Browsing by Subject "Nuclear Proteins"
Now showing 1 - 16 of 16
- Results Per Page
- Sort Options
Item Discovering GCNA: A Novel Regulator of Germline Genomic Stability(2018-10-15) Bhargava, Varsha; Mendell, Joshua T.; Buszczak, Michael; Olson, Eric N.; Tu, BenjaminGerm cells transfer genetic information across generations. Any change in germ line DNA is inherited by succeeding generations. Therefore, germ cell DNA must be protected from both internal and external assault. An advantage of sexual reproduction stems from the ability to generate variation by exchange of chromosomal segments during meiosis. During meiosis, hundreds of double-stranded DNA breaks are initiated at once, which if generated in most other cell types would introduce chromosomal aberrations. Germ cells, however, execute the formation of these breaks while preventing their deleterious effects from becoming pervasive throughout the genome. The mechanisms underlying the robustness of germ cells in the face of DNA damage, however, are poorly understood. We initiated an in vivo CRISPR-Cas9 knockout screen for genes highly enriched in the Drosophila female germ line. From this screen, we identified Germ Cell Nuclear Acidic Peptidase (GCNA) as a conserved regulator of genome stability across multiple species. Loss of GCNA results in replication stress, chromosomal instability, and an accumulation of DNA-protein crosslinks (DPCs). Disruption of GCNA leads to an accumulation of nuclear Top2 and Top2 DPCs. This work shows GCNA protects germ cells from damage and provides novel insights into the conserved networks that promote genome integrity across generations.Item Formation of an Exon-Defined A Complex Spliceosome Intermediate Results in CD45 Exon Repression(2007-12-17) House, Amy Elizabeth; Lynch, Kristen W.Alternative splicing is a common means for genetic regulation in higher eukaryotes, and variability in splicing patterns results in a significant increase in protein diversity. Of particular interest is how the splicing machinery is regulated to allow for alternative splicing while maintaining accurate splicing fidelity. CD45 is a trans-membrane tyrosine phosphatase that is expressed on the surface of hematopoetic cells. The CD45 gene contains three variable exons that are partially repressed in na?T cells and are preferentially skipped upon T cell activation. Importantly, it has been shown that the alternative splicing of CD45 pre-mRNA affects the resultant protein's function within the cell emphasizing the importance of this RNA processing event. As such, CD45 provides an excellent model system in which to study signal-responsive alternative splicing. Pre-mRNA splicing is catalyzed by the spliceosome, a macromolecular complex that assembles de novo on a pre-mRNA template in a stepwise and highly dynamic process. The inherent dynamic nature of the spliceosome suggests the potential for regulation at numerous points along the assembly pathway. Previously, a 60-nucleotide exonic silencer (ESS1) was identified within variable exon 4 of the CD45 gene. ESS1 is bound by at least three hnRNP proteins (hnRNP L, PTB, hnRNP E2) of which hnRNP L is the main functional component. Subsequent work showed that ESS1 does not regulate CD45 exon 4 splicing by the typical mechanism of preventing exon definition. Instead, spliceosome assembly is stalled after the exon has been recognized by the spliceosome such that both U1 and U2 are bound to the splice sites suggesting that the stalled complex represents an A-like exon-defined complex (AEC). Additionally, hnRNP L is sufficient to block spliceosome assembly at the AEC. Further biochemical analysis of the AEC suggests that the stall in assembly may be due to altered interactions between the spliceosomal snRNAs and/or proteins and the pre-mRNA substrate. Overall, this inhibition represents a new mechanism for splicing regulation, and suggests that the formation of an AEC intermediate is an important transition in the spliceosome assembly pathway.Item The Functional Roles for SWI/SNF Chromatin Remodeling Complexes in Physiology and Disease(2020-12-01T06:00:00.000Z) Celen, Cemre; Wu, Jiang I.; Zhu, Hao; Olson, Eric N.; Xu, JianSequencing studies have implicated multiple subunits of SWI/SNF complexes in human neurodevelopmental and psychiatric disorders, as well as in cancers. Particularly haploinsufficiency of ARID1B, a SWI/SNF chromatin-remodeling subunit, has been implicated in short stature, autism spectrum disorder, intellectual disability, and corpus callosum agenesis. In addition, ARID1B is the most common cause of Coffin-Siris Syndrome, a developmental delay syndrome characterized by some of the above abnormalities. However, its role in pathologies is not well characterized due to absence of in vivo models. Therefore, in the first part of this thesis, we generated Arid1b heterozygous mice, which showed social behavior impairment, altered vocalization, anxiety-like behavior, neuroanatomical abnormalities, and growth impairment. In the brain, Arid1b haploinsufficiency resulted in changes in the expression of SWI/SNF-regulated genes implicated in neuropsychiatric disorders. A focus on reversible mechanisms identified insulin-like growth factor deficiency with inadequate compensation by Growth Hormone Releasing Hormone and Growth Hormone, underappreciated findings in ARID1B patients. Therapeutically, GH supplementation was able to correct growth retardation and muscle weakness. This model functionally validates the involvement of ARID1B in human disorders and allows mechanistic dissection of neurodevelopmental diseases linked to chromatin-remodeling. ARID1A is a paralogous subunit that is commonly mutated in cancers and plays critical roles in liver regeneration. Chromatin remodeling mechanisms could be generally important for regeneration in other tissues. Since dynamic regulation of β-cell proliferation in pancreatic islets is poorly understood and better understanding could lead to therapeutic approaches for replenishing β-cell mass in type 1 and type 2 diabetes, in the second part of this thesis we focused on the role of ARID1A in β-cells. Arid1a is physiologically suppressed when β-cells proliferate during pregnancy or after pancreas resection. Whole-body Arid1a knockout mice were protected against streptozotocin induced diabetes. Cell-type and temporally specific genetic dissection showed that β-cell specific Arid1a deletion could potentiate β-cell regeneration in multiple contexts. Transcriptomic and epigenomic profiling of mutant islets revealed increased Neuregulin-ERBB-NR4A signaling. Functionally, chemical inhibition of ERBB or NR4A was able to block increased regeneration associated with Arid1a loss. Together, this work defined the role of ARID1A in β-cells and provided new insights into the molecular regulators of β-cell regeneration. Overall, we uncovered important roles of ARID1A and ARID1B-containing SWI/SNF complexes in physiological and disease states.Item GCNA: Guardian of the Genome(2020-05-01T05:00:00.000Z) Goldstein, Courtney DaVee; Abrams, John M.; Buszczak, Michael; Brekken, Rolf A.; Olson, Eric N.The propagation of species depends on the ability of germ cells to protect their genome in the face of numerous exogenous and endogenous threats. While germ cells employ a number of know repair pathways, specialized mechanisms that ensure high-fidelity replication, chromosome segregation, and repair of germ cell genomes remain incompletely understood. Here, we identify Germ cell nuclear acidic peptidase (GCNA) as a conserved regulator of genome stability in flies, worms, zebrafish and human germ cell tumors. GCNA contains an acidic intrinsically disordered region (IDR) and a protease-like SprT domain. In addition to chromosomal instability and replication stress, Gcna mutants accumulate DNA-protein crosslinks (DPCs). GCNA acts in parallel with a second SprT domain protein Spartan. Structural analysis reveals that while the SprT domain is needed to limit meiotic and replicative damage, much of GCNA's function maps to its IDR. This work shows GCNA protects germ cells from various sources of damage, providing novel insights into conserved mechanisms that promote genome integrity across generations.Item HP1BP3, A Chromatin Retention Factor for Co-Transcriptional MicroRNA Processing(2016-06-27) Liu, Haoming; Tu, Benjamin; Liu, Qinghua; Roth, Michael G.; Orth, KimRNA interference (RNAi) is a post-transcriptional gene silencing mechanism found in all eukaryotic organisms. It is characterized by a family of small non-coding RNAs, either endogenous (in the case of microRNAs) or exogenous (in the case of siRNAs), that inhibits gene expression post-transcriptionally. MicroRNAs (miRNAs) are a family of ~21-nt cellular RNAs that govern numerous pathological and physiological processes by mediating translational repression and deadenylation/decay of cognate mRNA. Dysregulation of miRNA expression have been associated with various types of cancer and developmental diseases. Typically, primary (pri-)miRNA transcripts are processed by Drosha complex into precursor (pre-)miRNAs, and then by cytoplasmic Dicer complex into mature miRNAs. The processing of pri-miRNAs is the most highly regulated step in the miRNA biogenesis pathway. Therefore, understanding the molecular mechanisms of pri-miRNA processing and its regulation represents a very important objective in the miRNA filed. Recent studies suggest that the Drosha-DGCR8 complex can be recruited to chromatin to catalyze co-transcriptional processing of primary microRNAs (pri-miRNAs) in mammalian cells. However, the molecular mechanism of co-transcriptional miRNA processing is poorly understood. Here, we find that HP1BP3, a histone H1-like chromatin protein, specifically associates with the Microprocessor and promotes global miRNA biogenesis in HeLa cells. Accordingly, chromatin immunoprecipitation (ChIP) studies reveal genome-wide co-localization of HP1BP3 & Drosha and HP1BP3-dependent Drosha binding to actively transcribed miRNA loci. Moreover, HP1BP3 exhibits a novel pri-miRNA binding activity and promotes the Drosha-pri-miRNA association in vivo. Knockdown of HP1BP3 compromises pri-miRNA processing by resulting in premature release of pri-miRNA transcripts from the chromatin. Taken together, these studies suggest that HP1BP3 promotes co-transcriptional miRNA processing via chromatin retention of nascent pri-miRNA transcripts. This work expands the functional repertoire of the H1 family of proteins and suggests a new concept of chromatin retention factor for widespread co-transcriptional miRNA processing.Item Mechanistic and Therapeutic Insights for Epigenetic Regulation in Cancer Development(2014-10-03) Patel, Amish Jayantibhai; Galindo, Rene; Martinez, Elisabeth; Brekken, Rolf A.; Le, Lu Q.Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are highly aggressive sarcomas that develop sporadically or in patients with Neurofibromatosis type 1 (NF1). Effective treatment options are lacking, and MPNSTs are typically fatal. To gain insights into MPNST pathogenesis, we utilized a novel MPNST mouse model that allowed us to study the evolution of these tumors at the transcriptome level. Strikingly, we found that progression to MPNST and loss of MPNST relevant tumor suppressors is associated with increased levels of chromatin regulator/BET bromodomain protein BRD4, and paradoxically, sensitivity and resistance to BET bromodomain inhibition with small molecule inhibitor JQ1. Indeed, genetic and pharmacological inhibition of BRD4 profoundly suppresses both growth and tumorigenesis of MPNSTs. Mechanistically, we uncovered that BET bromodomain inhibition leads to engagement of the ER stress/UPR pathway, and apoptosis through induction of pro-apoptotic effector molecule BIM and suppression of anti-apoptotic BCL-2 in MPNSTs. Moreover, we find that suppressed transcription of Cyclin D1 oncogene upon BRD4 inhibition correlates with reduced proliferation of MPNSTs. All together, this dual restraint on proliferation (via Cyclin D1 downregulation) and survival (via BIM induction) may indicate how BRD4 inhibition is exquisitely effective against MPNSTs and may represent a paradigm shift in therapy for MPNST patients. Moreover, these findings indicate an epigenetic mechanism underlying the balance of anti-/pro-apoptotic molecules, which suggests that BET bromodomain inhibition can shift this balance in favor of cancer cell death. Collectively, these studies provide new insights for developing strategies to overcome resistance to BET bromodomain inhibitor therapy for subverting cancer cell survival.Item Modulation of Nocturnin Phosphatase Activity through the Disordered Amino Terminus(August 2021) Wickramaratne, Anushka Christobel; Hibbs, Ryan E.; Green, Carla B.; Takahashi, Joseph; Conrad, NicholasThe endogenous circadian clock controls the rhythmicity of behavioral and physiological processes and this is entrained by the daily fluctuations in light and dark. Nocturnin (Noct) is a rhythmically expressed gene regulated by the circadian clock that belongs to the CCR4 family of endonuclease-exonuclease-phosphatase (EEP) enzymes. Its expression is induced by acute stimuli and loss of Nocturnin (Noct-/-) in mice results in resistance to diet-induced obesity on a high fat diet and confers a protective effect to oxidative stress in HEK cells. Modeling of full-length Nocturnin reveals a partially structured amino terminus that is disparate from its CCR4 family members. I show that Nocturnin functions as a phosphatase, catalyzing the removal of the 2′-phosphate from NADP(H). High sequence conservation of the leucine zipper (LZ)-like motif, the only structural element in the amino terminus, highlights the potential importance of this domain in modulating phosphatase activity. I use in vitro biochemical and biophysical techniques to demonstrate that the amino terminus and the LZ-like domain are necessary for preserving the active site cleft in an optimal conformation to promote efficient turnover of the substrate. This modulation occurs in cis and is additionally pivotal in maintaining the stability and conformational integrity of the enzyme. These new findings suggest an additional layer of modulating the activity of Nocturnin in addition to its rhythmicity in order to provide fine-tuned control over cellular levels of NADPH. This lays the essential groundwork necessary to further understand the role of the partially structured amino terminus in metabolism and the oxidative stress response through regulation of NADP(H) and NAD(H) levels.Item A Multi-Organ Role for Nocturnin in Post-Transcriptional Regulation of RNA(2018-04-16) Onder, Yasemin; Huber, Kimberly M.; Green, Carla B.; Takahashi, Joseph; Yu, Gang; Mishra, PrashantNocturnin is an RNA-specific nuclease, a circadian deadenylase first discovered in the retina of Xenopus laevis, and is conserved among eukaryotes. Nocturnin is widely expressed in the brain and in the periphery and is also an immediate early gene that is acutely induced in response to various stimuli. This study investigates Nocturnin's potential role in post-transcriptional regulation of mRNAs in the mitochondria and in two different tissues: brain and brown adipose tissue (BAT). Nocturnin has a predicted mitochondrial-localization signal (MLS) surrounded by two potential translation initiation sites (AUG codons) raising the possibility of dual translation initiation sites. Here we demonstrate that Nocturnin is present in the mitochondria and that it exhibits mitochondrial or cytoplasmic localization via the use of alternative translation initiation sites. I also show that Nocturnin is acutely induced in response to cold and mitochondrial-encoded mRNAs in Noc-/- BAT exhibit impaired stability upon cold exposure. Global analysis of cold-induced changes in the transcriptome of Noc-/- and wild type (WT) mice reveal down-regulation of glycan biosynthesis genes in the Noc-/- BAT. Strikingly, metabolomics analysis demonstrates robust alterations in key tricarboxylic acid metabolites like pyruvate and succinate in the Noc-/- mice BAT in response to a prolonged cold exposure. In summary, we propose a model that Nocturnin acts as a metabolic switch in response to cold by diverting glucose and free fatty acids (FFA) to the mitochondria. In this study, Nocturnin's potential role in post-transcriptional regulation of synaptic plasticity is also investigated. Activity-dependent local protein translation in the dendrites is thought to be an important component of synaptic plasticity. The mechanism of how translation of different mRNAs is differentially regulated in the dendrites is yet not clear. Here I demonstrate that Nocturnin is present in the dendrites as well as in the post-synaptic density as observed in cultured neurons and brain slices from cortex and hippocampus. I investigated mGluR-LTD in hippocampal slices from Noc-/- and WT littermates and observed an attenuation of mGluR-LTD in Noc-/- mice in the first cohort, but this result was not replicated in a second cohort. Further studies are needed to elucidate Nocturnin's role in the synapse.Item Nocturnin Regulates Metabolic Flux Through Maintenance of Poly(A) Tail Length Dynamics(2016-07-26) Stubblefield, Jeremy Joseph; Elmquist, Joel; Green, Carla B.; Mangelsdorf, David J.; Takahashi, JosephCyclic processes in both behavior and physiology are aligned to the external environment through the circadian clock. Nocturnin (Noc) is a rhythmically expressed gene regulated in part by the circadian clock and intimately linked to the metabolic state of an organism. Loss of Nocturnin (Noc-/-) in mice results in resistance to diet-induced obesity. Encoding a deadenylase, NOC protein is thought to regulate mRNA turnover through its ability to remove Poly(A) tails from mRNA transcripts. Though NOC has been linked with lipid and glucose metabolism, its specific targets have not been identified. I explored NOC's role in metabolism by exposing Wildtype (WT) and Noc-/- mice to nutrient challenges consisting of High Fat Diet (HFD) and fasting/refeeding. I demonstrated that Noc can be acutely reduced with a fast and induced with refeeding in WT mice. Hepatic Noc expression oscillates in WT mice fed a High Fat Diet (HFD) but with increased amplitude. I performed mRNA-seq from livers of Wildtype (WT) and Noc-/- mice and identified significant upregulation of both cholesterol and bile acid synthesis genes in Noc-/- mice under basal and nutrient-challenged conditions. This dysregulation results in Noc-/- mice having significantly increased gallbladder volumes during times of fasting. I subjected WT and Noc-/- to hyperinsulinemic-euglycemic clamps and found that HFD-fed Noc-/- mice develop more severe insulin resistance than HFD-fed WT mice. Under insulin-stimulated (clamped) conditions, HFD-fed Noc-/- mice fail to suppress endogenous glucose production and have reduced whole-body glucose turnover. Additionally, regular chow (RC) fed Noc-/- mice exhibit insulin resistance only during the dark phase. This deficit in glucose/insulin sensitivity can be partially rescued in Noc-/- mice through transgenic overexpression of WT NOC, but not a catalytically dead mutant NOC that cannot function as a deadenylase. The deadenylase activity of NOC is thus important for these metabolic phenotypes and I found that genes associated with bile acid, cholesterol and glucose metabolism have altered Poly(A) tail length regulation in Noc-/- mice and thus represent possible targets of NOC. This new understanding of the relationship between Nocturnin, the circadian system and metabolism will help guide the treatment of conditions such as obesity and diabetes.Item Novel Roles for BET Bromodomain Protein 4 (BRD4) in Cardiac Physiology and Disease(2020-08-01T05:00:00.000Z) Kim, Soo Young; Munshi, Nikhil; Chiang, Cheng-Ming; Rothermel, Beverly A.; Gupta, Rana K.; Hill, Joseph A.; Gillette, Thomas G.Bromodomain (BRD) protein of the BET (Bromodomain and Extra-Terminal) family are epigenetic reader proteins that have emerged as novel therapeutic targets in cardiovascular disease as well as in a variety of cancers. Small molecule BET inhibitors, such as JQ1, have demonstrated efficacy in reversing cardiac hypertrophy and heart failure in preclinical models. Yet, genetic studies elucidating the biology of BRD proteins in the heart have not been conducted to validate pharmacological findings and unveil potential side effects. Focusing on BRD4, we tested the hypothesis that cardiomyocyte BRD4 drives pathological cardiac remodeling in the setting of disease-related stress. To facilitate these studies, we engineered a cardiomyocyte-specific BRD4 knockout mouse. Using this model, we investigated the role of BRD4 in cardiac physiology and disease. To our surprise, loss of BRD4 protein triggered a spontaneous and progressive decline in myocardial contractile performance, culminating in dilated cardiomyopathy. Transcriptome analysis of BRD4 knockout mouse hearts showed early and specific disruption of genes essential to mitochondrial energy production and homeostasis. Functional analysis of isolated mitochondria confirmed that BRD4 ablation results in specific changes in protein levels and activity of the mitochondrial electron transport chain. Comparative analysis of the JQ1-altered transcriptome suggests that a BRD4-dependent effect of BET inhibition includes changes in transcription of nucleus-encoded mitochondrial genes, raising concerns for cardiotoxicity with potent pharmacological BET inhibition. Furthermore, we tested the roles of BRD4 isoforms, BRD4-L and BRD4-S(a), in cardiomyocyte biology. Isoform-specific knockdown of BRD4 using siRNAs in primary cardiomyocyte culture demonstrated that BRD4-S(a) is required for cardiomyocyte hypertrophy. BRD4-S(a) expression was low in naïve hearts, but it increased significantly in remodeling and failing hearts. Moreover, transgenic over-expression revealed that the BRD4-S(a) isoform is sufficient to induce hypertrophic remodeling and heart failure. In contrast, restoring BRD4-L expression partially rescued systolic dysfunction in animals with a cardiomyocyte specific deletion of BRD4. In conclusion, present study provides evidence that BRD4 regulates cardiomyocyte mitochondrial homeostasis, and that it is required for maintaining normal cardiac function in rodents. Moreover, we demonstrated that the BRD4 short isoform is a driver of pathological cardiac hypertrophy, whereas the long isoform may have a homeostatic role. In aggregate, we have identified novel roles of BRD4 in cardiomyocyte biology, unveiling critical insights - as well as caveats - regarding the therapeutic targeting of BRD proteins in heart failure.Item Protein Composition and Subcellular Localization of the De Novo Lipogenic Metabolon(2016-04-18) McKean, William Bennion, Jr.; DeBose-Boyd, Russell A.; Horton, Jay D.; Russell, David W.; Uyeda, KosakuFatty acids are the major components of triglycerides, phospholipids, and sphingolipids. Production of palmitate, the most abundant saturated fatty acid, involves the stepwise actions of three enzymes: ATP citrate lyase, acetyl-CoA carboxylase, and fatty acid synthase. Canonically each enzyme catalyzes discrete reactions, and it is thought that they localize diffusely in cellular cytoplasm separate from one another. If true, transfer of metabolic intermediates must occur through passive diffusion from one lipogenic enzyme to another. Such a model proposes an extremely inefficient and potentially hazardous method of palmitate production. We demonstrated that two related proteins - designated MIG12 and Spot 14 - modulate fatty acid synthesis and triglyceride production by regulating the polymerization and activity of acetyl-CoA carboxylase. To better characterize the relationship between these three proteins, biochemical properties of purified recombinant MIG12, Spot 14, and MIG12:Spot 14 heterodimer were assayed in combination with acetyl-CoA carboxylase. We found that Spot 14 abrogates the ability of MIG12 to polymerize and activate acetyl-CoA carboxylase. Co-immunoprecipitation studies using Spot 14 in rat liver revealed Spot 14 exists in a complex with fatty acid synthase and acetyl-CoA carboxylase. MIG12 and Spot 14 co-immunoprecipitation also revealed that ATP citrate lyase was in association with the complex, suggesting that these proteins can function as scaffolds for the three enzymes required for palmitate synthesis. Studies of the subcellular localization of these lipogenic proteins corroborated a functional interaction between these proteins. Confocal images of MIG12 and acetyl-CoA carboxylase in primary hepatocytes show filamentous structures that are immunofluorescent along junctions between the endoplasmic reticulum and mitochondria. Under high carbohydrate dietary conditions in which lipogenesis is stimulated, these structures expand to include fatty acid synthase, ATP citrate lyase, and Spot 14. They also co-localize around lipid droplets - storage organelles for excess triglycerides. Finally, the structural integrity of this lipogenic complex is shown to require microtubules. Blockade of microtubule formation inhibits proper formation of acetyl-CoA carboxylase structure and decreases total fatty acid synthesis in cells. Combined, these findings support the existence of a functional metabolon complex which facilitates the efficient channeling of fatty acid synthesis intermediates through an enzyme cascade that results in the production of palmitate at functionally relevant locations within the cell.Item Roles of MicroRNAs in Fetal Lung Development(2016-05-27) Guo, Wei; Liu, Qinghua; Mendelson, Carole R.; Minna, John D.; Mendell, Joshua T.Lung alveolar type II cells uniquely synthesize surfactant, a developmentally-regulated lipoprotein that is essential for breathing. Expression of the major surfactant protein, SP-A, in midgestation human fetal lung (HFL) is dramatically induced by cAMP. cAMP induction of SP-A expression is repressed by TGF-β and by hypoxia. In this study, we found that expression of the miR-29 family was significantly upregulated in epithelial cells isolated from mouse fetal lung during late gestation and in epithelial cells isolated from HFL explants during type II cell differentiation in culture. MiR-29 expression in cultured HFL epithelial cells was increased by cAMP and inhibited by hypoxia, whereas the miR-29 target, TGF-β2, was coordinately oppositely regulated. Knockdown of the miR-29 family in cultured HFL type II cells blocked cAMP-induced SP-A expression and accumulation of surfactant-containing lamellar bodies, suggesting its physiological relevance. This occurred through derepression of TGF-β signaling. Notably, cAMP increased binding of endogenous thyroid transcription factor-1 (TTF-1/Nkx2.1) to the miR-29ab1 promoter in HFL type II cells and TTF-1 increased miR-29ab1 promoter-driven luciferase activity in co-transfection assays. Together, these findings identify miR-29 family members as TTF-1-driven mediators of SP-A expression and type II cell differentiation through repression of TGF-β signaling.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 SMARCA4/BRG1-Inactivating Mutations as Potential Predictive Markers for Aurora Kinase A-Targeted Therapy in NSCLCs(2014-05-28) Tagal, Vural; White, Michael A.; Roth, Michael G.; Minna, John D.; Cobb, Melanie H.SMARCA4 encodes a catalytic subunit of the SWI/SNF chromatin remodeling complex, BRG1. Frequent occurrence of SMARCA4/BRG1-inactivating mutations and their mutually exclusive nature from EGFR and ALK lesions create one of the largest subsets of Non-Small Cell Lung Cancers (NSCLCs). Since these mutations have been identified as bona fide tumor suppressors, efforts have focused on understanding the pathology of cancer caused by SMARCA4/BRG1 aberrations. However, no therapeutic agent has been identified as synthetically lethal with SMARCA4/BRG1 loss. Utilizing genome-wide high-throughput small interfering RNA (siRNA)-based screening, we show here that Aurora kinase A (AURKA) activity is essential in NSCLCs carrying SMARCA4/BRG1-inactivating mutations. RNAi-mediated depletion or chemical inhibition of AURKA induces apoptosis and diminish cellular viability in SMARCA4/BRG1-mutant NSCLC cells in vitro and in mouse models. The relation between SMARCA4/BRG1 inactivation and increased requirement for AURKA appears to be due to the impairment of functional centrosomes. Thus, AURKA-centered, centrosome-independent, mitotic spindle assembly machinery becomes solely responsible for mitotic spindle formation and proper chromosome segregation during mitosis. DLG7, the only known protein specific to this centrosome-independent mitotic spindle assembly, is required for the survival and proliferation of cells with inactivated SMARCA4/BRG1. Depletion of DLG7 causes no effect in SMARCA4/BRG1-proficient cells, but significant decrease in cell viability occurs in SMARCA/BRG1-deficient NCI-H1819 cells and this cytotoxic effect can be rescued with the restoration of wild-type SMARCA4/BRG1 expression. Altogether, our findings identify AURKA inhibition with VX-680 as a candidate therapeutic strategy for biomarker-driven clinical studies to treat the NSCLCs harboring SMARCA4/BRG1 inactivation mutations, which account for approximately 35% of all NSCLC cases. Furthermore, these observations suggest a previously unrecognized concept of redundancy for mitotic spindle assembly machinery that has a potential use for cancer therapeutics.Item Spatiotemporal Regulation of the NADP(H) Phosphatase Nocturnin(August 2021) Laothamatas, Isara; Mishra, Prashant; Takahashi, Joseph; Conrad, Nicholas; Green, Carla B.Periodic changes in the environment are ubiquitous in the natural world. Among these, the most biologically relevant rhythm is the 24-hour geophysical day/night cycle. As an adaptive strategy, many organisms have evolved an endogenous biological clock to temporally organize their physiology and anticipate daily changes in the environment. At its core, the mammalian "circadian clock" is a molecular oscillator driven by a genetic transcription/translation feedback loop, which orchestrates the rhythmic expression of thousands of genes. An intimate link between circadian clocks and metabolism is established by the rhythmic transcription of output genes involved in almost every metabolic pathway. Among these oscillating genes, Nocturnin (also Noct; protein name: NOC) has one of the highest amplitude rhythms at the mRNA level. Mice with a loss-of-function in Noct possess metabolic phenotypes, where they are protected from high-fat diet-induced obesity and LPS-induced septic shock. However, the mechanism by which this occurs is not well-understood. Here, in collaboration with Green lab members and the Liou lab, I used both in vitro biochemical and in vivo cellular and mouse models to elucidate the molecular and physiological function of NOC. Even though NOC is highly-conserved with the endonuclease/exonuclease/phosphatase (EEP) domain-containing CCR4 family of deadenylases, we show that highly-purified recombinant NOC lacks ribonuclease activity. Instead, NOC catalyzes the dephosphorylation of NADP(H), and its activity level is associated with the cellular response to oxidative stress. Furthermore, we describe two isoforms of NOC and their spatiotemporal regulation in the mouse liver. Cytoplasmic NOC is constitutively-expressed throughout the day and associates externally with the endoplasmic reticulum and other membranes via N-terminal glycine myristoylation. In contrast, mitochondrial NOC levels are highly circadian with peak expression during the early dark phase. Overall, our work suggests that NOC links circadian clocks to metabolism by regulating local intracellular concentrations of NADP(H) in a manner that changes throughout the day.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.