggesting that the former enters the RNAi pathway at a later stage than double-strand siRNA, thereby requiring less time to exert its activity.

Tolerance for mutations and chemical modifications in a siRNA.

Short interfering RNA (siRNA), the active agent of RNA interference, shows promise of becoming a valuable tool in both basic and clinical research. We explore the tolerance to mutations and chemical modifications in various parts of the two 21-nt strands of a siRNA targeting the blood clotting initiator Tissue Factor. The mutations were G/C transversions. The chemical modifications were 2'-O-methylation, 2'-O-allylation and phosphorothioates. We found that siRNA generally tolerated mutations in the 5' end, while the 3' end exhibited low tolerance. This observation may facilitate the design of siRNA for specific targeting of transcripts containing single nucleotide polymorphisms. We further demonstrate that in our system the single antisense strand of the wild-type siRNA is almost as effective as the siRNA duplex, while the corresponding methylated M2+4 version of the antisense had reduced activity. Most of the chemically modified versions tested had near-wild-type initial activity, while the long-term activity was increased for certain siRNA species. Our results may improve the design of siRNAs for in vivo experiments.

Positional effects of short interfering RNAs targeting the human coagulation trigger Tissue Factor.

Chemically synthesised 21-23 bp double-stranded short interfering RNAs (siRNA) can induce sequence-specific post-transcriptional gene silencing, in a process termed RNA interference (RNAi). In the present study, several siRNAs synthesised against different sites on the same target mRNA (human Tissue Factor) demonstrated striking differences in silencing efficiency. Only a few of the siRNAs resulted in a significant reduction in expression, suggesting that accessible siRNA target sites may be rare in some human mRNAs. Blocking of the 3'-OH with FITC did not reduce the effect on target mRNA. Mutations in the siRNAs relative to target mRNA sequence gradually reduced, but did not abolish mRNA depletion. Inactive siRNAs competed reversibly with active siRNAs in a sequence-independent manner. Several lines of evidence suggest the existence of a near equilibrium kinetic balance between mRNA production and siRNA-mediated mRNA depletion. The silencing effect was transient, with the level of mRNA recovering fully within 4-5 days, suggesting absence of a propagative system for RNAi in humans. Finally, we observed 3' mRNA cleavage fragments resulting from the action of the most effective siRNAs. The depletion rate-dependent appearance of these fragments argues for the existence of a two-step mRNA degradation mechanism.

Mapping the global mRNA transcriptome during development of the murine first molar.

The main objective of this study was to map global gene expression in order to provide information about the populations of mRNA species participating in murine tooth development at 24 h intervals, starting at the 11th embryonic day (E11.5) up to the 7th post-natal day (P7). The levels of RNA species expressed during murine tooth development were mesured using a total of 58 deoxyoligonucleotide microarrays. Microarray data was validated using real-time RT-PCR. Differentially expressed genes (p < 0.05) were subjected to bioinformatic analysis to identify cellular activities significantly associated with these genes. Using ANOVA the microarray data yielded 4362 genes as being differentially expressed from the 11th embryonic day (E11.5) up to 7 days post-natal (P7), 1921 of these being genes without known functions. The remaining 2441 genes were subjected to further statistical analysis using a supervised procedure. Bioinformatic analysis results for each time-point studied suggests that the main molecular functions associated with genes expressed at the early pre-natal stages (E12.5-E18.5) were cell cycle progression, cell morphology, lipid metabolism, cellular growth, proliferation, senescence and apoptosis, whereas most genes expressed at post-natal and secretory stages (P0-P7) were significantly associated with regulation of cell migration, biosynthesis, differentiation, oxidative stress, polarization and cell death. Differentially expressed genes (DE) not described earlier during murine tooth development; Inositol 1, 4, 5-triphosphate receptor 3 (Itpr3), metallothionein 1(Mt1), cyclin-dependent kinase 4 (Cdk4), cathepsin D (Ctsd), keratin complex 2, basic, gene 6a (Krt2-6a), cofilin 1, non-muscle (Cfl1), cyclin 2 (Ccnd2), were verified by real-time RT-PCR.

Gene Expression Profiling during Murine Tooth Development.

The aim of this study was to describe the expression of genes, including ameloblastin (Ambn), amelogenin X chromosome (Amelx), and enamelin (Enam) during early (pre-secretory) tooth development. The expression of these genes has predominantly been studied at post-secretory stages. Deoxyoligonucleotide microarrays were used to study gene expression during development of the murine first molar tooth germ at 24 h intervals, starting at the 11th embryonic day (E11.5), and up to the 7th day after birth (P7). The profile search function of Spotfire software was used to select genes with similar expression profile as the enamel genes (Ambn, Amelx, and Enam). Microarray results where validated using real-time reverse transcription-polymerase chain reaction (real-time RT-PCR), and translated proteins identified by Western-blotting. In situ localization of the Ambn, Amelx, and Enam mRNAs were monitored from E12.5 to E17.5 using deoxyoligonucleotide probes. Bioinformatics analysis was used to associate biological functions with differentially expressed (DE; p ≤ 0.05) genes. Microarray results showed a total of 4362 genes including Ambn, Amelx, and Enam to be significant DE throughout the time-course. The expression of the three enamel genes was low at pre-natal stages (E11.5-P0) increasing after birth (P1-P7). Profile search lead to isolation of 87 genes with significantly similar expression to the three enamel proteins. These mRNAs were expressed in dental epithelium and epithelium derived cells. Although expression of Ambn, Amelx, and Enam were lower during early tooth development compared to secretory stages enamel proteins were detectable by Western-blotting. Bioinformatic analysis associated the 87 genes with multiple biological functions. Around 35 genes were associated with 15 transcription factors.

Loss of Caenorhabditis elegans UNG-1 uracil-DNA glycosylase affects apoptosis in response to DNA damaging agents.

The nematode Caenorhabditis elegans has been used extensively to study responses to DNA damage. In contrast, little is known about DNA repair in this organism. C. elegans is unusual in that it encodes few DNA glycosylases and the uracil-DNA glycosylase (UDG) encoded by the ung-1 gene is the only known UDG. C. elegans could therefore become a valuable model organism for studies of the genetic interaction networks involving base excision repair (BER). As a first step towards characterization of BER in C. elegans, we show that the UNG-1 protein is an active uracil-DNA glycosylase. We demonstrate that an ung-1 mutant has reduced ability to repair uracil-containing DNA but that an alternative Ugi-inhibited activity is present in ung-1 nuclear extracts. Finally, we demonstrate that ung-1 mutants show altered levels of apoptotic cell corpses formed in response to DNA damaging agents. Increased apoptosis in the ung-1 mutant in response to ionizing radiation (IR) suggests that UNG-1 contributes to repair of IR-induced DNA base damage in vivo. Following treatment with paraquat however, the apoptotic corpse-formation was reduced. Gene expression profiling suggests that this phenotype is a consequence of compensatory transcriptomic shifts that modulate oxidative stress responses in the mutant and not an effect of reduced DNA damage signaling.