Browsing by Subject "Cell Cycle Proteins"
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Item CDK5RAP2 Regulates Centriole Licensing to Restrict Centriole Duplication in Mice(2009-09-04) Barrera, Jose Anselmo; Megraw, TimothyCells division is a highly coordinated series of events that must occur with extreme precision. Defects during segregation of genetic material (DNA) can have adverse effects on the health of the cell, surrounding tissue, organ, and the organism as a whole. Accurate assembly of the bipolar mitotic spindle apparatus is crucial for precise chromosome segregation. Centrosomes play a crucial role in establishment of the mitotic spindle and therefore are vital to the maintenance of genetic stability. Centrosomes are composed of two centrioles that arrange a specialized conglomerate of proteins into a pericentriolar matrix. Centrosomes are highly regulated throughout the cell cycle, and duplicate only once per cell cycle ensuring that each cell inherits one centrosome after mitotic exit, and contains only two centrosomes at the following mitosis. Truncating mutations in the Cyclin-Dependent Kinase 5 Regulatory Associated Protein 2 gene (CDK5RAP2), which encodes a centrosomal protein, result in autosomal recessive primary microcephaly (MCPH, [MIM 251200]) in humans. The major phenotypic manifestation of this rare genetic disorder is a small head. Affected individuals have head circumferences at least 4 standard deviations below sex- and age-matched individuals and suffer mental retardation. In order to investigate how mutations in CDK5RAP2 affect centrosome structure and regulation, and how this leads to MCPH, we derived two distinct mouse mutant lines with truncating mutations within the CDK5RAP2 locus similar to those found in affected humans. We show that centriole engagement and cohesion, two distinct centriole-binding processes, are disrupted in CDK5RAP2 mutant cells. Partial disruption of CDK5RAP2 affected centriole cohesion, whereas complete CDK5RAP2 disruption deregulated the centriole duplication cycle leading to centriole/centrosome amplification. During mitosis amplified centrosomes in CDK5RAP2 mutant cells were potent microtubule organizing centers that drove formation of multipolar spindles. Furthermore, cells formed multiple primary cilia from multiple centrioles inherited from the previous cell cycle. Together these results define a role for CDK5RAP2 in the regulation of centriole duplication and also provide a basis for the development of MCPH.Item Cohesin Promotes the Myelination Transcriptional Program in Oligodendrocytes(December 2021) Cheng, Ningyan; O'Donnell, Kathryn A.; Buszczak, Michael; Olson, Eric N.; Yu, HongtaoThe cohesin complex is crucial for sister-chromatid cohesion and chromatin spatial organization in the interphase nucleus. Cohesin-extruded DNA loops have regulatory functions in gene expression. Mutations of cohesin subunits and regulators cause human developmental diseases termed cohesinopathies. The vertebrate cohesin consists of SMC1, SMC3, RAD21, and either STAG1 or STAG2. STAG1-cohesin and STAG2-cohesin are redundant in sister-chromatid cohesion, but appear to exert specific functions in gene regulation. How they achieve their functions in gene expression is poorly understood. I characterized the outcomes of Stag2 loss in the mouse nervous system. Conditional knockout (CKO) of Stag2 in the nervous system causes severe growth retardation, neurological defects, and premature death, in part due to insufficient myelination of nerve fibers. Expression profiling reveals that myelination-related genes are downregulated in oligodendrocytes of Stag2 CKO mice. Chromatin conformational capture experiments (Hi-C) reveal that Stag2-deficient oligodendrocytes contain fewer DNA loops than wild-type cells do. In particular, promoter-anchored DNA loops at downregulated genes are significantly reduced by Stag2 loss. Interestingly, downregulated genes exhibit promoter-anchored "stripes", indicative of strong loop extrusion. We propose that STAG2-cohesin generated promoter-anchored loops at myelination-promoting genes are critical for the proper gene expression during oligodendrocyte differentiation and brain development. Our study implicates defective myelination as a contributing factor to cohesinopathy and establishes oligodendrocytes as a relevant cell type to explore the mechanism by which cohesin regulates transcription.Item Defining Tumorigenic Contributions of Meiotic Cancer-Testis Antigens(2018-07-10) Nichols, Brandt Alan; Siegwart, Daniel J.; Cobb, Melanie H.; Westover, Kenneth D.; Whitehurst, Angelique WrightCancer Testis Antigens (CTAs) are expressed in testis and/or placenta and anomalously activated in a variety of tumors. However, the mechanistic contribution of CTAs to neoplastic phenotypes remains largely unknown. A cohort of CTAs are required for recombination events during meiosis, suggesting meiotic CTAs have potential to functionally contribute to the genomic stability of tumors. To assess the tumorigenic contributions of meiotic CTAs, I employed a targeted siRNA screen for five meiotic CTAs. Depletion of SYCP1 or HORMAD1 decreased tumor cell proliferation, while SYCE1 loss resulted in elevated DNA damage. Another meiotic CTA, SPO11, is a topoisomerase that induces DNA double-strand breaks during meiosis. I found that SPO11 expression not only correlates with elevated DNA damage in a variety of tumor cells, but ectopic SPO11 expression increases DNA double-strand breaks. A chemigenomics approach identified that a meiotic CTA, HORMAD1, correlates with resistance to piericidin A in non-small cell lung cancer (NSCLC). Resistance is due to a reductive intracellular environment that attenuates the accumulation of free radicals. In human lung adenocarcinoma (LUAD) tumors, patients expressing high HORMAD1 exhibit elevated mutation burden and reduced survival. Differential expression profiling revealed that HORMAD1 tumors are enriched for genes essential for homologous recombination (HR). Mechanistic studies find that HORMAD1 promotes RAD51-filament formation but not DNA resection during HR. Accordingly, HORMAD1 loss enhances sensitivity to gamma-irradiation and PARP inhibition. Furthermore, HORMAD1 depletion significantly reduces tumor growth in vivo. These results suggest that HORMAD1 expression specifies a novel subtype of LUAD which has adapted to mitigate DNA damage. Altogether, these finding indicate that meiotic CTAs play functional roles in altering the genomic stability of tumors and represent potential intervention strategies to enhance sensitivity to DNA damage agents and/or immunotherapies in patients.Item Function And Recruitment Of Centromeric Heterochromatin Protein 1(2011-02-01) Chaudhary, Jaideep; Yu, HongtaoDuring early mitosis, the sister chromatids are held together by Cohesin, a protein complex composed of Smc3, Smc1, Scc1/Rad21 and Scc3. Cohesin is first released from the arms of chromosomes, leaving it intact at the centromere. At the metaphase – anaphase transition, centromeric cohesin is cleaved, allowing the chromatids to segregate to two daughter cells. Shugoshin (Sgo-1) is a known protector of cohesin at the centromere. It prevents phosphorylation of cohesin complex by Plk1 before the metaphase – anaphase transition, which would otherwise lead to cohesin release, causing the two chromatids to separate untimely. In this study we show that Sgo1 localizes on centromeres through HP1 during interphase in human cells. Also, Sgo1 binds all three forms of HP1 (i.e. alpha, beta, gamma) through its chromoshadow domain. We have determined the dissociation constant of this interaction to be in the sub-micromolar range. We have shown conclusively that Sgo1 binds to HP1 chromoshadow domain via one PxVxL motif. We have further shown that, in mitosis, HP1 is recruited to centromeres by Incenp, a subunit of the chromosomal passenger complex via the chromoshadow domain of HP1. This interaction is most likely at the HP1 CSD dimer interface, where PxVxL motifsbind. Hence, it seems that Incenp may provide competition to Sgo1 for HP1 binding.Item Functional Analysis of the Human SMC5/6 Complex in Homologous Recombination and Telomere Maintenance(2008-05-13) Potts, Patrick Ryan; Yu, HongtaoDNA repair is required for the genomic stability and well-being of an organism. The structural maintenance of chromosomes (SMC) family of proteins has been implicated in the repair of DNA double-strand breaks (DSBs) by homologous recombination (HR). The SMC1/3 cohesin complex promotes HR by localizing to DSBs where it holds sister chromatids in close proximity to allow HR-induced strand invasion and exchange. The SMC5/6 complex is also required for DNA repair, but the mechanism by which it accomplishes this has been unclear. We have characterized the role of the human SMC5/6 complex in HRmediated DNA damage repair. The yeast SMC5/6 complex has been shown to be composed of the SMC5-SMC6 heterodimer and six non-SMC element (NSE) proteins. We show that the human homolog of one of these NSE proteins, MMS21/NSE2, is a ligase for small ubiquitin-like modifier (SUMO). Depletion of MMS21 by RNA interference (RNAi) sensitizes cells toward DNA damageinduced apoptosis. This hypersensitization of MMS21-RNAi cells is not due to a defect in DNA damage-induced cell cycle checkpoint, but rather in the kinetics of DNA damage repair. Since the yeast SMC5/6 complex has been implicated in HR-mediated DSB repair, we investigated the role of the human SMC5/6 complex in HR-mediated DSB repair. RNAi-mediated knockdown of the SMC5/6 complex components specifically decreases sister chromatid HR, but not non-homologous end-joining (NHEJ) or intra-chromatid, homologue, or extrachromosomal HR. We show that one potential mechanism by which the SMC5/6 complex specifically promotes sister chromatid HR is by facilitating the recruitment of the SMC1/3 cohesin complex to DSBs. We next examined whether the SMC5/6 complex is also required for sister chromatid HR of telomeres. Specific types of cancer cells, known as alternative lengthening of telomeres (ALT) cells, rely on telomere recombination for telomere lengthening and unlimited replicative potential. We show that the SMC5/6 complex promotes telomere recombination and lengthening in ALT cells by MMS21-dependent sumoylation of telomere-binding proteins. Sumoylation of these telomere-binding proteins relocalizes telomeres to nuclear PML bodies where HR proteins facilitate telomere recombination. These studies identify the human SMC5/6 complex and SUMO modification as critical mediators of sister chromatid HR.Item Macrophage PPAR-Gamma Inhibits Gpr132 to Mediate the Anti-Tumor Effects of Rosiglitazone(2016-04-04) Cheng, Wing Yin; Brekken, Rolf A.; Wan, Yihong; Minna, John D.; Pearson, Gray W.Tumor-associated macrophage (TAM) significantly contributes to tumorigenesis. Human cancer is enhanced by PPARgamma loss-of-function mutations, and inhibited by the thiazolidinedione (TZD) class of synthetic PPARgamma agonists and type 2 diabetes drugs such as rosiglitazone. However, it remains enigmatic whether and how macrophage contributes to PPARgamma tumor-suppressive functions. Here we uncover that macrophage PPARgamma deletion in mice exacerbates mammary tumor development by increasing the number and pro-inflammatory property of TAMs, which in turn stimulate cancer cell proliferation. Macrophage PPARgamma loss also impairs the anti-tumor effects of rosiglitazone. Mechanistically, we identify Gpr132 as a novel direct PPARgamma target in macrophage whose expression is enhanced by PPARgamma loss but repressed by PPARgamma activation. Functionally, macrophage Gpr132 is pro-inflammatory and protumor. Genetic Gpr132 deletion not only retards inflammation and cancer growth but also abrogates the anti-tumor effects of PPARgamma and rosiglitazone. Pharmacological Gpr132 inhibition significantly impedes mammary tumor malignancy. These findings identify macrophage PPARgamma and Gpr132 as critical TAM modulators, new cancer therapeutic targets, and essential mediators of TZD anti-cancer effects.Item The Multifunctional Kinase Bub1 Acts as a Signaling Hub for the Spindle Checkpoint(2015-11-12) Jia, Luying; De Martino, George; Yu, Hongtao; Cobb, Melanie H.; White, Michael A.The spindle checkpoint is an essential mechanism to ensure accurate chromosome segregation during mitosis. The checkpoint signal originates from the kinetochore, which is a huge protein assembly on centromeric chromatin. Kinetochore is also the receptor for spindle microtubules, which enables it to translate microtubule attachment status into spindle checkpoint signal. The separation of the sister chromatids and the progression from metaphase to anaphase requires the activation of an ubiquitin E3 ligase, anaphase-promoting complex or cyclosome (APC/C). Cdc20 is the mitosis-specific APC/C activator. The spindle checkpoint prevents premature sister chromatids separation by preventing Cdc20 from activating APC/C. Bub1 is a highly conserved spindle checkpoint protein that plays multiple roles in checkpoint signaling. On the kinetochore, Bub1 recruits other important checkpoint proteins like BubR1, Mad1 and Cdc20. We found phosphorylation on Bub1 serine 459 is essential for spindle checkpoint and for Bub1-Mad1 interaction. However, the majority of Mad1 still localize to the kinetochore in cells expressing Bub1-S459A mutant. These results suggest that the direct binding between Bub1 and Mad1 through Bub1-S459 may not be responsible for the localization of Mad1 to the kinetochore region. Instead, this interaction enables Mad1 to function in the checkpoint signaling pathway, possibly through regulating its interaction with Bub1-bound BubR1 and Cdc20. Bub1 is also a serine/threonine kinase. The only two identified substrates are histone H2A and Cdc20. Bub1 phosphorylates histone H2A threonine 120, which is important in recruiting Sgo1 and Aurora B kinase to the kinetochore. Bub1 also phosphorylates Cdc20 serine 153. It was shown in vitro that phosphorylation by Bub1 can inhibit APC/CCdc20. However, mouse embryonic fibroblasts (MEFs) expressing Bub1 kinase dead mutant only display mild checkpoint defect due to abnormal Aurora B localization. In addition, over-expression of Bub1 kinase dead mutant in HeLa cells can rescue the checkpoint defect caused by Bub1 depletion using siRNA. These results challenged the importance of Cdc20 phosphorylation by Bub1 in the spindle checkpoint. Here I show that Bub1 binds another kinase Plk1, forming a kinase complex. Phosphorylation of Cdc20 by Bub1-Plk1 not only inhibits APC/CCdc20 in vitro, but also is required for proper spindle checkpoint function in HeLa cells.Item Multiple Functions of BRD4 in E2 Mediated HPV Transciptional Regulation(2009-09-04) Lee, A-Young; Chiang, Cheng-MingHuman papillomaviruses (HPVs) are DNA viruses that cause benign and malignant tumors of epithelial origins. Expression of HPV-encoded E6 and E7 oncoproteins is controlled by the viral E2 protein, which plays a dual role in gene activation and repression. Recently, we identified bromodomain-containing protein 4 (Brd4) as a cellular corepressor for E2-mediated inhibition of HPV transcription. Brd4 contains two bromodomains which function as acetyl-lysine-binding modules that facilitate chromatin targeting via their interactions with acetylated histones. Although Brd4 has been known to be involved in E2-mediated transcriptional regulation, it was unclear how Brd4 regulates E2 function and whether the involvement of Brd4 in transactivation and transrepression is common to different types of E2 proteins. Here, we show that Brd4 enhances E2 binding to its cognate sequences through Brd4's bromodomains and the E2-interacting region in chromatin. We further demonstrate that the corepressor function of Brd4 is common to E2 proteins encoded by cancer-inducing high-risk HPV, wart-causing low-risk HPV, and bovine papillomavirus type 1. The general cofactor function of Brd4 on E2-mediated transcription is in part controlled by enhancing the protein stability of E2, which is normally degraded via the ubiquitin-dependent proteasome pathway. These findings indicate that a chromatin adaptor can enhance the binding of a sequence-specific transcription factor to chromatin and further promote the stability of a labile transcription factor via direct protein-protein interaction. Also, we identify two additional E2-interacting regions of Brd4: the E2-interacting domain (E2ID) and phosphorylation-dependent interacting domain (PDID). While E2ID binds to all different types of E2 proteins, PDID interacts only with high risk E2 (HRE2) in a casein kinase 2 (CK2) phosphorylation-dependent manner. In addition to HRE2-specific interaction, the phosphorylation of PDID also induces intramolecular interaction between PDID and E2ID, which blocks the E2-interaction of E2ID. Finally, we show that the PDID-HRE2 interaction is important for the HRE2-mediated transcriptional activation. Collectively, our data show that the posttranslational modification of the cellular protein Brd4 confers selective recognition of HRE2, thereby providing a unique regulation mechanism for the protein encoded by cancer-inducing HPV.Item On Sulfur Sensing in Saccharomyces cerevisiae(December 2021) Johnson, Zane Miller; Nijhawan, Deepak; De Martino, George; Yu, Hongtao; Tu, BenjaminThe unique chemistry available to sulfur compared to oxygen, such as the ability to exist in numerous oxidation states and greater nucleophilicity, makes many of the biochemical reactions requisite for cellular life possible. As a result of this critical importance, organisms have developed several mechanisms for sensing and maintaining levels of sulfur-containing metabolites. In the yeast Saccharomyces cerevisiae, regulation of sulfur metabolism can be distilled down to the actions of two proteins; the F-box protein Met30, and the transcriptional coactivator Met4. Met30 belongs to the family of SCF (Skp1-Cul1-F-box protein) E3 ubiquitin ligases, and negatively regulates the transcriptional activity of the master transcriptional activator of sulfur metabolism genes, Met4, via oligo-ubiquitination when sulfur metabolite levels are high. When yeast are starved of sulfur, Met30 ceases to ubiquitinate Met4, releasing it to be deubiquitinated and transcriptionally active to boost levels of a network of sulfur metabolic genes known as the MET regulon to restore sulfur metabolite levels. While the molecular activities of both Met30 and Met4 have been extensively studied over the last two decades, the biochemical basis for sulfur-sensing by the Met30 E3 ligase has remained unknown. Herein, I reveal the biochemical details by which Met30, the master regulator of sulfur metabolism, senses the availability of sulfur metabolites to modulate its E3 ligase activity to regulate sulfur metabolism in yeast. Utilizing a combination of yeast genetics and biochemical assays, I show that Met30 uses redox-active cysteine residues in its C-terminal WD-40 repeat region to modulate binding between itself and its substrate Met4 in accordance with the availability of sulfur metabolites. These insights represent significant advances in the understanding of sulfur metabolic regulation in yeast.Item Regulation and Mechanism of Bub1-Mediated Spindle Checkpoint Signaling(2006-12-20) Qi, Wei; Yu, HongtaoThe spindle checkpoint is a surveillance mechanism that ensures the fidelity of chromosome segregation during mitosis and meiosis. Bub1 is a highly conserved protein serine/threonine kinase that plays multiple roles in the spindle checkpoint. The regulation and mechanism of Bub1 in spindle checkpoint were investigated. Bub1 is degraded during mitotic exit and the degradation of it is mediated by APC/C in complex with its activator Cdh1 (APC/CCdh1). Overexpression of Cdh1 reduces the protein levels of ectopically expressed Bub1 whereas depletion of Cdh1 by RNA interference (RNAi) increases the level of the endogenous Bub1 protein. Two KEN-box motifs on Bub1 are required for its degradation in vivo and ubiquitination in vitro. A Bub1 mutant protein with both KEN-boxes mutated is stable in cells. Kinetochore is the origin of spindle checkpoint signal and contains the catalytic machinery for generating the signal. We identify an ATP-dependent APC/CCdc20 inhibitory activity on metaphase chromosomes with unattached kinetochores. The Cdc20-S153A that cannot be phosphorylated by Bub1 is not inhibited by metaphase chromosomes, suggesting Bub1 is likely responsible for the inhibitory activity. Bub1 on unattached kinetochores is hyperphosphorylated and activated. Furthermore, the kinase-dead mutant of Bub1 cannot restore spindle checkpoint in Bub1-RNAi cells, demonstrating that the kinase activity of Bub1 is required for the spindle checkpoint. Plk1 is required for the generation of the tension-sensing 3F3/2 kinetochore epitope and facilitates kinetochore localization of Mad2 and other spindle checkpoint proteins. We investigate the mechanism by which Plk1 is recruited to kinetochores. We show that Plk1 binds to Bub1 in mitotic cells. The Plk1-Bub1 interaction requires the polo-box domain (PBD) of Plk1 and is enhanced by Cdk1-mediated phosphorylation of Bub1 at T609. The PBD-dependent binding of Plk1 to Bub1 facilitates phosphorylation of Bub1 by Plk1 in vitro. Depletion of Bub1 in HeLa cells by RNAi diminishes the kinetochore localization of Plk1. Ectopic expression of the wild-type Bub1, but not the Bub1-T609A mutant, in Bub1-RNAi cells restores the kinetochore localization of Plk1. Our results suggest that phosphorylation of Bub1 at T609 by Cdk1 creates a docking site for the PBD of Plk1 and facilitates the kinetochore recruitment of Plk1.Item Regulation of Sister Chromatid by the Acetyltransferase Naa50(2016-07-25) Rong, Ziye; Seemann, Joachim; Yu, Hongtao; Li, Bing; Olson, Eric N.During the cell cycle, sister-chromatid cohesion tethers sister chromatids together from S phase to the metaphase-anaphase transition and ensures accurate chromosome segregation of chromatids into daughter cells. N-terminal acetylation is one of the most prevalent protein covalent modifications in eukaryotes and is mediated by a family of N-terminal acetyltransferases (NAT). Naa50 (also called San or NatE) has previously been shown to play a role in sister-chromatid cohesion in metazoans. The mechanism by which Naa50 contributes to cohesion is not understood, however. Here, I show that depletion of Naa50 in HeLa cells weakens the interaction between cohesin and its positive regulator sororin and causes cohesion defects in interphase, consistent with a role of Naa50 in cohesion establishment or maintenance. Strikingly, co-depletion of NatA, a heterodimeric NAT complex that physically interacts with Naa50, rescues the sister-chromatid cohesion defects and the resulting mitotic arrest caused by Naa50 depletion, indicating that NatA and Naa50 play antagonistic roles in cohesion. Purified recombinant NatA and Naa50 do not affect each other's NAT activity in vitro. Because NatA and Naa50 exhibit distinct substrate specificity, I propose that they modify different effectors and regulate sister-chromatid cohesion in opposing ways.Item Regulation of Sister-Chromatid Cohesion(2017-10-17) Zheng, Ge; Burma, Sandeep; Yu, Hongtao; Mendell, Joshua T.; Tu, BenjaminOrderly execution of two critical events during the cell cycle--DNA replication and chromosome segregation--ensures the stable transmission of genetic materials. The cohesin complex physically connects sister chromatids during DNA replication in a process termed sister-chromatid cohesion. Timely establishment and dissolution of sister-chromatid cohesion is a prerequisite for accurate chromosome segregation, and is tight regulated by the cell cycle machinery and cohesin-associated proteins. Errors in this process can lead to aneuploidy and promote tumorigenesis. Research in this dissertation has provided several key insights into the regulation of sister-chromatid cohesion during the mitotic cell cycle. First, we report the crystal structure and functional characterization of human Wapl, a key negative regulator of cohesin that promotes cohesin release from chromatin. Our results indicate that Wapl-mediated cohesin release from chromatin requires extensive physical contacts between Wapl and multiple cohesin subunits. Second, we have determined the crystal structure of human SA2-Scc1 cohesin subcomplex, which is the interaction hub for cohesin regulators. Further biochemical and functional analyses reveal the direct competition between Wapl and the cohesion protector Sgo1 for binding to a conserved site on SA2-Scc1. Our results implicate a role for this direct antagonism in centromeric cohesion protection. Third, we report the crystal structure of human Pds5B bound to a conserved peptide motif found in both Wapl and Sororin. Further biochemical and functional studies suggest that Pds5 has both positive and negative roles in cohesion regulation and establish the molecular basis for how Wapl and the cohesin-stabilizing factor Sororin antagonistically influence cohesin dynamics on chromosomes. The structure reveals inositol hexakisphosphate (IP6) as an unexpected cofactor of Pds5. The IP6-binding segment of Pds5B engages the N-terminal region of Scc1 and inhibits the binding of Scc1 to Smc3. Our results suggest a direct role of Pds5 in cohesin release from chromosomes by stabilizing a transient, open state of cohesin during its ATPase cycle. Finally, we show that cohesin loading onto chromosomes requires the phosphorylation of MCM2-7 by Cdc7-Dbf4 kinase (DDK) during early S phase, when a mega-complex composed of MCM2-7, Scc2/4 and cohesin is formed. At active replication forks, inactivation of multiple replisome components impairs cohesin loading, weakens MCM-Scc2/4-cohesin interaction and leads to cohesion defects. By contrast, interfering Okazaki fragment processing and nucleosome assembly during DNA replication do not impact interphase cohesion, suggesting that cohesion establishment occurs before Okazaki fragment maturation and histone deposition. Our results demonstrate that DNA replication-coupled cohesin loading is required for the establishment of sister-chromatid cohesion. In conclusion, combining structural, biochemical and cellular approaches, our studies advance the molecular understanding of spatial and temporal regulation of the establishment and dissolution of sister-chromatid cohesion.Item Regulation of the Insulin-like Growth Factor 1-Secretory Clusterin Expression Axis in Genomic Instability and Cell Stress(2009-09-04) Goetz, Eva Marie; Boothman, David A.Secretory clusterin (sCLU) is a pro-survival factor that is up-regulated in human tumors and after exposure to cell stress. Understanding the regulation of sCLU expression in cancer, and after exposure to therapeutic agents, could reveal new therapeutic targets for cancer treatment. A DNA damage induced signaling cascade leading from ATM to sCLU expression mediated by IGF-1/IGF-1R/MAPK activation was uncovered. IGF-1 ligand promoter activity, mRNA, and protein expression induced after exposure to ionizing radiation (IR), hydrogen peroxide, or topoisomerase I and II-alpha poisons matched sCLU expression. Elevated basal IGF-1-sCLU signaling was noted in genomically unstable cells, whether they were deficient in DNA repair factors or telomerase function. ATM function was necessary for induction of sCLU after IR, and for maintaining elevated expression of sCLU in genomically unstable cells. p53 suppressed IGF-1 promoter activity, leading to decreased mRNA and protein expression, and abrogated induction of IGF-1 and sCLU by IR. Loss of p53 by knockdown or knockout enhanced IGF-1 and sCLU induction. Mutations in the p53 DNA binding domain found in cancer did not repress IGF-1 and sCLU. An NF-Y binding site in the IGF-1 promoter was essential for p53 suppression, and both p53 and NF-YA bound to the IGF-1 promoter. Nutlin-3, an Mdm2-p53 inhibitor, stabilized p53 expression, leading to dramatically decreased sCLU expression. Nutlin-3 treatment sensitized wild-type p53 cells to IR exposure. Finally, exogenous IGF-1 exposure led to serine 1981 auto-phosphorylation of ATM, and enhanced DNA damage repair and abrogated cell death after IR exposure. These studies uncovered key molecules important for the regulation of IGF-1-sCLU expression axis after IR exposure, and supported the use of IGF-1 or sCLU expression inhibitors for cancer chemotherapy.Item Safeguard of Mitosis: The Spindle Checkpoint(2016-11-18) Ji, Zhejian; Chen, Zhijian J.; Cobb, Melanie H.; Rice, Luke M.; Yu, HongtaoIn mitosis, the kinetochore-microtubule attachment is under surveillance by the spindle checkpoint to ensure the fidelity of chromosome segregation. Defects in the checkpoint could lead to aneuploidy, which has been implicated in cancers, birth defects, and other human diseases. In presence of kinetochores that are not attached or improperly attached to microtubules, the checkpoint signals to assemble the mitotic checkpoint complex (MCC), which consists of BubR1-Bub3, Mad2, and Cdc20. The diffusible MCC inhibits the ubiquitin ligase activity of the anaphase-promoting complex or cyclosome bound to its co-activator Cdc20 (APC/C-Cdc20) to arrest cells in mitosis. Nevertheless, it remains unknown how the checkpoint monitors the status of the kinetochore-microtubule attachment. Neither is clear how MCC is assembled in an active checkpoint signaling. My graduate work has answered these two questions by revealing the critical functions of a checkpoint kinase, monopolar spindle 1 (Mps1), in both attachment sensing and checkpoint signaling. Of kinetochore proteins, the KMN network acts as both a critical microtubule receptor and a signaling platform for the spindle checkpoint. The human KMN contains the kinetochore null 1 complex (Knl1C), the minichromosome instability 12 complex (Mis12C), and the nuclear division cycle 80 complex (Ndc80C). In my first project, I have shown that the non-kinase domain of Mps1 directly binds to Ndc80C through two independent interactions. Both interactions involve the microtubule-binding surfaces of Ndc80C and are directly inhibited by microtubules. Elimination of one such interaction in human cells causes checkpoint defects expected from a failure in detecting unattached kinetochores. This competition between Mps1 and microtubules for Ndc80C binding thus constitutes a direct mechanism for unattached kinetochore detection. The next question is how the kinetochore-associated Mps1 kinase rules the checkpoint signaling. At kinetochore, Mps1 phosphorylates the scaffolding protein Knl1. Phosphorylated Knl1 (pKnl1) recruits checkpoint complexes budding uninhibited by benomyl 1-3 (Bub1-Bub3) and Bub1-related protein in complex with Bub3 (BubR1-Bub3) to kinetochores. My following work has demonstrated that Mps1 promotes the inhibition of APC/CCdc20 by MCC components in vitro through phosphorylating Bub1 and mitosis arrest deficiency 1 (Mad1). Phosphorylated Bub1 (pBub1) binds with Mad1-Mad2. Phosphorylated Mad1 (pMad1) directly interacts with Cdc20. Mutations of Mps1 phosphorylation sites in Bub1 or Mad1 abrogate the spindle checkpoint in human cells. Therefore, Mps1 promotes checkpoint activation through a pKnl1-pBub1-pMad1 phosphorylation cascade, in which phosphorylation of upstream components enables binding of downstream ones. We propose that this sequential multi-target phosphorylation cascade allows Mps1 to amplify checkpoint signals and makes the checkpoint highly responsive to Mps1, which itself is regulated by kinetochore-microtubule attachment. Taken together, my graduate work has solved two long-standing questions in spindle checkpoint regulation. Accordingly, Mps1 recognizes the unattached kinetochores via its non-kinase domain, while activates the checkpoint signaling through its kinase activity. The dual function of Mps1 couples checkpoint activation with unattached kinetochore detection, making checkpoint under the control of kinetochore-microtubule attachment.Item Spindle Checkpoint at Kinetochores(2014-07-24) Kim, Soonjoung; Sternweis, Paul C.; Cobb, Melanie H.; Rice, Luke M.; Yu, HongtaoThe kinetochore—a large protein assembly on centromeric chromatin—functions as the docking site for spindle microtubules and as a signaling hub for the spindle checkpoint. The Constitutive Centromere-Associated Network (CCAN) at the inner kinetochore nucleates the formation of the mature outer kinetochore during mitosis, including the recruitment of the KMN network that consists of Knl1, the Mis12 complex (Mis12C), and the Ndc80 complex (Ndc80C). The KMN is a critical receptor for microtubules, and provides a landing pad for various spindle checkpoint proteins and regulatory factors. The spindle checkpoint protein Mad2 has multiple conformations, including the inactive open Mad2 (O-Mad2) and the active closed Mad2 (C-Mad2). The kinetochore-bound checkpoint protein complex Mad1–Mad2 promotes the conformational activation of O-Mad2 and serves as a catalytic engine of checkpoint signaling. The activated C-Mad2 binds to and inhibits Cdc20, an activator of APC/C, to prevent precocious anaphase onset. Deficient spindle checkpoint signaling leads to premature sister-chromatid separation and aneuploidy. Research in this thesis has provided several key insights into spindle checkpoint signaling at kinetochores. First, we show that the conformational transition of Mad2 is regulated by phosphorylation of S195 in its C-terminal region. The phospho-mimicking Mad2S195D mutant and the phospho-S195 Mad2 protein do not form C-Mad2 on their own. Mad2 phosphorylation inhibits its function through differentially regulating its binding to Mad1 and Cdc20. Our results establish for the first time that the conformational change of Mad2 is regulated by posttranslational mechanisms. Second, we have studied how Mad1 is targeted to kinetochores. We have determined the crystal structure of the conserved C-terminal domain (CTD) of human Mad1. The structure reveals unexpected fold similarity between Mad1 CTD and known kinetochore-binding modules. Functional studies then validate a role of Mad1 CTD in kinetochore targeting and implicate Bub1 as its receptor. Interestingly, deletion of the CTD does not abolish Mad1 kinetochore localization. Non-overlapping Mad1 fragments retain detectable kinetochore targeting. Our results indicate that the CTD–Bub1 connection is one of several mechanisms of targeting Mad1 to kinetochores. Finally, we show that the proper assembly of KMN is required for generating the spindle checkpoint signal at kinetochores. We have developed several strategies to inactivate KMN at kinetochores in human cells, and demonstrate its requirement for the spindle checkpoint in the absence of microtubules. We further show that two quasi-independent pathways mediate the mitosis-specific assembly of KMN at kinetochores. In one pathway, the centromeric kinase Aurora B phosphorylates the Mis12C component Dsn1, and strengthens Mis12C binding to the CCAN component CENP-C. In the second pathway, CENP-T anchors the CENP-H/I/K sub-complex at kinetochores, which in turn recruits Ndc80C. Inactivation of both pathways abolishes KMN at kinetochores and causes gross spindle checkpoint defects. In conclusion, combining cell biology and structural biology methods, our studies have defined a new posttranslational mechanism of Mad2 regulation, uncovered a critical way for targeting Mad1 to kinetochores, and dissected assembly pathways of the KMN checkpoint sensor at kinetochores.Item Spindle Checkpoint Silencing by TRIP13(2017-10-30) Brulotte, Melissa Lynn; DeBose-Boyd, Russell A.; Yu, Hongtao; Luo, Xuelian; Burma, Sandeep; Roth, Michael G.The spindle checkpoint is important for maintaining genomic stability and preventing aneuploidy, a hallmark of cancer. The checkpoint ensures that chromosome segregation does not occur until all sister chromatids are correctly attached to the mitotic spindle during metaphase. When this requirement is met, the checkpoint must be silenced for the cell to proceed to anaphase. Thyroid hormone receptor interacting protein 13 (TRIP13) is a hexameric AAA+ ATPase involved in spindle checkpoint silencing. TRIP13 functions by initiating a conformational change in mitotic arrest deficient 2 (Mad2), a key component of the mitotic checkpoint complex (MCC). This TRIP13-mediated conformational change of Mad2 causes MCC disassembly and relieves inhibition of the anaphase promoting complex/cyclosome (APC/C). The interaction between TRIP13 and Mad2 is dependent on the p31comet adaptor protein. In my first project, I show that TRIP13-p31comet disrupts the MCC by local unfolding of Mad2. I identify a binding surface on human TRIP13 for p31comet-Mad2 and key TRIP13 residues involved in its conformational dynamics. I propose that the flexibility of the hinge region of TRIP13 is important for coupling its ATPase activity to substrate unfolding. The hinge region is conserved in other eukaryotic AAA+ ATPases, and may also be important for energetic coupling in those systems. I have also reconstituted the process of spindle checkpoint silencing in vitro. Importantly, I show that TRIP13 can disrupt the free MCC complex, but not MCC bound to APC/C, providing an explanation for the coordination of the multiple mechanisms that work together to achieve spindle checkpoint silencing. In my second project, to provide a tool for future mechanistic studies and to examine the oncogenic activity of TRIP13, I attempted to identify chemical inhibitors for TRIP13 through high-throughput screening. I identified a series of lead compounds that indirectly inhibited TRIP13 as pan-assay interference compounds. These compounds are redox cyclers that generate hydrogen peroxide, which covalently modifies protein residues such as cysteines and tryptophans. No other potent lead compounds were discovered. This study revealed that TRIP13 may be a difficult protein to target, and that large compound libraries should be prescreened for redox cyclers before they are used in high-throughput inhibitor screening.Item Studies of the Molecular Features of Brd4-Nut and P300 That Contribute to Condensate Formation and Transcriptional Regulation(2023-05-01T05:00:00.000Z) Kosno, Martyna Olga; Kohler, Jennifer J.; Tu, Benjamin; Henne, W. Mike; Brekken, Rolf A.; Liszczak, Glen; Rosen, Michael K.Aberrant formation of biomolecular condensates has been proposed to play a role in several cancers. The oncogenic fusion protein Brd4-Nut drives aberrant gene expression and forms condensates in Nut Carcinoma (NC). It has not been clear how these condensates form and whether they modulate gene expression. Here, I dissected the molecular features of Brd4-Nut and a histone acetyltransferase (HAT), p300, and analyzed their contribution to condensate formation and transcriptional changes. I determined that a minimal fragment of Nut (MIN) in fusion with Brd4 is necessary and sufficient for binding to p300, and for condensate formation. A Brd4-p300 fusion protein also forms condensates and drives a transcriptional profile similar to Brd4-Nut(MIN). The intrinsically disordered regions, transcription factor - binding domains, and HAT activity of p300 all collectively contribute to condensate formation. Conversely, only HAT activity appears to be necessary to mimic the transcriptional profile of cells expressing Brd4-Nut. My results suggest that interaction of Brd4-Nut with p300 is important for aberrant condensate formation, and that multiple, yet distinct, regions of p300 contribute to condensate formation and transcriptional regulation.