Genome-wide mapping of arsenic-activated Nrf2 reveals metabolic and epigenetic reprogramming in induced pluripotent stem cells

Abstract

Arsenic (As³⁺) is a well-established environmental carcinogen known to induce malignant transformation and cancer stem-like cell (CSC) properties in somatic cells, with Nrf2 functioning as a central regulator. However, the impact of chronic As³⁺ exposure on pluripotent stem cells, particularly through Nrf2-mediated epigenetic and metabolic reprogramming, remains largely unexplored. In this study, we chronically exposed human induced pluripotent stem cells (iPSCs, Nips-B2) to an environmentally relevant concentration of trivalent arsenic (0.25 μM, As³⁺) for three months. The tumorigenic potential of exposed iPSCs was evaluated using anchorage-independent growth assays and xenograft models, while mechanistic insights were gained via chromatin immunoprecipitation sequencing (ChIP-seq) for Nrf2 and key histone modifications (H3K4me3, H3K9me3, H3K27me3, H3K36me3, and H4K20me3), alongside transcriptomic profiling by RNA sequencing (RNA-seq). Prolonged exposure markedly enhanced tumor sphere formation in vitro and accelerated tumor growth in vivo, indicating the acquisition of CSC-like traits. Integrated ChIP-seq and RNA-seq analyses revealed widespread Nrf2 chromatin binding and global epigenetic remodeling, characterized by increased levels of H3K27me3, H3K36me3, and H4K20me3, a modest rise in H3K9me3, and reduced H3K4me3. Notably, As³⁺ exposure enhanced Nrf2 binding at loci regulating glycolysis, cholesterol biosynthesis, self-renewal, and oncogenesis. Functional analyses confirmed that transcriptional and metabolic changes were Nrf2-driven and closely linked to H3K36me3 and H3K27me3 dynamics. Collectively, our findings demonstrate that chronic As³⁺ exposure reprograms iPSCs through Nrf2 activation and coordinated epigenetic remodeling, revealing a novel mechanism by which environmental carcinogens exploit stem cell plasticity to initiate CSC-like transformation.

Keywords: Arsenic (As(3+)); CSCs; Histone methylation; Nrf2; iPSCs.

Graphical abstract

Fig. 1

Chronic low-dose As³⁺ exposure enhances sphere formation and tumorigenicity of iPSCs. A Bright-field images of iPSCs (Nips-B2) after continuous exposure to 0.25 μM As³⁺ for 1, 2, and 3 months. Cells maintained normal morphology and confluency, indicating tolerance to prolonged low-dose As³⁺ treatment. B Western blot analysis of stemness markers CD44 and NANOG in iPSCs at passage 24 (day 91) and passage 26 (day 105) following 0.25 μM As³⁺ exposure. As³⁺ treatment reduced full-length CD44 while markedly increased its cleaved form; effects on NANOG expression were variable. CIn vitro sphere formation assay showing increased sphere size in iPSCs treated with As³⁺ for 3 months, indicating enhanced self-renewal capacity under anchorage-independent conditions. Data are presented as mean ± SD (N = 10 per group). D Tumor growth curves in NSG mice subcutaneously injected with control or 3-month As³⁺-treated iPSCs (1 × 10⁶ cells/mouse, N = 5 per group). Tumors derived from As³⁺-transformed iPSCs grew significantly larger between days 32 and 39 post-injection. Data are presented as mean ± SD. E Representative H&E-stained tumor sections from control and As³⁺-transformed iPSC-derived xenografts. Control tumors exhibited typical teratoma architecture with ectodermal, mesodermal, and endodermal elements. In contrast, tumors from As³⁺-transformed iPSCs showed malignant features, including keratinized epithelium, mucin-filled cysts, pigment deposition, and nests of neoplastic squamous cells.

Fig. 2

Chronic As³⁺ exposure alters histone methylation landscapes and Nrf2 chromatin binding in iPSCs. A ChIP-seq analysis showing average enrichments of H3K4me3, H3K9me3, H3K27me3, H3K36me3, H4K20me3, and Nrf2 in control and 0.25 μM As³⁺-treated iPSCs after 3 months. Chronic As³⁺ treatment led to a global reduction in H3K4me3, accompanied by increases in H3K27me3, H3K36me3, H4K20me3, and Nrf2 occupancy. B Representative genome browser views of the TBCK locus (left panel) showing gene-specific enrichment of H3K4me3 and Nrf2 at the promoter and H3K36me3 across the gene body in control and As³⁺-transformed iPSCs. A conserved Nrf2 binding element in the Nrf2 peak region, TGAGTGA, inserted in the panel. The right panel displays genome-wide histone methylation profiles—H3K4me3, Nrf2, H3K27me3, H3K36me3, H3K9me3, and H4K20me3—across chromosome 22. Red filled triangles indicate regions of broad H3K9me3 and H4K20me3 enrichment predominantly localized to gene-poor heterochromatic domains, including pericentromeric regions and the distal ends of chromosome arms. These repressive clusters become further intensified following As³⁺ exposure.

Fig. 3

Chronic As³⁺ exposure reshapes H3K4me3 enrichment at genes regulating cell differentiation and stemness. A ChIP-seq analysis showing the average profile of merged H3K4me3 peaks across regions spanning -5 kb to +5 kb relative to gene loci in control and As³⁺-transformed iPSCs. The right panel illustrates a global reduction in H3K4me3 peak intensities across chromosomes 3 and 10 in iPSCs following 3 months of chronic 0.25 μM As³⁺ exposure. A subset of peaks displayed increased intensity post-treatment, highlighted by red triangles. B WikiPathways enrichment analysis of 9325 genes with decreased H3K4me3 levels (log₂ fold change < -0.1) revealed top pathways related to dopaminergic neurogenesis, norepinephrine signaling, and cell differentiation. The right panel shows genome browser tracks with reduced H3K4me3 enrichment at several tumor suppressor genes, including MYOD1, IRF4, ID2, CDKN2A, CDKN2B, and BAP1, in As³⁺-transformed iPSCs. C WikiPathways analysis of 1050 genes with increased H3K4me3 levels (log₂ fold change >0.1) highlighted enrichment in RNA processing and metabolic pathways (left). The right panel (highlighted by red triangles) displays representative genome browser views showing elevated H3K4me3 enrichment at core stemness genes such as NANOG, SLC2A3, DPPA4, NR6A1, and TDGF1 in As³⁺-transformed iPSCs.

Fig. 4

Overlap of As³⁺-induced Nrf2 activation with H3K36me3 and H3K27me3 enrichment in iPSCs. A Pathway enrichment analysis of 5943 genes marked by H3K36me3 following chronic 0.25 μM As³⁺ exposure revealed activation of signaling pathways including TXA2R, LPAR, CXCR3, PTP1B, Hedgehog, endothelin, RhoA, EPHB, EPHA, and SNCA. B Comparative pathway analysis showing that 8 of the top 10 pathways enriched in H3K36me3-marked genes also overlapped with those enriched in H3K27me3-marked genes (6511 genes), suggesting epigenetic crosstalk (left). Right: Venn diagrams showing that ∼54 % of H3K36me3-marked genes and ∼49 % of H3K27me3-marked genes were co-enriched for both modifications in As³⁺-transformed iPSCs. C Transcription factor perturbation analysis revealed that 6 of the top 10 pathways enriched in H3K27me3-targeted genes are regulated by Nrf2 signaling, supporting a potential link between Nrf2 activity and repressive chromatin remodeling. D ChIP-seq analysis showing average Nrf2 peak distribution across ±5 kb of gene loci in control and As³⁺-treated iPSCs. Right: Genome browser views highlighting amplified Nrf2 peaks and de novo As³⁺-specific Nrf2 peaks (red triangles), not present in control. E Genomic distribution of Nrf2 binding sites before and after 3-month As³⁺ exposure. Chronic As³⁺ treatment reduced Nrf2 occupancy at promoter and 5′ UTR regions while increasing its binding within exons and introns, indicating a redistribution of Nrf2 across the genome. F WikiPathways enrichment analysis of 1905 genes with increased Nrf2 binding following As³⁺ treatment revealed significant associations with Nrf2 signaling, glycolysis, aryl hydrocarbon receptor (AHR) signaling, oxidative stress response, and amino acid metabolism—highlighting widespread transcriptional reprogramming induced by As³⁺.

Fig. 5

Enrichment analysis of genes co-targeted by Nrf2 and H3K4me3 following As³⁺ exposure. A ChEA pathway enrichment analysis of genes with concurrent Nrf2 binding and H3K4me3 enrichment in iPSCs after chronic As³⁺ treatment. Identified transcriptional regulators include FOXP3, SOX2, EST1, and MYC, implicating their potential roles in promoting stemness and tumorigenic features. B ChEA analysis of bivalent chromatin states in As³⁺-transformed iPSCs. Left: Venn diagram showing minimal overlap (<1 %) between genes marked by both H3K4me3 and H3K27me3. Right: Enrichment analysis of 47 genes exhibiting a bivalent chromatin state (co-enrichment of H3K4me3 and H3K27me3), revealing strong association with pluripotency-related factors such as SOX2 and NANOG, suggesting these genes are poised for developmental regulation. C Genome browser snapshot showing the alignment of Nrf2 binding peaks with clusters of H3K27me3 and H3K36me3, as well as selected overlap with H3K9me3 (indicated by green-filled circles). These Nrf2-enriched regions are largely excluded from domains marked by H3K9me3 and H4K20me3, as highlighted in the yellow box. Right panels illustrate rare instances of Nrf2 occupancy within H3K9me3-enriched regions, all of which are located in gene-poor areas or near long noncoding RNA (lncRNA) loci.

Fig. 6

Oncogenic transcriptional reprogramming in As³⁺-transformed iPSCs. A Heatmap showing the top 20 differentially expressed genes in iPSCs chronically exposed to 0.25 μM As³⁺ for 3 months, as identified by RNA-seq. Significantly upregulated genes include ALPPL2, XYLT1, TRNP1, XXYLT1, and PLA2G2A. B Gene Ontology (GO) analysis using EnrichNet revealed significant enrichment of upregulated genes in pathways related to cholesterol biosynthesis, isoprenoid metabolism, and vesicle trafficking (COPI/COPII coating and intra-Golgi transport). C MSigDB enrichment analysis showing upregulated genes are associated with oncogenic and stemness-related pathways, including Myc signaling, glycolysis, hypoxia response, and cholesterol metabolism. D Transcription factor perturbation analysis of downregulated genes revealed enrichment for targets of GATA6, SPDEF, ATF6, ETV2, and FOXQ1, indicating suppression of key regulators involved in lineage specification and differentiation. E MSigDB analysis of downregulated genes showing significant association with tumor suppressive and differentiation-related pathways, including p53 signaling, apoptosis, and myogenesis, suggesting impaired differentiation potential and enhanced self-renewal in As³⁺-transformed iPSCs.

Fig. 7

Nrf2-mediated metabolic reprogramming in As³⁺-transformed iPSCs. A RNA-seq analysis reveals significant upregulation of key Nrf2 target genes in As³⁺-treated iPSCs, including Nrf2 regulators (VCP, SQSTM1, KLF2) and antioxidant defense genes (PRDX1, FTL, HMOX1, NQO1), confirming robust activation of Nrf2 signaling. B ChIP-seq analysis demonstrates strong Nrf2 binding at the promoter regions of SQSTM1, VCP and PRDX1 in As³⁺-transformed iPSCs, highlighted by red arrows, suggesting a positive feedback loop that amplifies Nrf2 signaling. No Nrf2 binding was detected at the promoter region of TXNRD2, indicating selective regulation. C Bar graph showing significant upregulation of key enzymes involved in glycolysis and glycolytic shunt pathways, including the pentose phosphate pathway (PPP) and hexosamine biosynthetic pathway (HBP). Key enzymes such as HK2 and TKT (glycolysis) and G6PD, PGLS, PGD, GFPT, PGM3, and UAP1 (PPP/HBP) are markedly upregulated in As³⁺-transformed iPSCs. D ChIP-seq analysis shows significant Nrf2 binding and H3K36me3 histone modification at the gene bodies and first exons of key metabolic genes in the PPP (G6PD, PGLS, PGD) and HBP (GFPT, PGM3, UAP1), supporting transcriptional activation through both Nrf2 signaling and histone modification. GNPNAT1 in the HBP pathway was not induced.

Fig. 8

Nrf2 directly regulates cholesterol biosynthesis and oncogenic stemness transcriptional programs in As³⁺-transformed iPSCs. A Schematic representation of the cholesterol biosynthetic pathway, with genes upregulated in As³⁺-transformed iPSCs highlighted in red. B Bar graph showing RNA-seq-based differential expression of key cholesterol biosynthetic genes in control and As³⁺-transformed iPSCs. Gene expression is presented as average log₂ fold change. C ChIP-seq tracks illustrating Nrf2 occupancy at promoter or gene body regions of representative cholesterol biosynthetic genes—including ACAT2, HMGCS1, MMAB-MVK, and FDPS—in control and As³⁺-transformed iPSCs. D RNA-seq analysis showing upregulation of oncogenic stemness-associated transcription factors, including PRDM14, KLF4, NR6A1, STAT5A, NANOG, and SOX2, in As³⁺-transformed iPSCs. Gene expression is shown as average log₂ fold change. E ChIP-seq tracks of the STAT5 gene cluster showing specific Nrf2 enrichment at the STAT5A locus (indicated by red arrows), which correlates with increased expression. No Nrf2 binding or expression changes were observed at neighboring genes STAT5B or STAT3. F Pie chart depicting the altered expression profiles of genes involved in DNA methylation and histone modification in As³⁺-transformed iPSCs compared to controls, including upregulated genes such as DNMT3B, DNMT3L, TET, GADD45G, EZH2, SETD4, and members of the KDM4 family.

Fig. 9

Increased expression of cholesterol biosynthesis genes is associated with poorer prognosis of lung cancer. A First Progression (Progression Free, FP) survival of lung cancer patients (n = 2167; data from the Kaplan-Meier Plotter) stratified by expression levels of key cholesterol biosynthesis genes. The optimal cutoff was used to define high and low expression groups for survival comparison. B Schematic diagram showing contribution of the indicated CYP family members to cholesterol metabolism. C FP survival curves of lung cancer patients stratified by expression levels of the indicated CYP genes, based on Kaplan–Meier analysis.

Fig. 10

Schematic representation of potential mechanisms underlying Nrf2-mediated metabolic rewiring in response to As³⁺exposurein iPSCs. As³⁺ induces Nrf2-dependent metabolic reprogramming that coordinates a network of interconnected pathways—including enhanced glycolysis, nucleotide biosynthesis, protein glycosylation, cholesterol metabolism, epigenetic modifications, and weakened mitochondrial citric acid cycle (TCA or Krebs cycle). Together, these alterations contribute to cancer development and promote the acquisition of CSC properties.