Glycobiology Advance Access originally published online on February 27, 2006
Glycobiology 2006 16(6):551-563; doi:10.1093/glycob/cwj096
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Disrupting the enzyme complex regulating O-GlcNAcylation blocks signaling and development
2 Department of Cell Biology, 3 Medical Scientist Training Program, 4 Department of Pharmacology and Toxicology, and 5 Department of Medicine, University of Alabama at Birmingham, Birmingham, AL 35294; and 6Gene Expression Laboratory, Salk Institute for Biological Studies, La Jolla, CA 92037
1 To whom correspondence should be addressed; e-mail: kudlow{at}uab.edu
Received on January 18, 2006; revised on February 14, 2006; accepted on February 21, 2006
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Abstract |
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Although the knowledge that nuclear and cytoplasmic proteins are modified with N-acetylglucosamine has existed for decades, little has been shown as to its function until recently. There are now substantial data highlighting the significance of proper regulation of this modification in multiple cellular processes. Currently, only two enzymes are known that regulate this modification. O-GlcNAc transferase (OGT) modifies protein substrates posttranslationally by adding the N-acetylglucosamine. Bifunctional nuclear/cytoplasmic O-GlcNAcase and acetyl transferase (NCOAT) is responsible for cleaving the modification from target proteins. Here, we demonstrate for the first time an unusual association of these two opposing enzymes into a single O-GlcNAczyme complex. NCOAT and OGT associate strongly through specific domains such that NCOAT accompanies OGT, with histone deacetylases (HDACs), into transcription corepression complexes. Exclusion of NCOAT activities from OGT association blocks proper estrogen-dependent cell signaling as well as mammary development in transgenic mice. This demonstrates that NCOAT is in a strategic position to rapidly counteract OGT and HDAC without requiring its recruitment.
Key words: corepressor / estrogen / HAT / O-GlcNAcase / OGT
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Introduction |
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Transcription in multicellular organisms, whether plant or animal, is controlled in precise spatial and temporal patterns, resulting in proper differentiation, development, and homeostasis. Although, expression of certain genes in differentiated tissue is forbidden, others can be activated by environmental or hormonal signals. One way to accomplish this homeostatic control rapidly and thoroughly is through cooperative posttranslational modifications of existing proteins.
Differentiation of tissues results from the expression of a subset of tissue-specific genes that are driven by their promoter. These promoters consist of generalized basal transcription machinery, general activators, plus more specific activators that bind to cis elements in the DNA (Hahn, 1998
). Coactivators, residing within protein complexes, bind to the DNA-binding activators by protein–protein interaction partly to relay biological signals to the basal transcription machinery (Bjorklund et al., 1999). A common feature of coactivator complexes is the histone acetyltransferase (HAT) activity (Struhl, 1998) that acetylates neighboring nucleosomes, facilitating the opening of the DNA to allow access of RNA polymerase II holoenzyme.
This activation of genes can be reversed in some cases by corepressor complexes that must replace activating complexes. These corepressor complexes contain enzymes that remove acetate groups from histones (Glass and Rosenfeld, 2000) to close the DNA template. These histone deacetylases (HDACs) are brought to the DNA within these complexes that are nucleated by a number of corepressors: N-CoR, SMRT, and mSin3A/B (Glass and Rosenfeld, 2000). It is the exchange of the activation complex with a repression complex that results in a dramatic change in gene expression. Nevertheless, repression of several genes cannot be accounted for entirely by HDAC activity (Perissi and Rosenfeld, 2005). Furthermore, the composition of the corepressor enzyme complexes appears to be quite variable (Ishizuka and Lazar, 2003) which allows for a wide range of promoters to be regulated while maintaining the required specificity. Generally, though, the chromatin structure must be enzymatically modified by the HDACs to contribute to the repression of promoter activity.
Metazoans need more stringent gene repression. One way to accomplish this stringency is through the synergy arising from the cooperation of more than one posttranslational modification acting at different sites. Recently, our laboratory added the O-GlcNAc posttranslational modification to the HDACs in the cooperative repression of transcription (Yang et al., 2002). The corepressor mSin3A recruits both HDAC and O-GlcNAc transferase (OGT), the enzyme responsible for this latter modification (Roos and Hanover, 2000), to repressed genes. The O-GlcNAc posttranslational modification occurs in both multicellular plants (Hartweck et al., 2002) and animals (O’Donnell, 2002). In higher animals, O-GlcNAcylation of the ubiquitous Sp1 transcriptional activator makes this GC-binding factor incapable of activating transcription (Roos et al., 1997; Yang et al., 2001), even though it can still bind to DNA (Jackson and Tjian, 1988; Sekinger and Gross, 2001). But even in the absence of a GC box, OGT targeted to DNA can repress basal transcription, perhaps by modifying the tail of RNA polymerase II (Comer and Hart, 2001). Through these effects, and effects on proteasome activity, signal transduction, and secretion, O-GlcNAc can regulate development and other vital cellular processes (Gao et al., 2001; Wells et al., 2001; Yang et al., 2001; Zhang et al., 2003; Liu et al., 2004). By cooperating with the HDACs that change chromatin structure, the physical coassociation of OGT allows it to modify the transcriptional apparatus. By turning the transcription off, O-GlcNAcylation can add to gene repression through an HDAC-independent mechanism. The corepressor mSin3A, by recruiting both HDAC and OGT through defined domains only to the repressed genes, ensures the specificity of both these modifiers. Furthermore, the stringent repression of gene expression occurs through the synergy of these repressive modifications acting in concert.
A bifunctional enzyme, formerly called Mgea5/O-GlcNAcase (Gao et al., 2001), is the only protein known to remove the O-GlcNAc modification with its N-terminal O-GlcNAcase domain (Clifford Toleman, personal communication). In addition, this protein, like many others, has a HAT domain (Toleman et al., 2004) and has been accordingly termed the nuclear/cytoplasmic O-GlcNAcase and acetyl transferase (NCOAT). Because NCOAT has these two domains, one that reverses the OGT modification and the other that reverses the HDAC modification, NCOAT fulfils the requirement of reversibility of gene repression. To test this idea, we extended our prior studies on estrogen signals in the nucleus (Yang et al., 2002). In the course of our studies, we learned that NCOAT associates with its opposite, OGT, very tightly. This unusual association of opposite enzymes also gives a means by which NCOAT can be as gene specific as OGT, and, by residing with OGT in the repression complex, it is strategically placed to reverse both repressive modifications rapidly: O-GlcNAcylation and deacetylation. However, this association of opposing enzymes is a biological conundrum, because it provides the possibility of a futile cycle. However, in this article, we show that such a cycle of O-GlcNAcylation does not occur probably because both NCOAT activities are regulated (Toleman et al., 2004), and its unique O-GlcNAcase activity is converse to that of its association partner, OGT.
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NCOAT associates with corepressors
Antibodies were raised against NCOAT, and an OGT antibody was obtained (Kreppel et al., 1997
) to characterize in vivo interactions. Antibodies were affinity purified and checked for specificity by western blot analysis (supplementary data, Figure 1). Although raised against recombinant full-length NCOAT purified from Escherichia coli, two antisera were generated, one of which [
-NCOAT(P)] was state specific and did not recognize phosphorylated NCOAT. Multiple forms of NCOAT were detected by both antibodies. These represent various isoforms and modified versions of NCOAT and OGT (Comtesse et al., 2001; Love et al., 2003; Lazarus et al., 2006).
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Protein complexes were immunoprecipitated from chinese hamster ovary (CHO) cells using normal rabbit IgG, -OGT, -NCOAT, and -mSin3A antibodies. The immunoprecipitated complexes and whole-cell lysate (WCL) were analyzed by western blot probed for OGT, NCOAT, and mSin3A (Figure 1A). -OGT complexes contained NCOAT and mSin3A, -NCOAT complexes contained OGT and mSin3A, and -mSin3A complexes contained NCOAT and OGT. These proteins were not detected in the control IgG as expected. The data indicate the in vivo associations of OGT, NCOAT, and mSin3A. Complexes were unaffected by the phosphorylation state of NCOAT (supplemental data, Figure 2).
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In vitro associations between NCOAT and mSin3A were not seen (data not shown); therefore, we mapped the regions of OGT and NCOAT required for the observed in vivo association. By in vitro pull-down assays using GST–OGT fusion proteins (supplemental data, Figure 3) and full-length NCOAT, we found that OGT strongly interacted with NCOAT through the N-terminus and first six tetratricopeptide repeat (TPR) elements of OGT (Figure 1B). This region of OGT was both necessary and sufficient for the interaction. The necessity of the N-terminus is, so far, unique to the OGT interaction with NCOAT, because all other interactions with OGT only require the TPR motifs.
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To conversely map the interaction domain (ID) in NCOAT, we incubated GST–OGT with various length deletion constructs of NCOAT (supplemental data, Figure 2) (Figures 1C and D). The region in NCOAT encompassing amino acids 404–548 [NCOAT(ID)] was found to be necessary, and amino acids 336–548 [NCOAT(ID+)] were sufficient for the interaction. Interactions across species were tested, showing that NCOAT and NCOAT(ID+) from humans, rats, and mice interchangeably interacted with rat OGT (Figure 1D). A summary of the association between NCOAT and OGT along with their catalytic domains is shown in Figure 1E.
To demonstrate the strength of association between NCOAT and OGT, we compared in vitro pull-downs with transcription factors HDAC1, N-CoR, SMRT, and p53 to OGT and NCOAT. [35S]methionine-labeled proteins were incubated with equal amounts of GST–OGT, GST–NCOAT, GST–NCOAT(ID+), and GST alone (Figure 1F). All three corepressors were able to interact with GST–OGT and GST–NCOAT(ID+) but not with GST alone. HDAC1 showed a stronger interaction with GST–NCOAT than with GST–OGT. SMRT had a stronger interaction with GST–NCOAT(ID+) than either full-length proteins. Although p53 can be modified with O-GlcNAc (Shaw et al., 1996), it did not interact directly with any of the proteins. The interaction of NCOAT with itself is likely representative of intramolecular interactions between the termini (data not shown). Notably, the association between NCOAT and OGT was the strongest demonstrated by the ratio of input to pulled-out protein with equal exposure. Most of the labeled OGT or NCOAT was pulled out by its opposite, emphasizing a strong and direct association between them.
To test the associations of naturally occurring NCOAT splice variants (Toleman et al., 2004) with OGT and corepressors, BSC40 cells were transfected with tagged NCOAT constructs for these variants (Figure 1G). Transfection allowed us to control which variant was interacting and the dependence on the OGT/NCOAT interaction for binding with corepressors. Cells transfected with both HA–OGT and GST–NCOAT demonstrated strong association regardless of the splice variants of NCOAT used in the GST–NCOAT construct. These variants all contained NCOAT(ID); however, the SD variant does not contain amino acids 336–403 found in NCOAT(ID+). When the complex was precipitated via endogenous mSin3A, the transfected HA–OGT and GST–NCOAT were detected. Significantly, OGT interacted with endogenous mSin3A (Yang et al., 2002), but the interaction was much more efficient if NCOAT was present. NCOAT also interacted with endogenous mSin3A despite the lack of direct in vitro association. When more OGT was present, the association was more efficient. This preference of binding the NCOAT/OGT complex was especially notable with the GK variant, which is inactive as an O-GlcNAcase (Toleman et al., 2004). The need for both NCOAT and OGT proteins, constituting the O-GlcNAczyme, for stable complex formation with mSin3A is evidence for the specificity of the interactions and aids in complete and correct corepressor complex formation.
NCOAT and OGT locate to repressed promoters
It was previously shown that mSin3A associates with repressed promoters along with a higher level of O-GlcNAc as compared with activated promoters (Yang et al., 2002). We performed chromatin immunoprecipitation (ChIP) assays to identify the recruitment of both OGT and NCOAT, as a complex, to promoters. Cell lysates from MCF7 cells, and MCF7 cells transfected with HA–OGT and Flag–NCOAT were collected for western blot analysis or ChIP assays (Shang et al., 2000). Western blot analysis showed Flag–NCOAT expression as well as OGT (data not shown) and mSin3A associations (Figure 2A). ChIP assay for cathepsin D (CatD), PS2, and p21 gene promoters were immunoprecipitated using the indicated antibodies to endogenous or tagged proteins showing association with the target proteins (Figure 2B). The promoters of the two estradiol (E2)-responsive genes, CatD and PS2, showed an inverse E2-dependent association with these targets. However, the association with the E2-independent gene, p21, was not significantly effected by E2. These results confirmed earlier experiments showing that, in the absence of estrogen, repressed promoters are hyper-O-GlcNAcylated and that this modification is removed from these promoters with the addition of E2 (Yang et al., 2002). Furthermore, the enzyme responsible for the addition of this modification (OGT) is resident on the repressed promoters along with other corepressors. Primers against a downstream section of the PS2 gene showed no association with the corepressors OGT, mSin3A, and HDAC1 and showed a sustained level of O-GlcNAc modification independent of E2.
As expected, based on corepressor associations, rather than associating with the activated promoters, NCOAT was among the proteins on the repressed promoters. It responded to E2 in a similar fashion to that of OGT, mSin3A, and HDAC1. The control primers against a downstream section of the PS2 gene showed a slight increase in associated Flag–NCOAT, but not endogenous NCOAT, with the addition of E2. These observations confirm that NCOAT specifically associates with repressed promoters rather than with activated promoters.
NCOAT activities should reverse the actions of its partners, OGT and HDAC1, but is found in a region of high-protein O-GlcNAcylation, suggesting that NCOAT activity is off during repression. NCOAT, the only known protein with the needed O-GlcNAcase activity to yield the decrease seen on activated promoters, must be specifically activated at these promoters in response to E2 and likely acetylates neighboring histones (Toleman et al., 2004), before its exit from, and the activation of, the promoter. The fact that the O-GlcNAcase activity of NCOAT must be suppressed on repressed promoters implies that for O-GlcNAcylation, there is no futile cycle.
NCOAT can recruit corepressors to repress transcription
Although NCOAT has the requisite enzyme activity for reversing the hyper-O-GlcNAcylation of proteins seen on repressed promoters, we have provided compelling evidence that NCOAT is itself associated with repressed promoters as a component of the O-GlcNAczyme. The question, therefore, remained as to whether NCOAT could reverse repression by undoing this modification or whether its dominant feature was to nucleate the corepressor complexes. To determine which property of NCOAT dominated, activation by removal of O-GlcNAcylation or repression through association with corepressors, the protein was fused to the Gal4 DNA-binding domain (Gal4-DBD) and cotransfected along with a Gal4–luciferase reporter plasmid (Figure 3A). Putting NCOAT on this synthetic promoter caused a dose-dependent repression, rather than activation, of the reporter. The amount of Gal4–NCOAT, determined by western blot analysis, was proportional to the amount of plasmid transfected, whereas mSin3A as a loading control remained the same (Figure 3A, middle panel). The Gal4–GFP fusion had no significant effect on transcription, whereas the Gal4-DBD was mildly stimulatory. Gal4–NCOAT was as potent as Gal4–OGT in repression (Figure 3C).
With previous evidence that the activity of the transcriptional activator, Sp1, can be down-regulated by O-GlcNAcylation (Roos et al., 1997; Yang et al., 2001, 2002), reporter constructs were tested which contained Sp1-binding sites upstream or downstream of the Gal4-binding sites. These constructs would allow Sp1 to be modified and thus tests a specifically activated promoter rather than just a basal promoter. Again, repression with Gal4–NCOAT was observed regardless of the position of the Sp1-binding sites relative to the Gal4 sites (Figure 3B).
This repression by Gal4–NCOAT was additive with OGT (Figure 3C). Even when catalytically inactive OGT (1–286) was expressed, some additivity remained because this fragment of OGT could recruit both NCOAT (Figure 1E) and other corepressors, including wild-type OGT, to the promoter (Yang et al., 2002). When OGT was tethered to the DNA using the Gal4-DBD, free NCOAT gave additional repression (Figure 3C, lanes 8 and 9) but only if NCOAT could interact with OGT (Figure 3C, lanes 10 and 11). A deletion of NCOAT that prevented interaction with OGT despite maintaining potential enzymatic activities (Toleman et al., 2004) (Figure 3C, lane 12) was unable to repress or activate basal transcription activity. Thus, the dominant feature of NCOAT in this assay was transcriptional repression through its association with OGT and repression complexes. The data suggest that NCOAT is not a classical activator, but rather exists as a member of the repression complex, poised to derepress as needed.
A modified ChIP assay was preformed to confirm that the transcription repression by the O-GlcNAczyme results from the recruitment of corepressors to the DNA of the reporter, as can be done by OGT alone (Yang et al., 2002). MCF7 cells were transfected with a Gal4–luc reporter (Figure 3A) and plasmids encoding Gal4–DBD fused to mSin3A, NCOAT, or OGT proteins. Detection of the immunoprecipitated DNA was by Southern blot probed for the presence of the luciferase reporter (Figure 3D). The -Gal4–DBD precipitated significant amounts of cross-linked DNA in all samples containing transfected DBD and DBD fusions. A significant difference in the ability of -N-CoR to precipitate reporter DNA with endogenous N-CoR was only seen when the transfected fusion proteins, Gal4–mSin3A, Gal4–NCOAT, and Gal4–OGT, were present. These results confirm previous reports of an association between N-CoR and mSin3A (Heinzel et al., 1997; Nagy et al., 1997; Wong and Privalsky, 1998).
The ability of both Gal4–NCOAT and Gal4–OGT to nucleate endogenous corepressors like N-CoR to the DNA explains the observed repression with both fusion proteins. This result implies that NCOAT is not a passive transcription activator, but rather that its enzymes must be specifically activated to reverse repression.
Regulating E2 response with O-GlcNAc
NCOAT(ID+) alone is responsible for the binding of NCOAT to OGT. To test whether the binding is saturable, [35S]-labeled NCOAT(wt) and NCOAT(ID+) were incubated with GST–OGT on beads in differing ratios where the amount of NCOAT(wt) and GST–OGT remained constant. The inputs show how much protein is used in the assay and are representative of 1 on the ratios given in the panel. In the presence of an excess of NCOAT(ID+), wild-type binding to GST–OGT was competed away (Figure 4A), indicating that NCOAT(ID+) is sufficient to compete NCOAT(wt) from OGT. NCOAT(ID+) has no enzymatic activities, and OGT/NCOAT binding is qualitatively saturable. Therefore, NCOAT(ID+) would act as a location-dominant negative by disrupting endogenous NCOAT association into corepressor complexes rather than a classical dominant negative which would disrupt enzymatic activity.
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The O-GlcNAczyme, formed by the interaction between OGT and NCOAT, binds corepressors better than the respective enzymes alone. Furthermore, NCOAT(ID+) binds these proteins even better than NCOAT(wt) (Figure 1G). The lack of enzymatic activities coupled with the greater interactions with corepressors would predict that addition of NCOAT(ID+) to OGT would lead to a similar or greater repression than NCOAT(wt). This was tested using the Gal4–luc assay for transcription control (Figure 4B). The NCOAT(ID+) indeed repressed transcription better than NCOAT(wt). The repression with Gal4–OGT and NCOAT(ID+) is in stark contrast to the activation seen when NCOAT(ID+) is expressed along with the Gal4–DBD alone, again highlighting the interaction between OGT and NCOAT(ID+). This result supports the proposed location-dominant negative potential of the NCOAT(ID+) on promoter regulation.
With only one gene for OGT and one gene for NCOAT, knockouts (O’Donnell et al., 2004) and drug-based inhibition are lethal (Liu et al., 2000; Konrad et al., 2002). Furthermore, because the OGT, bound to NCOAT, binds to its accompanying corepressors better than OGT alone, we chose to use a dominant-negative approach to obviate problems inherent with knocking out the expression of NCOAT. Because the binding of NCOAT to OGT is saturable, this approach was possible. We used the ability of the NCOAT(ID+) to inhibit formation of completely active O-GlcNAczyme complexes with NCOAT activities to test its role in the regulation of estrogen-responsive genes. MCF7 cells were transfected with indicated constructs at 
85% transfection efficiency in estrogen-free media. Cells were treated with E2 for the indicated times, 24 h after transfection. Northern blot analysis of the RNA with probes to CatD, CAD (Khan et al., 2003), and glyseraldehyde-3-phosphate dehydrogenase (GAPDH) was performed (Figure 4C). Overexpression of NCOAT(wt) and NCOAT
ID did not repress the initiation of transcription but did have an effect on sustained transcription in the presence of E2. On the contrary, NCOAT(ID+) yielded an almost complete block of the E2-induced transcription of the CatD and CAD genes.
A naturally occurring NCOAT splice variant was cloned from the Goto-Kakizaki rat [NCOAT(GK)] (Toleman et al., 2004). This particular NCOAT variant is able to interact with OGT (Figure 1G) and has HAT activity in vitro but lacks O-GlcNAcase activity (Toleman et al., 2004). Therefore, this variant could be used as a location-dominant negative in the O-GlcNAczyme complex specific for O-GlcNAcase activity. We transfected MCF7 cells with Flag–NCOAT(wt), Flag–NCOAT(GK), or Flag–GFP. Western blot analysis showed significantly higher levels of expression for the GK variant over endogenous NCOAT to yield the theoretical location-dominant negative (Figure 4D). The transfected cells were treated as before to perform ChIP assays. To distinguish between nontransfected and transfected cells, we performed a two-step immunoprecipitation (IP). The cell lysate was incubated with -Flag beads to obtain promoters with Flag associations. Following elution of the bead-bound complexes, a second IP was performed with the indicated antibodies (Figure 4E). Cells transfected with the Flag–NCOAT again showed the E2-dependent promoter occupancy by Flag–NCOAT, and secondary IP indicated the Flag–NCOAT complexes contained OGT and O-GlcNAc which exited with the addition of E2 (Figure 4E, top panel). On the contrary, cells transfected with the GK variant showed stable promoter occupancy in the presence of E2 for Flag–NCOAT(GK), OGT, and O-GlcNAc (Figure 4E, middle panel). These data indicate that NCOAT(GK), lacking only O-GlcNAcase activity, does indeed behave as a location-dominant negative. Interestingly, unlike NCOAT(wt), the Flag–NCOAT(GK) complexes were not found to be associated with the p21 promoter, and ongoing studies hope to elucidate this difference in promoter regulation. As well, Flag–GFP was not associated with any of the tested promoters (Figure 4E, bottom panel).
O-GlcNAcase activity in the O-GlcNAczyme complex is essential for proper estrogen signaling
GFP and GFP–NCOAT(GK) were expressed in MCF7 cells and imaged by fluorescent microscopy (Van Tine et al., 2004). Although expression of GFP–NCOAT(GK) (Figure 5A) shows a predominantly cytosolic location, the presence of NCOAT(GK) on the DNA (Figure 4E) and the nuclear pattern seen with GFP–NCOAT(GK) imply sufficient amounts are in the nucleus to regulate gene expression.
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To test the role of the O-GlcNAcase activity in the O-GlcNAczyme, a conditional transgenic animal model was used. A TetRE–NCOAT(GK) transgenic mouse line was crossed with an MMTV–rtTA transgenic line to yield mammary-specific expression of the NCOAT(GK) splice variant in the presence of doxycycline (Dox) (Roh et al., 2001, 2005; Xie et al., 1999) (Figure 5B). Prior studies have confirmed that this system is effective in dominant-negative expression analysis (Roh et al., 2001).
A bitransgenic MMTV–rtTA/TetRE–LacZ line was shown to express ß-Galactosidase in the ductal epithelia of mammary glands, following Dox induction (data not shown). The MMTV–rtTA and TetRE–NCOAT(GK) mice were bred to result in eight female pups, three of which were bitransgenic. The NCOAT(GK) transgene was activated by administering Dox at 3 weeks of age and treatment continued until the animals were sacrificed at 8 weeks of age, several weeks beyond onset of puberty. Northern blot analysis confirmed transgene mRNA expression (Figure 5C). Whole-mount analysis demonstrated reduced mammary ductal side branching in comparison with littermate controls (Figure 5D [1 and 2]). These mice also demonstrated reduced end buds (Figure 5D [3 and 4]), and H & E-stained sections of these glands showed less ducts (Figure 5D [5 and 6]). Immunohistochemical examination of mammary ducts demonstrated reduced expression of CatD (red) in bitransgenic females receiving Dox in comparison with controls (Figure 5D [7–12]) consistent with the results shown in Figure 4C. Transgenic animals are very complex, making individual relationships hard to decipher. Although activation of one E2-dependent gene was significantly inhibited, this inhibition could have resulted from the nonpromoter-specific effects NCOAT(GK) could play on hormone signaling (Nadal et al., 2001). However, the absence of O-GlcNAcase activity in the O-GlcNAczyme does demonstrate an in vivo location-dominant-negative phenotype, consistent with the previous data, blocking hormone-dependent signaling and E2-induced gene activation.
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OGT, which modifies protein with O-GlcNAc, directly associates with NCOAT, which reverses the modification with its O-GlcNAcase domain, both in vivo and in vitro. NCOAT also has a HAT (Toleman et al., 2004
) domain putting it in the same complex as an HDAC, reversing at least part of the acetylation state of histone tails. This association of opposite enzymes is unusual in biology, because, on the surface, it would set up futile cycles for both O-GlcNAcylation and histone acetylation. However, both enzymatic activities can be modified in NCOAT (Toleman et al., 2004) in vitro raising the potential that the OGT and O-GlcNAcase and the HDAC and HAT activities are not on simultaneously. Furthermore, the ChIP assays reveal that on repressed DNA, there is detectably more O-GlcNAc than on activated DNA. This observation implies that during repression, at least the OGT dominates the O-GlcNAcase activity, and during gene activation, this situation is reversed. Thus, in vivo, with natural levels as well as with overexpression of the enzymes, there is no O-GlcNAc futile cycle.
Although, the targets of OGT were not addressed here, we have shown previously that O-GlcNAc modification of at least one of the activation domains of the ubiquitous transcription activator, Sp1, makes it incapable of activating transcription (Yang et al., 2001). However, other sites of O-GlcNAcylation on Sp1 could activate this transcription factor in different cells and on different promoters (Goldberg et al., 2006). Also, the tail of RNA polymerase II itself is modified by OGT at phosphorylation sites, and the cycling of this enzyme may require O-GlcNAc as one of the modifications (Comer and Hart, 2001). Regardless of whether GC boxes for Sp1 binding are present, basal transcription is repressed by OGT that is recruited to the promoter (Yang et al., 2002). Removal of this modification from Sp1, RNA polymerase II, and perhaps other proteins (Jiang and Hart, 1997) by the O-GlcNAcase domain of NCOAT would thus be required to permit a reversal of gene repression. Residing in the repression complex through association with OGT and other proteins, NCOAT is in the correct place to reverse the repressive modification by OGT. Furthermore, NCOAT may stabilize the structure of the repression complex. When combined with OGT, the two proteins bind the corepressor molecules better than OGT alone and this may apply to other complex associations as well. In addition, the presence of both domains in one protein, NCOAT, lends further credence to the role of O-GlcNAc in complementing HDAC-mediated gene repression.
For a promoter to transition from repression to activation requires the recruitment of signaling posttranslational modifiers (Bjorklund et al., 1999; Perissi et al., 2004), although the substrates of these modifiers have not yet been described. However, the derepressor NCOAT, by residing in corepression complexes, does not itself require recruitment like these other modifiers. Rather, NCOAT, which is strategically preplaced so that gene derepression can occur rapidly, must be posttranslationally modified so that its enzymatic activities can be activated. When activation of both NCOAT enzymes cannot occur, gene transition from repression to activation cannot occur in response to the hormone. In particular, NCOAT contains the only known O-GlcNAcase encoded in the genome. When it is replaced in the repression complex with a splice variant of NCOAT putatively lacking only the O-GlcNAcase activity, there is a failure to remove the O-GlcNAc modification. In association with this failure to remove the O-GlcNAc modification on the genes, an estrogen-responsive gene in breast ducts is not activated during puberty. Other genes in the mammary gland must be off as well, because the breast ducts do not develop properly in transgenic mice expressing this same splice variant. Although wild-type NCOAT is expressed at normal levels, it is ineffective at overcoming the dominant-negative NCOAT. This observation implies that free NCOAT, not directed to the correct location through OGT association, is ineffective in the system. This also suggests that the removal of this O-GlcNAc modification by NCOAT is required to allow this transition to activation by hormone-responsive genes.
The O-GlcNAcase of NCOAT has other functions. Relevant to gene activation, its O-GlcNAcase activates the adenosine triphosphatase (ATPase) of the 19S cap of the proteasomes (Zhang et al., 2003). The active ATPases are required by the proteasomes-allowing corepressors to be vectored to their destruction (Li et al., 2003). The local degradation by the proteasome of these repressor proteins, by decreasing their concentrations, could also contribute to gene activation. Although the NCOAT enzymes can be activated (Toleman et al., 2004), probably by posttranslational modification, the details of how they are activated remain unknown at this time. The epitope masking of the initial state-specific NCOAT antibody that can be relieved by phosphatases implies that at least phosphorylation is one modification. We speculate that these phosphorylations of NCOAT may at least be part of the signal that activates the enzymes of NCOAT. Whether phosphorylations control the localization of NCOAT remains to be determined, but the antibody studies indicate that NCOAT’s association with OGT and other corepressors is not affected by the phosphorylation blocking the antibody. Nevertheless, the involvement of the O-GlcNAczyme, composed of OGT and NCOAT, in genes with estrogen receptors imparts the necessary properties of reversibility, rapidity, and thoroughness required for homeostasis and suggests that the O-GlcNAc modification is more central to gene regulation than simply providing a subtle nutritional input.
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Plasmids
Mammalian expression plasmids pCMX–mSin3A, pCMX–Flag–SMRT, and pCMX–Flag–N-CoR were generously provided by R. Evans. pcDNA3–Flag–HDAC1 was kindly provided by E. Seto. pcDNA3–OGT and G5–luc reporter constructs were described previously (Yang et al., 2001
). pcDNA3.1–HA3–OGT and Sp1–G5–luc reporter constructs were described previously (Yang et al., 2002). pcDNA3.1–NCOAT was provided by G. Hart. 3xFlag–TEV–2xMyc tag was a kind gift from D. Crawford. The 3xFlag–TEV–2xMyc tag was moved to pcDNA3.1–NCOAT to create the Flag–NCOAT expression vector. For protein expression in mammalian cells, the indicated mutants were subcloned into pcDNA3.1 (Invitrogen, Carlsbad, CA), whereas pGEX-2T (Amersham Biosciences Corp., Piscataway, NJ) was used for GST fusion protein expression in E. coli.
Cell culture and transient transfection
Cells were maintained under standard conditions. MCF7 cells were estrogen deprived in Phenol Red-free media supplemented with 10% charcoal-stripped fetal bovine serum (FBS). Transient transfection of HepG2 cells was performed by electroporation (Yang et al., 2001). Luciferase activities were assayed 18–24 h later. Transfection efficiencies were normalized using a cotransfected ß-galactosidase plasmid. Each transfection was done in triplicate and repeated 3–5 times. Transient transfection of BSC40 cells was by electroporation, followed by infection with vTF7 (Moss et al., 1990) to induce protein expression. Cells were harvested 12–18 h later for protein purification. MCF7 cells were transfected with Lipofectamine 2000 (Invitrogen).
GST pull-down assays and coimmunoprecipitation assay
GST pull-downs and coimmunoprecipitations were performed as previously described (Yang et al., 2002). Precipitation was carried out using ANTI-FLAG M2 affinity Gel (Sigma A2220, St. Louis, MO) or antibody followed by 1:1 protein A: protein G beads (Amersham). Antibodies used were -HA (Roche 12CA5, Indianapolis, IN), -mSin3A (Santa Cruz K-20, Santa Cruz, CA), -GST (Sigma G1160), -Flag (Sigma F3165), -N-CoR (Santa Cruz N-19), -OGT (provided by G. W. Hart; Kreppel et al., 1997), and -NCOAT (raised in rabbits against the intact protein and affinity purified as previously described; Shin et al., 1992). Phosphatase cocktail contained equal units of protein phosphatase 1- (Sigma), protein phosphatase 1-
(Calbiochem, SanDiego, CA), and protein phosphatase 2A (Upstate, Charlottesville, VA). Treatment consisted of a 30 min incubation at 30°C in low stringency binding buffer (LSB).
Chromatin immunoprecipitation
MCF-7 cells transfected using Lipofectamine 2000 (Invitrogen) were treated with 100 nM 17ß-estradiol (Sigma) for 2 h. Cells were processed as previously described (Yang et al., 2002). Where indicated, DNA quantity was determined by Southern blot analysis. Linearity of polymerase chain reaction (PCR) amplification for the indicated genes was demonstrated by serial 3-fold dilutions of the input DNA.
Northern/Southern blot analysis
Northern and Southern blot analyses were performed as previously described (Roh et al., 2001). Hybond-N+ nylon membrane (Amersham Pharmacia Biotech) was used as described in the manufacturer’s protocol. The membranes were probed using 32P-labeled probes as in the figure.
ß-Galactosidase staining of mammary tissue
Confirmation of rtTA transgene expression was carried out by breeding the three transgenic founder lines with a tetracycline-responsive lacZ transgenic mouse line (tetOLacZ) obtained from Dr J. Segre. Mating partners, each containing the transgenes for MMTV–rtTA and tetOLacZ gave rise to mice consisting of a mixture of genotypes. Postpubertal mice containing both transgenes were fed Diet-Dox (2 mg/mL Dox [Sigma-Aldrich] containing 1 mL "Sweet’N Low" per 100 mL drinking water) for 3 days at which point, they were sacrificed and the mammary tissue was collected and processed by established methods.
Mammary gland whole mounts and tissue section immunofluorescence
Inguinal and thoracic mammary glands were dissected from mice at 8 weeks of age and prepared as previously described (Bowe et al., 2002). Immunostaining was performed using rabbit anticathepsin D 1:500 (Santa Cruz Biotechnology) followed by chicken antirabbit Alexa 594 at 1:1000 (Molecular Probes, Carlsbad, CA). Nuclei were counterstained with DAPI (Sigma) and mounted using Vectasheild (Vector Laboratories, Burlingame, CA). Normal goat IgG (Santa Cruz) was used as a negative control (data not shown). Sections were analyzed using fluorescence Leica (Nussloch, Germany) DMRB microscope with image captured by a Leica DFC480 camera and standard filter set.
Generation of TetRE–NCOAT(GK) transgenic mice
The TetRE–NCOAT(GK) transgenic mouse line was constructed as a chimera of the "short GK" NCOAT sequence (350–1110) inserted into the corresponding mouse sequence (350–1400) using unique BlpI–NcoI restriction sites. The cDNA for the mouse brain NCOAT was cloned as described above for the GK rat. The full-length 2.75 bp PCR product was cloned into the TetRE–SV40 transgene cassette used previously (Roh et al., 2001) into XbaI sites. A 5'-UTR sequence was synthesized from a mouse brain cDNA library using the First Choice 5' RLM-RACE kit from Ambion (Austin, TX). The 5' oligo included a BamHI restriction site in the "nested" oligo and the 3' antisense oligo was designed to anneal to mNCOAT, 350 bases downstream from the ATG; 5'-CATGGTACCTCGTGCAGCAGAGATCAGAGTC-3'. The resulting 550-bp PCR fragment was digested with BamHI/BlpI and inserted between these same sites in the TetRE–mNCOAT transgene, providing a 5'UTR of about 100 bp. The 760-bp GK sequence was inserted into the mouse sequence to complete the chimeric transgene which was then excised from the plasmid with SphI–ClaI and microinjected into BL/6xSJL mouse zygotes as described above. Identification of four founders out of 22 mice was performed as above using oligonucleotides to TetRE (Roh et al., 2001) and the reverse primer for NCOAT (5'-AAG TTG CTC AGC TTC TTC CAC TG-3'), resulting in a 460-bp PCR fragment. Northern blot analysis showed good expression of the NCOAT(GK) transgene transcript.
Generation of MMTV–rtTA transgenic mice
The MMTV-ß-globin transgene cassette, originally provided to us by Dr H.L. Moses (Pierce et al., 1993), has been described earlier in the construction of the MMTV–mEGFtr transgene (Xie et al., 1997). The 1 kb rtTA sequence (Clontech, Palo Alto, CA) was inserted into the EcoRI site of ß-globin exon 3, replacing EGFtr. The complete 3.7 kb XhoI transgene fragment was purified by electrophoresis on 1% agarose, elution, and extraction using the QIAquick Gel Extraction Kit (Qiagen, Valencia, CA). The transgene DNA was microinjected into one-cell BL/6xSJL mouse zygotes at a concentration of 2 ng/µL at the University of Alabama at Birmingham Transgenic animal facility under the direction of Dr Carl Pinkert. Twenty-nine potential transgenic founders were derived and their tail DNA was isolated as previously described (McAndrew et al., 1995). Gene-positive animals were determined by PCR analysis using oligonucleotides designed to anneal to the MMTV sense sequence, 5'-TGCAACAGTCCTAACATTCA-3', and rtTA antisense sequence, 5'-TGAATGTTAGGACTGTTGCA-3'. A DNA thermal cycler (PE Applied Biosystems GeneAmp pcr System 9700) was used under the following conditions for 36 cycles: 94°C for 30 sec, 55°C for 45 sec, and 72°C for 45 sec. Three gene-positive founder mice were identified as a 1345 bp PCR product on 1% agarose. Expression of rtTA was determined by northern blot analysis of isolated MMTV-targeted tissues. Two founder lines (4–1 and 6–2) gave appropriately sized message (1.7–1.8 kb) corresponding to the ß-globin and the rtTA sequences.
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Supplementary data |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary dataConflicts of interest statementAcknowledgmentsReferences |
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Supplementary data are available at Glycobiology online (http://glycob.oxfordjournals.org/).
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Conflicts of interest statement |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary dataConflicts of interest statementAcknowledgmentsReferences |
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None declared.
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Acknowledgments |
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TopAbstractIntroductionResultsDiscussionMaterials and methodsSupplementary dataConflicts of interest statementAcknowledgmentsReferences |
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This work was supported by NIH and the Ruth Lawson Hanson endowment to J.E.K. and the UAB Comprehensive Cancer Center.
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Abbreviations |
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ChIP, chromatin immunoprecipitation; CHO, Chinese hamster ovary; E2, estradiol; Gal4-DBD, Gal4 DNA-binding domain; HAT, histone acetyltransferase; HDAC, histone deacetylase; ID, interaction domain; IP, immunoprecipitation; LSB, low stringency binding buffer; NCOAT, nuclear/cytoplasmic O-GlcNAcase and acetyl transferase; OGT, O-GlcNAc transferase; PCR, polymerase chain reaction
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