Three-Dimensional Gene Regulation Network in Glioblastoma Ferroptosis

Ferroptosis is an iron-dependent form of cell death, which is reported to be associated with glioma progression and drug sensitivity. Targeting ferroptosis is a potential therapeutic approach for glioma. However, the molecular mechanism of glioma cell ferroptosis is not clear. In this study, we profile the change of 3D chromatin structure in glioblastoma ferroptosis by using HiChIP and study the 3D gene regulation network in glioblastoma ferroptosis. A combination of an analysis of HiChIP and RNA-seq data suggests that change of chromatin loops mediated by 3D chromatin structure regulates gene expressions in glioblastoma ferroptosis. Genes that are regulated by 3D chromatin structures include genes that were reported to function in ferroptosis, like HDM2 and TXNRD1. We propose a new regulatory mechanism governing glioblastoma cell ferroptosis by 3D chromatin structure.


Introduction
Glioma is a common intracranial primary tumor.About 80% of malignant primary brain tumors are glioblastoma (GBM), which is the most malignant glioma and has a high mortality rate [1,2].Currently, the clinical treatment methods for glioma are limited, and patients' median survival time is only 14-17 months [3].Despite the fact that glioma therapeutic approaches have improved in recent years, the clinical effects and prognosis are still not satisfactory.Finding the accurate drug target is important, and problems such as drug resistance and immunosuppression in glioma treatment have not yet been fully solved.Tumor cell death is key in tumor therapy, and elucidating the molecular mechanisms of tumor cell death is important to the development and improvement of therapeutic approaches.However, the molecular mechanisms of glioma cell death remain unclear.
Ferroptosis is a newly defined form of cell death (2012), which is caused by ironinduced oxidative damage, the disruption of the cell membrane, and cell lysis [4,5].Ferroptosis is related to oncogenesis and tumor development and is regulated by multiple cancer-associated signaling pathways [6].Recent studies have shown that ferroptosis inhibits the growth of glioma cells, which is associated with the survival of patients and the clinical outcome of radiotherapy and chemotherapy [7], but the mechanisms underlying this are not clear.
The development of 3D genomic technologies reveals that gene regulatory elements regulate distal target genes' expression through the 3D structure of chromatin, and the 3D gene regulatory network can regulate physiological and biochemical processes in cells [8][9][10].Several studies reported that change in the 3D structure of chromatin regulates the expression of oncogenes and tumor suppressor genes and affects tumor initiation and development [10][11][12][13][14][15][16].As an important physiological process that affects glioma initiation and progression, ferroptosis involves multiple important signaling pathways, and the related gene regulation may be associated with the change of the 3D chromatin architecture.In this study, H3K27ac HiChIP was performed to investigate the change of active enhancer-related 3D chromatin structures in glioblastoma cell ferroptosis.By combining an analysis of RNA-seq and ChIP-seq data, this study found a 3D enhancer-gene regulation network in glioblastoma cell ferroptosis.

Distribution of Active Enhancers in Ferroptotic Glioblastoma Cells
Erastin is a small molecular compound that inhibits the system Xc-and prevents the import of cystine, reducing glutathione (GSH) biosynthesis and glutathione peroxidas 4 (GPX4) activity and finally inducing cell death via ferroptosis [17].To establish a model of Erastin-induced ferroptosis in glioblastoma cells, U87MG cells were treated for different concentrations of Erastin.Significant survival inhibition of U87MG was detected by MTS assay under the treatment of Erastin, and higher concentrations or longer treatment times of Erastin induced a more significant decrease in cell viability (Figures 1A,B and S1A,B).U87MG cell survival inhibition reached lower than 50% after 72 h of treatment with 10 µM Erastin (Figures 1A and S1A).Malondialdehyde (MDA) is the product of polyunsaturated fatty acid peroxidation.The level of MDA in cells indicates the degree of cellular oxidation, which can be used to quantify ferroptosis.A significant increase of intracellular MDA levels in U87MG glioblastoma cells was detected after Erastin treatment (10 µM, 72 h) compared to control (Figures 1C and S1C).The depletion of glutathione (GSH) is associated with an increase in cell ferroptosis, and significant GSH depletion was shown in the Erastin (10 µM, 72 h)-treated U87MG cells (Figures 1D and S1D).In addition, the accumulation of intracellular Fe 2+ is a marker of increased ferroptosis, and the level of Fe 2+ significantly increased in Erastin (10 µM, 72 h)-treated U87MG cells compared to control (Figures 1E  and S1E).These data suggest significant ferroptosis in U87MG cells treated with 10 µM Erastin for 72 h.
H3K27ac is known as a marker of active enhancers in mammals [18,19].Enrichment of H3K27ac modification is associated with the upregulation of gene expression.H3K27ac ChIP-seq was performed in control glioblastoma cells and glioblastoma cells that were treated with Erastin (10 µM, 72 h) to investigate the distribution of active enhancers in control glioblastoma cells and ferroptotic glioblastoma cells.In both control glioblastoma cells and ferroptotic glioblastoma cells, ~50% of all H3K27ac peaks are at promoters, ~40% are at non-coding regions, such as intergenic regions and introns, and ~5% appeared in exon regions (Figure 1F).No significant global changes in H3K27ac enrichment were detected in ferroptotic glioblastoma cells relative to control cells (Figure 1F).H3K27ac peaks are mainly enriched near the transcriptional start sites of the genes in both control glioblastoma cells and ferroptotic glioblastoma cells (Figure 1G).

HiChIP Identifies the Change of 3D Chromatin Structure in Glioblastoma Cell Ferroptosi
To investigate functions of 3D genome structure in glioblastoma cell ferropto H3K27ac HiChIP was performed to identify enhancer-related 3D chromatin structure control glioblastoma cells and ferroptotic glioblastoma cells.HiChIP identified 66172

HiChIP Identifies the Change of 3D Chromatin Structure in Glioblastoma Cell Ferroptosis
To investigate functions of 3D genome structure in glioblastoma cell ferroptosis, H3K27ac HiChIP was performed to identify enhancer-related 3D chromatin structures in control glioblastoma cells and ferroptotic glioblastoma cells.HiChIP identified 66172 and 30722 high-confidence, reproducible chromatin loops in control glioblastoma cells and Erastin (10 µM, 72 h)-treated ferroptotic glioblastoma cells, respectively.HiChIP data indicated the change of 3D chromatin structure in ferroptotic glioblastoma cells compared to control (Figure 2A,B).MAP1LC3B and CHAC1 were reported to function in ferroptosis [20][21][22][23].To determine whether the change of 3D chromatin structures induced a transcription change of genes in ferroptosis, RNA-seq was performed in U87MG cells and Erastin (10 µM, 72 h)-treated U87MG cells to detect the transcriptome change in ferroptotic glioblastoma cells.HiChIP data indicated increased chromatin interactions near MAP1LC3B and CHAC1 loci in ferroptotic glioblastoma cells.
Chromatin loops that are identified by H3K27ac HiChIP include enhancer-enhancer interactions (EEI), enhancer-promoter interactions (EPI), and promoter-promoter interactions (PPI) (Figure 3A,B).Among all enhancer and promoter interaction networks in control U87MG glioblastoma cells, 56.2% were EEI, 35.1% were EPI, and 8.7% were PPI, while in Erastin (10 µM, 72 h)-treated U87MG glioblastoma cells, PPI and EPI increased to 10.3% and 40.1%, respectively, and EEI decreased to 49.6% (Figure 3C).Moreover, the median length of chromatin interaction loops was longer in Erastin (10 µM, 72 h)-treated U87MG glioblastoma cells compared to control, and more long-range chromatin interactions were detected in Erastin-treated U87MG cells (Figure 3D), suggesting global alterations of 3D chromatin structure in ferroptosis.To determine whether the change of 3D chromatin structures induced a transcription change of genes in ferroptosis, RNA-seq was performed in U87MG cells and Erastin (10 µM, 72 h)-treated U87MG cells to detect the transcriptome change in ferroptotic glioblastoma cells.HiChIP data indicated increased chromatin interactions near MAP1LC3B and CHAC1 loci in ferroptotic glioblastoma cells.

Transcriptome Change in Ferroptotic Glioblastoma Cells Is Associated with Changes of 3D Chromatin Structure
RNA-seq data indicated 917 genes were significantly upregulated (log 2 FC > 1; p adj < 0.05) and 1422 genes were significantly downregulated (log 2 FC ≤ −1; p adj < 0.05) in ferroptotic cells compared to control (Figure 4A).Gene ontology (GO) analysis of significantly upregulated gene revealed that positive regulation of apoptotic process, apoptotic process, regulation of autophagy and negative regulation of cell proliferation-related biological processes were enriched in ferroptotic glioblastoma cells (Figure 4B).These cell signaling pathways or biological processes are associated with ferroptosis [24][25][26].GO analysis of significantly downregulated genes (log 2 FC ≤ −1, p adj < 0.05) showed that the top pathway altered was cell division (Figure 4C).In addition, the Gene Set Enrichment Analysis (GSEA) indicated that the ferroptosis-related genes [27] are highly enriched in Erastin (10 µM, 72h)-treated U87MG cells (Figure 4D).
The genes that were significantly upregulated in Erastin (10 µM, 72 h)-treated U87MG cells and gained chromatin loops at their respective loci can be ranked by the p adj value of the transcription fold change; among these, HDM2 and TXNRD1 emerged as the top two genes.Western blot was performed to validate the expression levels of HDM2 and TXNRD1 in control and U87MG glioblastoma cells that were treated with 10 µM Erastin for 72 h (Figure S2).HDM2 is a negative regulator of tumor suppressor p53 and binds to MDMX to facilitate ferroptosis in cells [30].HiChIP data showed a new chromatin loop at HDM2 locus in Erastin (10 µM, 72 h)-treated U87MG cells, and RNA-seq data showed that HDM2 is significantly upregulated (log 2 FC = 2.44, p adj = 2.91 × 10 −239 ) in Erastin (10 µM, 72 h)-treated U87MG cells, suggesting function of the gained chromatin loop in regulation of HDM2 (Figures 5E and 6A).TXNRD1 encodes thioredoxin reductase 1, which regulates cellular redox homeostasis [31].TXNRD1 was reported to function in regulating the ferroptosis of liver cancer and chronic myeloid leukemia cells [32,33].HiChIP data showed a chromatin loop that links a distal enhancer and promoter of TXNRD1 was strengthened in Erastin (10 µM, 72 h)-treated U87MG cells (Figure 6B), and a new chromatin loop that links an enhancer and promoter of TXNRD1 was identified in Erastin (10 µM, 72 h)-treated U87MG cells (Figure 6B).In addition, TXNRD1 was upregulated (log 2 FC = 2.04, p adj = 1.26 × 10 −216 ) compared to control (Figure 5E), which indicated that the gained chromatin loop is related to an increase of TXNRD1 expression.In addition, the genes that were significantly downregulated in Erastin (10 µM, 72 h)-treated U87MG cells and lost chromatin loops at their respective loci can be ranked by the p adj value of the transcription fold change; among these, MKI67 and TOP2A emerged as the top two genes.MKI67 is a marker of cell proliferation that encodes the nuclear protein Ki67 [34].HiChIP data showed that MKI67 locus lost two loops that link the MKI67 promoter and two distal enhancers in Erastin (10 µM, 72 h)-treated U87MG cells, and RNA-seq data showed that MKI67 is significantly downregulated (log 2 FC = −3.89,p adj = 0) in Erastin (10 µM, 72 h)-treated U87MG cells (Figures 5G and 6C).TOP2A encodes a nuclear enzyme that resolves entanglements and relieves the torsional stress of DNA double strands [35].TOP2A is related to the cell cycle in embryonic stem cells [36].A chromatin loop that links the TOP2A promoter and a distal enhancer was lost in Erastin (10 µM, 72 h)-treated U87MG cells, and another chromatin loop that links the distal enhancer and promoter of TOP2A was weakened (Figure 6D).TOP2A is also significantly downregulated (log 2 FC = −3.31,p adj = 0) in Erastin-treated U87MG cells (Figure 5G).These data suggest the association of loss of chromatin loops and downregulation of MKI67 and TOP2A expressions.
cycle in embryonic stem cells [36].A chromatin loop that links the TOP2A promoter a a distal enhancer was lost in Erastin (10 µM, 72 h)-treated U87MG cells, and another ch matin loop that links the distal enhancer and promoter of TOP2A was weakened (Figu 6D).TOP2A is also significantly downregulated (log2FC = -3.31,padj = 0) in Erastin-treat U87MG cells (Figure 5G).These data suggest the association of loss of chromatin loo and downregulation of MKI67 and TOP2A expressions.

Discussion
The 3D structure of chromatin is reported to play important roles in cell cycle, transcriptional activation, DNA replication, and cell differentiation [37][38][39].Abnormal 3D chromatin structures and chromatin interactions were found in tumor cells and were reported to be related to cancer progression and other diseases [15,40,41].Previous studies have shown that CTCF-s activates the expression of IFI6 by disrupting the 3D spatial conformation of classical CTCF in the IFI6 region and promoting the interaction between a distal enhancer and the promoter of IFI6, leading to apoptosis of HeLa-S3 cells [42].Recently, several studies have reported that ferroptosis functions in cancer progression and treatment; however, the 3D gene regulation network in ferroptotic cancer cells that mediated by higher-order chromatin structures is not clear.Here in this study, H3K27ac HiChIP was performed and indicated a global change of enhancer-related 3D chromatin structures in ferroptotic glioblastoma cells and revealed that long-range chromatin loops were significantly alternated in ferroptosis.One hundred sixty-two genes that were significantly upregulated in ferroptotic glioblastoma cells correspond to the gain of chromatin loops at their respective loci, and 302 genes that were significantly downregulated in ferroptotic glioblastoma cells correspond to the loss of chromatin loops at their respective loci.These genes include genes that were reported to function in ferroptosis, like HDM2 and TXNRD1 (Figure 7).Data in this study suggest a new mechanism by which the change in the 3D chromatin structure in ferroptosis regulates gene expression and functions in glioblastoma cell ferroptosis.
tin loops at their respective loci, and 302 genes that were significantly downregulated i ferroptotic glioblastoma cells correspond to the loss of chromatin loops at their respectiv loci.These genes include genes that were reported to function in ferroptosis, like HDM and TXNRD1 (Figure 7).Data in this study suggest a new mechanism by which the chang in the 3D chromatin structure in ferroptosis regulates gene expression and functions i glioblastoma cell ferroptosis.This study focused on enhancer connectome that was induced by 3D chromatin structures.Enhancer-enhancer interactions were important to the regulation of gene expression in biological processes and diseases [43,44].It has been shown that enhancer interactions in the short distance can ensure high expression of genes through the additive effects [45], and enhancer interactions over the long distance confer functional robustness of gene expression [46].Recent studies showed that some enhancer interactions can form enhancer hubs, which are connected with key gene promoters and contribute to gene expression in Type 2 diabetes [47].We noticed 5563 gained enhancer-enhancer interactions in ferroptotic glioblastoma cells (Figure 5A).These new E-E interactions may form enhancer hubs or important regulatory domains and regulate ferroptosis-related genes.
HDM2 is one of the target genes in the 3D gene regulation network.HDM2 binds to p53 and forms a complex to prevent p53 transcriptional activation.On the other hand, HDM2 is also an E3 ubiquitination ligase, which leads to the degradation of p53 through ubiquitination [48,49].The HDM2-p53 hub is regulated by different cellular stress signals, and HDM2 is highly expressed in many types of tumors and is closely related to the proliferation, invasion, apoptosis, and chemotherapy resistance of tumor cells [50][51][52][53][54][55].Prior studies have shown that HDM2 and MDMX antagonists combined with TMZ have better antitumor activity in experimental animals [56].In addition, HDM2 was also reported to function in promoting tumor progression in a p53-dependent pathway in glioma cells [48,57,58].There have been studies showing that HDM2, and likely the HDM2-MDMX complex, are able to increase the sensitivity of glioblastoma cells to ferroptosis [30].HDM2 antagonist MEL23 can inhibit RSL3-induced cell death in the cells with high expression of HDM2 but cannot change the sensitivity of the cells with normal expression of HDM2 to ferroptosis or inhibit RSL3-induced cell death [30].The function of HDM2 in glioma ferroptosis is related to the E3 ligase activity of the HDM2-MDMX complex.HDM2 and MDMX form complex and regulate lipids by altering the activity of PPARα, which may be a downstream target of E3 ligase activity of HDM2 [30].Similar mechanisms may also exist in glioblastoma ferroptosis.In our study, HiChIP and RNA-seq results showed that the HDM2 promoter interacts with an enhancer by a new chromatin loop induced by glioblastoma ferroptosis (Figure 6A), and the expression of HDM2 is upregulated (Figure 5E), which suggests that change of chromatin loops induced change of HDM2 expression and then affected glioblastoma ferroptosis.In addition to HDM2, TXNRD1 is another target gene in the 3D gene regulation network; it is an important enzyme with extensive reducing activity in the thioredoxin system [59].TXNRD1 is highly expressed in many tumors and is associated with the poor prognosis of tumors [60][61][62][63].Our results showed that in ferroptotic glioblastoma cells, new enhancer-promoter loops might induce upregulation of TXNRD1 (Figures 5E and 6B).However, previous studies have shown that the downregulation of TXNRD1 can promote the production of reactive oxygen species, thereby promoting ferroptosis in tumor cells [64].We reckon that some rescue pathways may simultaneously work in ferroptotic glioblastoma cells, or complex functions of TXNRD1 in the regulation of ferroptosis work in different kinds of cells or at different cell stages.
Ferroptotic cell death is characterized by the accumulation of reactive oxygen species in cells and increased oxidative stress [65], and excessive reactive oxygen species in cells can affect many cell physiological processes, including cell cycle arrest, cell proliferation inhibition, and apoptosis [66][67][68][69].Several studies have indicated that MKI67 and TOP2A are important genes involved in cell proliferation and cell cycle regulation [70][71][72][73].HiChIP data in our study indicated attenuated interactions between promoters of MKI67 and TOP2A and enhancers (Figure 6C,D), and RNA-seq data showed downregulation of these two genes (Figure 5G).Downregulation of MKI67 and TOP2A is related to the decrease of cell proliferation [35,[73][74][75], which is consistent with the phenotype of ferroptosis.Recent studies show that MKI67 and TOP2A were contained within a network of genes that are upregulated in NK-cell repertoires in patients with neutropenia, which is associated with apoptosis and cell cycle [71].Though ferroptosis is mechanistically and morphologically different from apoptosis [76], genes known to be involved in apoptosis could also function in ferroptosis, such as TP53 [77,78].However, the underlying mechanism of MKI67 and TOP2A involved in ferroptosis is currently not well known.It is not clear whether the downregulation of MKI67 and TOP2A are just induced by increased oxidative stress in ferroptotic glioblastoma cells or whether these two genes also take part in the regulation of ferroptosis.Though bioinformatic analyses suggest that TOP2A may be associated with ferroptosis [79,80], further studies are needed to figure out the whole gene regulation network that contains MKI67 and TOP2A in glioblastoma ferroptosis.
Prior studies reported that in p53 wild-type GBM cell line U87MG and p53-mutated GBM cell line U251, P62 plays a dual role in glioma ferroptosis [81], suggestive of different function mechanisms of ferroptosis due to the heterogeneity of glioma cells.In addition, previous studies reported that T98G cells were extremely responsive to Erastin, while U251MG was more resistant to ferroptosis [82].These studies suggest that the mechanism of ferroptosis may vary in different cell lines.Our study reveals one of the regulation mechanisms in glioblastoma ferroptosis.Further studies are needed to investigate the function of 3D chromatin structures in ferroptosis in view of the heterogeneity of glioma cells.
Temozolomide (TMZ) is a first-line treatment drug for glioma, and a prior study revealed that TMZ inhibits glioma cell growth by inducing ferroptosis by regulating the expression of DMT1 [83], which indicates that the regulation of ferroptosis may affect the drug sensitivity of glioma cells.A recent study designed a nanoscale antibody vector, S-biAb/dEGCG@NPs, which effectively cleared GBM cells in mice by enhancing the effects of ferroptosis and enhancing immune checkpoint blocking (ICB) immunotherapy [84].A nanodrug, Au (I)-based NIR-II ferrotoxin nanoparticles (TBTP Au NPs), was reported to induce ferroptosis of glioma cells and prolong the survival time of glioma-bearing mice [85].In addition, ferroptosis was also reported to play an important role in the radiotherapy of glioma [86,87].Ferroptosis is reported to be associated with various ions [88,89].Studies of ion metabolism in ferroptosis indicate that ion channels may be a new therapeutic target in glioma [88][89][90][91].These studies suggest targeting ferroptosis could be an alternative method to improve the poor clinical prognosis of glioma treatment.Our study reveals a new mechanism by which the 3D chromatin structure regulates gene expression in glioblastoma ferroptosis.Thus, specific enhancers or enhancer-promoter interactions may be new therapeutic targets in glioblastoma treatment.Curaxins were reported to target specific 3D chromatin structures in tumors and exert antitumor effects, which indicates the important significance of exploring drug targets based on the 3D structure of chromatin [92,93].The technology of modification of 3D chromatin structures has been reported [94][95][96], which suggests targeting specific 3D chromatin structures or regulation elements to affect glioblastoma cell ferroptosis could be an alternative method to treat glioblastoma.The findings in this study are helpful in further exploring the mechanism of glioblastoma ferroptosis and discovering new therapeutic targets to treat glioblastoma.

Cell Culture
The glioblastoma cell line U87MG was obtained from the National Infrastructure of Cell Line Resource (Beijing, China).Glioblastoma cells were incubated at 37 • C in 90% humidity and 5% CO 2 and cultured in DMEM (GIBCO), supplemented with 10% fetal bovine serum (BI) and 1% penicillin/streptomycin (GIBCO).

MTS Assay
Cell viability was measured using the MTS Assay Kit (Promega, Madison, WI, USA).Briefly, cells were cultured in 96-well plates at a density of 1500 cells/well in a growth medium for 12 h.Cells were subsequently incubated with ferroptosis-inducing compounds Erastin (MCE) at the indicated concentrations in a growth medium for an additional 24 h, 48 h, 72 h, and 96 h.In total, 20 µL (per 100 µL medium) of MTS reagent was added to each well, and absorbance was measured at 490 nm using a microplate reader.

Lipid Peroxidation Assay
The activity level of MDA was assayed following the protocol of the Lipid Peroxidation MDA Assay Kit (Beyotime, Shanghai, China).A total of 200 µL MDA working solution was added to 100 µL of supernatant.The mixture was heated to 100 • C for 15 min.After cooling to room temperature, the mixture was centrifuged at 1000× g for 10 min, and 200 µL of supernatant was detected at 532 nm by a microplate reader.

Glutathione and Iron Assay
The intracellular concentration of total GSH was assessed using a GSH Assay Kit (Beyotime, Shanghai, China) according to the manufacturer's instructions.The content of iron in glioblastoma cells was measured using a Cell Ferrous Iron Colorimetric Assay Kit (Elabscience, Hubei, China) according to the manufacturer's instructions.

RNA-Seq
Total RNA was extracted from Erastin-induced glioblastoma cells and control cells using TRIzol according to the manufacturer's protocol.RNA was sequenced by the Novogene.Clean reads were mapped to the Ensemble hg38 human genome using Hisat2 with default parameters.The number of reads mapped to genes was determined with htseq-count [98].DEGs between treatment and control samples were identified with DEseq2.Genes were considered significantly altered with |log 2 FC|≥ 1 and p adj < 0.05.Gene ontology (GO) analysis was carried out using the online tool DAVID (https://david.ncifcrf.gov/accessed on 10 January 2023).GSEA analysis was performed using GSEA software version 4.3.2[99].

ChIP-Seq
ChIP-seq assay was performed as previously described [100].Cells were cross-linked with 1% formaldehyde at RT for 10 min and quenched in 125 mM glycine for 5 min.Cells were resuspended in SDS lysis buffer for 5 min and sonicated to generate DNA fragments averaging 100-500 bp in length.After centrifugation, chromatin fragments were immunoprecipitated with antibodies overnight (H3K27ac, H3K4me1, Abcam, Boston, MA, USA).The precipitated DNA and input DNA were purified and then sequenced.Bowtie2 was used to map ChIP-seq raw reads to the hg38 human reference genome.The MACS2 program was used to call peaks of ChIP-seq data with the corresponding input data as control with default parameters [101][102][103].

HiChIP
HiChIP was performed as previously described [10] using an antibody against H3K27ac (Abcam).Briefly, 1 × 10 7 cells were cross-linked, and chromatin was digested using MboI restriction enzyme (NEB).Then, biotin-14-dATP was used to fill in the restriction fragment overhangs and mark the DNA ends.After ligation and sonication, the genomic DNA was incubated with H3K27ac antibody at 4 • C overnight.Immunocomplexes were captured by protein A magnetic beads.The beads were subsequently washed three times with low salt wash buffer, high salt wash buffer, and LiCl wash buffer.DNA was eluted with ddH 2 O and purified with AMPure XP Beads (Beckman Coulter Genetics, Danvers, MA, USA).Biotinylated DNA beads were captured by Streptavidin C-1.QIAseq FX DNA Library Kits (QIAGEN, Hilden, Germany) was used to generate the sequencing library according to the manufacturer's protocol.The libraries were validated for size distribution of 300-700 bp using AMPure XP beads (Beckman Coulter Genetics, Danvers, MA, USA).The DNA was subjected to 2 × 150 bp paired-end sequencing.The respective paired-end reads were aligned to the hg38 genome by the HiC-Pro pipeline [104].Default settings were used for the removal of duplicated reads, assignment to MboI restriction fragments, and filtering for valid interactions.For each cell type, we merged the valid read pairs of individual samples.Hichipper was applied to call loops with default parameters [105].Reads between samples were normalized to the total number of valid reads.Loops with FDR < 0.05 and supported by at least 5 pairedend tags (PETS) were kept for further analysis.HiC-Pro was used to generate the HiChIP interaction maps [104].HiChIP interaction maps were then visualized by JuicerBox software version 1.11.08 [106] at 50, 10, and 5 kb resolution as indicated.

Conclusions
In summary, our study shows a new mechanism of ferroptosis that enhancers regulate glioblastoma ferroptosis by 3D chromatin structure-mediated gene regulation networks.
Int. J. Mol.Sci.2023, 24, x FOR PEER REVIEW 4 of 18 the change of 3D chromatin structure in ferroptotic glioblastoma cells compared to control (Figure 2A,B).MAP1LC3B and CHAC1 were reported to function in ferroptosis [20-23].
,D), suggesting a significant change of long-range enhancer connectome in ferroptosis.

Figure 5 .
Figure 5. Transcriptome change in ferroptotic glioblastoma cells is associated with changes matin structures.(A) The numbers of EEI, EPI, and PPI of "gained" and "lost" chroma U87MG cells that were treated with Erastin (10 µM, 72 h).(B) Percentages of EEI, EPI, an "gained" and "lost" loops in U87MG cells that were treated with Erastin (10 µM, 72 h).distances of "gained" chromatin loops in U87MG cells that were treated with Erastin (10 "In number" indicates gained chromatin loops that increased in loop number, and "in stre cates gained chromatin loops that increased in interaction strength.(D) Median distanc chromatin loops in U87MG cells that were treated with Erastin (10 µM, 72 h)."In numbe lost chromatin loops that decreased in loop number, and "in strength" indicates lost chro that decreased in interaction strength.(E) Comparison of gene expression in control U87MG trol_1 and Control_2) and Erastin (10 µM, 72 h)-treated U87MG cells (Erastin_1 and Heatmap shows clustering of "gained" chromatin loop-associated upregulated genes (log < 0.05) in control U87MG cells and Erastin (10 µM, 72 h)-treated U87MG cells.(F) GO

Figure 5 .
Figure 5. Transcriptome change in ferroptotic glioblastoma cells is associated with changes in 3D chromatin structures.(A)The numbers of EEI, EPI, and PPI of "gained" and "lost" chromatin loops in U87MG cells that were treated with Erastin (10 µM, 72 h).(B) Percentages of EEI, EPI, and PPI in all "gained" and "lost" loops in U87MG cells that were treated with Erastin (10 µM, 72 h).(C) Median distances of "gained" chromatin loops in U87MG cells that were treated with Erastin (10 µM, 72 h)."In number" indicates gained chromatin loops that increased in loop number, and "in strength" indicates gained chromatin loops that increased in interaction strength.(D) Median distances of "lost" chromatin loops in U87MG cells that were treated with Erastin (10 µM, 72 h)."In number" indicates lost chromatin loops that decreased in loop number, and "in strength" indicates lost chromatin loops that decreased in interaction strength.

Figure 6 .
Figure 6.Genes that were regulated by 3D chromatin structures in glioblastoma ferroptosis.(A-H3K4me1 and H3K27ac enrichment for control U87MG cells and U87MG cells that were trea

Figure 6 .
Figure 6.Genes that were regulated by 3D chromatin structures in glioblastoma ferroptosis.(A-D) H3K4me1 and H3K27ac enrichment for control U87MG cells and U87MG cells that were treated with Erastin (10 µM, 72 h), as well as HiChIP interaction loops between promoters of HDM2 (A), TXNRD1 (B), MKI67 (C), and TOP2A (D) and enhancers.Promoters are shaded in red; enhancers are shaded in blue.Black curves indicate chromatin interaction loops in control U87MG cells, and red curves indicate chromatin interaction loops in U87MG cells that were treated with Erastin (10 µM, 72 h).Black dashed curves indicate weaker chromatin interactions in control U87MG cells compared to U87MG cells that were treated with Erastin (10 µM, 72 h).Red dashed curves indicate weaker chromatin interactions in U87MG cells that were treated with Erastin (10 µM, 72 h) compared to control.The red track is H3K4me1 ChIP-seq signal from control U87MG cells.The light blue track is H3K4me1 ChIP-seq signal from U87MG cells that were treated with Erastin (10 µM, 72 h).The blue track is H3K27ac ChIP-seq signal from control U87MG cells.The purple track is H3K27ac ChIP-seq signal from U87MG cells that were treated with Erastin (10 µM, 72 h).

Figure 7 .
Figure 7. Schematics showed that some ferroptosis-related genes were upregulated and gaine chromatin interaction loops between their respective loci and enhancers during Erastin-induced fe roptosis in glioblastoma.En: Enhancer.Blue and yellow rounds represent enhancers, red squar

Figure 7 .
Figure7.Schematics showed that some ferroptosis-related genes were upregulated and gained chromatin interaction loops between their respective loci and enhancers during Erastin-induced ferroptosis in glioblastoma.En: Enhancer.Blue and yellow rounds represent enhancers, red square represents ferroptosis-related gene, "↓" in the boxes represents transcription, "↑" below the boxes represents up-regulated expression.