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Transcriptome analysis of the circadian clock gene BMAL1 deletion with opposite carcinogenic effects

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Abstract

We have previously reported that the deletion of BMAL1 gene has opposite effects in respect to its contribution to the pathways that are effective in the multistage carcinogenesis process. BMAL1 deletion sensitized nearly normal breast epithelial (MCF10A) and invasive breast cancer cells (MDA-MB-231) to cisplatin- and doxorubicin-induced apoptosis, while this deletion also aggravated the invasive potential of MDA-MB-231 cells. However, the mechanistic relationship of the seemingly opposite contribution of BMAL1 deletion to carcinogenesis process is not known at genome-wide level. In this study, an RNA-seq approach was taken to uncover the differentially expressed genes (DEGs) and pathways after treating BMAL1 knockout (KO) or wild-type (WT) MDA-MB-231 cells with cisplatin and doxorubicin to initiate apoptosis. Gene set enrichment analysis with the DEGs demonstrated that enrichment in multiple genes/pathways contributes to sensitization to cisplatin- or doxorubicin-induced apoptosis in BMAL1-dependent manner. Additionally, our DEG analysis suggested that non-coding transcript RNA (such as lncRNA and processed pseudogenes) may have role in cisplatin- or doxorubicin-induced apoptosis. Protein-protein interaction network obtained from common DEGs in cisplatin and doxorubicin treatments revealed that GSK3β, NACC1, and EGFR are the principal genes regulating the response of the KO cells. Moreover, the analysis of DEGs among untreated BMAL1 KO and WT cells revealed that epithelial-mesenchymal transition genes are up-regulated in KO cells. As a negative control, we have also analyzed the DEGs following treatment with an endoplasmic reticulum (ER) stress-inducing agent, tunicamycin, which was affected by BMAL1 deletion minimally. Collectively, the present study suggests that BMAL1 regulates many genes/pathways of which the alteration in BMAL1 KO cells may shed light on pleotropic phenotype observed.

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References

  • Aboussekhra A, Biggerstaff M, Shivji MK, Vilpo JA, Moncollin V, Podust VN, Protic M, Hubscher U, Egly JM, Wood RD (1995) Mammalian DNA nucleotide excision repair reconstituted with purified protein components. Cell 80(6):859–868

    CAS  PubMed  Google Scholar 

  • Antoch MP, Gorbacheva VY, Vykhovanets O, Toshkov IA, Kondratov RV, Kondratova AA, Lee C, Nikitin AY (2008) Disruption of the circadian clock due to the Clock mutation has discrete effects on aging and carcinogenesis. Cell Cycle 7(9):1197–1204

    CAS  PubMed  Google Scholar 

  • Bindea G, Mlecnik B, Hackl H, Charoentong P, Tosolini M, Kirilovsky A, Fridman WH, Pages F, Trajanoski Z, Galon J (2009) ClueGO: a Cytoscape plug-in to decipher functionally grouped gene ontology and pathway annotation networks. Bioinformatics 25(8):1091–1093

    CAS  PubMed  PubMed Central  Google Scholar 

  • Bolger AM, Lohse M, Usadel B (2014) Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30(15):2114–2120

    CAS  PubMed  PubMed Central  Google Scholar 

  • Bozulic L, Surucu B, Hynx D, Hemmings BA (2008) PKBalpha/Akt1 acts downstream of DNA-PK in the DNA double-strand break response and promotes survival. Mol Cell 30(2):203–213

    CAS  PubMed  Google Scholar 

  • Cavga AD, Tardu M, Korkmaz T, Keskin O, Ozturk N, Gursoy A, Kavakli IH (2019) Cryptochrome deletion in p53 mutant mice enhances apoptotic and anti-tumorigenic responses to UV damage at the transcriptome level. Funct Integr Genomics 19(5):729–742

    CAS  PubMed  Google Scholar 

  • Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, Batut P, Chaisson M, Gingeras TR (2013) STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29(1):15–21

    CAS  PubMed  Google Scholar 

  • Doench JG, Fusi N, Sullender M, Hegde M, Vaimberg EW, Donovan KF, Smith I, Tothova Z, Wilen C, Orchard R, Virgin HW, Listgarten J, Root DE (2016) Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol 34(2):184–191

    CAS  PubMed  PubMed Central  Google Scholar 

  • Dong Z, Zhang G, Qu M, Gimple RC, Wu Q, Qiu Z, Prager BC, Wang X, Kim LJY, Morton AR, Dixit D, Zhou W, Huang H, Li B, Zhu Z, Bao S, Mack SC, Chavez L, Kay SA, Rich JN (2019) Targeting glioblastoma stem cells through disruption of the circadian clock. Cancer Discov 9(11):1556–1573

    CAS  PubMed  PubMed Central  Google Scholar 

  • Dunlap JC, Loros JJ, Liu Y, Crosthwaite SK (1999) Eukaryotic circadian systems: cycles in common. Genes Cells 4(1):1–10

    CAS  PubMed  Google Scholar 

  • Ekman D, Light S, Bjorklund AK, Elofsson A (2006) What properties characterize the hub proteins of the protein-protein interaction network of Saccharomyces cerevisiae? Genome Biol 7(6):R45

    PubMed  PubMed Central  Google Scholar 

  • Filipski E, King VM, Li X, Granda TG, Mormont MC, Liu X, Claustrat B, Hastings MH, Levi F (2002) Host circadian clock as a control point in tumor progression. J Natl Cancer Inst 94(9):690–697

    PubMed  Google Scholar 

  • Fu L, Pelicano H, Liu J, Huang P, Lee C (2002) The circadian gene Period2 plays an important role in tumor suppression and DNA damage response in vivo. Cell 111(1):41–50

    CAS  PubMed  Google Scholar 

  • Gasiunas G, Barrangou R, Horvath P, Siksnys V (2012) Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc Natl Acad Sci U S A 109(39):E2579–E2586

    CAS  PubMed  PubMed Central  Google Scholar 

  • Gauger MA, Sancar A (2005) Cryptochrome, circadian cycle, cell cycle checkpoints, and cancer. Cancer Res 65(15):6828–6834

    CAS  PubMed  Google Scholar 

  • Gospodinov A, Vaissiere T, Krastev DB, Legube G, Anachkova B, Herceg Z (2011) Mammalian Ino80 mediates double-strand break repair through its role in DNA end strand resection. Mol Cell Biol 31(23):4735–4745

    CAS  PubMed  PubMed Central  Google Scholar 

  • Jensen LJ, Kuhn M, Stark M, Chaffron S, Creevey C, Muller J, Doerks T, Julien P, Roth A, Simonovic M, Bork P, von Mering C (2009) STRING 8--a global view on proteins and their functional interactions in 630 organisms. Nucleic Acids Res 37(Database issue):D412–D416

    CAS  PubMed  Google Scholar 

  • Kao GD, McKenna WG, Guenther MG, Muschel RJ, Lazar MA, Yen TJ (2003) Histone deacetylase 4 interacts with 53BP1 to mediate the DNA damage response. J Cell Biol 160(7):1017–1027

    CAS  PubMed  PubMed Central  Google Scholar 

  • Kavakli IH, Baris I, Tardu M, Gul S, Oner H, Cal S, Bulut S, Yarparvar D, Berkel C, Ustaoglu P, Aydin C (2017) The photolyase/cryptochrome family of proteins as DNA repair enzymes and transcriptional repressors. Photochem Photobiol 93(1):93–103

    CAS  PubMed  Google Scholar 

  • Korkmaz T, Aygenli F, Emisoglu H, Ozcelik G, Canturk A, Yilmaz S, Ozturk N (2018) Opposite carcinogenic effects of circadian clock gene BMAL1. Sci Rep 8(1):16023

    PubMed  PubMed Central  Google Scholar 

  • Lee JH, Sancar A (2011) Regulation of apoptosis by the circadian clock through NF-kappaB signaling. Proc Natl Acad Sci U S A 108(29):12036–12041

    CAS  PubMed  PubMed Central  Google Scholar 

  • Li HF, Kim JS, Waldman T (2009) Radiation-induced Akt activation modulates radioresistance in human glioblastoma cells. Radiat Oncol 4:43

    PubMed  PubMed Central  Google Scholar 

  • Li W, Liu W, Kakoki A, Wang R, Adebali O, Jiang Y, Sancar A (2019) Nucleotide excision repair capacity increases during differentiation of human embryonic carcinoma cells into neurons and muscle cells. J Biol Chem 294(15):5914–5922

    CAS  PubMed  PubMed Central  Google Scholar 

  • Li H, Yang Y, Hong W, Huang M, Wu M, Zhao X (2020) Applications of genome editing technology in the targeted therapy of human diseases: mechanisms, advances and prospects. Signal Transduct Target Ther 5(1):1

    PubMed  PubMed Central  Google Scholar 

  • Liccardi G, Hartley JA, Hochhauser D (2011) EGFR nuclear translocation modulates DNA repair following cisplatin and ionizing radiation treatment. Cancer Res 71(3):1103–1114

    CAS  PubMed  PubMed Central  Google Scholar 

  • Liu Z-H, Wang M-H, Ren H-J, Qu W, Sun L-M, Zhang Q-F, Qiu X-S, Wang E-H (2014) Interleukin 7 signaling prevents apoptosis by regulating bcl-2 and bax via the p53 pathway in human non-small cell lung cancer cells. International journal of clinical and experimental pathology 7(3):870–881

    PubMed  PubMed Central  Google Scholar 

  • Ma T, Wang Z, Guo Y, Pei D (2009) The C-terminal pentapeptide of Nanog tryptophan repeat domain interacts with Nac1 and regulates stem cell proliferation but not pluripotency. J Biol Chem 284(24):16071–16081

    CAS  PubMed  PubMed Central  Google Scholar 

  • Miller BH, McDearmon EL, Panda S, Hayes KR, Zhang J, Andrews JL, Antoch MP, Walker JR, Esser KA, Hogenesch JB, Takahashi JS (2007) Circadian and CLOCK-controlled regulation of the mouse transcriptome and cell proliferation. Proc Natl Acad Sci U S A 104(9):3342–3347

    CAS  PubMed  PubMed Central  Google Scholar 

  • O'Driscoll M, Jeggo PA (2006) The role of double-strand break repair - insights from human genetics. Nat Rev Genet 7(1):45–54

    CAS  PubMed  Google Scholar 

  • Ozturk N (2017) Phylogenetic and functional classification of the photolyase/cryptochrome family. Photochem Photobiol 93(1):104–111

    CAS  PubMed  Google Scholar 

  • Ozturk N, Lee JH, Gaddameedhi S, Sancar A (2009) Loss of cryptochrome reduces cancer risk in p53 mutant mice. Proc Natl Acad Sci U S A 106(8):2841–2846

    CAS  PubMed  PubMed Central  Google Scholar 

  • Qin Y, Li L, Wang F, Zhou X, Liu Y, Yin Y, Qi X (2018) Knockdown of Mir-135b sensitizes colorectal cancer cells to oxaliplatin-induced apoptosis through increase of FOXO1. Cell Physiol Biochem 48(4):1628–1637

    CAS  PubMed  Google Scholar 

  • Qiu H, Zhang X, Ni W, Shi W, Fan H, Xu J, Chen Y, Ni R, Tao T (2016) Expression and clinical role of Cdc5L as a novel cell cycle protein in hepatocellular carcinoma. Dig Dis Sci 61(3):795–805

    CAS  PubMed  Google Scholar 

  • Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F (2013) Genome engineering using the CRISPR-Cas9 system. Nat Protoc 8(11):2281–2308

    CAS  PubMed  PubMed Central  Google Scholar 

  • Rapaport F, Khanin R, Liang Y, Pirun M, Krek A, Zumbo P, Mason CE, Socci ND, Betel D (2013) Comprehensive evaluation of differential gene expression analysis methods for RNA-seq data. Genome Biol 14(9):R95

    PubMed  PubMed Central  Google Scholar 

  • Reppert SM, Weaver DR (2002) Coordination of circadian timing in mammals. Nature 418(6901):935–941

    CAS  PubMed  Google Scholar 

  • Rijo-Ferreira F, Takahashi JS (2019) Genomics of circadian rhythms in health and disease. Genome Med 11(1):82

    PubMed  PubMed Central  Google Scholar 

  • Saini N, Sarin A (2020) Nucleolar localization of the Notch4 intracellular domain underpins its regulation of the cellular response to genotoxic stressors. Cell Death Discov 6:7

    PubMed  PubMed Central  Google Scholar 

  • Sanjana NE, Shalem O, Zhang F (2014) Improved vectors and genome-wide libraries for CRISPR screening. Nat Methods 11(8):783–784

    CAS  PubMed  PubMed Central  Google Scholar 

  • Sarayloo E, Tardu M, Unlu YS, Simsek S, Cevahir G, Erkey C, Kavakli IH (2017) Understanding lipid metabolism in high-lipid-producing Chlorella vulgaris mutants at the genome-wide level. Algal Research 28:244–252

    Google Scholar 

  • Shibue T, Weinberg RA (2017) EMT, CSCs, and drug resistance: the mechanistic link and clinical implications. Nature Reviews Clinical Oncology 14(10):611–629

    PubMed  PubMed Central  Google Scholar 

  • Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA, Paulovich A, Pomeroy SL, Golub TR, Lander ES, Mesirov JP (2005) Gene set enrichment analysis: a knowledge-based approach for interpreting genome-wide expression profiles. Proc Natl Acad Sci U S A 102(43):15545–15550

    CAS  PubMed  PubMed Central  Google Scholar 

  • Surucu B, Bozulic L, Hynx D, Parcellier A, Hemmings BA (2008) In vivo analysis of protein kinase B (PKB)/Akt regulation in DNA-PKcs-null mice reveals a role for PKB/Akt in DNA damage response and tumorigenesis. J Biol Chem 283(44):30025–30033

    CAS  PubMed  PubMed Central  Google Scholar 

  • Szklarczyk D, Franceschini A, Wyder S, Forslund K, Heller D, Huerta-Cepas J, Simonovic M, Roth A, Santos A, Tsafou KP, Kuhn M, Bork P, Jensen LJ, von Mering C (2015) STRING v10: protein-protein interaction networks, integrated over the tree of life. Nucleic Acids Res 43(Database issue):D447–D452

    CAS  PubMed  Google Scholar 

  • Takahashi JS, Hong HK, Ko CH, McDearmon EL (2008) The genetics of mammalian circadian order and disorder: implications for physiology and disease. Nat Rev Genet 9(10):764–775

    CAS  PubMed  PubMed Central  Google Scholar 

  • Tardu M, Bulut S, Kavakli IH (2017) MerR and ChrR mediate blue light induced photo-oxidative stress response at the transcriptional level in Vibrio cholerae. Sci Rep 7:40817

    CAS  PubMed  PubMed Central  Google Scholar 

  • Tardu M, Dikbas UM, Baris I, Kavakli IH (2016) RNA-seq analysis of the transcriptional response to blue and red light in the extremophilic red alga, Cyanidioschyzon merolae. Funct Integr Genomics 16(6):657–669

    CAS  PubMed  Google Scholar 

  • Trapnell C, Roberts A, Goff L, Pertea G, Kim D, Kelley DR, Pimentel H, Salzberg SL, Rinn JL, Pachter L (2012) Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat Protoc 7(3):562–578

    CAS  PubMed  PubMed Central  Google Scholar 

  • Vasquez KM, Christensen J, Li L, Finch RA, Glazer PM (2002) Human XPA and RPA DNA repair proteins participate in specific recognition of triplex-induced helical distortions. Proc Natl Acad Sci U S A 99(9):5848–5853

    CAS  PubMed  PubMed Central  Google Scholar 

  • Wang J, Rao S, Chu J, Shen X, Levasseur DN, Theunissen TW, Orkin SH (2006) A protein interaction network for pluripotency of embryonic stem cells. Nature 444(7117):364–368

    CAS  PubMed  Google Scholar 

  • Warshamana-Greene GS, Litz J, Buchdunger E, García-Echeverría C, Hofmann F, Krystal GW (2005) The insulin-like growth factor-I receptor kinase inhibitor, NVP-ADW742, sensitizes small cell lung cancer cell lines to the effects of chemotherapy. Clinical cancer research : an official journal of the American Association for Cancer Research 11(4):1563–1571

    CAS  Google Scholar 

  • Wold MS (1997) Replication protein A: a heterotrimeric, single-stranded DNA-binding protein required for eukaryotic DNA metabolism. Annu Rev Biochem 66:61–92

    CAS  PubMed  Google Scholar 

  • Yu H, Kim PM, Sprecher E, Trifonov V, Gerstein M (2007) The importance of bottlenecks in protein networks: correlation with gene essentiality and expression dynamics. PLoS Comput Biol 3(4):e59

    PubMed  PubMed Central  Google Scholar 

  • Zhang N, Kaur R, Akhter S, Legerski RJ (2009) Cdc5L interacts with ATR and is required for the S-phase cell-cycle checkpoint. EMBO Rep 10(9):1029–1035

    CAS  PubMed  PubMed Central  Google Scholar 

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Funding

This work was supported by the Turkish Scientific and Technical Research Council (TUBITAK) grant 114S446 (to N.O.) and Gebze Technical Research Program grant G.T.Ü. BAP 2018-A-105-39 (to N.O.)

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Author notes

  1. Fatih Aygenli

    Present address: Institute of Medical Psychology, Faculty of Medicine, LMU Munich, Munich, Germany

  2. Handan Emisoglu Kulahli and Seref Gul contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular Biology and Genetics, Gebze Technical University, Gebze, Kocaeli, Turkey

    Handan Emisoglu-Kulahli, Fatih Aygenli, Saba Selvi & Nuri Ozturk

  2. Department of Molecular Biology and Genetics, Koc University, Istanbul, Turkey

    Seref Gul & Ibrahim Halil Kavakli

  3. Department of Chemical and Biological Engineering, Koc University, Istanbul, Turkey

    Seref Gul, Onur Ozcan & Ibrahim Halil Kavakli

  4. Department of Biology, Istanbul University, Istanbul, Turkey

    Hande Morgil

  5. Istanbul University Centre for Plant and Herbal Products Research-Development, 34126, Istanbul, Turkey

    Hande Morgil

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  1. Handan Emisoglu-Kulahli
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Correspondence to Nuri Ozturk.

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ESM 1

Supp. Fig. 1 Confirmation of authenticity of MDA-MB-231 WT and KO cells A) The BMAL1 protein level was detected in parental (+), BMAL1 knockout (KO) and mock-infected (WT) MDA-MB-231 cells. B) In agreement with previous findings, BMAL1 deletion sensitized MDA-MB-231 cells to cisplatin (CIS)- and C) doxorubicin (DOX)- induced apoptosis as probed for c-PARP. D) The cells were also treated with tunicamycin (TUN) and spliced (active) form of XBP1 (sXBP1) was detected as the output marker for tunicamycin-induced ER stress response. Since sXPB1 signal and a cross-reacting band were merged, we run the samples for a longer period to confirm that sXBP1 signal was not much different between WT and KO cells in agreement with the transcriptome data that revealed much less DEGs in compared to CIS and DOX treatments. Supp. Fig. 2 Statistical analysis of migration rates of WT and KO cells. A) Migration properties of KO and WT MDA-MB-231 cells were analyzed using wound-healing assay. Representative images show the scratch (wound) at t = 0 h, t = 24 h and t = 48 h. Marks (red lines) were placed to locate the open area on the scratch. Images were taken by using Leica inverted microscope. Magnification 5X. B) The graph shows the percentage of wound healing in WT, and KO cells under starvation condition. The open area was calculated at the indicated time points by ImageJ Software (Free Accessible Image Processing Program, www.imagej.net). All results are representative of four independent experiments. Statistical Analysis was done with 24-h and 48-h data by one-way ANOVA followed by Tukey’s post hoc tests for pairwise comparison. (****P < 0.0001). Supp. Fig. S3 Gene Set Enrichment Analysis after TUN treatment. In agreement with similar sXBP1 formation in both WT and KO cells, no significant GSEA was detected after TUN treatment. TUN, tunicamycin. Supp. Fig. S4 Gene Set Enrichment Analysis and KEGG GO term of DEGs without treatment. In order to reveal the genes enriched in KO cells, DEGs were used for GSEA (KO vs WT). A) In agreement with the observed phenotype, HALLMARK_EPITHELIAL_MESENCHYMAL_TRANSITION was the most significant one. B) BMAL1 gene deletion affected many genes as KEGG GO term of DEGs was shown. (PDF 701 kb)

ESM 2

(DOCX 32 kb)

Table S1.

List of proteasomal or polymerase-related genes excluded from network analysis (XLSX 18 kb)

Table S2.

List of common DEGs in CIS and DOX treatments used to construct PPI network (XLSX 33 kb)

Table S3.

List of additional DEGs after 0-FPKM correction (XLSX 57 kb)

Table S4.

List of oligos used for qPCR analysis (XLSX 12 kb)

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Emisoglu-Kulahli, H., Gul, S., Morgil, H. et al. Transcriptome analysis of the circadian clock gene BMAL1 deletion with opposite carcinogenic effects. Funct Integr Genomics 21, 1–16 (2021). https://doi.org/10.1007/s10142-020-00757-6

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