Fig. 5: HIF1α bears a nucleolar localization domain and binds to rDNA promoter.

A Schematic representation of HIF1α protein domains. The predicted nucleolar localization signal (NoLS; residues 5–34) and the engineered mutant (Mut-NoLS) are indicated. Additional functional domains include MP-NLS (monopartite nuclear localization signal), BP-NLS (bipartite nuclear localization signal), bHLH (basic helix–loop–helix), PAS-A, PAS-B (Per/ARNT/Sim domains), ODD (oxygen-dependent degradation domain), and CAD (C-terminal activation domain). B, C The predicted NoLS directs nucleolar accumulation of EGFP. SUM1315 and T47D cells were transfected with wild-type or mutant HIF1α NoLS fused to pEGFP-N1. Representative live-cell images show EGFP (green) and DAPI (blue). Data shown are from one representative of two independent experiments. Scale bar = 5 μm. D Schematic diagram of the human 45S rDNA promoter region, indicating locations of predicted hypoxia response elements (HREs) and external transcribed spacer (ETS). E, F Chromatin immunoprecipitation (ChIP)-qPCR analysis showing HIF1α binding to two regions (site A and site B) of the 45S rDNA promoter in SUM1315 (E) and SKBR3 (F) cells exposed to 8-h hypoxia. Isotype IgG served as negative control. Data are presented as mean ± SEM from n = 3 technical replicates. The experiment was independently performed two times, each yielding consistent results; data from one representative independent experiment is shown. G Flowchart outlining proteomic screening strategy for HIF1α-associated nucleolar proteins interacting with RNA Pol I subunit RPA194, including nucleolar fractionation, immunoprecipitation, and LC-MS analysis in SUM1315 and T47D cells. H Pol I activity in SUM1315 cells after NPM1 knockdown under 8-h hypoxia. 5′ ETS qRT-PCR was performed; data are mean ± SEM from n = 3 technical replicates. The experiment was independently performed two times, each yielding similar results; data from one representative independent experiment is shown. I Proposed model illustrating NPM1 interaction with HIF1α and RPA194, promoting Pol I activity under hypoxic conditions.