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BubR1 in the Coordination of Kinetochore-Microtubule Attachment and Spindle Assembly Checkpoint Signaling
DC Field | Value | Language |
---|---|---|
dc.contributor.advisor | 이현숙 | - |
dc.contributor.author | 박인애 | - |
dc.date.accessioned | 2017-07-14T00:51:18Z | - |
dc.date.available | 2019-04-18 | - |
dc.date.issued | 2016-02 | - |
dc.identifier.other | 000000132184 | - |
dc.identifier.uri | https://hdl.handle.net/10371/121436 | - |
dc.description | 학위논문 (박사)-- 서울대학교 대학원 : 생명과학부, 2016. 2. 이현숙. | - |
dc.description.abstract | BubR1 is a key protein constituting the mitotic checkpoint complex (MCC). During mitosis, the spindle assembly checkpoint (SAC) acts to delay anaphase onset until all chromosomes are attached to mitotic spindles at kinetochores. The SAC works through generation of MCC, which inhibits the anaphase-promoting complex/cyclosome E3 ligase (APC/C) in the cytoplasm.
This study addresses on first, how BubR1 coordinated kinetochore-microtubule (KT-MT) attachments and SAC signaling. Secondly, it also addresses on how a regulator in mitosis, tumor suppressor BRCA2 serves as a signaling platform during SAC. In order to understand the physiological role of BubR1 acetylation, acetylation deficient knock-in mice (K243R/+) were generated. After 60 weeks, 38% of mice spontaneously developed tumors at various tissues. K243R/+ Mouse Embryonic Fibroblasts (MEFs) were highly aneuploid and had weakened SAC. At kinetochores, unstable KT-MT attachments were observed due to reduced recruitment of PP2AB56a. Insufficient amount of PP2AB56a could not counterbalance the excessive Aurora B activity at kinetochores. All together, unstable KT-MT attachment and failure in maintaining MCC formed in mitosis led to accumulation of chromosome instability (CIN) in K243R/+. These CIN manifested in various forms in the acetylation deficient mice generated a mutation-prone cellular environment favoring tumorigenesis. Previous works have shown that in prometaphase, BubR1 acetylation occurs at kinetochores, only if BRCA2 is present to support the BubR1-P300/CBP-associated factor (PCAF) interaction (Choi et al., 2012). My research showed that BubR1 was deacetylated when SAC was silenced. Deacetylation of BubR1 was a cue to SAC silencing, as cells expressing acetylation mimetic form of BubR1 could not exit mitosis after the metaphase delay. Also, acetylation of BubR1 diminished when SAC silencing was mimicked in mitotic cells. Deacetylation of BubR1 was mediated by class I HDACs, and BRCA2 was needed for HDACs to interact with BubR1 at prometaphase. Hence, one can conclude that BRCA2 not only regulates BubR1 acetylation, but it presents the acetylated BubR1 to HDACs at prometaphase for deacetylation. Analysis on the BRCA2 complex in mitosis suggested that BRCA2 acts as a signaling platform within the SAC signaling network by specifying the interaction and localization of essential proteins at kinetochores. In essence, my study provides further insight into the following key questions in mitosis. First, the question of how KT-MT attachment is sensed and relayed to SAC was further explained by elucidating the role of BubR1 in coordinating KT-MT attachment and SAC signaling. Second, the question of how complex SAC signaling is punctually regulated by multiple proteins during metaphase to anaphase transition was addressed through elucidating the role of BRCA2 as a scaffold for BubR1 acetylation/deacetylation at kinetochores. | - |
dc.description.tableofcontents | I INTRODUCTION 9
I-1. Mitosis and chromosome instability (CIN) 9 I-2. Mouse models for CIN 14 I-3. Spindle assembly checkpoint (SAC) activity and regulation 18 I-4. Sensors in SAC 25 I-5. Roles of BubR1 in mitosis 29 I-6. A Novel function of BRCA2 in mitosis 37 I-7. SAC silencing 39 II. MATERIALS AND METHODS 42 II-1. Genotyping 42 II-2. Cell culture, drugs and transfection 42 II-3. Constructs, antibodies and siRNA 43 II-4. Immunoprecipitation (IP) and western blot (WB) 45 II-5. Immunofluorescence assay (IFA) 45 II-6. Chromosome spreads 46 II-7. Histopathology 46 II-8. Microscope image acquisition and processing 47 II-9. Cold stable microtubule assay and scoring of the immunofluorescence intensity 47 II-10. Cell cycle analysis 48 III. RESULTS 49 BUBR1 ACETYLATION IS REQUIRED FOR KINETOCHORE-MICROTUBULE ATTACHMENT AND STABLE MITOTIC CHECKPOINT COMPLEX FORMATION 49 III-1. Loss of BubR1 acetylation leads to spontaneous tumorigenesis in mice 49 III-2. CIN in BubR1 acetylation deficient mice 58 III-3. SAC is weakened in BubR1 acetylation deficient mice 62 III-4. Intact initialization of SAC in BubR1 acetylation deficient mice 65 III-5. BubR1 acetylation deficiency leads to premature mitotic checkpoint complex (MCC) disassembly 68 III-6. BubR1 acetylation deficiency leads to abrupt kinetochore-microtubule attachment 72 III-7. Cell cycle profile of cells with BubR1 acetylation deficiency 86 EXTENDED STUDY ON THE ROLE OF BRCA2 IN MITOSIS: SPINDLE ASSEMBLY CHECKPOINT SIGNALING REGULATING BUBR1 91 III-8. BRCA2 complex in mitosis 91 III-9. BRCA2 mediated BubR1 acetylation is required for the BubR1-HDAC interaction 94 III-10. BubR1 deacetylation is a cue to SAC silencing 99 IV. DISCUSSION 113 IV-1. Dual roles of BubR1 acetylation and tumorigenesis 113 IV-1-1. BubR1 acetylation deficiency results in premature disassembly of MCC 116 IV-1-2. BubR1 acetylation deficiency disrupts stable kinetochore-microtubule attachment 117 IV-2. BubR1 acetyl/deacetylation in SAC signaling network 120 IV-3. BRCA2 is a hub for SAC silencing: a hub for BubR1 modulation 122 V. REFERENCE 127 국문 초록 135 | - |
dc.format | application/pdf | - |
dc.format.extent | 2456534 bytes | - |
dc.format.medium | application/pdf | - |
dc.language.iso | en | - |
dc.publisher | 서울대학교 대학원 | - |
dc.subject | Spindle assembly checkpoint | - |
dc.subject | BubR1 | - |
dc.subject | tumorigenesis | - |
dc.subject | SAC signaling | - |
dc.subject | KT-MT attachment | - |
dc.subject.ddc | 570 | - |
dc.title | BubR1 in the Coordination of Kinetochore-Microtubule Attachment and Spindle Assembly Checkpoint Signaling | - |
dc.type | Thesis | - |
dc.description.degree | Doctor | - |
dc.citation.pages | 134 | - |
dc.contributor.affiliation | 자연과학대학 생명과학부 | - |
dc.date.awarded | 2016-02 | - |
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