Binding of Carbonic Anhydrase IX to 45S rDNA Genes Is Prevented by Exportin-1 in Hypoxic Cells

Figure 3

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 values for pairwise comparisons of ChIP data were in the 0.001 to 0.01 range (Student’s -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 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 (, not significant) (Student’s -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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