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A role for insulator elements in the regulation of gene expression response to hypoxia.

Tiana M, Villar D, Pérez-Guijarro E, Gómez-Maldonado L, Moltó E, Fernández-Miñán A, Gómez-Skarmeta JL, Montoliu L, del Peso L - Nucleic Acids Res. (2011)

Bottom Line: Analysis of the transcriptional response of chimeric constructs derived from the intergenic region revealed an inhibitory sequence whose deletion allowed RUVBL2 induction by HIF.Hence, in this model, the selective response to HIF is achieved with the aid of insulator elements.This is the first report suggesting a role for insulators in the regulation of differential gene expression in response to environmental signals.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica, Universidad Autónoma de Madrid and Instituto de Investigaciones Biomedicas Alberto Sols, CSIC-UAM, 28029 Madrid, Spain.

ABSTRACT
Hypoxia inducible factor (HIF) up-regulates the transcription of a few hundred genes required for the adaptation to hypoxia. This restricted set of targets is in sharp contrast with the widespread distribution of the HIF binding motif throughout the genome. Here, we investigated the transcriptional response of GYS1 and RUVBL2 genes to hypoxia to understand the mechanisms that restrict HIF activity toward specific genes. GYS1 and RUVBL2 genes are encoded by opposite DNA strands and separated by a short intergenic region (~1 kb) that contains a functional hypoxia response element equidistant to both genes. However, hypoxia induced the expression of GYS1 gene only. Analysis of the transcriptional response of chimeric constructs derived from the intergenic region revealed an inhibitory sequence whose deletion allowed RUVBL2 induction by HIF. Enhancer blocking assays, performed in cell culture and transgenic zebrafish, confirmed the existence of an insulator element within this inhibitory region that could explain the differential regulation of GYS1 and RUVBL2 by hypoxia. Hence, in this model, the selective response to HIF is achieved with the aid of insulator elements. This is the first report suggesting a role for insulators in the regulation of differential gene expression in response to environmental signals.

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Related in: MedlinePlus

Differential regulation of GYS1 and RUVBL2 by hypoxia. (A) Schematic diagram depicting the human (hg18 assembly) genomic region containing the intergenic region between GYS1 and RUVBL2 and its sequence conservation across mammals [adapted from UCSC genomic browser, http://genome.ucsc.edu/(53)]. The boxes below the diagram represent the different blocks identified within this region according to their evolutionarily conservation and solid lines indicate the regions cloned to generate reporter constructs, cG, cR and cG+cR. (B) Effect of hypoxia or the hypoxia-mimetic deferoxamine (GSE5579) on GYS1 and RUVBL2 expression extracted from gene expression profiles of human breast carcinoma cell line MCF7 (GSE3188), mouse embryo fibroblast (GSE3196), human B lymphocyte P493-6 cells (GSE4086), human monocyte-derived macrophages (GDS2036), human lymphatic endothelial cells (GSE5579), human aortic smooth muscle cells (GSE4725), human colon adenocarcinoma cell line HT29 cells (GSE9234), mouse hepatocytes (GDS1648), human embryonic kidney cell line HEK293 (GSE2020), human astrocytes (GSE3045) and human epithelial cervical cancer cell line HeLa (GSE3051) exposed to hypoxia. Asterisks indicate that the set of data values was significantly different (one sample t-test, t = 3.6988, df = 11, P = 0.003509) from the value of zero (no induction). (C) c2c12 myoblast were exposed to normoxia or hypoxia for 12–24 h and GYS1 and RUVBL2 expression was determined by quantitative PCR from total RNA samples. Data were calculated relative to β-actin and expressed as fold change relative to normoxia. Data shown are the results of three independent experiments and their mean (bar). The relative induction of both mRNAs was significantly different (t-test, t = 4.9995, df = 2, P = 0.03776). (D) A variety of cell lines (HepG2 and HepaC1, mouse hepatocarcinoma cell lines; primary mouse hepatocytes; NIH3T3, mouse fibroblast cell line; HeLa; N2a, mouse neuroblastoma cell line; HEK293; A549, human lung adenocarcinoma cell line) were exposed to normoxia or hypoxia and the levels of GYS1 and RUVBL2 mRNA determined and represented as indicated in C. Asterisks indicate that the set of data values was significantly different (one sample t-test, t = 4.4522, df = 7, P = 0.002964) from the value of one (no induction). (E) HeLa cells were transiently transfected with reporter constructs containing the indicated region (cG, cR, cGcR or cRcG, see Figure 1A) upstream a firefly luciferase gene and exposed to normoxia, hypoxia or the hypoxia mimetic DMOG for 12 h. The graphs represent the corrected luciferase activity values of each construct in cells exposed to hypoxia/DMOG and represented as fold change over the activity obtained in normoxic cells. Bars represent the average of values obtained in three independent experiments and error bars their standard deviation. Statistically significant differences between pairs of constructs are indicated by asterisks (one-way ANOVA, F9,20 = 36.704, P = 1.6 × 10−10, followed by Tukey's multiple comparison test).
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gkr842-F1: Differential regulation of GYS1 and RUVBL2 by hypoxia. (A) Schematic diagram depicting the human (hg18 assembly) genomic region containing the intergenic region between GYS1 and RUVBL2 and its sequence conservation across mammals [adapted from UCSC genomic browser, http://genome.ucsc.edu/(53)]. The boxes below the diagram represent the different blocks identified within this region according to their evolutionarily conservation and solid lines indicate the regions cloned to generate reporter constructs, cG, cR and cG+cR. (B) Effect of hypoxia or the hypoxia-mimetic deferoxamine (GSE5579) on GYS1 and RUVBL2 expression extracted from gene expression profiles of human breast carcinoma cell line MCF7 (GSE3188), mouse embryo fibroblast (GSE3196), human B lymphocyte P493-6 cells (GSE4086), human monocyte-derived macrophages (GDS2036), human lymphatic endothelial cells (GSE5579), human aortic smooth muscle cells (GSE4725), human colon adenocarcinoma cell line HT29 cells (GSE9234), mouse hepatocytes (GDS1648), human embryonic kidney cell line HEK293 (GSE2020), human astrocytes (GSE3045) and human epithelial cervical cancer cell line HeLa (GSE3051) exposed to hypoxia. Asterisks indicate that the set of data values was significantly different (one sample t-test, t = 3.6988, df = 11, P = 0.003509) from the value of zero (no induction). (C) c2c12 myoblast were exposed to normoxia or hypoxia for 12–24 h and GYS1 and RUVBL2 expression was determined by quantitative PCR from total RNA samples. Data were calculated relative to β-actin and expressed as fold change relative to normoxia. Data shown are the results of three independent experiments and their mean (bar). The relative induction of both mRNAs was significantly different (t-test, t = 4.9995, df = 2, P = 0.03776). (D) A variety of cell lines (HepG2 and HepaC1, mouse hepatocarcinoma cell lines; primary mouse hepatocytes; NIH3T3, mouse fibroblast cell line; HeLa; N2a, mouse neuroblastoma cell line; HEK293; A549, human lung adenocarcinoma cell line) were exposed to normoxia or hypoxia and the levels of GYS1 and RUVBL2 mRNA determined and represented as indicated in C. Asterisks indicate that the set of data values was significantly different (one sample t-test, t = 4.4522, df = 7, P = 0.002964) from the value of one (no induction). (E) HeLa cells were transiently transfected with reporter constructs containing the indicated region (cG, cR, cGcR or cRcG, see Figure 1A) upstream a firefly luciferase gene and exposed to normoxia, hypoxia or the hypoxia mimetic DMOG for 12 h. The graphs represent the corrected luciferase activity values of each construct in cells exposed to hypoxia/DMOG and represented as fold change over the activity obtained in normoxic cells. Bars represent the average of values obtained in three independent experiments and error bars their standard deviation. Statistically significant differences between pairs of constructs are indicated by asterisks (one-way ANOVA, F9,20 = 36.704, P = 1.6 × 10−10, followed by Tukey's multiple comparison test).

Mentions: The muscle glycogen synthase gene, GYS1, is regulated by HIF as part of the cellular metabolic reprogramming required for the adaptation to hypoxia (27). The regulation of human GYS1 by hypoxia is mediated by a functional RCGTG element located 255 bp upstream its transcription start site (TSS) (27). Very close to GYS1, but encoded by the opposite DNA strand, is located the RUVBL2 gene (Figure 1A). The relative position, intergenic distance and orientation of these two genes are conserved across mammals (data not shown). The TSS of RUVBL2 is located at 288 bp of the HRE driving GYS1 expression in response to hypoxia (location of the HRE is shown by a black box in the ‘blat’ track, Figure 1A), raising the possibility of a coordinated regulation of these two genes by HIF. In fact, the intergenic region between GYS1 and RUVBL2 can be considered a bidirectional promoter (Figure 1A, ‘Elnitski bidirectional promoters’ prediction track). To study this possibility, we analyzed publicly available gene expression profiles of cells exposed to hypoxia and found that, whereas GYS1 mRNA levels were induced by hypoxia in most of the profiles, the expression of RUVBL2 remained constant or was even repressed under low oxygen tension (Figure 1B). To confirm these results we exposed myotubes to hypoxia and determined its effect on GYS1 and RUVBL2 mRNA levels. As shown in Figure 1C, GYS1 mRNA level significantly increased in response to hypoxia, in agreement with published results (27), but the treatment did not induce RUVBL2 mRNA. In order to rule out a cell-type specific effect, we determined the effect of hypoxia on the expression of GYS1 and RUVBL2 in a variety of cell types (Figure 1D). These analyses confirmed that, whereas GYS1 expression was increased by hypoxia in virtually all cell lines studied, RUVBL2 remained largely unaffected. In agreement with this conclusion, a meta-analysis of gene expression profile experiments suggested that, unlike GYS1, RUVBL2 is not significantly modulated by hypoxia (10). These results indicated that hypoxia specifically affects GYS1, but not RUVBL2 transcription. To confirm this possibility, we investigated the effect of hypoxia on a set of reporter constructs derived from this locus. As shown in Figure 1E, a reporter construct containing the region flanking GYS1 gene and including the HRE (region cG spanning residues +84 to −429 relative to GYS1 TSS, Figure 1A), was robustly induced by hypoxia and the hypoxia mimetic DMOG in HeLa cells, as expected from the presence of the evolutionarily conserved block containing the HRE within this cG region. In contrast, the analogous region upstream of RUVBL2 (cR, spanning −396 to +12 relative to RUVBL2 TSS) did not respond to HIF activation in spite of including the same HRE-containing region (Figure 1E). Importantly, this same result was obtained when the whole intergenic region, maintaining the original genomic structure, was used to drive luciferase expression from the GYS1 (cRcG) or the RUVBL2 (cGcR) promoters (Figure 1E).Figure 1.


A role for insulator elements in the regulation of gene expression response to hypoxia.

Tiana M, Villar D, Pérez-Guijarro E, Gómez-Maldonado L, Moltó E, Fernández-Miñán A, Gómez-Skarmeta JL, Montoliu L, del Peso L - Nucleic Acids Res. (2011)

Differential regulation of GYS1 and RUVBL2 by hypoxia. (A) Schematic diagram depicting the human (hg18 assembly) genomic region containing the intergenic region between GYS1 and RUVBL2 and its sequence conservation across mammals [adapted from UCSC genomic browser, http://genome.ucsc.edu/(53)]. The boxes below the diagram represent the different blocks identified within this region according to their evolutionarily conservation and solid lines indicate the regions cloned to generate reporter constructs, cG, cR and cG+cR. (B) Effect of hypoxia or the hypoxia-mimetic deferoxamine (GSE5579) on GYS1 and RUVBL2 expression extracted from gene expression profiles of human breast carcinoma cell line MCF7 (GSE3188), mouse embryo fibroblast (GSE3196), human B lymphocyte P493-6 cells (GSE4086), human monocyte-derived macrophages (GDS2036), human lymphatic endothelial cells (GSE5579), human aortic smooth muscle cells (GSE4725), human colon adenocarcinoma cell line HT29 cells (GSE9234), mouse hepatocytes (GDS1648), human embryonic kidney cell line HEK293 (GSE2020), human astrocytes (GSE3045) and human epithelial cervical cancer cell line HeLa (GSE3051) exposed to hypoxia. Asterisks indicate that the set of data values was significantly different (one sample t-test, t = 3.6988, df = 11, P = 0.003509) from the value of zero (no induction). (C) c2c12 myoblast were exposed to normoxia or hypoxia for 12–24 h and GYS1 and RUVBL2 expression was determined by quantitative PCR from total RNA samples. Data were calculated relative to β-actin and expressed as fold change relative to normoxia. Data shown are the results of three independent experiments and their mean (bar). The relative induction of both mRNAs was significantly different (t-test, t = 4.9995, df = 2, P = 0.03776). (D) A variety of cell lines (HepG2 and HepaC1, mouse hepatocarcinoma cell lines; primary mouse hepatocytes; NIH3T3, mouse fibroblast cell line; HeLa; N2a, mouse neuroblastoma cell line; HEK293; A549, human lung adenocarcinoma cell line) were exposed to normoxia or hypoxia and the levels of GYS1 and RUVBL2 mRNA determined and represented as indicated in C. Asterisks indicate that the set of data values was significantly different (one sample t-test, t = 4.4522, df = 7, P = 0.002964) from the value of one (no induction). (E) HeLa cells were transiently transfected with reporter constructs containing the indicated region (cG, cR, cGcR or cRcG, see Figure 1A) upstream a firefly luciferase gene and exposed to normoxia, hypoxia or the hypoxia mimetic DMOG for 12 h. The graphs represent the corrected luciferase activity values of each construct in cells exposed to hypoxia/DMOG and represented as fold change over the activity obtained in normoxic cells. Bars represent the average of values obtained in three independent experiments and error bars their standard deviation. Statistically significant differences between pairs of constructs are indicated by asterisks (one-way ANOVA, F9,20 = 36.704, P = 1.6 × 10−10, followed by Tukey's multiple comparison test).
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gkr842-F1: Differential regulation of GYS1 and RUVBL2 by hypoxia. (A) Schematic diagram depicting the human (hg18 assembly) genomic region containing the intergenic region between GYS1 and RUVBL2 and its sequence conservation across mammals [adapted from UCSC genomic browser, http://genome.ucsc.edu/(53)]. The boxes below the diagram represent the different blocks identified within this region according to their evolutionarily conservation and solid lines indicate the regions cloned to generate reporter constructs, cG, cR and cG+cR. (B) Effect of hypoxia or the hypoxia-mimetic deferoxamine (GSE5579) on GYS1 and RUVBL2 expression extracted from gene expression profiles of human breast carcinoma cell line MCF7 (GSE3188), mouse embryo fibroblast (GSE3196), human B lymphocyte P493-6 cells (GSE4086), human monocyte-derived macrophages (GDS2036), human lymphatic endothelial cells (GSE5579), human aortic smooth muscle cells (GSE4725), human colon adenocarcinoma cell line HT29 cells (GSE9234), mouse hepatocytes (GDS1648), human embryonic kidney cell line HEK293 (GSE2020), human astrocytes (GSE3045) and human epithelial cervical cancer cell line HeLa (GSE3051) exposed to hypoxia. Asterisks indicate that the set of data values was significantly different (one sample t-test, t = 3.6988, df = 11, P = 0.003509) from the value of zero (no induction). (C) c2c12 myoblast were exposed to normoxia or hypoxia for 12–24 h and GYS1 and RUVBL2 expression was determined by quantitative PCR from total RNA samples. Data were calculated relative to β-actin and expressed as fold change relative to normoxia. Data shown are the results of three independent experiments and their mean (bar). The relative induction of both mRNAs was significantly different (t-test, t = 4.9995, df = 2, P = 0.03776). (D) A variety of cell lines (HepG2 and HepaC1, mouse hepatocarcinoma cell lines; primary mouse hepatocytes; NIH3T3, mouse fibroblast cell line; HeLa; N2a, mouse neuroblastoma cell line; HEK293; A549, human lung adenocarcinoma cell line) were exposed to normoxia or hypoxia and the levels of GYS1 and RUVBL2 mRNA determined and represented as indicated in C. Asterisks indicate that the set of data values was significantly different (one sample t-test, t = 4.4522, df = 7, P = 0.002964) from the value of one (no induction). (E) HeLa cells were transiently transfected with reporter constructs containing the indicated region (cG, cR, cGcR or cRcG, see Figure 1A) upstream a firefly luciferase gene and exposed to normoxia, hypoxia or the hypoxia mimetic DMOG for 12 h. The graphs represent the corrected luciferase activity values of each construct in cells exposed to hypoxia/DMOG and represented as fold change over the activity obtained in normoxic cells. Bars represent the average of values obtained in three independent experiments and error bars their standard deviation. Statistically significant differences between pairs of constructs are indicated by asterisks (one-way ANOVA, F9,20 = 36.704, P = 1.6 × 10−10, followed by Tukey's multiple comparison test).
Mentions: The muscle glycogen synthase gene, GYS1, is regulated by HIF as part of the cellular metabolic reprogramming required for the adaptation to hypoxia (27). The regulation of human GYS1 by hypoxia is mediated by a functional RCGTG element located 255 bp upstream its transcription start site (TSS) (27). Very close to GYS1, but encoded by the opposite DNA strand, is located the RUVBL2 gene (Figure 1A). The relative position, intergenic distance and orientation of these two genes are conserved across mammals (data not shown). The TSS of RUVBL2 is located at 288 bp of the HRE driving GYS1 expression in response to hypoxia (location of the HRE is shown by a black box in the ‘blat’ track, Figure 1A), raising the possibility of a coordinated regulation of these two genes by HIF. In fact, the intergenic region between GYS1 and RUVBL2 can be considered a bidirectional promoter (Figure 1A, ‘Elnitski bidirectional promoters’ prediction track). To study this possibility, we analyzed publicly available gene expression profiles of cells exposed to hypoxia and found that, whereas GYS1 mRNA levels were induced by hypoxia in most of the profiles, the expression of RUVBL2 remained constant or was even repressed under low oxygen tension (Figure 1B). To confirm these results we exposed myotubes to hypoxia and determined its effect on GYS1 and RUVBL2 mRNA levels. As shown in Figure 1C, GYS1 mRNA level significantly increased in response to hypoxia, in agreement with published results (27), but the treatment did not induce RUVBL2 mRNA. In order to rule out a cell-type specific effect, we determined the effect of hypoxia on the expression of GYS1 and RUVBL2 in a variety of cell types (Figure 1D). These analyses confirmed that, whereas GYS1 expression was increased by hypoxia in virtually all cell lines studied, RUVBL2 remained largely unaffected. In agreement with this conclusion, a meta-analysis of gene expression profile experiments suggested that, unlike GYS1, RUVBL2 is not significantly modulated by hypoxia (10). These results indicated that hypoxia specifically affects GYS1, but not RUVBL2 transcription. To confirm this possibility, we investigated the effect of hypoxia on a set of reporter constructs derived from this locus. As shown in Figure 1E, a reporter construct containing the region flanking GYS1 gene and including the HRE (region cG spanning residues +84 to −429 relative to GYS1 TSS, Figure 1A), was robustly induced by hypoxia and the hypoxia mimetic DMOG in HeLa cells, as expected from the presence of the evolutionarily conserved block containing the HRE within this cG region. In contrast, the analogous region upstream of RUVBL2 (cR, spanning −396 to +12 relative to RUVBL2 TSS) did not respond to HIF activation in spite of including the same HRE-containing region (Figure 1E). Importantly, this same result was obtained when the whole intergenic region, maintaining the original genomic structure, was used to drive luciferase expression from the GYS1 (cRcG) or the RUVBL2 (cGcR) promoters (Figure 1E).Figure 1.

Bottom Line: Analysis of the transcriptional response of chimeric constructs derived from the intergenic region revealed an inhibitory sequence whose deletion allowed RUVBL2 induction by HIF.Hence, in this model, the selective response to HIF is achieved with the aid of insulator elements.This is the first report suggesting a role for insulators in the regulation of differential gene expression in response to environmental signals.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica, Universidad Autónoma de Madrid and Instituto de Investigaciones Biomedicas Alberto Sols, CSIC-UAM, 28029 Madrid, Spain.

ABSTRACT
Hypoxia inducible factor (HIF) up-regulates the transcription of a few hundred genes required for the adaptation to hypoxia. This restricted set of targets is in sharp contrast with the widespread distribution of the HIF binding motif throughout the genome. Here, we investigated the transcriptional response of GYS1 and RUVBL2 genes to hypoxia to understand the mechanisms that restrict HIF activity toward specific genes. GYS1 and RUVBL2 genes are encoded by opposite DNA strands and separated by a short intergenic region (~1 kb) that contains a functional hypoxia response element equidistant to both genes. However, hypoxia induced the expression of GYS1 gene only. Analysis of the transcriptional response of chimeric constructs derived from the intergenic region revealed an inhibitory sequence whose deletion allowed RUVBL2 induction by HIF. Enhancer blocking assays, performed in cell culture and transgenic zebrafish, confirmed the existence of an insulator element within this inhibitory region that could explain the differential regulation of GYS1 and RUVBL2 by hypoxia. Hence, in this model, the selective response to HIF is achieved with the aid of insulator elements. This is the first report suggesting a role for insulators in the regulation of differential gene expression in response to environmental signals.

Show MeSH
Related in: MedlinePlus