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Binding of carbonic anhydrase IX to 45S rDNA genes is prevented by exportin-1 in hypoxic cells.

Sasso E, Vitale M, Monteleone F, Boffo FL, Santoriello M, Sarnataro D, Garbi C, Sabatella M, Crifò B, Paolella LA, Minopoli G, Winum JY, Zambrano N - Biomed Res Int (2015)

Bottom Line: Accordingly, we described complex subcellular localization for this enzyme in human cells, as well as the redistribution of a carbonic anhydrase IX pool to nucleoli during hypoxia.We highlighted the binding of carbonic anhydrase IX to nucleolar chromatin, which is regulated by oxygen levels.Both conditions were associated with increased representation of carbonic anhydrase IX/exportin-1 complexes in nucleoli. 45 S rRNA transcript levels were accordingly downrepresented.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università di Napoli Federico II, 80131 Napoli, Italy ; CEINGE Biotecnologie Avanzate, Via Gaetano Salvatore 486, 80145 Napoli, Italy ; Associazione Culturale DiSciMuS RFC, 80026 Casoria, Italy.

ABSTRACT
Carbonic anhydrase IX (CA IX) is a surrogate marker of hypoxia, involved in survival and pH regulation in hypoxic cells. We have recently characterized its interactome, describing a set of proteins interacting with CA IX, mainly in hypoxic cells, including several members of the nucleocytoplasmic shuttling apparatuses. Accordingly, we described complex subcellular localization for this enzyme in human cells, as well as the redistribution of a carbonic anhydrase IX pool to nucleoli during hypoxia. Starting from this evidence, we analyzed the possible contribution of carbonic anhydrase IX to transcription of the 45 S rDNA genes, a process occurring in nucleoli. We highlighted the binding of carbonic anhydrase IX to nucleolar chromatin, which is regulated by oxygen levels. In fact, CA IX was found on 45 S rDNA gene promoters in normoxic cells and less represented on these sites, in hypoxic cells and in cells subjected to acetazolamide-induced acidosis. Both conditions were associated with increased representation of carbonic anhydrase IX/exportin-1 complexes in nucleoli. 45 S rRNA transcript levels were accordingly downrepresented. Inhibition of nuclear export by leptomycin B suggests a model in which exportin-1 acts as a decoy, in hypoxic cells, preventing carbonic anhydrase IX association with 45 S rDNA gene promoters.

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XPO1 controls binding of CA IX to 45S rDNA gene promoters. (a) Chromatin immunoprecipitation analysis of CA IX and UBF1 binding to 45S rDNA precursor gene clusters in a triplicate set of samples of normoxic and hypoxic HEK293 cells. Hypoxic cells were also exposed to leptomycin B (LMB) for 4 hours. Calculated P values for pairwise comparisons of ChIP data were in the 0.001 to 0.01 range (Student's t-test). (b) HEK293 cells from a triplicate set of samples exposed to normoxia, hypoxia, and hypoxia in the presence of leptomycin B were lysed for RNA extraction and qRT-PCR analysis of CA IX and LDHA mRNAs and 45S rRNA transcripts, as indicated. Calculated P values for pairwise comparisons of qRT-PCR data were in the 0.0005 to 0.01 range, with the exception of the CA IX transcript levels in normoxic and hypoxic/+LMB condition (P = 0.4, not significant) (Student's t-test). (c) Cells subjected to the same treatments, as in (a) and (b), were fixed, permeabilized, and exposed to antibodies for CA IX (green) and XPO1 (red) immunofluorescence analysis. Confocal images from representative fields were taken. White arrowheads in hypoxic cells (middle panels) indicate strong colocalization of CA IX and XPO1 in some nucleolar and subnucleolar areas. White arrowheads in hypoxic cells treated with leptomycin B (LMB) indicate the nucleoli, devoid of CA IX/XPO1 complexes. White bars: 10 μm.
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fig3: XPO1 controls binding of CA IX to 45S rDNA gene promoters. (a) Chromatin immunoprecipitation analysis of CA IX and UBF1 binding to 45S rDNA precursor gene clusters in a triplicate set of samples of normoxic and hypoxic HEK293 cells. Hypoxic cells were also exposed to leptomycin B (LMB) for 4 hours. Calculated P values for pairwise comparisons of ChIP data were in the 0.001 to 0.01 range (Student's t-test). (b) HEK293 cells from a triplicate set of samples exposed to normoxia, hypoxia, and hypoxia in the presence of leptomycin B were lysed for RNA extraction and qRT-PCR analysis of CA IX and LDHA mRNAs and 45S rRNA transcripts, as indicated. Calculated P values for pairwise comparisons of qRT-PCR data were in the 0.0005 to 0.01 range, with the exception of the CA IX transcript levels in normoxic and hypoxic/+LMB condition (P = 0.4, not significant) (Student's t-test). (c) Cells subjected to the same treatments, as in (a) and (b), were fixed, permeabilized, and exposed to antibodies for CA IX (green) and XPO1 (red) immunofluorescence analysis. Confocal images from representative fields were taken. White arrowheads in hypoxic cells (middle panels) indicate strong colocalization of CA IX and XPO1 in some nucleolar and subnucleolar areas. White arrowheads in hypoxic cells treated with leptomycin B (LMB) indicate the nucleoli, devoid of CA IX/XPO1 complexes. White bars: 10 μm.

Mentions: The data obtained so far support the hypothesis that CA IX acts as a positive modulator of transcription for rDNA 45S genes. In fact, decreased CA IX binding to nucleolar chromatin observed in hypoxic cells was associated with decreased 45S rRNA precursor transcript levels. What was also occurring in hypoxic cells was the increased representation of CA IX/XPO1 complexes in nucleoli [17]. Thus, XPO1 and its interaction with CA IX in the nucleoli of hypoxic cells may act as a decoy mechanism to prevent 45S rDNA genes' transcription during hypoxia. To validate this hypothesis, we evaluated 45S rDNA promoter binding by CA IX, 45S rRNA transcript levels, and CA IX/XPO1 colocalization in hypoxic HEK293 cells treated with the XPO1 inhibitor leptomycin B. As shown in Figure 3, CA IX binding to rDNA gene promoters (Figure 3(a)) was significantly decreased, and rRNA 45S transcript levels (Figure 3(b)) were underrepresented in hypoxic cells. As expected, the transcripts of the HIF1α targets, CA IX and LDHA, were upregulated in hypoxic cells (Figure 3(b)). Accordingly, some hypoxic cells showed increased representation of CA IX/XPO1 complexes, as demonstrated by the appearance of yellow nucleoli or nucleolar spots in the merged immunofluorescence experiment shown in Figure 3(c). Treatment of hypoxic cells with leptomycin B led to disappearance of these complexes (Figure 3(c)) and to increased representation of CA IX on rDNA 45S genes (Figure 3(a)). The levels of rRNA 45S transcripts were extremely low under this condition (Figure 3(b)), in agreement with the loss of UBF1 binding (Figure 3(a)).


Binding of carbonic anhydrase IX to 45S rDNA genes is prevented by exportin-1 in hypoxic cells.

Sasso E, Vitale M, Monteleone F, Boffo FL, Santoriello M, Sarnataro D, Garbi C, Sabatella M, Crifò B, Paolella LA, Minopoli G, Winum JY, Zambrano N - Biomed Res Int (2015)

XPO1 controls binding of CA IX to 45S rDNA gene promoters. (a) Chromatin immunoprecipitation analysis of CA IX and UBF1 binding to 45S rDNA precursor gene clusters in a triplicate set of samples of normoxic and hypoxic HEK293 cells. Hypoxic cells were also exposed to leptomycin B (LMB) for 4 hours. Calculated P values for pairwise comparisons of ChIP data were in the 0.001 to 0.01 range (Student's t-test). (b) HEK293 cells from a triplicate set of samples exposed to normoxia, hypoxia, and hypoxia in the presence of leptomycin B were lysed for RNA extraction and qRT-PCR analysis of CA IX and LDHA mRNAs and 45S rRNA transcripts, as indicated. Calculated P values for pairwise comparisons of qRT-PCR data were in the 0.0005 to 0.01 range, with the exception of the CA IX transcript levels in normoxic and hypoxic/+LMB condition (P = 0.4, not significant) (Student's t-test). (c) Cells subjected to the same treatments, as in (a) and (b), were fixed, permeabilized, and exposed to antibodies for CA IX (green) and XPO1 (red) immunofluorescence analysis. Confocal images from representative fields were taken. White arrowheads in hypoxic cells (middle panels) indicate strong colocalization of CA IX and XPO1 in some nucleolar and subnucleolar areas. White arrowheads in hypoxic cells treated with leptomycin B (LMB) indicate the nucleoli, devoid of CA IX/XPO1 complexes. White bars: 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig3: XPO1 controls binding of CA IX to 45S rDNA gene promoters. (a) Chromatin immunoprecipitation analysis of CA IX and UBF1 binding to 45S rDNA precursor gene clusters in a triplicate set of samples of normoxic and hypoxic HEK293 cells. Hypoxic cells were also exposed to leptomycin B (LMB) for 4 hours. Calculated P values for pairwise comparisons of ChIP data were in the 0.001 to 0.01 range (Student's t-test). (b) HEK293 cells from a triplicate set of samples exposed to normoxia, hypoxia, and hypoxia in the presence of leptomycin B were lysed for RNA extraction and qRT-PCR analysis of CA IX and LDHA mRNAs and 45S rRNA transcripts, as indicated. Calculated P values for pairwise comparisons of qRT-PCR data were in the 0.0005 to 0.01 range, with the exception of the CA IX transcript levels in normoxic and hypoxic/+LMB condition (P = 0.4, not significant) (Student's t-test). (c) Cells subjected to the same treatments, as in (a) and (b), were fixed, permeabilized, and exposed to antibodies for CA IX (green) and XPO1 (red) immunofluorescence analysis. Confocal images from representative fields were taken. White arrowheads in hypoxic cells (middle panels) indicate strong colocalization of CA IX and XPO1 in some nucleolar and subnucleolar areas. White arrowheads in hypoxic cells treated with leptomycin B (LMB) indicate the nucleoli, devoid of CA IX/XPO1 complexes. White bars: 10 μm.
Mentions: The data obtained so far support the hypothesis that CA IX acts as a positive modulator of transcription for rDNA 45S genes. In fact, decreased CA IX binding to nucleolar chromatin observed in hypoxic cells was associated with decreased 45S rRNA precursor transcript levels. What was also occurring in hypoxic cells was the increased representation of CA IX/XPO1 complexes in nucleoli [17]. Thus, XPO1 and its interaction with CA IX in the nucleoli of hypoxic cells may act as a decoy mechanism to prevent 45S rDNA genes' transcription during hypoxia. To validate this hypothesis, we evaluated 45S rDNA promoter binding by CA IX, 45S rRNA transcript levels, and CA IX/XPO1 colocalization in hypoxic HEK293 cells treated with the XPO1 inhibitor leptomycin B. As shown in Figure 3, CA IX binding to rDNA gene promoters (Figure 3(a)) was significantly decreased, and rRNA 45S transcript levels (Figure 3(b)) were underrepresented in hypoxic cells. As expected, the transcripts of the HIF1α targets, CA IX and LDHA, were upregulated in hypoxic cells (Figure 3(b)). Accordingly, some hypoxic cells showed increased representation of CA IX/XPO1 complexes, as demonstrated by the appearance of yellow nucleoli or nucleolar spots in the merged immunofluorescence experiment shown in Figure 3(c). Treatment of hypoxic cells with leptomycin B led to disappearance of these complexes (Figure 3(c)) and to increased representation of CA IX on rDNA 45S genes (Figure 3(a)). The levels of rRNA 45S transcripts were extremely low under this condition (Figure 3(b)), in agreement with the loss of UBF1 binding (Figure 3(a)).

Bottom Line: Accordingly, we described complex subcellular localization for this enzyme in human cells, as well as the redistribution of a carbonic anhydrase IX pool to nucleoli during hypoxia.We highlighted the binding of carbonic anhydrase IX to nucleolar chromatin, which is regulated by oxygen levels.Both conditions were associated with increased representation of carbonic anhydrase IX/exportin-1 complexes in nucleoli. 45 S rRNA transcript levels were accordingly downrepresented.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Medicina Molecolare e Biotecnologie Mediche, Università di Napoli Federico II, 80131 Napoli, Italy ; CEINGE Biotecnologie Avanzate, Via Gaetano Salvatore 486, 80145 Napoli, Italy ; Associazione Culturale DiSciMuS RFC, 80026 Casoria, Italy.

ABSTRACT
Carbonic anhydrase IX (CA IX) is a surrogate marker of hypoxia, involved in survival and pH regulation in hypoxic cells. We have recently characterized its interactome, describing a set of proteins interacting with CA IX, mainly in hypoxic cells, including several members of the nucleocytoplasmic shuttling apparatuses. Accordingly, we described complex subcellular localization for this enzyme in human cells, as well as the redistribution of a carbonic anhydrase IX pool to nucleoli during hypoxia. Starting from this evidence, we analyzed the possible contribution of carbonic anhydrase IX to transcription of the 45 S rDNA genes, a process occurring in nucleoli. We highlighted the binding of carbonic anhydrase IX to nucleolar chromatin, which is regulated by oxygen levels. In fact, CA IX was found on 45 S rDNA gene promoters in normoxic cells and less represented on these sites, in hypoxic cells and in cells subjected to acetazolamide-induced acidosis. Both conditions were associated with increased representation of carbonic anhydrase IX/exportin-1 complexes in nucleoli. 45 S rRNA transcript levels were accordingly downrepresented. Inhibition of nuclear export by leptomycin B suggests a model in which exportin-1 acts as a decoy, in hypoxic cells, preventing carbonic anhydrase IX association with 45 S rDNA gene promoters.

Show MeSH
Related in: MedlinePlus