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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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Chromatin immunoprecipitation analysis of CA IX and UBF1 binding to 45S rDNA precursor gene clusters. Normoxic or hypoxic HEK293 (a) and SHSY-5Y (b) cells were subjected to ChIP analysis with UBF1 or CA IX monoclonal antibodies, as indicated (upper charts). Binding of the proteins to 45S rDNA genes was evaluated via quantitative PCR analysis from triplicate samples. Calculated P values for pairwise comparisons of ChIP data were in the 0.0038 to 0.03 range (Student's t-test). The middle panels show the results of qRT-PCR analysis of CA IX or 45S rRNA transcripts from normoxic or hypoxic HEK293 (a) and SHSY-5Y (b) cells. Calculated P values for pairwise comparisons of qRT-PCR data were in the 0.0027 to 0.04 range. Lower panels show the hypoxia-induced increases in HIF1α and CA IX protein levels in normoxic (N) or hypoxic (H) HEK293 (a) and SHSY-5Y (b) cells. Filters were reprobed to β-actin, which was used as a loading control.
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fig2: Chromatin immunoprecipitation analysis of CA IX and UBF1 binding to 45S rDNA precursor gene clusters. Normoxic or hypoxic HEK293 (a) and SHSY-5Y (b) cells were subjected to ChIP analysis with UBF1 or CA IX monoclonal antibodies, as indicated (upper charts). Binding of the proteins to 45S rDNA genes was evaluated via quantitative PCR analysis from triplicate samples. Calculated P values for pairwise comparisons of ChIP data were in the 0.0038 to 0.03 range (Student's t-test). The middle panels show the results of qRT-PCR analysis of CA IX or 45S rRNA transcripts from normoxic or hypoxic HEK293 (a) and SHSY-5Y (b) cells. Calculated P values for pairwise comparisons of qRT-PCR data were in the 0.0027 to 0.04 range. Lower panels show the hypoxia-induced increases in HIF1α and CA IX protein levels in normoxic (N) or hypoxic (H) HEK293 (a) and SHSY-5Y (b) cells. Filters were reprobed to β-actin, which was used as a loading control.

Mentions: One additional feature, emerging from our previous analysis of CA IX interactome, was the increased abundance of CA IX/XPO1 complexes and their peculiar enrichment in nucleoli in hypoxic cells. Thus, we hypothesized a nucleolar function for CA IX and for its complexes with XPO1. Nucleoli are the districts in which 45S rRNA synthesis and processing occur to allow ribosome biogenesis. 45S rRNA precursor is actively transcribed from arrays of 45S rDNA genes in nucleoli. We then evaluated, by chromatin immunoprecipitation (ChIP) analysis, the potential binding of CA IX to nucleolar chromatin in normoxic and hypoxic HEK293 and SHSY-5Y cells. ChIP analysis was also performed for UBF1, an architectural factor regulating RNA polymerase I transcription. The data shown in the panel A of Figure 2 demonstrated that both UBF1 and CA IX are actually bound to rDNA 45S genes in normoxic HEK293 cells. Even in the presence of increases in CA IX protein levels (lower panels of Figure 2(a)), the exposure of the cells to a hypoxic environment resulted in a decreased association of CA IX to nucleolar chromatin, while UBF1 was stabilized on 45S rDNA genes. This was associated with decreased levels of 45S rRNA in hypoxic cells, as shown by the results of qRT-PCR in the middle panel of Figure 2(a). These data were consistently replicated in SHSY-5Y cells (Figure 2(b)). As previously demonstrated [17], CA IX levels and its nucleolar presence are increased in hypoxic cells, as for its participation in molecular complexes with XPO1. These novel results show that the increased formation of CA IX/XPO1 complex in the nucleoli of hypoxic cells is indeed associated with a decreased representation of CA IX on nucleolar chromatin and with a decreased transcription of 45S rDNA genes, both in HEK293 and in SHSY-5Y cells. These data support the hypothesis that in human cell lines CA IX might be associated with active transcription of 45S rDNA genes in normoxic condition.


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)

Chromatin immunoprecipitation analysis of CA IX and UBF1 binding to 45S rDNA precursor gene clusters. Normoxic or hypoxic HEK293 (a) and SHSY-5Y (b) cells were subjected to ChIP analysis with UBF1 or CA IX monoclonal antibodies, as indicated (upper charts). Binding of the proteins to 45S rDNA genes was evaluated via quantitative PCR analysis from triplicate samples. Calculated P values for pairwise comparisons of ChIP data were in the 0.0038 to 0.03 range (Student's t-test). The middle panels show the results of qRT-PCR analysis of CA IX or 45S rRNA transcripts from normoxic or hypoxic HEK293 (a) and SHSY-5Y (b) cells. Calculated P values for pairwise comparisons of qRT-PCR data were in the 0.0027 to 0.04 range. Lower panels show the hypoxia-induced increases in HIF1α and CA IX protein levels in normoxic (N) or hypoxic (H) HEK293 (a) and SHSY-5Y (b) cells. Filters were reprobed to β-actin, which was used as a loading control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4352447&req=5

fig2: Chromatin immunoprecipitation analysis of CA IX and UBF1 binding to 45S rDNA precursor gene clusters. Normoxic or hypoxic HEK293 (a) and SHSY-5Y (b) cells were subjected to ChIP analysis with UBF1 or CA IX monoclonal antibodies, as indicated (upper charts). Binding of the proteins to 45S rDNA genes was evaluated via quantitative PCR analysis from triplicate samples. Calculated P values for pairwise comparisons of ChIP data were in the 0.0038 to 0.03 range (Student's t-test). The middle panels show the results of qRT-PCR analysis of CA IX or 45S rRNA transcripts from normoxic or hypoxic HEK293 (a) and SHSY-5Y (b) cells. Calculated P values for pairwise comparisons of qRT-PCR data were in the 0.0027 to 0.04 range. Lower panels show the hypoxia-induced increases in HIF1α and CA IX protein levels in normoxic (N) or hypoxic (H) HEK293 (a) and SHSY-5Y (b) cells. Filters were reprobed to β-actin, which was used as a loading control.
Mentions: One additional feature, emerging from our previous analysis of CA IX interactome, was the increased abundance of CA IX/XPO1 complexes and their peculiar enrichment in nucleoli in hypoxic cells. Thus, we hypothesized a nucleolar function for CA IX and for its complexes with XPO1. Nucleoli are the districts in which 45S rRNA synthesis and processing occur to allow ribosome biogenesis. 45S rRNA precursor is actively transcribed from arrays of 45S rDNA genes in nucleoli. We then evaluated, by chromatin immunoprecipitation (ChIP) analysis, the potential binding of CA IX to nucleolar chromatin in normoxic and hypoxic HEK293 and SHSY-5Y cells. ChIP analysis was also performed for UBF1, an architectural factor regulating RNA polymerase I transcription. The data shown in the panel A of Figure 2 demonstrated that both UBF1 and CA IX are actually bound to rDNA 45S genes in normoxic HEK293 cells. Even in the presence of increases in CA IX protein levels (lower panels of Figure 2(a)), the exposure of the cells to a hypoxic environment resulted in a decreased association of CA IX to nucleolar chromatin, while UBF1 was stabilized on 45S rDNA genes. This was associated with decreased levels of 45S rRNA in hypoxic cells, as shown by the results of qRT-PCR in the middle panel of Figure 2(a). These data were consistently replicated in SHSY-5Y cells (Figure 2(b)). As previously demonstrated [17], CA IX levels and its nucleolar presence are increased in hypoxic cells, as for its participation in molecular complexes with XPO1. These novel results show that the increased formation of CA IX/XPO1 complex in the nucleoli of hypoxic cells is indeed associated with a decreased representation of CA IX on nucleolar chromatin and with a decreased transcription of 45S rDNA genes, both in HEK293 and in SHSY-5Y cells. These data support the hypothesis that in human cell lines CA IX might be associated with active transcription of 45S rDNA genes in normoxic condition.

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