bioRxiv preprint doi: https://doi.org/10.1101/2020.11.03.367268; this version posted November 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 Long-range gene regulation network of the MGMT enhancer in modulating glioma cell 2 sensitivity to temozolomide 3 4 5 Anshun He1, †, Bohan Chen1, †, Jinfang Bi1, Wenbin Wang1, Jun Chen1, Yuyang Qian1, 6 Tengfei Shi1, Zhongfang Zhao1, Jiandang Shi1, Hongzhen Yang1, Lei Zhang1,*, Wange Lu1,* 7 8 9 1State Key Laboratory of Medicinal Chemical Biology and College of Life Sciences, Nankai 10 University, 94 Weijin Road, 300071, Tianjin, China. 11 12 13 † These authors contributed equally to this work. 14 * Corresponding authors: Wange Lu and Lei Zhang 15 E-mail: [email protected]; [email protected] 16 Running title: Network of MGMT enhancer associated with TMZ sensitivity 17 18 19 Key words: MGMT enhancer; glioma; temozolomide, 3D chromatin structure, long-range 20 interactions 21 22 23 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.03.367268; this version posted November 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 24 Abstract 25 Acquired resistance to temozolomide (TMZ) is a major obstacle in glioblastoma treatment. 26 MGMT, a DNA repair protein, and the methylation at its gene promoter, plays an important 27 role in TMZ resistance. However, some evidences have suggested a MGMT-independent 28 mechanisms underlying TMZ resistance. Here, we used MGMT enhancer as a model and 29 discovered that its deletion in glioma cells of low MGMT expression induced increased 30 sensitivity to temozolomide. Analysis of a combination of RNA-seq and Capture Hi-C further 31 suggested multiple long-range target genes regulated by the MGMT enhancer and that 32 interactions may play important roles in glioma cell sensitivity to TMZ. This study reveals a 33 novel mechanism of regulation of TMZ sensitivity in glioma cells. 34 35 Introduction 36 Approximately 80% of primary tumors in the central nervous system are malignant glioma 37 (1), and >50% of those are diagnosed as glioblastoma (GBM), the most aggressive glioma. 38 Despite treatment advances, glioblastoma prognosis is unsatisfying, and median survival time 39 of patients is only 14~17 months (2). 40 41 Temozolomide (TMZ) is a reagent frequently used in the clinic as chemotherapy for GBM 42 (3,4), due to its high bioavailability and relatively low side effects. However, development of 43 chemo-resistance to TMZ significantly decreases its efficacy and remains a major treatment 44 obstacle. Thus it is proposed that increasing glioma cells’ sensitivity to TMZ could improve 45 prognosis of patients with GBM. Several previous studies report that 46 O-6-methylguanine-DNA methyltransferase (MGMT) promotes TMZ resistance in glioma 47 (5,6) by removing cytotoxic O-6-methylguanine-DNA lesions generated by TMZ. Other 48 factors associated with TMZ resistance include poly (ADP-ribose) polymerase (7), 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.03.367268; this version posted November 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 49 ALDH1A1 (8), and P4HB (9). Moreover, Gaspar and colleagues report that phosphoinositide 50 3-kinase-mediated HOXA9/HOXA10 expression is associated with MGMT-independent 51 TMZ-resistance in glioblastoma cells (10). In addition, patients whose tumor cells show no 52 apparent expression of MGMT are susceptible to TMZ resistance (11), indicating that other 53 factors may also underlie TMZ resistance. These findings are supported in part by the fact 54 that MGMT inhibitors show little effect against glioma in clinical trials (12,13). 55 56 A previously identified MGMT enhancer was reported to be associated with methylation of 57 MGMT promoter and expression of MGMT (14,15). However, inconsistency of methylation 58 level of MGMT promoter and MGMT expression were also found in GBM (16-18). Thus, 59 MGMT expression may be regulated by other enhancers or factors (19), or the MGMT 60 enhancer may regulate genes other than MGMT, which may contribute to its role in TMZ 61 resistance. 62 63 High-order chromatin structure studies have revealed that regulatory elements like enhancers 64 can regulate long-range gene expression (20-23). Thus we asked whether the MGMT 65 enhancer may not only regulate MGMT but also other genes through long-range interactions 66 and in so doing alter TMZ sensitivity of glioma cells. Here, we report that in glioma cells of 67 low MGMT expression a MGMT enhancer regulates glioma cell sensitivity to TMZ through 68 long-range regulation of multiple target genes, including MKI67, providing a novel 69 regulatory mechanism underlying glioma cells TMZ sensitivity. 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.03.367268; this version posted November 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 70 71 Results 72 MGMT enhancer deletion in glioma cells showing low MGMT expression increases their 73 sensitivity to TMZ 74 To determine whether the MGMT enhancer is associated with glioma cell sensitivity to TMZ 75 we used the CRISPR/Cas9 system to delete the MGMT enhancer region in U251 glioma cells, 76 whose MGMT expression is low (24). Two enhancer KO lines (KO-1, lacking 874bp, and 77 KO-2, lacking 573bp of the enhancer region) were generated (Fig. 1A, Supplementary Fig. 78 S1). Wickstrom et al. previously reported Western blot data showing no MGMT expression in 79 five of six typical glioma lines, including U251, and proposed this deficit was due to MGMT 80 promoter methylation (24). Our RT-qPCR analysis confirmed low MGMT transcription levels 81 in wild-type (WT) U251 cells treated 72 hours with TMZ (1mM) or control vehicle 82 (Supplementary Fig. S2A). Moreover, MGMT enhancer deletion did not significantly alter 83 MGMT transcription in either KO line (Supplementary Fig. S2B). We then treated both 84 enhancer KO lines and WT U251 cells with TMZ (1mM) for 72 hours and assessed potential 85 cytotoxicity by performing lactate dehydrogenase (LDH) and caspase 3/7 activity assays (Fig. 86 1B). Cytosolic LDH is released to the medium by dying cells, and caspase 3/7 activity is an 87 apoptotic marker. Compared with TMZ-treated WT U251 cells, LDH levels in culture 88 medium were significantly increased by 2-3 fold (after 24h) and 4-5 fold (after 48h) in 89 MGMT enhancer KO glioma cells after TMZ treatment (Fig. 1C,D). Caspase 3/7 activities in 90 KO cells also significantly increased (~3-fold) relative to WT U251 cells after TMZ 91 treatment at 48h and 72h (Fig. 1E,F). Given that MGMT is not detectable in U251 cells 92 treated with or without TMZ (1mM, 72h), these data suggest that the MGMT enhancer 93 modulates glioma cell TMZ sensitivity through genes other than MGMT. 94 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.03.367268; this version posted November 4, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 95 Long-range interactions of the MGMT enhancer with target genes 96 Studies of high-order chromatin structure indicate that enhancers can regulate long-range 97 targets though 3D genome structure. Hi-C data analysis in glioma cells treated with or 98 without TMZ showed that TMZ treatment altered high-order chromatin structure (Fig. 2A). 99 Specifically, a Z-score map showed global changes in interaction frequencies in the MGMT 100 enhancer region (Fig. 2A), suggesting that long-range interactions are associated with drug 101 sensitivity. To investigate potential MGMT enhancer target genes, we performed Capture 102 Hi-C in U251 glioma cells. We used a 1.8 kb region containing the MGMT enhancer as a 103 “bait” region (Fig. 2B) and performed Capture Hi-C based on a previously reported protocol 104 (25,26). Interactions between the bait region and target chromatin regions from two 105 biological replicates are depicted as curves in Circos plots shown in Fig. 2C,D (27). In cis 106 interacting sites comprised 73.3% and 69.8% of respective biological replicates. When we 107 mapped sites to the human genome, we identified 89 target genes, 84 in cis and 5 are in trans, 108 which physically contacted with the MGMT enhancer in both biological replicates. RNA-seq 109 data in WT and KO-1 and KO-2 cells revealed global transcriptional changes in enhancer KO 110 relative to WT U251 cells (Fig. 3A). Gene Ontology (GO) analysis of significantly changed 111 genes (q <0.05) between enhancer KO and WT U251 cells identified focal adhesion as the 112 top altered signaling pathway (Fig. 3B), an observation consistent with a previous report that 113 focal adhesion activity is associated with glioma cell TMZ sensitivity (28). We then 114 performed combination analysis of RNA-seq and Capture Hi-C to confirm direct target genes 115 potentially regulated by the MGMT enhancer. Genes with a positive signal in Capture Hi-C 116 data are shown in Fig. 3C. Moreover, Fig. 3C shows the names of genes (KIAA1549L, 117 SH3XPD2A, ADAM12 and MKI67) with significant altered mRNA expression in the MGMT 118 enhancer KO relative to WT U251 cells (WT/KO fold change > 1.5; q value < 0.05). 119 SH3XPD2A, ADAM12 and MKI67 were in cis targets and KIAA1549L in trans (on 5 bioRxiv preprint doi: https://doi.org/10.1101/2020.11.03.367268; this version posted November 4, 2020.