Additional file 2 of SATB2 dysregulation generates a novel circular RNA and drives KRAS-like transcriptional reprogramming and transformation-associated phenotypes

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Supplementary Material 2: Fig S1. General experimental methods. A, General location of ON-TARGETplus small interfering (si) RNAs for transiently knocking down SATB2 mRNA or circ3915. Numbers correspond to SATB2 exons. C+L = probe for circular and linear RNA. B, General location of Millipore Sigma short hairpin (sh) RNAs for stably knocking down SATB2 mRNA or circ3915. C, Cloning strategy for expressing SATB2 mRNA (top) or circ3915 (bottom) from the pcDNA3.1(+) vector in non-transformed BEAS-2B cells. Once transcribed, the inverted Alu repeats in the pre-mRNA form a loop that promotes back-splicing. D, Position of the tag (HiBiT or 3×FLAG) in the lentiviral circ3915 expression vectors. The tag is placed upstream of the putative start codon to ensure that any peptide is derived from the back-spliced transcript. E, RT-qPCR primer design strategy for specifically detecting linear SATB2 mRNA, circ3915, or both transcripts. F, NanoString probe design for detecting linear (top, red fluor) and circular (bottom, green fluor) RNAs. RNA isoforms are counted with complementary capture and reporter probes designed to hybridize to the linear splice and circular back-splice junctions. RNA isoforms were counted via a unique barcode assigned to each reporter probe. Images created with BioRender. Fig S2. Inorganic arsenic (iAs) exposure promotes transformation by driving oncogenic gene expression programs. Data were generated from the rapid (12-week) exposure model. A, Principal component analysis of RNA-seq expression profiles from non-transformed (NT, pink) and iAs-transformed BEAS-2B cells (iAsT, blue). B, The top 30 differentially expressed genes (DEGs) in iAs-transformed (iAsT) cells relative to non-transformed (NT) controls. C, Correlation between RNA-seq and NanoString gene expression levels for 191 DEGs. Dashed lines = 95% confidence interval. D, The top enriched biological processes reflected in the iAsT DEGs. E, The top enriched KEGG pathways in the iAsT DEGs. F, The top enriched Reactome pathways in the iAsT DEGs. G, The top enriched MSigDB hallmarks in the iAsT DEGs. Fig S3. Inorganic arsenic induces global changes in chromatin accessibility. iAs-transformed cells were generated with the rapid (12-week) exposure model. A, DARs between non-transformed (NT) and iAs-transformed (iAsT) cells. B, Principal component analysis of differentially accessible regions in non-transformed (NT, black) and iAs-transformed (brown, iAsT) BEAS-2B cells. C, Genomic distance between ATAC-seq peaks and promoters (left) or gene bodies (right) in iAsT DARs. D, Congruence between genes associated with open chromatin and closed chromatin in iAsT cells. E, The top enriched biological processes reflected in the iAsT DARs. F, The top enriched KEGG pathways in the iAsT DARs. G, The top enriched Reactome pathways in the iAsT DARs. H, The top enriched MSigDB hallmarks in the iAsT DARs. I, the top enriched C6 oncogenic signatures in the iAsT DARs. Fig S4. circRNA detection in iAs-transformed cells. A, Microarray-based detection of differential circRNA expression in pre-transformed (0.5 µM iAs for 17 weeks) and fully transformed (+ 2.0 µM iAs for another 10 weeks) BEAS-2B cells relative to non-treated cells. Upregulated circRNAs were defined by p < 0.05 and fold change (FC) >1.2. Downregulated circRNAs were defined by p <0.05 and FC < -1.2. B, Overlapping circRNA expression in pre-transformed and fully transformed BEAS-2B cells, as measured by Arraystar microarray. C, SATB2 mRNA and circ3915 expression in 16HBE cells after 48 hr exposure to increasing concentrations of iAs. D, SATB2 mRNA and circ3915 expression in 16HBE cells in the two-hit exposure model. * p < 0.05, *** p < 0.001, **** p < 0.00001. E, Validation of circ3915 using convergent and divergent primers. Primers were designed to amplify the backsplice junction (targeting exons 3-6; 211 bp) and a linear region common to SATB2 and circ3915 (Exon 3, 130 bp). Convergent primers amplify cDNA and gDNA, while divergent primers only amplify cDNA. The bands with diverging primers on cDNA were cut, purified, and sequenced to confirm the presence of the backsplice junction (chromatograms are shown above the agarose gel images). Fig S5. Predicted interactions between SATB2 and circ3915p. A, 3×FLAG (blue) tagged circ3915 expression vector showing the backsplice junction (yellow), and start codon (green). The predicted circ3915p amino acid sequence is shown in Fig 3B; note the numerous lysines (K) present in the backsplice junction. Peptides detected by LC-MS/MS are shown beneath the construct, with a representative mass spectrum shown in B. C, Puromycin treatment shifts endogenous circ3915 translation towards light and free polysomes. CHX = cycloheximide. D, In contrast, a known but untranslated endogenous circRNA (hsa_circ_0008928) does not associate with polysomes and does not show a shift in polysome association when treated with puromycin. E, Alphafold3 predicted structure and space-filling model for the SATB2-circ3915p complex (SATB2 UniProt accession Q9UPW6-1). F, Same as panel E, except color coding indicates the predicted local Distance Difference Test (plDDT), or the quality of the predicted interactions. Structured regions are reflected in blue, and disordered regions are reflected in orange. G, Same as panels E and F, except colors now reflect the SATB2 domains. The red square box is enlarged in panel H, and suggests that SATB2 and circ3915p interact through their respective ubiquitin-like domains. I, J. Western blots for SATB2-IP (I) and circ3915p (FLAG-IP; J), validating IP-MS data from Table S4. Primary antibodies are found in the Key Resources table. Fig S6. Differentially expressed genes (DEGs) in non-transformed BEAS-2B cells that express SATB2. A, Differentially expressed genes (DEGs) in non-transformed BEAS-2B cells that express SATB2 (SATB2). In total, 5178 genes were upregulated, and 5492 were downregulated at padj < 0.05. B, Top 30 differentially expressed genes from panel A. Control BEAS-2B cells contained an empty expression vector (EV). C, Correlation between RNA-seq and NanoString gene expression levels for 191 DEGs in non-transformed BEAS-2B cells that express SATB2. Dashed lines = 95% confidence interval; FC = fold-change D, Overlapping up- and downregulated genes in iAs-transformed (iAsT) BEAS-2B cells and non-transformed BEAS-2B cells expressing SATB2. iAsT cells were generated with the rapid (12-week) transformation model. Fig S7. Differentially accessible regions (DARs) in non-transformed BEAS-2B cells that express SATB2. A, Genomic distribution of DARs (top) and ENCODE candidate cis-regulatory elements (cCREs; bottom) in non-transformed BEAS-2B cells expressing SATB2. DARs were defined by FDR <0.01 and Shrunken Log2(FC) >1 for opened DARs, and Shrunken Log2(FC) <-1 for closed DARs. PLS = promoter-like signatures; pELS = proximal enhancer-like signatures; dELS = distal enhancer-like sequences. In total, there were 6963 opened chromatin regions, and 2089 closed regions. B, MSigDB oncogenic signatures in non-transformed BEAS-2B cells expressing SATB2 that were commonly enriched in DARs and DEGs. ATAC UP = opened chromatin; ATAC DN = closed chromatin; RNA UP = upregulated transcripts; RNA DN = downregulated transcripts. C, SATB2 opens chromatin at KRAS signature genes, increasing their gene expression. Shown is an integrative genomics viewer (IGV) snapshot of normalized average peak distributions for KRAS_UP_UP signature genes. NT = non-transformed BEAS-2B cells. iAsT = iAs-transformed BEAS-2B cells generated with the rapid (12-week) transformation model; EV = non-transformed BEAS-2B cells containing an empty expression vector; SATB2 = non-transformed BEAS-2B cells expressing SATB2. Encode cCREs are shown in yellow (dELSs), orange (pRLSs), and red (PLS). D, RNA expression of 161 genes from the MSigDB oncogenic NFE2L2 signature. E, Top enriched MSigDB Hallmark pathways in non-transformed BEAS-2B cells that expre

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