Timothy A Bolger

Interests

Research

Regulation of mRNP dynamics in gene expression:Eukaryotic gene expression is a fundamental cellular activity that is critical for cellular identity, function, and physiology. During gene expression, a messenger RNA (mRNA) is generated by transcription and undergoes a number of different steps, including splicing and nuclear processing, nucleocytoplasmic export and localization, translation, and decay. These steps result in dynamic changes to the RNA sequence, structure, and the cohort of proteins bound to the mRNA. Furthermore, these changes need to occur with the proper timing and in the correct sequence to avoid aberrant expression. Therefore elaborate regulation of mRNP dynamics is required for proper gene expression.At virtually every step in gene expression, members of a highly conserved protein family called the DEAD-box proteins are required for facilitating mRNP transitions by acting either as RNA helicases or as ribonucleoprotein (RNP) remodeling enzymes. Furthermore, by regulating their activity, the potential exists to control mRNAs in different subsets and in response to different conditions. Thus we hypothesize that the DEAD-box proteins exert overarching control of mRNP dynamics in gene expression.Our research in the Bolger lab has dual goals: 1. addressing fundamental biological questions, and 2. utilizing this knowledge to advance human health. The Bolger laboratory uses the budding yeast Saccharomyces cerevisiae as a model system, and takes advantage of the combination of genetics, biochemistry, and cell biology allowed by yeast work. Our long-term goal in fundamental biology is to uncover the regulation of mRNP dynamics. Specifically, we are focusing on the in vivo roles, molecular targets, and regulation of DEAD-box proteins. Our research will not only greatly increase our understanding of how these factors function in the control of gene expression but also may open up new avenues for therapies, either for cancer or for the other pathologies related to this research.

Courses

Scholarly Contributions

Chapters

• Bolger, T. A., Cohen, T., & Yao, T. (2006). HATs and HDACs. In Gene Expression and Regulation (ed. J Ma)(pp p111-133). Beijing, China: Higher Education Press and Springer.

Journals/Publications

• Aryanpur, P. P., Mittelmeier, T. M., & Bolger, T. A. (2022). The RNA Helicase Ded1 Regulates Translation and Granule Formation during Multiple Phases of Cellular Stress Responses. Molecular and cellular biology, 42(1), e0024421.
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  Ded1 is a conserved RNA helicase that promotes translation initiation in steady-state conditions. Ded1 has also been shown to regulate translation during cellular stress and affect the dynamics of stress granules (SGs), accumulations of RNA and protein linked to translation repression. To better understand its role in stress responses, we examined Ded1 function in two different models: overexpression and oxidative stress. overexpression inhibits growth and promotes the formation of SGs. A mutant lacking the low-complexity C-terminal region (), which mediates Ded1 oligomerization and interaction with the translation factor eIF4G1, suppressed these phenotypes, consistent with other stresses. During oxidative stress, a mutant was defective in growth and in SG formation compared to wild-type cells, although SGs were increased rather than decreased in these conditions. Unlike stress induced by direct TOR inhibition, the phenotypes in both models were only partially dependent on eIF4G1 interaction, suggesting an additional contribution from Ded1 oligomerization. Furthermore, examination of the growth defects and translational changes during oxidative stress suggested that Ded1 plays a role during recovery from stress. Integrating these disparate results, we propose that Ded1 controls multiple aspects of translation and RNP dynamics in both initial stress responses and during recovery.
• Brown, N. P., Vergara, A. M., Whelan, A. B., Guerra, P., & Bolger, T. A. (2021). Medulloblastoma-associated mutations in the DEAD-box RNA helicase DDX3X/DED1 cause specific defects in translation. The Journal of Biological Chemistry, 100296.
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  Medulloblastoma is the most common pediatric brain cancer, and sequencing studies identified frequent mutations in DDX3X, a DEAD-box RNA helicase primarily implicated in translation. Forty-two different sites were identified, suggesting that the functional effects of the mutations are complex. To investigate how these mutations are affecting DDX3X cellular function, we constructed a full set of equivalent mutant alleles in DED1, the S. cerevisiae ortholog of DDX3X, and characterized their effects in vivo and in vitro. Most of the medulloblastoma-associated mutants in DDX3X/DED1 (ded1-mam) showed substantial growth defects, indicating that functional effects are conserved in yeast. Further, while translation was affected in some mutants, translation defects affecting bulk mRNA were neither consistent nor correlated with the growth phenotypes. Likewise, increased formation of stress granules in ded1-mam mutants was common but did not correspond to the severity of the mutants' growth defects. In contrast, defects in translating mRNAs containing secondary structure in their 5' untranslated regions (UTRs) were found in almost all ded1-mam mutants and correlated well with growth phenotypes. We thus conclude that these specific translation defects, rather than generalized effects on translation, are responsible for the observed cellular phenotypes and likely contribute to DDX3X-mutant medulloblastoma. Examination of ATPase activity and RNA binding of recombinant mutant proteins also did not reveal a consistent defect, indicating that the translation defects are derived from multiple enzymatic deficiencies. This work suggests that future studies into medulloblastoma pathology should focus on this specific translation defect, while taking into account the wide spectrum of DDX3X mutations.
• Fernandes, N., Nero, L., Lyons, S. M., Ivanov, P., Mittelmeier, T. M., Bolger, T. A., & Buchan, J. R. (2020). Stress Granule Assembly Can Facilitate but Is Not Required for TDP-43 Cytoplasmic Aggregation. Biomolecules, 10(10).
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  Stress granules (SGs) are hypothesized to facilitate TAR DNA-binding protein 43 (TDP-43) cytoplasmic mislocalization and aggregation, which may underly amyotrophic lateral sclerosis pathology. However, much data for this hypothesis is indirect. Additionally, whether P-bodies (PBs; related mRNA-protein granules) affect TDP-43 phenotypes is unclear. Here, we determine that induction of TDP-43 expression in yeast results in the accumulation of SG-like foci that in >90% of cases become the sites where TDP-43 cytoplasmic foci first appear. Later, TDP-43 foci associate less with SGs and more with PBs, though independent TDP-43 foci also accumulate. However, depleting or over-expressing yeast SG and PB proteins reveals no consistent trend between SG or PB assembly and TDP-43 foci formation, toxicity or protein abundance. In human cells, immunostaining endogenous TDP-43 with different TDP-43 antibodies reveals distinct localization and aggregation behaviors. Following acute arsenite stress, all phospho-TDP-43 foci colocalize with SGs. Interestingly, in SG assembly mutant cells (), TDP-43 is enriched in nucleoli. Finally, formation of TDP-43 cytoplasmic foci following low-dose chronic arsenite stress is impaired, but not completely blocked, in cells. Collectively, our data suggest that SG and PB assembly may facilitate TDP-43 cytoplasmic localization and aggregation but are likely not essential for these events.
• Aryanpur, P. P., Renner, D. M., Rodela, E., Mittelmeier, T. M., Byrd, A., & Bolger, T. A. (2019). The DEAD-box RNA helicase Ded1 has a role in the translational response to TORC1 inhibition. Molecular biology of the cell, 30(17), 2171-2184.
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  Ded1 is a DEAD-box RNA helicase with essential roles in translation initiation. It binds to the eukaryotic initiation factor 4F (eIF4F) complex and promotes 48S preinitiation complex assembly and start-site scanning of 5' untranslated regions of mRNAs. Most prior studies of Ded1 cellular function were conducted in steady-state conditions during nutrient-rich growth. In this work, however, we examine its role in the translational response during target of rapamycin (TOR)C1 inhibition and identify a novel function of Ded1 as a translation repressor. We show that C-terminal mutants of are defective in down-regulating translation following TORC1 inhibition using rapamycin. Furthermore, following TORC1 inhibition, eIF4G1 normally dissociates from translation complexes and is degraded, and this process is attenuated in mutant cells. Mapping of the functional requirements for Ded1 in this translational response indicates that Ded1 enzymatic activity and interaction with eIF4G1 are required, while homo-oligomerization may be dispensable. Our results are consistent with a model wherein Ded1 stalls translation and specifically removes eIF4G1 from translation preinitiation complexes, thus removing eIF4G1 from the translating mRNA pool and leading to the codegradation of both proteins. Shared features among orthologues suggest that this role is conserved and may be implicated in pathologies such as oncogenesis.
• Aryanpur, P. P., Regan, C. A., Collins, J. M., Mittelmeier, T. M., Renner, D. M., Vergara, A. M., Brown, N. P., & Bolger, T. A. (2017). Gle1 regulates RNA binding of the DEAD-box helicase Ded1 in its complex role in translation initiation. Molecular and cellular biology, 37(21), e00139-17.
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  DEAD-box proteins (DBPs) are required in gene expression to facilitate changes to ribonucleoprotein complexes, but the cellular mechanisms and regulation of DBPs are not fully defined. Gle1 is a multifunctional regulator of DBPs with roles in mRNA export and translation. In translation, Gle1 modulates Ded1, a DBP required for initiation. However,overexpression causes defects, suggesting that Ded1 can promote or repress translation in different contexts. Here we show thatexpression suppresses the repressive effects of, and Gle1 counteracts Ded1 in translation assaysFurthermore, Ded1 and Gle1 both affect assembly of pre-initiation complexes. Through mutation analysis and binding assays, we show that Gle1 inhibits Ded1 by reducing its affinity for RNA. Our results are consistent with a model wherein active Ded1 promotes translation, but inactive or excess Ded1 leads to translation repression. Gle1 can inhibit either role of Ded1, positioning it as a gatekeeper to optimize Ded1 activity to the appropriate level for translation. This study suggests a paradigm for finely controlling the activity of DEAD-box proteins to optimize their function in RNA-based processes. It also positions the versatile regulator Gle1 as a potential node for the coordination of different steps of gene expression.
• Bolger, T. A., & Wente, S. R. (2011). Gle1 is a multifunctional DEAD-box protein regulator that modulates Ded1 in translation initiation. Journal of Biological Chemistry, 286(46), 39750-39759.
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  PMID: 21949122;PMCID: PMC3220593;Abstract: DEAD-box protein (Dbp) family members are essential for gene expression; however, their precise roles and regulation are not fully defined. During messenger (m)RNA export, Gle1 bound to inositol hexakisphosphate (IP 6) acts via Dbp5 to facilitate remodeling of mRNA-protein complexes. In contrast, here we define a novel Gle1 role in translation initiation through regulation of a different DEAD-box protein, the initiation factor Ded1. We find that Gle1 physically and genetically interacts with Ded1. Surprisingly, whereas Gle1 stimulates Dbp5, it inhibits Ded1 ATPase activity in vitro, and IP 6 does not affect this inhibition. Functionally, a gle1-4 mutant specifically suppresses initiation defects in a ded1-120 mutant, and ded1 and gle1 mutants have complementary perturbations in AUG start site recognition. Consistent with this role in initiation, Gle1 inhibits translation in vitro in competent extracts. These results indicate that Gle1 has a direct role in initiation and negatively regulates Ded1. Together, the differential regulation of two distinct DEAD-box proteins by a common factor (Gle1) establishes a new paradigm for controlling gene expression and coupling translation with mRNA export. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.
• Alcázar-Román, A. R., Bolger, T. A., & Wente, S. R. (2010). Control of mRNA export and translation termination by inositol hexakisphosphate requires specific interaction with Gle. Journal of Biological Chemistry, 285(22), 16683-16692.
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  PMID: 20371601;PMCID: PMC2878036;Abstract: The unidirectional translocation of messenger RNA (mRNA) through the aqueous channel of the nuclear pore complex (NPC) is mediated by interactions between soluble mRNA export factors and distinct binding sites on the NPC. At the cytoplasmic side of the NPC, the conserved mRNA export factors Gle1 and inositol hexakisphosphate (IP6) play an essential role in mRNA export by activating the ATPase activity of the DEAD-box protein Dbp5, promoting localized messenger ribonucleoprotein complex remodeling, and ensuring the directionality of the export process. In addition, Dbp5, Gle1, and IP 6 are also required for proper translation termination. However, the specificity of the IP6-Gle1 interaction in vivo is unknown. Here, we characterize the biochemical interaction between Gle1 and IP6 and the relationship to Dbp5 binding and stimulation. We identify Gle1 residues required for IP6 binding and show that these residues are needed for IP6-dependent Dbp5 stimulation in vitro. Furthermore, we demonstrate that Gle1 is the primary target of IP6 for both mRNA export and translation termination in vivo. In Saccharomyces cerevisiae cells, the IP 6-binding mutants recapitulate all of the mRNA export and translation termination defects found in mutants depleted of IP6. We conclude that Gle1 specifically binds IP6 and that this interaction is required for the full potentiation of Dbp5 ATPase activity during both mRNA export and translation termination. © 2010 by The American Society for Biochemistry and Molecular Biology, Inc.
• Bolger, T. A., Folkmann, A. W., Tran, E. J., & Wente, S. R. (2008). The mRNA Export Factor Gle1 and Inositol Hexakisphosphate Regulate Distinct Stages of Translation. Cell, 134(4), 624-633.
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  PMID: 18724935;PMCID: PMC2601711;Abstract: Gene expression requires proper messenger RNA (mRNA) export and translation. However, the functional links between these consecutive steps have not been fully defined. Gle1 is an essential, conserved mRNA export factor whose export function is dependent on the small molecule inositol hexakisphosphate (IP6). Here, we show that both Gle1 and IP6 are required for efficient translation termination in Saccharomyces cerevisiae and that Gle1 interacts with termination factors. In addition, Gle1 has a conserved physical association with the initiation factor eIF3, and gle1 mutants display genetic interactions with the eIF3 mutant nip1-1. Strikingly, gle1 mutants have defects in initiation, whereas strains lacking IP6 do not. We propose that Gle1 functions together with IP6 and the DEAD-box protein Dbp5 to regulate termination. However, Gle1 also independently mediates initiation. Thus, Gle1 is uniquely positioned to coordinate the mRNA export and translation mechanisms. These results directly impact models for perturbation of Gle1 function in pathophysiology. © 2008 Elsevier Inc. All rights reserved.
• Bolger, T. A., Zhao, X., Cohen, T. J., Tsai, C., & Yao, T. (2007). The neurodegenerative disease protein ataxin-1 antagonizes the neuronal survival function of myocyte enhancer factor-2. Journal of Biological Chemistry, 282(40), 29186-29192.
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  PMID: 17646162;Abstract: Ataxin-1 is a neurodegenerative disorder protein whose mutant form causes spinocerebellar ataxia type-1 (SCA1). Evidence suggests that ataxin-1 may function as a transcription repressor. However, neither the importance of this putative transcriptional repression activity in neural cytotoxicity nor the transcriptional targets of ataxin-1 are known. Here we identify the MEF2-HDAC4 transcriptional complex involved in neuron survival as a target of ataxin-1. We show that ataxin-1 binds specifically to histone deacetylase-4 (HDAC4) and MEF2 and colocalizes with them in nuclear inclusion bodies. Significantly, these interactions are greatly reduced by the S776A mutation, which largely abrogates the cytotoxicity of ataxin-1. Supporting the importance of these interactions, we show that wild type ataxin-1 represses MEF2-dependent transcription, whereas the S776A mutant is less potent. Furthermore, overexpression of MEF2 can partially reverse cytotoxicity caused by ataxin-1. Our results identify the MEF2-HDAC4 complex as a target for ataxin-1 transcriptional repression activity and suggest a novel pathogenic mechanism whereby ataxin-1 sequesters and inhibits the neuronal survival factor MEF2. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.
• Tran, E. J., Bolger, T. A., & Wente, S. R. (2007). SnapShot: Nuclear Transport. Cell, 131(2), 420.e1-420.e2.
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  PMID: 17956740;
• Bolger, T. A., & Yao, T. (2005). Intracellular trafficking of histone deacetylase 4 regulates neuronal cell death. Journal of Neuroscience, 25(41), 9544-9553.
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  PMID: 16221865;Abstract: Histone deacetylase 4 (HDAC4) undergoes signal-dependent shuttling between the cytoplasm and nucleus, which is regulated in part by calcium/calmodulin- dependent kinase (CaMK)-mediated phosphorylation. Here, we report that HDAC4 intracellular trafficking is important in regulating neuronal cell death. HDAC4 is normally localized to the cytoplasm in brain tissue and cultured cerebellar granule neurons (CGNs). However, in response to low-potassium or excitotoxic glutamate conditions that induce neuronal cell death, HDAC4 rapidly translocates into the nucleus of cultured CGNs. Treatment with the neuronal survival factor BDNF suppresses HDAC4 nuclear translocation, whereas a proapoptotic CaMK inhibitor stimulates HDAC4 nuclear accumulation. Moreover, ectopic expression of nuclear-localized HDAC4 promotes neuronal apoptosis and represses the transcriptional activities of myocyte enhancer factor 2 and cAMP response element-binding protein, survival factors in neurons. In contrast, inactivation of HDAC4 by small interfering RNA or HDAC inhibitors suppresses neuronal cell death. Finally, an increase of nuclear HDAC4 in granule neurons is also observed in weaver mice, which harbor a mutation that promotes CGN apoptosis. Our data identify HDAC4 and its intracellular trafficking as key effectors of multiple pathways that regulate neuronal cell death. Copyright © 2005 Society for Neuroscience.
• Zhao, X., Sternsdorf, T., Bolger, T. A., Evans, R. M., & Yao, T. (2005). Regulation of MEF2 by histone beacetylase 4- and SIRT1 deacetylase-mediated lysine modifications. Molecular and Cellular Biology, 25(19), 8456-8464.
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  PMID: 16166628;PMCID: PMC1265742;Abstract: The class II deacetylase histone deacetylase 4 (HDAC4) negatively regulates the transcription factor MEF2. HDAC4 is believed to repress MEF2 transcriptional activity by binding to MEF2 and catalyzing local histone deacetylation. Here we report that HDAC4 also controls MEF2 by a novel SUMO E3 ligase activity. We show that HDAC4 interacts with the SUMO E2 conjugating enzyme Ubc9 and is itself sumoylated. The overexpression of HDAC4 leads to prominent MEF2 sumoylation in vivo, whereas recombinant HDAC4 stimulates MEF2 sumoylation in a reconstituted system in vitro. Importantly, HDAC4 promotes sumoylation on a lysine residue that is also subject to acetylation by a MEF2 coactivator, the acetyltransferase CBP, suggesting a possible interplay between acetylation and sumoylation in regulating MEF2 activity. Indeed, MEF2 acetylation is correlated with MEF2 activation and dynamically induced upon muscle cell differentiation, while sumoylation inhibits MEF2 transcriptional activity. Unexpectedly, we found that HDAC4 does not function as a MEF2 deacetylase. Instead, the NAD+-dependent deacetylase SIRT1 can potently induce MEF2 deacetylation. Our studies reveal a novel regulation of MEF2 transcriptional activity by two distinct classes of deacetylases that affect MEF2 sumoylation and acetylation. Copyright © 2005, American Society for Microbiology. All Rights Reserved.
• Fazio, I. K., Bolger, T. A., & Gill, G. (2001). Conserved Regions of the Drosophila Erect Wing Protein Contribute Both Positively and Negatively to Transcriptional Activity. Journal of Biological Chemistry, 276(22), 18710-18716.
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  PMID: 11278998;Abstract: Genetic studies of the Drosophila erect wing (ewg) gene have revealed that ewg has an essential function in the embryonic nervous system and is required for the specification of certain muscle cells. We have found that EWG is a site-specific transcriptional activator, and we report here that evolutionarily conserved regions of EWG contribute both positively and negatively to transcriptional activity. Using gel mobility shift assays, we have shown that an EWG dimer binds specifically to DNA. In transfection assays, EWG activated expression of a reporter gene bearing specific binding sites. Analysis of deletion mutants and fusions of EWG to the Gal4 DNA binding domain has identified a transcriptional activation domain in the C terminus of EWG. Deletion analysis also revealed a novel inhibitory region in the N terminus of EWG. Strikingly, both the activation domain and the inhibitory region are conserved in EWG homologs including human nuclear respiratory factor 1 (NRF-1) and the sea urchin P3A2 protein. The strong conservation of elements that determine transcriptional activity suggests that the EWG, NRF-1, and P3A2 family of proteins shares common mechanisms of action and has maintained common functions across evolution.
• Zhao, X., Ito, A., Kane, C. D., Liao, T., Bolger, T. A., Lemrow, S. M., Means, A. R., & Yao, T. (2001). The Modular Nature of Histone Deacetylase HDAC4 Confers Phosphorylation-dependent Intracellular Trafficking. Journal of Biological Chemistry, 276(37), 35042-35048.
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  PMID: 11470791;Abstract: In C2C12 myoblasts, endogenous histone deacetylase HDAC4 shuttles between cytoplasmic and nuclear compartments, supporting the hypothesis that its subcellular localization is dynamically regulated. However, upon differentiation, this dynamic equilibrium is disturbed and we find that HDAC4 accumulates in the nuclei of myotubes, suggesting a positive role of nuclear HDAC4 in muscle differentiation. Consistent with the notion of regulation of HDAC4 intracellular trafficking, we reveal that HDAC4 contains a modular structure consisting of a C-terminal autonomous nuclear export domain, which, in conjunction with an internal regulatory domain responsive to calcium/calmodulin-dependent protein kinase IV (CaMKIV), determines its subcellular localization. CaMKIV phosphorylates HDAC4 in vitro and promotes its nuclear-cytoplasmic shuttling in vivo. However, although 14-3-3 binding of HDAC4 has been proposed to be important for its cytoplasmic retention, we find this interaction to be independent of CaMKIV. Rather, the HDAC4·14-3-3 complex exists in the nucleus and is required to confer CaMKIV responsiveness. Our results suggest that the subcellular localization of HDAC4 is regulated by sequential phosphorylation events. The first event is catalyzed by a yet to be identified protein kinase that promotes 14-3-3 binding, and the second event, involving protein kinases such as CaMKIV, leads to efficient nuclear export of the HDAC4·14-3-3 complex.

Presentations

• Bolger, T. A. (2015, March). D-E-A-D and Alive: Controlling mRNA-Protein Dynamics in Normal and Cancer Cells. Basic Medical Sciences Seminar Series, College of Medicine-Phoenix. Phoenix, AZ.
• Bolger, T. A. (2004, Sept). Histone deacetylase 4 promotes neuronal cell death.. Department Annual Symposium. Wilmington, NC: Department of Pharmacology and Cancer Biology, Duke University School of Medicine.

Poster Presentations

• Bolger, T. A. (2009, Aug). Functional coupling of mRNA export and translation for control of gene expression. International Meeting on Nuclear Trafficking. Banff, Alberta, Canada.