![DFO treatment causes an acute rise in NCOA4 mRNA and protein. (A) NCOA4 mRNA expression relative to ACTB in Hep3B cells that were treated with CM supplemented with DFO (+DFO) or with CM alone (−DFO) for the indicated time periods. The mean mRNA ratio from cells not treated with DFO and harvested at 3 hours was normalized to 1. n = 6 per group. **P < .01 and ****P < .0001 by 2-way ANOVA. n.s., not significant. (B) Immunoblotting analyses of NCOA4, HIF-1α, HIF-2α, and β-actin in protein lysates harvested from Hep3B cells that were treated with CM supplemented with DFO (+DFO) or with CM alone (−DFO) for the indicated time periods. Numbers on the right indicate the position of molecular weight markers in kiloDaltons. For panels A and B, Hep3B cells were seeded at 2.5 × 105 cells/well in CM in 6-well plates. Twenty-four hours later (time 0 hr), some wells received additional DFO-supplemented CM to achieve a final DFO concentration of 100 μM (+DFO), whereas other wells received additional CM without DFO (− DFO). RNA and proteins were harvested 3, 6, 9, and 12 hours later. (C) Genomic binding profile of HIF-1α, HIF-2α, and HIF-1β (ARNT) subunits at the NCOA4 gene locus in HepG2 cells subjected to hypoxia. (Ci) A 50-kb genomic region of chromosome 10 containing the NCOA4 locus. (Cii) The genomic region shaded in gray from subpanel i is expanded to provide a high-resolution view of the NCOA4 promoter region. ChIP-seq data (Gene Expression Omnibus [GEO] DataSet GSE120885; GEO sample accession shown for each track),40 and genomic coordinates of the HIF-1α binding region identified by ChIP-on-chip (GEO DataSet GSE16347)41 were visualized in the UCSC Genome Browser (http://genome.ucsc.edu).64 The orange line indicates the genomic position of an HRE motif (chr10:51 563,614-51 563,618 on GRCh37/hg19 assembly). Also shown are DNase I hypersensitivity sites (DHS) identified in HepG2 cells and human hepatocytes by ENCODE65,66; chromatin accessibility has been proposed as a major factor in predicting HIF binding to the HRE motif.67 (D) Proposed model of HIF-mediated NCOA4 regulation in hepatocyte iron homeostasis. Under conditions of hypoxia and/or iron deficiency, such as after acute blood loss, the stabilized HIF-α subunit heterodimerizes with the HIF-β subunit and binds an HRE motif in the NCOA4 promoter region to enhance NCOA4 expression. This maintains a supply of NCOA4 protein to support ferritinophagy, making iron available for cellular export to meet extrahepatic iron demands.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/136/23/10.1182_blood.2020006321/2/bloodbld2020006321f7.png?Expires=1722721108&Signature=K0H088MNI8PL9uP-EuTppjQ7wDeSb4FZB2IXgjc0XnaUzqp2UWMju6al~zjnJybEHDWGynGLQ1nrZ4IJ1U1XUNHhXR7cwmhEiz2Z6l2LKY6swbVEbVXXNYpkZZSfPEQAGUd~1NCblIw49yxn-zuVMSuJorN5HYg5hhHIai7bRALebSMJpdKbIzCavLRFXTfGcmjayxmw2I9EQixDy6aROypZ3kzNzbpF-tPEAB9M3WTt-S7sPxrXE-nF82X~jXK42k0DP8kQ0VUoV-gCnsEGJYQeNu8MieSBCUo4maO47KkSoGZ4e9NhRD7fb0PNlUKgcZ7t-17zxRwHJgvg-tHPfA__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
DFO treatment causes an acute rise in NCOA4 mRNA and protein. (A) NCOA4 mRNA expression relative to ACTB in Hep3B cells that were treated with CM supplemented with DFO (+DFO) or with CM alone (−DFO) for the indicated time periods. The mean mRNA ratio from cells not treated with DFO and harvested at 3 hours was normalized to 1. n = 6 per group. **P < .01 and ****P < .0001 by 2-way ANOVA. n.s., not significant. (B) Immunoblotting analyses of NCOA4, HIF-1α, HIF-2α, and β-actin in protein lysates harvested from Hep3B cells that were treated with CM supplemented with DFO (+DFO) or with CM alone (−DFO) for the indicated time periods. Numbers on the right indicate the position of molecular weight markers in kiloDaltons. For panels A and B, Hep3B cells were seeded at 2.5 × 105 cells/well in CM in 6-well plates. Twenty-four hours later (time 0 hr), some wells received additional DFO-supplemented CM to achieve a final DFO concentration of 100 μM (+DFO), whereas other wells received additional CM without DFO (− DFO). RNA and proteins were harvested 3, 6, 9, and 12 hours later. (C) Genomic binding profile of HIF-1α, HIF-2α, and HIF-1β (ARNT) subunits at the NCOA4 gene locus in HepG2 cells subjected to hypoxia. (Ci) A 50-kb genomic region of chromosome 10 containing the NCOA4 locus. (Cii) The genomic region shaded in gray from subpanel i is expanded to provide a high-resolution view of the NCOA4 promoter region. ChIP-seq data (Gene Expression Omnibus [GEO] DataSet GSE120885; GEO sample accession shown for each track),40 and genomic coordinates of the HIF-1α binding region identified by ChIP-on-chip (GEO DataSet GSE16347)41 were visualized in the UCSC Genome Browser (http://genome.ucsc.edu).64 The orange line indicates the genomic position of an HRE motif (chr10:51 563,614-51 563,618 on GRCh37/hg19 assembly). Also shown are DNase I hypersensitivity sites (DHS) identified in HepG2 cells and human hepatocytes by ENCODE65,66 ; chromatin accessibility has been proposed as a major factor in predicting HIF binding to the HRE motif.67 (D) Proposed model of HIF-mediated NCOA4 regulation in hepatocyte iron homeostasis. Under conditions of hypoxia and/or iron deficiency, such as after acute blood loss, the stabilized HIF-α subunit heterodimerizes with the HIF-β subunit and binds an HRE motif in the NCOA4 promoter region to enhance NCOA4 expression. This maintains a supply of NCOA4 protein to support ferritinophagy, making iron available for cellular export to meet extrahepatic iron demands.