Decoding the gene regulatory response to iron through multiomics Free

Background: Normal cellular functions rely on iron, making dietary intake crucial for maintaining iron levels. Iron deficiency (ID) from low intake, blood loss, or pregnancy causes symptoms like fatigue, brain fog, and anemia. Intracellular iron homeostasis primarily involves post-transcriptional control by RNA-binding proteins, iron regulatory protein (IRP)-1 and IRP2, binding mRNAs of genes responsible for regulating iron levels. Emerging studies have highlighted the involvement of additional RNA-binding proteins, such as ZFP36 and SRSF7, that exhibit translational and alternative splicing control in response to low iron availability. Further, we recently uncovered iron-sensitive alternative splicing by PCBP1 and PCBP2—known intracellular iron chaperones. This budding body of evidence indicates ID triggers a broader post-transcriptional response than previously understood.

Aims: We aim to study how iron deficiency impacts the transcriptome and proteome in key iron-sensitive cell types. By conducting a comparative analysis across cell lines, we seek to highlight conserved and novel mechanisms underlying the transcriptomic and proteomic changes associated with ID stress, thereby advancing our understanding of ID at a molecular level.

Methods: Two well-established cell lines, HepG2 and K562, were treated with either 10 micromolar iron chelator, 21H7, or 100% DMSO overnight (n=3), and isolated RNA was polyA-selected and prepared for Illumina sequencing (mRNAseq). We characterized changes in overall gene expression and alternative splicing and evaluated effected pathways with gene ontology analysis. Parallel cell lysate samples were used for label-free whole cell mass spectrometry. Hypergeometric tests evaluated the significance of overlapping datasets, considering only co-detected genes or events as background. Spearman's rank correlation test was employed for correlation analysis. Chi-square tests were used to ascertain significant discrepancies between observed and expected results.

Results: At the mRNA level, 1294 genes were upregulated and 706 downregulated for HepG2, while K562 showed 1899 genes up- and 586 downregulated. Both cell transcriptomes demonstrated more significant changes than expected by chance (p<2e-16). Comparing HepG2 and K562 revealed 407 genes (87%, p<8e-167), sharing a similar regulation profile, with a positive correlation (R=0.43, p<2e-16). Gene ontology (GO) analysis of differentially expressed genes in both cell lines identified pathways including leukocyte differentiation, nephron development, and connective tissue development, highlighting the broad role of iron in pathways for growth and development. In HepG2, we detected about 1950 alternative splicing events, with 66% of cassette exons being more excluded upon 21H7 treatment (p<2e-16). In K562, we identified nearly 5,000 alternative splicing events, indicating an unprecedented impact on RNA splicing. Unlike HepG2, only 46% of cassette exons were more excluded (p=4e-10). Between both datasets, 303 cassette exon events were found to overlap (p=7e-78). Close to 300 proteins were differentially expressed in K562, vs 137 in HepG2, leading to 43 proteins overlapping (p=3e-21). Common GO terms for differential proteins in both cell lines included response to oxygen levels and hypoxia. However, K562 GO terms uniquely included 'anchoring junction’ while HepG2 unique GO terms included muscle cell proliferation, highlighting the different responses between cell types.

Conclusion: Our results show that posttranscriptional gene regulation is a major step in the cellular response to ID. Many genes were found to change in their gene expression and alternative splicing, showing wide sensitivity to iron level changes. Given the diverse metabolic demands across tissues, sensitivity to ID may vary significantly. Additionally, many alternative splicing is cell-type and tissue-specific. Thus, identifying a subset of cassette exons with significant overlap between cell types suggesting a conserved response to iron deficiency via alternative splicing. Our findings extend the role iron plays in gene regulation, especially alternative splicing.