Browsing by Author "Yu, Hongtao"
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Item Analysis of Aurora B Regulation and Signaling(2006-05-16) Oncel, Dilhan; Yu, HongtaoAurora B is a serine/threonine kinase that functions in a complex with two other chromosomal passenger proteins called INCENP and Survivin. Its function is implicated in a variety of processes related to mitosis, such as chromosome condensation, regulation of arm cohesion, spindle assembly, chromosome bi-orientation and cytokinesis. During the cell cycle, the level of this protein is tightly controlled and its deregulated abundance is suspected to contribute to aneuploidy. The cell cycle profile for Aurora B is reminiscent of those for substrates of the anaphase-promoting complex/cyclosome (APC/C), an ubiquitin ligase essential for mitotic progression. Here, we showed that Aurora B is a substrate of APC/C both in vitro and in vivo. Aurora B is efficiently ubiquitinated iv in an in vitro reconstituted system by APC/C that had been activated by Cdh1. The recognition of Aurora B by APC/CCdh1 is specific as it requires the presence of a conserved KEN-box motif at the amino terminus of Aurora B. Degradation of Aurora B at the end of mitosis requires Cdh1 in vivo as the reduction of Cdh1 level by RNA interference stabilizes Aurora B protein. We conclude that, as a key mitotic regulator, Aurora B is degraded by APC/CCdh1 in late mitosis. Aurora B lies at the heart of the cellular mechanism that resolves synthelic and merotelic attachments. A failure to eliminate such events results in gain or loss of chromosomes. Therefore, identifying the physiological substrates of Aurora B is of pivotal importance for research. We screened Aurora B substrates using an in vitro expression cloning system. However, the methodology we employed didn't lead to candidate substrates to be further validated by more rigorous in vivo approaches. The use of high concentrations of misfolded recombinant Aurora B was partially responsible for the loss of specificity. Therefore, purifying active recombinant Aurora B has become a primary goal for future biochemical and structural work. Two molecular chaperones Hsp90 and Cdc37 assist the folding of a variety of kinases in vivo, among which Aurora B is also a candidate. This gave us the final idea of expressing Aurora B-INCENP complexes in bacteria via the coexpression of Hsp90-Cdc37 molecular chaperones.Item CDC14 Coordinates Cyclin Destruction With the Onset of Cytokinesis(2004-08-19) Bembenek, Joshua Nathaniel; Yu, HongtaoThe Cdc14 family of protein phosphatases operate during the final stages of mitosis in various organisms. The Cdc14 phosphatases are downstream components of two homologous signaling pathways: the mitotic exit network (MEN) of S. cerevisiae and septation initiation network (SIN) of S. pombe. Studies of these pathways have revealed divergent roles of Cdc14. In the MEN pathway, Cdc14 is required for cyclin degradation by dephosphorylating Cdh1. The dephosphorylated form of Cdh1 binds to and activates a ubiquitin ligase known as the anaphase-promoting complex (APC/C), which then ubiquitinates mitotic cyclins, targeting them for degradation by the 26S proteosome. In contrast, Cdc14 of the SIN is dispensable for cyclin degradation, but plays an important role during cytokinesis. Two Cdc14 homologues are found in vertebrates, hCdc14A and hCdc14B. I have investigated the regulation of Cdc14 phosphatases to obtain insights into the mechanisms of mitotic exit in higher eukaryotes. Biochemical studies demonstrate that recombinant hCdc14A and hCdc14B can dephosphorylate human Cdh1 and stimulate APC/CCdh1 ligase activity in vitro. Since both the MEN and SIN pathways control Cdc14 localization, I have examined the regulation of the subcellular localization of hCdc14A, hCdc14B and the budding yeast Cdc14. In HeLa cells, hCdc14A localizes to the centrosome whereas hCdc14B is nucleolar during interphase. Both hCdc14 homologues localize to the centrosome and midbody during mitosis. In budding yeast, Cdc14p localizes to the nucleolus during most of the cell cycle and is released in late anaphase when it localizes to the centrosome and the bud neck. The subcellular localization the Cdc14 homologues in HeLa cells is regulated by a nuclear export signal. S. cerevisiae strains carrying only NES mutant CDC14 alleles are capable of degrading mitotic cyclins and escaping mitosis. However, they exhibit a temperature-sensitive phenotype at 37°C because they fail to complete cytokinesis and lack centrosome and bud neck localization of Cdc14. This demonstrates that the Cdc14 phosphatases are regulated by nucleocytoplasmic shuttling. Collectively, my work strongly suggests that the Cdc14 phosphatases play a conserved role in coordinating the destruction of mitotic cyclins with the execution of cytokinesis.Item Function And Recruitment Of Centromeric Heterochromatin Protein 1(2011-02-01T19:34:12Z) 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 Generating the Spindle Assembly Checkpoint Signal at the Kinetochore(2004-08-19) Bharadwaj, Rajnish; Yu, HongtaoTo avoid missegregation of chromosomes during mitosis cells employ a surveillance mechanism termed Spindle assembly checkpoint that senses the lack of tension/attachment on the kinetochores and consequently blocks anaphase onset by inhibiting an E3 ubiquitin ligase called anaphase-promoting complex. The roles of two kinases- BubR1 and Mps1, implicated in spindle assembly checkpoint were investigated. A checkpoint complex containing BubR1 and Bub3 has been purified from mitotic human cells. BubR1 directly interacts with Cdc20 and inhibits the activity of APC in vitro,much more efficiently than Mad2. Surprisingly, the kinase activity of BubR1 or association with Bub3 is not required for the inhibition of APCCdc20. Furthermore, BubR1 restores the mitotic arrest in Cdc20-overexpressing cells treated with nocodazole. Mps1 is a dual specificity kinase that localizes to kinetochores in mitosis. Depletion of Mps1 by RNAi leads to the abrogation of spindle assembly checkpoint. The kinetochore proteins involved in the recruitment of checkpoint proteins and the generation of wait-anaphase signal have not been identified. Kinetochores also provide the attachment sites for spindle microtubules and are required for the alignment of chromosomes at the metaphase plate (chromosome congression). Components of the conserved Ndc80 complex have been implicated in both these function. To better understand the function of the Ndc80 complex, we have identified two novel subunits of the human Ndc80 complex, termed human Spc25 (hSpc25) and human Spc24 (hSpc24), using an immuno-affinity approach. Human Spc25 interacts with Hec1 (human Ndc80) throughout the cell cycle and localizes to kinetochores during mitosis. RNAi-mediated depletion of hSpc25 in HeLa cells causes aberrant mitosis followed by cell death, a phenotype similar to that of cells depleted for Hec1. Loss of hSpc25 also causes multiple spindle aberrations, including elongated, multipolar, and fractured spindles. In the absence of hSpc25, Mad1 and Hec1 fail to localize to kinetochores during mitosis whereas the kinetochore localization of Bub1 and BubR1 is largely unaffected. Interestingly, the kinetochore localization of Mad1 in cells with a compromised Ndc80 function is restored upon microtubule depolymerization. Thus, hSpc25 is an essential kinetochore component that plays a significant role in proper execution of mitotic events.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 Chromatin-Associated Proteins by Sumoylation(2007-08-08) Cocke, Christian Burris; Yu, HongtaoSmall Ubiquitin-like Modifier (SUMO) regulates diverse cellular processes through its reversible, covalent attachment to target proteins. Many SUMO substrates are involved in transcription and chromatin structure. Sumoylation appears to regulate the functions of target proteins by changing their subcellular localization, increasing their stability, and/or mediating their binding to other proteins. Using an In Vitro Expression Cloning (IVEC) approach, we have identified 40 human SUMO1 substrates. We have validated the sumoylation of 24 substrates in living cells. We show that one of these substrates, Mef2C, is coordinately regulated by phosphorylation and sumoylation. The spectrum of human SUMO1 substrates identified in our screen suggests general roles of sumoylation in transcription, chromosome structure, and RNA processing. Moreover, multiple subunits of a given chromatin-associated complex are targets for SUMO-conjugation. For example, a substrate identified in our screen, lysinespecific demethylase 1 (LSD1), is part of a complex that also contains Histone Deacetylase 1 (HDAC1), that is a SUMO substrate. This prompted me to study the function of this complex and its regulation by SUMO. Histone methylation regulates diverse chromatin-templated processes, including transcription. Many transcriptional corepressor complexes contain LSD1 and CoREST that collaborate to demethylate mono- and di-methylated histone H3 lysine 4 (H3K4) of nucleosomes. We have determined the crystal structure of the LSD1-CoREST complex. LSD1-CoREST forms an elongated structure with a long stalk connecting the catalytic domain of LSD1 and the CoREST SANT2 domain. LSD1 recognizes a large segment of the H3 tail through a deep, negatively charged pocket at the active site and possibly a shallow groove on its surface. CoREST SANT2 interacts with DNA. Disruption of the SANT2-DNA interaction diminishes CoREST-dependent demethylation of nucleosomes by LSD1. The shape and dimension of LSD1-CoREST suggest its bivalent binding to nucleosomes, allowing efficient H3-K4 demethylation. This spatially separated, multivalent nucleosome-binding mode may apply to other chromatin-modifying enzymes that generally contain multiple nucleosomebinding modules. The core CoREST corepressor complex, consisting of CoREST, LSD1, and HDAC1/2, represses transcription by coordinately removing histone modifications associated with gene activation. ZNF198 and other MYM-type zinc-finger proteins are also components of this complex. ZNF198, HDAC1, and LSD1 are SUMO substrates, and ZNF198 binds to SUMO non-covalently. We show that ZNF198 and its homologues do not regulate REST-responsive genes. Consistently, binding of REST and ZNF198 to CoREST are mutually exclusive. However, these MYM-domain proteins are required for tethering LSD1 to nuclear compartments and for repression of E-cadherin, a non-REST responsive gene. ZNF198 interacts efficiently only with the intact LSD1-CoREST-HDAC1 ternary complex, but not its individual subunits. ZNF198 also binds specifically to sumoylated, but not unsumoylated HDAC1. These interactions are mediated by tandem zinc-fingers of ZNF198. HDAC1 activity is not stimulated by sumoylation or ZNF198 binding. Sumoylated HDAC1 does not interact with CoREST, and LSD1 sumoylation is inhibited by CoREST binding. Therefore, ZNF198, through its unique and diverse protein-protein interactions, helps to maintain the intact CoREST complex on specific promoters.Item Role for NIP45 in Telomere Recruitment to PML Bodies in ALT Cancer Cells(2013-05-14) Farley, Demetra Dannielle; Cobb, Melanie; Yu, Hongtao; Corey, David; Scaglioni, PierTelomere length maintenance is critical for continued cell proliferation. The SMC5/6 complex, required for double-strand break (DSB) repair in both yeast and humans, has been implicated in the maintenance of telomere length in certain cancer cells. In the absence of active telomerase, SMC5/6 complex-dependent homologous recombination is utilized to maintain telomere length at PML bodies, a mechanism referred to as alternative lengthening of telomeres (ALT). Sumoylation of several telomere-binding proteins is required for the localization of telomeres to PML bodies in G2 phase cells (APBs). We demonstrate that NIP45, a SUMO-like domain (SLD) containing protein, also affects telomere targeting in ALT cells. Loss of endogenous NIP45 protein results in decreased localization of telomeres to PML bodies in a manner independent of the SMC5/6 complex. NIP45 stimulates telomere binding protein sumoylation, as knockdown of the NIP45 protein negatively affects their sumoylation. Importantly, the NIP45 C-terminal SUMO-like domain (SLD2) is sufficient to rescue both APB formation and telomere-binding-protein sumoylation. NIP45 localizes to PML bodies, but not telomeres, in log phase cells, yet interacts efficiently with TIN2, a sumoylatable telomere binding protein. Additionally, a fragment of NIP45 containing the functional SLD2 domain is sufficient to maintain TIN2 binding. We predict, then, that NIP45 might act to recruit telomeres to the PML bodies via its interaction with TIN2, ultimately allowing for SMC5/6 complex-dependent telomere maintenance in G2 phase cells. In keeping with this hypothesis, loss of endogenous TIN2 protein also negatively affects localization of telomeres to PML bodies, even in the presence of NIP45, supporting a requirement for the TIN2-NIP45 interaction in telomere localization to PML bodies. Through this work, we have defined a role for the NIP45 protein in ALT cancer cell telomere length maintenance, further detailing the mechanism by which telomerase-negative cancer subtypes achieve unlimited replicative potential.