CREB3/ATF6 family members act as karyoptosis factors

다국어 초록 (Multilingual Abstract)

The nuclear membrane is a double-membrane system composed of two lipid bilayers that compartmentalize and protect the genome, playing a crucial role in regulation of DNA replication and gene transcription. Beyond functioning as a physical barrier, the nuclear membrane integrates structural maintenance and signaling pathway through dynamic interactions among various nuclear membrane components, such as lamina, LINC complexes and nucleoporins. Based on recent studies of CREB3 that revealed its role in regulating the nuclear integrity, cleavage of CREB3 disrupts the nuclear mechanical force equilibrium, consequently leading to karyoptosis. In this study, it was found that type Ⅱ membrane proteins containing a bZIP domain, including CREB3, CREB3L1, CREB3L2, CREB3L4, CREB3L4 (CREB3L1~L4), ATF6α and ATF6β, function as constituents of the inner nuclear membrane (INM). These proteins are classically known to be located at the ER/Golgi and the N-terminal fragments, which are produced by the sequential cleavage of S1P and S2P, are translocated into the nucleus, where they act as transcription factors. In contrast to this canonical concept, I found that the full-length (FL) forms of CREB3/ATF6 family members are localized to the INM, whereas they are tightly bound to chromatin. Importantly, overexpression of CREB3/ATF6 family members-CFs leads to karyoptosis, by deriving of nuclear membrane rupture, genome leakage and cell death. Mechanism studies showed that CREB3/ATF6 family members form homo- and heterodimers or multimers, thereby regulating the genome-wide chromatin tethering to the INM. Notably, the overexpression of each CREB3/ATF6 family members-cleaved forms strongly suppressed foci formation and cell proliferation in cancer cells. The LDH assay results supported that this suppression was caused by the cell death of cancer cells. Taken together, this study demonstrated that CREB3/ATF6 family act as key factors to regulate nuclear scaffold and to stabilize the nuclear membrane integrity via chromatin tethering into the INM. Therefore, the dysregulated cleavage of these factors at the INM collapses the nuclear force’s equilibrium, resulting in karyoptosis. These findings will lead to a novel conceptual framework for cancer therapy. Key words: bZIP family, type Ⅱ membrane-bound transcription factor, nuclear skeleton, regulated cell death, karyoptosis

목차 (Table of Contents)

• Ⅰ. Abstract ⅵ
• Ⅱ. Introduction 1
• 1. Regulated cell death and cancer therapy 3
• 1.1 Autophagy 8
• 1.2 Apoptosis 10
• 1.3 Necroptosis 12
• 2. Karyoptosis 14
• 3. Nuclear morphology 16
• 4. Nuclear membrane structure 20
• 5. bZIP family 25
• 5.1 AP-1 family 26
• 5.2 PAR family 27
• 5.3 CNC family 27
• 5.4 MAF family 28
• 5.5 C/EBP family 29
• 5.6 CREB family 29
• Ⅲ. Materials and Methods 31
• Ⅳ. Results 52
• 1. CREB3/ATF6 family members are membrane-bound bZIP-
• containing transcription factors 52
• 2. Profiling of CREB3/ATF6 family mRNA expression across normal
• and tumor tissues 55
• 3. CREB3/ATF6-FLs are nuclear membrane proteins 57
• 4. BFA induced cleavage of the CREB3/ATF6-FLs 61
• 5. Overexpression of CREB3/ATF6-CFs induces loss of nuclear
• integrity 63
• 6. Comparative analysis of signaling pathways between
• CREB3/ATF6-CFs–induced cell death and apoptosis, autophagy
• and necroptosis 66
• 7. CREB3/ATF6-CFs induce karyoptosis 69
• 8. CREB3/ATF6-CFs induces DNA damage 73
• 9. Homodimerization of CREB3/ATF6 family 75
• 10. Heterodimerization of CREB3/ATF6 family 78
• 11. CREB3/ATF6-FLs form homo-/heterodimers at the INM 81
• 12. Overexpression of CREB3/ATF6-CFs suppress cancer cell
• proliferation 84
• Ⅴ. Discussion 87
• Ⅵ. Conclusion 92
• Ⅶ. References 93
• Ⅷ. 국문초록 105