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Hypoxic transcription gene profiles under the modulation of nitric oxide in nuclear run on-microarray and proteomics.

Igwe EI, Essler S, Al-Furoukh N, Dehne N, Brüne B - BMC Genomics (2009)

Bottom Line: In addition, both array and proteomics data supported a consistent repression of hypoxia-regulated targets by NO.By eliminating the interference of steady state mRNA in gene expression profiling, we obtained a smaller number of significantly regulated transcripts in our study compared to published microarray data and identified previously unknown hypoxia-induced targets.Gene analysis profiling corroborated the interplay between NO- and hypoxia-induced signaling.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Biochemistry I/ZAFES, Faculty of Medicine, Goethe-University Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany. emekaigwe@zbc.kgu.de

ABSTRACT

Background: Microarray analysis still is a powerful tool to identify new components of the transcriptosome. It helps to increase the knowledge of targets triggered by stress conditions such as hypoxia and nitric oxide. However, analysis of transcriptional regulatory events remain elusive due to the contribution of altered mRNA stability to gene expression patterns as well as changes in the half-life of mRNAs, which influence mRNA expression levels and their turn over rates. To circumvent these problems, we have focused on the analysis of newly transcribed (nascent) mRNAs by nuclear run on (NRO), followed by microarray analysis.

Results: We identified 196 genes that were significantly regulated by hypoxia, 85 genes affected by nitric oxide and 292 genes induced by the cotreatment of macrophages with both NO and hypoxia. Fourteen genes (Bnip3, Ddit4, Vegfa, Trib3, Atf3, Cdkn1a, Scd1, D4Ertd765e, Sesn2, Son, Nnt, Lst1, Hps6 and Fxyd5) were common to all treatments but with different levels of expression in each group. We observed that 162 transcripts were regulated only when cells were co-treated with hypoxia and NO but not with either treatment alone, pointing to the importance of a crosstalk between hypoxia and NO. In addition, both array and proteomics data supported a consistent repression of hypoxia-regulated targets by NO.

Conclusion: By eliminating the interference of steady state mRNA in gene expression profiling, we obtained a smaller number of significantly regulated transcripts in our study compared to published microarray data and identified previously unknown hypoxia-induced targets. Gene analysis profiling corroborated the interplay between NO- and hypoxia-induced signaling.

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Venn diagram of grouped genes. Analysis of gene expression in RAW cells subjected to hypoxia (1% O2) and/or 0.5 mM DETA-NO for 6 h. After removing double and unknown transcripts data represent total number of genes regulated by each treatment (in parentheses) as well as overlapping genes shared by treatments. A complete list of genes for each treatment is provided in the supplementary tables 1-3. Detailed information of genes from the intersections is given in table 1-5.
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Figure 1: Venn diagram of grouped genes. Analysis of gene expression in RAW cells subjected to hypoxia (1% O2) and/or 0.5 mM DETA-NO for 6 h. After removing double and unknown transcripts data represent total number of genes regulated by each treatment (in parentheses) as well as overlapping genes shared by treatments. A complete list of genes for each treatment is provided in the supplementary tables 1-3. Detailed information of genes from the intersections is given in table 1-5.

Mentions: The intention of this study was to identify hypoxia- or nitric oxide-regulated genes by a systematic analysis of de novo (nascent) transcription and to question whether regulation of HIF1α by hypoxia or nitric oxide would generate overlapping gene profiles. We chose to analyze newly synthesized mRNA obtained by NRO. This allowed detection of bona fide transcriptionally regulated genes, thus reducing the interference of steady-state mRNA turnover on the pool of expressed mRNA common to conventional microarray analysis. Therefore, RAW 264.7 cells were exposed to hypoxia (1% O2), DETA-NO (0.5 mM), or a combination of hypoxia and NO for 6 h. Transcriptional changes were systematically assessed using Affymetrix microarray analysis, which allowed following 32000 transcripts. [26]. Most gene transcripts (> 99% of genes on the array) remained unchanged by either hypoxia or NO, as well as in the combination of both. Only 196 genes were regulated by hypoxia with the majority being upregulated [see Additional file 1]. The greater number of the transcripts regulated by hypoxia is linked to energy consumption through glycolysis. Nitric oxide regulated a set of 85 genes shown in supplementary table 2 [see Additional file 2], while treatment of cells with both hypoxia and NO regulated 292 transcripts [see Additional file 3]. Figure 1 gives an overview of all groups with their number of regulated genes. Comparing gene profiles following treatments with hypoxia and/or NO, we observed that only 14 genes (Bnip3, Ddit4, Vegfa, Trib3, Atf3, Cdkn1a, Scd1, D4Ertd765e, Sesn2, Son, Nnt, Lst1, Hps6, and Fxyd5) were common to all treatments. It is not surprising that many of these genes have roles in cell death, DNA damage and apoptosis since under stress conditions cells try to avoid cell demise. Most of these genes had been previously described as hypoxia or NO regulated [27-29], underscoring the validity of this approach. Interestingly, 9 of the 14 common genes (Bnip3, Ddit4, Vegfa, Trib3, Atf3, Cdkn1a, Scd1, D4Ertd765e, Sesn2) were upregulated by hypoxia and/or NO, whereas 5 (Son, Nnt, Lst1, Hps6, and Fxyd5) were downregulated (Table 1). Most genes upregulated by hypoxia or NO were more strongly regulated under hypoxia, with the exception of Scd1 and Sesn2, whose levels were slightly higher with NO. For the commonly downregulated genes 4 out of the 5 identified ones were regulated to the same extent by hypoxia or NO. Interestingly, only one gene being downregulated (Fxyd5 a glycoprotein containing a Na+- K+-ATPase domain) revealed different levels of inhibition between hypoxia and NO, albeit being the strongest suppressed gene in the list. Hypoxia and NO downregulated Fxyd5 4.2-folds and 18.27-folds compared to controls. Contrary to the finding in this study, Fxyd5 has been shown to be upregulated in the mouse carotid body in response to 10% hypoxia [30], suggesting a different regulation pattern for this protein at different oxygen pressures. Fxyd5 affects the ion transport system of the blood brain barrier and modulates Na+ absorption during cystic fibrosis [31]. The notion that NO decreased chloride adsorption by reducing Na+-K+-2Cl- cotransporter activity and inhibits transepithelial ion movement in cystic fibrosis [32] corroborates our finding on repression of Fxyd5. Fxyd5 interacts with the Na+-K+-ATPase and modulates its properties after NO-treatment [33].


Hypoxic transcription gene profiles under the modulation of nitric oxide in nuclear run on-microarray and proteomics.

Igwe EI, Essler S, Al-Furoukh N, Dehne N, Brüne B - BMC Genomics (2009)

Venn diagram of grouped genes. Analysis of gene expression in RAW cells subjected to hypoxia (1% O2) and/or 0.5 mM DETA-NO for 6 h. After removing double and unknown transcripts data represent total number of genes regulated by each treatment (in parentheses) as well as overlapping genes shared by treatments. A complete list of genes for each treatment is provided in the supplementary tables 1-3. Detailed information of genes from the intersections is given in table 1-5.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2743718&req=5

Figure 1: Venn diagram of grouped genes. Analysis of gene expression in RAW cells subjected to hypoxia (1% O2) and/or 0.5 mM DETA-NO for 6 h. After removing double and unknown transcripts data represent total number of genes regulated by each treatment (in parentheses) as well as overlapping genes shared by treatments. A complete list of genes for each treatment is provided in the supplementary tables 1-3. Detailed information of genes from the intersections is given in table 1-5.
Mentions: The intention of this study was to identify hypoxia- or nitric oxide-regulated genes by a systematic analysis of de novo (nascent) transcription and to question whether regulation of HIF1α by hypoxia or nitric oxide would generate overlapping gene profiles. We chose to analyze newly synthesized mRNA obtained by NRO. This allowed detection of bona fide transcriptionally regulated genes, thus reducing the interference of steady-state mRNA turnover on the pool of expressed mRNA common to conventional microarray analysis. Therefore, RAW 264.7 cells were exposed to hypoxia (1% O2), DETA-NO (0.5 mM), or a combination of hypoxia and NO for 6 h. Transcriptional changes were systematically assessed using Affymetrix microarray analysis, which allowed following 32000 transcripts. [26]. Most gene transcripts (> 99% of genes on the array) remained unchanged by either hypoxia or NO, as well as in the combination of both. Only 196 genes were regulated by hypoxia with the majority being upregulated [see Additional file 1]. The greater number of the transcripts regulated by hypoxia is linked to energy consumption through glycolysis. Nitric oxide regulated a set of 85 genes shown in supplementary table 2 [see Additional file 2], while treatment of cells with both hypoxia and NO regulated 292 transcripts [see Additional file 3]. Figure 1 gives an overview of all groups with their number of regulated genes. Comparing gene profiles following treatments with hypoxia and/or NO, we observed that only 14 genes (Bnip3, Ddit4, Vegfa, Trib3, Atf3, Cdkn1a, Scd1, D4Ertd765e, Sesn2, Son, Nnt, Lst1, Hps6, and Fxyd5) were common to all treatments. It is not surprising that many of these genes have roles in cell death, DNA damage and apoptosis since under stress conditions cells try to avoid cell demise. Most of these genes had been previously described as hypoxia or NO regulated [27-29], underscoring the validity of this approach. Interestingly, 9 of the 14 common genes (Bnip3, Ddit4, Vegfa, Trib3, Atf3, Cdkn1a, Scd1, D4Ertd765e, Sesn2) were upregulated by hypoxia and/or NO, whereas 5 (Son, Nnt, Lst1, Hps6, and Fxyd5) were downregulated (Table 1). Most genes upregulated by hypoxia or NO were more strongly regulated under hypoxia, with the exception of Scd1 and Sesn2, whose levels were slightly higher with NO. For the commonly downregulated genes 4 out of the 5 identified ones were regulated to the same extent by hypoxia or NO. Interestingly, only one gene being downregulated (Fxyd5 a glycoprotein containing a Na+- K+-ATPase domain) revealed different levels of inhibition between hypoxia and NO, albeit being the strongest suppressed gene in the list. Hypoxia and NO downregulated Fxyd5 4.2-folds and 18.27-folds compared to controls. Contrary to the finding in this study, Fxyd5 has been shown to be upregulated in the mouse carotid body in response to 10% hypoxia [30], suggesting a different regulation pattern for this protein at different oxygen pressures. Fxyd5 affects the ion transport system of the blood brain barrier and modulates Na+ absorption during cystic fibrosis [31]. The notion that NO decreased chloride adsorption by reducing Na+-K+-2Cl- cotransporter activity and inhibits transepithelial ion movement in cystic fibrosis [32] corroborates our finding on repression of Fxyd5. Fxyd5 interacts with the Na+-K+-ATPase and modulates its properties after NO-treatment [33].

Bottom Line: In addition, both array and proteomics data supported a consistent repression of hypoxia-regulated targets by NO.By eliminating the interference of steady state mRNA in gene expression profiling, we obtained a smaller number of significantly regulated transcripts in our study compared to published microarray data and identified previously unknown hypoxia-induced targets.Gene analysis profiling corroborated the interplay between NO- and hypoxia-induced signaling.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Biochemistry I/ZAFES, Faculty of Medicine, Goethe-University Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany. emekaigwe@zbc.kgu.de

ABSTRACT

Background: Microarray analysis still is a powerful tool to identify new components of the transcriptosome. It helps to increase the knowledge of targets triggered by stress conditions such as hypoxia and nitric oxide. However, analysis of transcriptional regulatory events remain elusive due to the contribution of altered mRNA stability to gene expression patterns as well as changes in the half-life of mRNAs, which influence mRNA expression levels and their turn over rates. To circumvent these problems, we have focused on the analysis of newly transcribed (nascent) mRNAs by nuclear run on (NRO), followed by microarray analysis.

Results: We identified 196 genes that were significantly regulated by hypoxia, 85 genes affected by nitric oxide and 292 genes induced by the cotreatment of macrophages with both NO and hypoxia. Fourteen genes (Bnip3, Ddit4, Vegfa, Trib3, Atf3, Cdkn1a, Scd1, D4Ertd765e, Sesn2, Son, Nnt, Lst1, Hps6 and Fxyd5) were common to all treatments but with different levels of expression in each group. We observed that 162 transcripts were regulated only when cells were co-treated with hypoxia and NO but not with either treatment alone, pointing to the importance of a crosstalk between hypoxia and NO. In addition, both array and proteomics data supported a consistent repression of hypoxia-regulated targets by NO.

Conclusion: By eliminating the interference of steady state mRNA in gene expression profiling, we obtained a smaller number of significantly regulated transcripts in our study compared to published microarray data and identified previously unknown hypoxia-induced targets. Gene analysis profiling corroborated the interplay between NO- and hypoxia-induced signaling.

Show MeSH
Related in: MedlinePlus