Role of the Skp1 prolyl-hydroxylation/glycosylation pathway in oxygen dependent submerged development of Dictyostelium
© Xu et al.; licensee BioMed Central Ltd. 2012
Received: 28 June 2012
Accepted: 11 October 2012
Published: 25 October 2012
Oxygen sensing is a near universal signaling modality that, in eukaryotes ranging from protists such as Dictyostelium and Toxoplasma to humans, involves a cytoplasmic prolyl 4-hydroxylase that utilizes oxygen and α-ketoglutarate as potentially rate-limiting substrates. A divergence between the animal and protist mechanisms is the enzymatic target: the animal transcriptional factor subunit hypoxia inducible factor-α whose hydroxylation results in its poly-ubiquitination and proteasomal degradation, and the protist E3SCFubiquitin ligase subunit Skp1 whose hydroxylation might control the stability of other proteins. In Dictyostelium, genetic studies show that hydroxylation of Skp1 by PhyA, and subsequent glycosylation of the hydroxyproline, is required for normal oxygen sensing during multicellular development at an air/water interface. Because it has been difficult to detect an effect of hypoxia on Skp1 hydroxylation itself, the role of Skp1 modification was investigated in a submerged model of Dictyostelium development dependent on atmospheric hyperoxia.
In static isotropic conditions beneath 70-100% atmospheric oxygen, amoebae formed radially symmetrical cyst-like aggregates consisting of a core of spores and undifferentiated cells surrounded by a cortex of stalk cells. Analysis of mutants showed that cyst formation was inhibited by high Skp1 levels via a hydroxylation-dependent mechanism, and spore differentiation required core glycosylation of Skp1 by a mechanism that could be bypassed by excess Skp1. Failure of spores to differentiate at lower oxygen correlated qualitatively with reduced Skp1 hydroxylation.
We propose that, in the physiological range, oxygen or downstream metabolic effectors control the timing of developmental progression via activation of newly synthesized Skp1.
KeywordsProlyl 4-hydroxylase Glycosyltransferase Oxygen sensing Hypoxia Hydroxyproline Cellular slime mold
To address this issue, and to investigate the generality of O2 regulation of development, we turned to a previously described submerged development model in which terminal cell differentiation depends on high (≥70%) atmospheric O2[22, 23]. The wider range of O2 concentrations presented to cells in this setting may facilitate analysis of the dependence of Skp1 hydroxylation on O2, and absence of the morphogenetic movements of culmination might reveal later developmental steps that are dependent on Skp1 and its modifications. In a static adaptation of the previous shaking cultures, we observed that terminal cell differentiation occurs in a novel radially symmetrical fashion in multicellular cyst-like structures. Under these conditions, we find that O2 is apparently rate-limiting for Skp1 hydroxylation, and that cyst formation and terminal spore differentiation that require high O2 also depend on normal levels of Skp1 and both its hydroxylation and glycosylation. This expands the role of Skp1 and its modifications in developmental regulation, and supports the model that O2 regulates its modification in cells.
Dictyosteliumcell strains and growth
The normal D. discoideum strain Ax3 and its derivatives with the following genotypes were described previously: phyA–, ecmA::PhyA-myc/phyA–, cotB::PhyA-myc/phyA–, PKA(cat)/phyA–, pgtA–, PgtA-N/pgtA–, agtA–, gmd–, ecmA::Skp1A.1/Ax3, ecmA::Skp1A.2/Ax3, cotB::Skp1A.1/Ax3, cotB::Skp1A.3/Ax3, cotB::Skp1A3.H2/Ax3, ecmA::Skp1B.2/phyA–, cotB::Skp1A.2/phyA – , cotB::Skp1A.3/phyA–. Note that the number before the decimal point represents alleles, and the number after represents clones that may vary in expression level. Cells were grown in shaking HL-5 axenic medium at 22°C , and collected before their density reached 0.8 × 107/ml.
Cells were harvested by centrifugation (2000 g × 1 min) at 4°C, resuspended in PDF buffer (33 mM NaH2PO4, 10.6 mM Na2HPO4, 20 mM KCl, 6 mM MgSO4, pH 5.8), re-centrifuged and resuspended in PDF at 108/ml, and deposited on 0.45 μm pore Millipore cellulose nitrate filters for standard development at an air-water interface . For submerged development, washed cells were resuspended in PDF at 2 × 107/ml and 1.4 ml was deposited into each well of a 6-well bacteriological or tissue culture plate (3 cm diameter wells). Plates were incubated for up to 72 h in a sealed plastic box, with inlet and outlet ports for gas flow, under room fluorescent lights at 22°C. The inlet valve was connected via a bubbling water humidifier to a compressed gas tank formulated with the indicated percentage of O2, with the balance made up of N2. Previously it was shown that inclusion of 1% CO2 did not affect the O2 dependence of culmination . The outlet tube was connected to a Pasteur pipette held under water to monitor gas flow. Cultures were kept unstirred to prevent contact of cells or cell aggregates with the buffer surface, which led to polarization and/or floating fruiting bodies (data not shown). Volume and cell density were optimized for maximal spore differentiation at 100% O2 (data not shown). Alternate buffers, including KP (17 mM potassium phosphate, pH 6.5), or Agg buffer (0.01 M NaPO4, pH 6.0, 0.01 M KCl, 0.005 M MgCl2), yielded lower spore numbers.
Cell aggregates were visualized in a stereomicroscope using transmitted light, or using phase contrast illumination on an inverted microscope. For detection of cellulosic cell walls, samples were analyzed under epifluorescence illumination in the presence of 0.1% (v/v) Calcofluor White ST (American Cyanamid) in 10 mM potassium phosphate (pH 8.0), using DAPI-filters. Multiphoton confocal microscopy was performed at the OUHSC Imaging Laboratory on a Leica SP2 MP Confocal microscope.
For determining spore numbers, samples were supplemented with 0.2% NP-40, and spores were counted in a hemacytometer. Spores were identified based on their resistance to detergent, shape, refractility, and labeling with Calcofluor White ST or anti-spore coat Abs. Spore plating efficiency was determined by spreading an aliquot of detergent-treated spores on SM agar in association with Klebsiella aerogenes, and dividing the number of colonies by the counted number of input spores.
Spores were released from cysts by probe sonication in 0.2% NP-40 in KP, centrifuged at 13,000 g × 10 s, and resuspended in KP buffer. Spores were recovered from fruiting bodies on non-nutrient agar by slapping the inverted Petri plate on a counter and washing the spores from the lid, and processed in parallel. An aliquot was treated with 6 M urea, 1% (v/v) 2-mercaptoethanol in TBS (10 mM Tris–HCl, pH 7.4, 150 mM NaCl) for 3 min at 100°C prior to dilution in cold TBS and recovery by centrifugation. Spore suspensions (2 × 106/50 μl) were deposited on glass slides onto which had been dried a 50-μl volume of 10 μg/ml poly-L-lysine in H2O. After 15 min, non-bound spores were removed by aspiration and washing with TBS. The monolayer was incubated in 4 mg/ml hemoglobin in TBS for 5 min, 1 μg/ml mAb 83.5  in 4 mg/ml hemoglobin in TBS for 1 h, TBS (5 washes), 2 μg/ml Alexa 568-conjugated Rabbit anti-mouse IgG (Molecular Probes/Invitrogen) in 3% (w/v) bovine serum albumin in TBS, TBS (5 washes), and Vectashield mounting medium. Samples were analyzed through a 40× (N.A. 0.75) lens via the TRITC-channel of an Olympus epifluorescence microscope, and images were identically recorded using a SPOT Flex camera (Diagnostic Instruments) and processed using Photoshop CS3.
Developing cells were collected by centrifugation at 2000 g × 1.5 min at 4°C and boiled for 2 min in Laemmli sample buffer containing 50 mM DTT. Low O2 samples were first supplemented with 2 mM sodium dithionite  to minimize possible hydroxylation during sample preparation. Whole cell lysates were resolved by SDS-PAGE on a 4-12% gradient gel (NuPAGE Novex, Invitrogen), and transferred to nitrocellulose membrane using an iBlot system (Invitrogen). Blots were probed with primary and fluorescent secondary Abs as described . Blots were blocked in, and Abs were dissolved in, 5% non-fat dry milk in 20 mM Tris–HCl (pH 7.4), 150 mM NaCl, 0.02% NaN3, and Alexa 680 fluorescence was imaged using a Li-Cor Odyssey scanner. Prespore cell differentiation was probed using mAbs 5F5 and 83.5 , and Skp1 isoforms were detected using pAb UOK87 , pAb UOK85 , mAb 4H2 , mAb 1C9 , and mAb 4E1 . Affinity-purified anti-actin was from Sigma Chemical Co.
Images were analyzed densitometrically using NIH Image J. mAb 4E1 was used in its linear response range  to obtain the fraction of Skp1 that was not modified. Initially, values for each upper and lower band were corrected for general background by subtraction of a blank intensity value obtained from the vicinity of the band of interest. Studies using pAb UOK87, which selectively recognizes unmodified Skp1, showed that 5% of Skp1 was unmodified at 100% O2 based on comparison with a phyA– sample (not shown). The remaining density in the lower band of the 100% O2 sample is of uncertain identity but, since its level was observed to be proportionate to the level of the upper band (not shown), its value (as a fraction of the upper band) was subtracted from each sample in the O2 series. The fraction of unmodified Skp1 was determined by dividing the corrected intensity of the lower Skp1 band by the sum of the intensities of the lower and upper bands.
Terminal differentiation at an air-water interface
D. discoideum amoebae develop to form fruiting bodies when dispersed in a low ionic strength buffer on a moist surface (Figure 2A). About 75% of the cells become aerial spores and the remainder form the structural stalk. At reduced O2 levels (2.5-10%), the slug intermediate continues to migrate on the surface without culminating . When returned to the ambient O2 level (21%), culmination then occurs within about 5 h. To determine the minimal time required for exposure to ambient O2, slugs were exposed to 21% O2 for varying times before returning to low O2. Figure 2B shows that exposure to high O2 can be as brief as 1 h, though up to 4 h is required for maximal culmination based on spore counts. The requirement for high O2 appeared to be selective for induction of culmination, because terminal cell differentiation occurred normally even within the fruiting bodies formed after only 1 h of exposure to normoxia (data not shown). The effect of O2 appears to be mediated at least in part by prolyl 4-hydroxylation of Skp1, because elevated O2 levels are required by phyA– and Skp1-overexpression strains, and lower O2 is required by PhyA overexpression and Skp1B– cells [10, 24]. To further explore the role of Skp1 modification in O2 sensing and the importance of culmination as the target of regulation, we turned to a previously described submerged development model [22, 23, 30], in which progress beyond the loose aggregate stage is strictly dependent on elevated atmospheric O2, and terminal differentiation bypasses the morphogenetic movements of culmination.
Terminal differentiation in submerged cultures
Under 21% O2, stalk cells and spores were rarely observed in the less compacted aggregates that form under these conditions. When present they occurred as clusters or single cells (not shown). At 40% O2, larger aggregates were formed but they lacked dense cores observed at higher O2 levels. These cyst-like aggregates possessed a stalk cell cortex but their interior cells produced few spores, as visualized after squashing (Figures 3C,E). Though spores were not detected in this example, variable numbers were observed over the 5 independent trials as quantitated in Figure 4C. The variation suggests that 40% O2 is close to the threshold required for sporulation whose exact value is likely influenced by other factors, as observed for culmination . To address the differentiation status of cells at the lower O2 levels, extracts were Western blotted for the spore coat precursor proteins SP85, SP96 and SP75 that are markers of prespore cell differentiation . Whereas all 3 glycoproteins appeared in Ax3 cells by 24 h at 70% O2, negligible expression occurred at 20% after 3 d (Figure 4E). Thus increasing O2 levels were required for tight aggregate formation, terminal stalk cell differentiation, and differentiation of the interior prespore cells into spores. It is likely that metabolic O2 consumption results in intracyst hypoxia in these unstirred cultures which, in the submerged state, is not adequately replenished by O2 diffusion. The finding that elevated O2 tension in the atmosphere above the medium can rescue terminal differentiation indicates that O2 availability is the limiting factor for terminal cell differentiation in this setting. It is not evident whether the higher O2 level required for spore compared to stalk cell differentiation reflects a higher O2 threshold requirement for spore differentiation or lower O2 in the aggregate centers.
Requirement of PhyA for sporulation in submerged conditions
A previously described mutant strain disrupted at its phyA locus  was analyzed to determine the involvement of Skp1 prolyl 4-hydroxylation in submerged development. phyA– cells formed cyst-like structures at 40-100% O2 with outer layers of differentiated stalk cells, similar to the normal Ax3 strain (Figure 3C, D). However, interior cells failed to differentiate as spores, even after extended periods, as shown in the side-by-side comparisons in Figures 3B, D, 4A, and D. Instead, they remained as prespore cells, based on Western blot analysis showing abundant expression of the spore coat precursors (Figure 4E). Failure to sporulate was due to the PhyA deficiency, because phyA – cells complemented with ecmA::phyA or cotB::phyA, which overexpress PhyA activity in prestalk or prespore cells respectively , were rescued at high O2 (Figure 4B). ecmA::phyA/phyA – cells formed normal numbers of spores compared to Ax3, while cotB::phyA/phyA – only partially rescued spore formation to about 30% of Ax3 levels. The difference suggests that prestalk cells may be important in mediating the role of PhyA in sporulation, consistent with evidence for a role of prestalk cells in processing or mediating sporulation signals during normal culmination [32–34]. While overexpression in prespore cells (cotB promoter) was also partially effective, the possibility that PhyA signals autonomously in prespore cells is not proved because on filters, cotB::PhyAoe cells tend to migrate to the tip in chimeras with normal cells . Successful complementation from these developmental promoters confirmed that cells had differentiated into prestalk and prespore cells in the absence of PhyA, and showed that PhyA is required only after their appearance. Since spore formation selectively depended on high O2 and the threshold for spore (but not stalk cell) differentiation was specifically affected by the absence of PhyA, PhyA activity appears to have a novel function in mediating O2 regulation of spore differentiation.
Since overexpression of PhyA in a phyA+ (wild-type) background reduces the O2 level required for culmination on filters , the effect of PhyA overexpression on sporulation was investigated. As shown in Figure 4C, modestly increased sporulation was observed at 70% O2 when PhyA was overexpressed in prespore cells. However, overexpression in prestalk cells inhibited sporulation, without affecting cyst formation per se. As noted above, PhyA overexpression under the ecmA promoter in a phyA– background rescued sporulation better than under the cotB promoter, so the inhibitory effect of overexpression in phyA+ cells appears to be depend on a complex interplay between relative levels of expression in the different cell types rather than a cell autonomous effect on prestalk cells.
Skp1 modification is O2dependent
Role of glycosylation in submerged development
Role of Skp1 and its modifications in submerged development
In comparison, the requirement of Skp1 glycosylation for sporulation suggests that for this later developmental step, Skp1 contributes to the breakdown of a hypothetical inhibitor of sporulation. Without modification, Skp1 is not activated and the inhibitor accumulates. However, overexpression of Skp1 in the phyA– background (thereby bypassing the block to cyst formation) allows sporulation, which can be interpreted as providing additional activity to compensate for lack of activation by modification (Figure 7B, blue bars and inset; data not shown). Similar effects were observed irrespective of the promoter used, or whether wild-type Skp1A or B, or mutant Skp1, was overexpressed (data not shown). However, overexpression of Skp1 at very high levels did not rescue sporulation in phyA– cells as well, which might reflect a dominant negative effect toward SCF-complex formation. Separate effects on activators and inhibitors may depend on involvement of distinct F-box proteins.
Three novel observations regarding development under submerged conditions are presented here: i) In the presence of high O2 and absence of stirring, cell differentiation occurs in a radially symmetrical rather than the typical linearly polarized pattern. With their outer husk-like cortex and interior germinative cells, these structures have the organization of multicellular cysts as occur in animal tissues. The cyst-like structures are distinct from other terminal states formed by Dictyostelium, including the dormant unicellular microcyst and the multinucleated macrocyst . Although conditions leading to the formation of cyst-like structures are not known to occur naturally, its O2 dependence is likely to be relevant to interpreting O2 signaling in normoxia as outlined below. ii) Skp1 hydroxylation is limited by O2 availability. iii) Certain developmental transitions that occur during submerged development, including tight aggregate formation and terminal spore differentiation, critically rely on hydroxylation and glycosylation of Skp1. Together, these findings reinforce a role for environmental O2 for influencing polarity and key developmental transitions, and strongly implicate the Skp1 modification pathway in decoding the O2 signal.
Significance of O2for control of polarity and terminal differentiation
NH3, a volatile metabolite released during the massive breakdown of protein during development , has also been implicated as a polarity factor and inhibits the slug-to-fruit switch . Since NH3 is expected to diffuse away most at the same surfaces that O2 is expected to diffuse in, the two compounds may play complementary inhibitory and activating roles that tune developmental decisions. Thus, while hypoxic or phyA– preculminants may still form tips at the air-water interface  due to the NH3 effect, the spherical shapes assumed by phyA– slugs after long periods of migration  might reflect eventual depletion of the NH3 signal as protein is finally consumed. The isotropic environment during static submerged development may thwart formation of orienting NH3 as well thereby resulting in radial polarization, and high NH3 in the interior is expected to promote sporulation . Since NH3-signaling is mediated in part by NH3-transporter/sensors [16, 17], investigation of genetic interactions with phyA may allow understanding of the interplay with Skp1 modification.
Role of Skp1 prolyl hydroxylation in tight aggregate formation
Tight aggregate formation depended on an elevated O2 level of ≥40%, but this was inhibited when Skp1 (either isoform) was overexpressed under either developmental promoter (Figure 7A). This correlates with the 7-hr delay of the loose-to-tight aggregate transition of these overexpression strains at the air-water interface . Interestingly, inhibition of tight aggregate formation was partially relieved when Skp1 was overexpressed in a phyA-mutant background, which also relieved the delay on filters. Consistent with a requirement for modification, overexpression of Skp1A3(P143A), which cannot be hydroxylated, is not inhibitory (Figure 7A, B). The opposing effects of Skp1 overexpression and inhibiting its modification are consistent with a model in which modification activates Skp1 and its role in polyubiquitination and breakdown of a hypothetical activator of cyst formation.
Role of Skp1 prolyl hydroxylation and glycosylation in sporulation
A second function of the pathway was revealed by the essentially complete failure of the interior prespore cells to differentiate in the phyA– strain, whereas stalk cell differentiation was qualitatively unaffected (Figures 3, 4). The blockade was overcome when PhyA was overexpressed in prestalk and to a lesser extent prespore cells (Figure 4B), so control by O2 may be mediated via prestalk cells. This is consistent with evidence that prestalk cells can regulate sporulation via processing of spore differentiation factor-1 and −2 [33, 34]. However, the role of PhyA appears complex because overexpression in prestalk cells in the phyA+ (wild-type) background inhibited sporulation, as if relative levels of O2 signaling between cell types could be important. The blockade was also partially overcome when PKA activity was promoted by overexpression of its catalytic domain under its own promoter (Figure 4B). Since PKA expression in prespore cells was previously shown to be sufficient for activating sporulation , PhyA may signal upstream of PKA as suggested for its role in culmination on filters .
Hydroxylated Skp1 is a substrate for Gnt1 that in turn generates a substrate for PgtA, and then AgtA, resulting in formation of the pentasaccharide on Hyp143 (Figure 6A). Mutants lacking enzymes to extend to the trisaccharide state were also unable to sporulate at high O2 (Figures 6B,C), suggesting that hydroxylation supports extension of the glycan chain to three or more sugars to trigger sporulation. Though the preceding culmination step (on filters) exhibited more modest dependence on addition of the first two sugars (at lower O2 levels) , the more dramatic difference in the static submerged model may simply result from failure to achieve a critical threshold of O2 in the cyst interior. The greater difference was in the role of AgtA, whose contribution was almost as important for culmination as PhyA  but was unnecessary for submerged sporulation. Thus the role of AgtA appears to be specialized for culmination compared to sporulation.
The requirement of PhyA for sporulation was partially overcome by overexpression of Skp1 (Figure 7). This suggests that PhyA action normally promotes Skp1 activity, and its absence can be bypassed by excess Skp1. A related effect was observed on filter development, where Skp1 overexpression inhibited sporulation at high O2 levels that allowed culmination, but removal of PhyA blocked inhibition , indicating that PhyA tunes Skp1 activity. This is consistent with activation of Skp1 poly-ubiquitination activity toward an inhibitor. In comparison, the effect of Skp1 modification on culmination implied inhibition of Skp1 breakdown activity toward a hypothetical activator [10, 11], and the effects on cyst formation (assessed morphologically) above suggested activation of breakdown activity toward an activator. These disparate effects are consistent with what is known about the SCF family of E3 ubiquitin-ligases, which polyubiquitinate different substrates depending on which F-box protein is present. Furthermore, these Ub-ligases can have opposite effects via auto-polyubiquitination of the F-box protein itself, which results in protection of the substrate receptor [6, 7]. Conceivably, Skp1 modification may selectively affect these different activities.
O2is limiting for Skp1 hydroxylation in submerged culture and mechanistic implications
In submerged development, substantial levels of unmodified Skp1 (Figure 5D) accumulated at 5% and 21% O2. Since i) there is no evidence for enzymatic reversal of hydroxylation or glycosylation, ii) the level of Skp1 was similar at different O2 levels, and iii) Skp1 turns over with a half-life of 12–18 h , it is likely that appearance of unmodified Skp1 was due to failure to hydroxylate nascent Skp1. Since the total Skp1 pool becomes 95% hydroxylated at ≥40% O2 (Figure 5D), O2 is likely rate-limiting for Skp1 prolyl hydroxylation. This is consistent with co-expression evidence that PhyA is rate limiting for Skp1 hydroxylation . Since sporulation is minimal at 40% O2 even though the steady-state pool of Skp1 appears fully modified, it may be that O2 and PhyA have additional or alternative mechanisms for controlling sporulation. However, it should also be considered that a several hour delay in the hydroxylation of nascent Skp1, which might be most important for partnering with nascent F-box proteins, would have escaped detection against the background of total Skp1 using our methods.
Since the Skp1/F-box protein complex is characterized by a high affinity  that is increased by hydroxylation as suggested in Figure 1B (M.O. Sheikh and C.M. West, unpublished data), we propose that even transient accumulation of unmodified Skp1 will influence the spectrum of complexes with one or more of the ~38 predicted F-box proteins that are strongly up and/or down-regulated at various times during development based on RNAseq data  (unpublished studies). This in turn may affect the timing of developmental transitions via effects on the stability of F-box proteins and hypothetical F-box protein substrates (activators and inhibitors) that normally control aggregation, slug formation, culmination and sporulation [e.g., . Figure 2B shows that O2 exposure of 1–3 h can rescue culmination of hypoxic slugs, consistent with a transient role that might correlate with expression of a specific F-box protein. Current studies are focused on how Skp1 modification influences E3SCFubiquitin-ligase assembly and activity.
These findings in social amoebae may be pertinent to numerous protist groups, including other amoebae (e.g., Acanthamoeba), plant pathogens (Phytophthora), diatoms (brown algae), green algae (Chlamydomonas), ciliates (Tetrahymena), and apicomplexans including Toxoplasma, whose O2 dependence have been little studied but whose genomes harbor Skp1 modification pathway-like genes . For example, recent studies  showed that the related Skp1 modification pathway supports growth of Toxoplasma in cultured fibroblasts especially at low O2.
In an isotropic submerged environment under high O2, starved Dictyostelium cells form cyst-like structures in which terminal differentiation occurs in a radially symmetrical pattern consisting of external stalk cells and internal spores. Low O2 is rate-limiting for the hydroxylation and subsequent glycosylation of Skp1, which correlates qualitatively with inhibition of spore differentiation. Genetic perturbations indicate the importance of Skp1 hydroxylation and glycosylation for activating Skp1 activity in regulating cyst formation and sporulation, in addition to previous evidence for its inhibition in regulating culmination at an air-water interface. The findings support a model in which environmental control of Skp1 modification differentially influences sequential developmental transitions via polyubiquitination and degradation of F-box proteins and their respective regulatory factor substrates.
(4R,2S)-hydroxyproline (aka 4(trans)-hydroxy-L-proline)
Prolyl 4-hydroxylase-1 from D. discoideum
Protein kinase A
E3 ubiquitin ligase sub-complex consisting of Skp1, a cullin-1, an F-box protein, and Rbx1
Standard error of the mean
We are grateful to Jim Henthorn at the Flow & Image Cytometry Lab at OUHSC for assistance in the confocal imaging. Haitham Abd El-Moaty provided valuable assistance early in the project. Funding was provided by NIH grants RO1 GM037539 and R01 GM084383, grant HR10-181 from the Oklahoma Center for the Advancement of Science and Technology (OCAST), and a grant to the Summer Undergraduate Research Program (SURE) program from the OUHSC Graduate School and the Provost’s Office.
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