YY1 Is Regulated by O-Linked N-Acetylglucosaminylation (O-GlcNAcylation)*

Makoto HiromuraDagger , Chu H. ChoiDagger , Nicaulas A. SabourinDagger , Heath JonesDagger , Dimcho Bachvarov§, and Anny UshevaDagger

From the Dagger  Beth Israel Deaconess Medical Center, Department of Medicine, Endocrinology, Harvard Medical School, Boston, Massachussetts 02215 and the § Centre Hospitalier Universitaire de Québec (CHUQ)-Centre de Recherche, Hopital L'Hôtel-Dieu de Québec et Université Laval, Québec G1R 2J6, Canada

Received for publication, January 23, 2003, and in revised form, February 12, 2003

    ABSTRACT
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INTRODUCTION
MATERIALS AND METHODS
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YY1 is a zinc finger DNA-binding transcription factor that influences expression of a wide variety of cellular and viral genes. YY1 is essential for the development of mammalian embryos. It regulates the expression of genes with important functions in DNA replication, protein synthesis, and cellular response to external stimuli during cell growth and differentiation. How YY1 accomplishes such a variety of functions is unknown. Here, we show that a subset of the nuclear YY1 appears to be O-GlcNAcylated regardless of the differentiation status of the cells. We found that glucose strongly stimulates O-linked N-acetylglucosaminylation (O-GlcNAcylation) on YY1. Glycosylated YY1 no longer binds the retinoblastoma protein (Rb). Upon dissociation from Rb, the glycosylated YY1 is free to bind DNA. The ability of the O-glycosylation on YY1 to disrupt the complex with Rb leads us to propose that O-glycosylation might have a profound effect on cell cycle transitions that regulate the YY1-Rb heterodimerization and promote the activity of YY1. Our observations provide strong evidence that YY1-regulated transcription is very likely connected to the pathway of glucose metabolism that culminates in the O-GlcNAcylation on YY1, changing its function in transcription.

    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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DISCUSSION
REFERENCES

YY1 is a zinc finger DNA-binding transcription factor that influences the expression of a wide variety of cellular and viral genes (1-4). It can function as an activator or as a repressor, depending on the context of its binding site within a promoter, its relative concentration, and the presence of other cellular factors (2). It acts as a transcriptional initiator when bound at the initiator element of the adeno-associated virus P5 promoter (5, 6). Deletion of the murine YY1 gene results in embryonic lethality, suggesting an essential function for YY1 in development (7). YY1 is highly conserved in vertebrates, and a functional homolog was found in Drosophila melanogaster (8).

YY1 participates in checkpoint functions that regulate cell cycle transitions by physical and functional association with the retinoblastoma protein (Rb)1 (9). Both PCAF and p300 interact and acetylate the protein in HeLa cells (10). YY1 interacts with the basic transcription factors TBP, TFIIB, and RNA polymerase II (5, 6). It also interacts with transcription regulators such as Sp1 (11, 12) and E2Fs (13), regulating the expression of genes with important functions in DNA replication, protein synthesis, and cellular response to external stimuli (14). Under conditions of cellular DNA damage, YY1 was found in a complex with the nuclear enzyme poly(ADP-ribose) polymerase 1 (PARP-1), stimulating the ADP-ribosylation activity of the enzyme in DNA repair (15). Exactly how YY1 accomplishes such a variety of functions is unknown.

Post-translational YY1 modifications could potentially change the YY1 functions in transcription by altering protein stability and binding affinity to DNA and switching protein partners. It has been found that, in HeLa cells, acetylation and deacetylation might regulate the YY1-DNA interaction through a complex mechanism (10). Phosphorylation on YY1 was also reported; however, its role in cellular YY1 functions was not further investigated (16). The carbohydrate addition known as O-linked N-acetylglucosaminylation (O-GlcNAc modification or O-GlcNAcylation) has been observed lately in several proteins with nuclear localization (17). O-GlcNAc is formed by the enzymatic addition of N-acetylglucosamine (GlcNAc) to serine and threonine residues by nucleocytoplasmic glycosyltransferase (18, 19). This reversible post-translational modification (20) is present in a variety of proteins, including numerous chromatin-associated proteins and several transcription factors (21). The ubiquitous transcription factors Sp1 (22, 23), members of the AP-1 family (22), P53 (24), the serum response factor (SRF) (25), the estrogen receptor (ER), Pax-6 (26), and c-Myc (27) all carry O-GlcNAc residues. A subset of the nuclear RNA polymerase II itself is also O-GlcNAc-modified (28) at the carboxyl-terminal moiety (CTD) of the largest subunit. O-GlcNAcylation is highly dynamic, with rapid cycling in response to cellular signals or cellular stages. It was found to be reciprocal to phosphorylation on some well studied phosphoproteins, including the CTD of RNA polymerase II. Although the precise function of O-GlcNAc is not exactly determined, recent data suggest a wide variety of key cellular events, including gene transcription (21).

In this study, we demonstrate that a subset of the nuclear YY1 appears to be O-GlcNAcylated regardless of the differentiation status of the cells. We show that the amount of O-glycosylated YY1 changes in response to external stimuli. We have determined that high glucose stimulates the O-GlcNAc on YY1 in cells. In glucose-stimulated cultures, the O-glycosylated YY1 is mostly free of Rb. The YY1-Rb complex is significantly more abundant in glucose-deprived cultures where YY1 is mostly not O-glycosylated. We observed a correlation between the activated YY1 O-glycosylation and the formation of DNA-YY1 complexes in nuclear extracts. The results led us to propose that O-GlcNAc on YY1 favors dissociation with Rb and association with consensus DNA promoter elements that may repress or activate gene expression depending on the context of its binding site within a promoter. The dynamic and reversible O-GlcNAcylation further expands the possible molecular and functional range of YY1.

    MATERIALS AND METHODS
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Cell Lines-- Primary human coronary artery smooth muscle cells (CASMC) (Clonetics) were grown as recommended by the supplier.

Protein Purification and Nuclear Extracts Isolation and Manipulation-- Recombinant YY1 was purified from Escherichia coli (6). HeLa YY1 was purified from HeLa nuclear extract as described previously except that the pooled DNA affinity column fractions were next loaded onto a wheat germ agglutinin (WGA)-agarose (Vector) matrix and eluted from this matrix at pH 3 in the presence of 1 M GlcNAc as recommended by the supplier. The pooled WGA fractions were applied onto Superose 12 support (Amersham Biosciences) in 6 M guanidine HCl, pH 6. The purified guanidine HCl-denatured YY1 was next folded as described previously. The molecular mass of YY1 was determined by protein chip tandem mass spectroscopy (Ciphergen). Rb was purified from Sf9 cells (29). To test for a YY1-Rb interaction in solution, Rb (50 µg) and YY1 (30 µg) were incubated alone or in a mixture for 30 min at 22 °C and then separated by gel filtration on Superose 6 (Amersham Biosciences) as described (9). Chymotrypsinogen A (18 kDa), bovine serum albumin (68 kDa), and myosin (206 kDa) were used as markers. To test for a YY1-Rb interaction in cells, nuclear extracts were prepared (30) from cells that were growth-arrested by serum starvation for 48 h in 30 mM of glucose or from cells that were growth-arrested in the absence of glucose. YY1, Rb, and control pre-immune IgG immunoaffinity matrices were prepared and used as described (9). For Western blot assays, YY1 was localized using a rabbit polyclonal and mouse monoclonal antibody, Rb was identified with a monoclonal antibody to Rb (a gift from E. Harlow, Harvard Medical School), and TFIIB was monitored with rabbit polyclonal antibody.

Galactosyltransferase Assay-- The assay was conducted as described previously (31, 32).

beta -Elimination Reaction-- [3H]galactose-labeled YY1 or protein fractions were mixed and incubated with 0.1 M NaOH at 22 °C for no longer than 6 h. Incubation longer than 6 h was avoided because of strong YY1 degradation.

Peptide N-Glycosidase F (PNGase F) Digestion-- [3H]galactose-labeled protein samples were systematically digested with PNGase F (BioLabs) according to the manufacturer's recommendations overnight at 37 °C.

Gel Shift Assays-- The gel shift assays and in vitro transcription reactions were performed as described previously (6). Synthetic oligonucleotides were purchased from Oligos Etc. Inc.

    RESULTS
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INTRODUCTION
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RESULTS
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Labeling of YY1 with Galactosyl Transferase in Vitro-- The apparent molecular weight of the major cellular YY1 variant is greater than that of a peptide encoded by the open reading frame of the human YY1. Similar observations were reported for the O-GlcNAcylated forms of other proteins with functions in transcription, such as Sp1, Pax-6, and c-Myc. Accordingly, we tested for the presence of glycosylated nuclear YY1, applying in vitro labeling procedure with bovine galactosyl transferase (GTase). This enzyme catalyzes the transfer of galactose from UDP-[3H]galactose to glycoproteins with accessible terminal residue (32), creating a covalently bound radioactive marker for the presence of GlcNAc (Fig. 1A). When affinity purified YY1 from HeLa cells (Fig. 1B, lane 1) or smooth muscle cell nuclear extract (Fig. 1B, lane 3) were incubated with galactosyl transferase and UDP-[3H]galactose and then analyzed by SDS-PAGE followed by fluorography, a 3H-labeled product migrating at the position of YY1 was observed (Fig. 1C, lanes 1 and 3). In a Western blot, the migration position of the 3H-labeled product was found to overlap with the position of the immunologically reactive YY1 in both HeLa-purified YY1 and nuclear extract from smooth muscle cells (Fig. 1D, lanes 1 and 3). In contrast, the bacterially produced recombinant His6-YY1 (Fig. 1D, lane 2) and the majority of proteins present in the extract from smooth muscle cells (Fig. 1B, lane 3) were not labeled (Fig. 1C, lanes 2 and 3).


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Fig. 1.   Cellular YY1 is a galactosyl transferase substrate. A, schematic representation of the enzymatic [3H]galactose transfer from UDP-[3H]galactose to YY1 Ser/Thr hydroxyl groups with GlcNAc residues. B, silver-stained 10% SDS gel containing 1 µg of HeLa-purified YY1 (lane 1), 0.4 µg of purified E. coli-expressed human YY1 (lane 2), and 40 µg of crude nuclear extract from smooth muscle cells growth arrested in high glucose (lane 3). C, after labeling with [3H]galactose, aliquots of the reactions with HeLa YY1 (lane 1), E. coli YY1 (lane 2), and smooth muscle cells nuclear extract (lane 3) were separated on 10% SDS gels and subjected to fluorography. D, after labeling, aliquots of the reactions were loaded in the same order as in B and C, separated on 10% SDS gels, and analyzed for YY1 content by Western blot with anti-YY1 antibody (alpha YY1). The identity of the analyzed probes is indicated at the top of the lanes. E, YY1 affinity-capturing after [3H]galactose-labeling reaction with nuclear extracts from smooth muscle cells. After 60 min of [3H]galactose labeling, nuclear extracts (300 µg of protein) from growth-arrested CASMC in high glucose were mixed and incubated with an affinity matrix containing covalently attached monoclonal antibody to YY1. Captured proteins were analyzed by fluorography and Western blot with alpha YY1 (identified at the top of the panel). Samples are as follows: 30% of input (IN) (lanes 1 and 5); not bound (NB) (lanes 2 and 6); bound and eluted by boiling in buffer containing 1% SDS (lanes 3 and 7); and captured with pre-immune mouse polyclonal antibody (Pre) (lanes 4 and 8). The migration of the molecular markers is indicated to the right of the panels.

To confirm 3H-labeling of YY1, we performed YY1-immuno affinity capturing (Fig. 1E). [3H]galactose-labeled nuclear extract (NE) from human CASMC was incubated with YY1-specific IgG covalently coupled to activated Sepharose beads. The unbound and the captured products were visualized by fluorography and Western blot with YY1-specific antibodies and HCL reaction. We observed that the YY1 affinity beads specifically captured 3H-labeled YY1 (Fig. 1E, lanes 3 and 7). Less than 2% of the initial 3H-labeled YY1 amount in the labeled NE was found to bind to the control matrix with pre-immune rabbit IgG (Fig. 1E, lanes 4 and 8).

The results strongly suggest that the nuclear YY1 carries covalently attached terminal GlcNAc. Determining by mass spectroscopy the amount of [3H]galactose incorporated into a known quantity of purified HeLa YY1 and assuming that each molecule of the present YY1 was a substrate for labeling, we have estimated that each YY1 molecule in the HeLa purified YY1 carries, on average, approximately two GlcNAc moieties.

YY1 Is Specifically Recognized by WGA in Cellular Nuclear Extracts-- To confirm that YY1 is glycosylated, we employed a WGA-capturing experiment. Lectins have been used to isolate and characterize transcription factors based on their glycosylation profile (34). For instance, the O-GlcNAc-modified transcription factor Sp1 has been isolated and detected by its association with WGA, which is dependent on the presence of beta -O-GlcNAc groups (35). Association with WGA has also been used to detect O-GlcNAc modification on the largest subunit of polymerase II (28). To test whether this lectin would bind cellular YY1, WGA affinity beads were mixed together with a fraction of smooth muscle cell nuclear extract in the presence of 0.5 M NaCl. After removing the unbound fraction, the specifically captured proteins were eluted with 1 M GlcNAc and 0.5% SDS under reducing conditions (22, 28). Bound proteins were then visualized by Western blot with YY1-specific antibodies (Fig. 2). YY1 was present in the WGA-captured fraction even after stringent washing with salt (Fig. 2A, lane 2). Preincubation of the YY1 fraction with different concentrations of GlcNAc before adding it to the affinity matrix reduced the amount of captured YY1 4-6-fold (Fig. 2A, lanes 3 and 4). No detectable YY1 retention was observed on a control Sepharose matrix lacking WGA (Fig. 2A, lane 5). Preincubation of the YY1 fraction with WGA also reduced significantly the amount of WGA-affinity matrix-captured YY1 (Fig. 2B, lane 3), therefore confirming the WGA matrix-glycoprotein retention specificity. The lectin concanavalin A (ConA) was also probed as a competitor for the YY1-WGA interaction. ConA is a lectin with high specificity for mannose-containing sugars (34). It was found previously that Sp1 and RNA polymerase II do not bind ConA specifically (22, 28). Similarly we observed that ConA was not a competitor for the WGA-YY1 interaction, and most of the YY1 remained captured on the WGA affinity matrix in the presence of ConA (Fig. 2B, lane 4). Similar results were obtained when HeLa YY1 (Fig. 1A, lane 1) and HeLa nuclear extract were used in the experiments (not shown).


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Fig. 2.   YY1 binds to WGA. Nuclear extracts from growth-arrested CASMC in high glucose (200 µg of protein) were mixed with an affinity matrix containing covalently attached WGA. A, WGA affinity binding reactions were assembled, and different concentrations of GlcNAc were added as a competitor for the protein-WGA binding (indicated at the top of the lanes). Captured proteins were eluted by boiling in a buffer containing 1% SDS and analyzed by Western blot with anti-YY1 antibody (alpha YY1). Samples are as follows: 100% of input (IN) (lane 1); bound in the absence of competitor conditions (B) (lane 2); bound in the presence of 0.5 M GlcNAc (0.5) (lane 3); bound in the presence of 1 M GlcNAc (0.1) (lane 4); and captured with affinity matrix containing BSA instead of WGA (C) (lane 5). B, WGA affinity binding reactions were assembled with nuclear extracts and WGA affinity beads in the presence of WGA protein or ConA as competitors (indicated at the top of the lanes). Captured proteins were eluted by boiling in 1% SDS and analyzed by Western blot with alpha YY1. Samples are as follows: 100% of input (IN) (lane 1); bound in the absence of competitor conditions (B) (lane 2); bound in the presence of 1 mg of WGA protein (lane 3); bound in the presence of 1 mg of ConA protein (lane 4). C, nuclear extract was first denatured under reducing conditions in the presence of 1% SDS and then mixed with WGA affinity matrix. Bound proteins were eluted by boiling in 1% SDS and analyzed by Western blot with alpha YY1. Lane 1, 100% of input (IN); lane 2, bound fraction (B); lane 3, captured with affinity matrix containing covalently attached BSA instead of WGA (C). The position of the molecular marker proteins is indicated at the right side of the panels. In all Western blots we used only 20% of the fractions. The relative amount of YY1 in the fractions is shown in the diagrams on the right side of the panels. The amount of YY1 in the input was used as a base for comparison. The calculations were conducted in NIH Image 1.31 and Photoshop programs. Data were collected from three independent experiments.

YY1 forms complexes with other WGA targets such as Sp1, c-Myc, and the CTD of the polymerase II; therefore, it could be these and not YY1 glycosylation that are causing the retention of YY1 on the WGA matrix. To eliminate this possibility, we repeated the capturing experiments with denatured nuclear extract fractions treated with 0.5% SDS and beta -mercaptoethanol before incubation with the WGA matrix (Fig. 2C). We found that ~25% of the YY1 from CASMC extract was retained on the WGA matrix (Fig. 2C, lane 2). Less than 1% was captured on the control beads without WGA (Fig. 2C, lane 3), confirming the existence of a specific and direct interaction between YY1 and WGA.

WGA is known to be specific for terminal GlcNAc moieties and GlcNAc oligomers (36). The WGA-YY1 interaction strongly suggests that a subset of the nuclear YY1 is a glycoprotein with terminal GlcNAc. High GlcNAc concentration, SDS, and reducing conditions are needed to strip the WGA-captured YY1. The high affinity of WGA for YY1 supports the idea that YY1 is likely to be glycosylated at multiple sites, as was reported for the human Sp1 (22) and the CTD of the RNA polymerase II (28).

The Carbohydrate Moieties on YY1 Are Attached via an O-Glycosidic Linkage-- Carbohydrates are attached to proteins in two broad groups, i.e. linked to the nitrogen of Asn in the consensus sequence NXT (N-linked) and linked to the oxygen of Ser or Thr in Ser/Thr-rich regions (O-linked) (37). To determine the linkage by which the carbohydrate moiety is attached to YY1, we made use of the fact that the O-linked sugars, but not the N-glycosidic linkage, is susceptible to removal by reductive beta -elimination reaction under mild alkaline conditions (Fig. 3A). The beta -elimination reaction was previously used to characterize the O-glycosylation on Sp1 (22). When HeLa YY1 was subjected to beta -elimination at 37 °C in the presence of 0.1 M NaOH for 3 and 6 h, significantly less 3H label was found attached to YY1 (Fig. 3B, lanes 2 and 3). Possibly because of the lack of customary borohydride in our system, Western blotting surprisingly shows little or no protein degradation, even after 6 h.


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Fig. 3.   YY1 carries O-linked GclNAc moieties. HeLa [3H]galactose-YY1 treatment with 0.1 M NaOH, known as beta -elimination, was used to probe for the presence of O-linked carbohydrates. A, schematic representation of the beta -elimination reaction. B, HeLa YY1, purified and [3H]galactose-labeled (2 µg of protein), was incubated with 0.1 M NaOH for 3 and 6 h as indicated at the top of the lanes. To probe for N-linked carbohydrates, YY1 was incubated with PNGase F for 14 h. The reaction products were separated on 10% SDS gels and analyzed by fluorography for 3H-labeling and Western blot with anti-YY1 antibodies (alpha YY1) for YY1 protein content as indicated at the top of the panels. Samples are as follows: input (IN) 3H-labeled YY1 (lane 1); YY1 after 3 h incubation with NaOH (3) (lane 2); YY1 after 6 h incubation with NaOH (6) (lane 3); YY1 after incubation for 14 h with PNGase F (lane 4). The migration of a 72 kDa protein standard is indicated at the left side of the panels.

We also analyzed for the presence of N-linked carbohydrates, which can be deglycosylated by the enzyme PNGase F. This enzyme specifically releases N-linked carbohydrates by cleaving between the first internal GlcNAc residue and the asparaginyl moiety. The [3H]galactose-labeled YY1 was found to be resistant to overnight digestion with PNGase F, as the [3H]galactose-labeling of YY1 remained essentially unaffected after the reaction (Fig. 3B, lane 4). The amount of YY1 before and after the reactions was monitored by analyzing aliquots of the reactions in a Western blot (Fig. 3B). We did not observe a significant change in the YY1 protein presence as a result of the enzymatic and NaOH treatment (Fig. 3B), suggesting that the reduced amount of 3H-labeled YY1 after alkali treatment was not because of protein degradation. This clearly indicates that the glycosylation of YY1 consists of carbohydrates attached via O-linkages to serine and/or threonine residues.

High Glucose Stimulates the O-GlcNAc on YY1 in the Golgi-- It was recently reported that activation of the cellular hexosamine pathway by hyperglycemia results in a significant enhancement of O-linked protein glycosylation (38). We asked whether there would be a difference in the level of O-glycosylated YY1 in glucose-deprived and glucose-stimulated cells. Nuclear extracts were prepared from both CASMC stimulated with 30 mM glucose (NE-HG) or deprived of glucose (NE-NG) for 48 h. Aliquots of both extracts with equal amounts of protein were SDS-denatured and finally mixed with WGA affinity beads. Unbound protein was removed, and the beads were incubated in 1 M glucosamine and 0.5% SDS to remove bound proteins. The eluted fractions were assayed for YY1 by Western blot (Fig. 4). When NE-NG was applied to the WGA beads, <20% of the initial YY1 remained in the bound fraction (Fig. 4A, lane 3). In contrast, the WGA affinity matrix captured >60% of the YY1 in NE-HG (Fig. 4B). Slightly reduced levels of YY1 were also observed in NE-NG (Fig. 4A, lane1) compared with YY1 in NE-HG (Fig. 4B, lane 1). The results clearly demonstrate that high glucose stimulates O-GlcNAcylation of YY1 in the CASMC.


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Fig. 4.   High glucose stimulates the O-GclN acylation of YY1 in the Golgi. The presence of O-GclNAc-YY1 in nuclear extracts from growth-arrested CASMC in high glucose and in the absence of glucose cells was evaluated by comparing the amount of YY1 bound to a WGA affinity matrix. The nuclear extracts were treated under reducing conditions with 1% SDS before mixing with the WGA beads. A, WGA affinity binding with extracts from CASMC in the absence of glucose (100 µg in protein). B, WGA affinity binding with extracts from growth-arrested CASMC in high glucose (100 µg in protein). In both experiments captured proteins were eluted by boiling in 1% SDS and analyzed by Western blot with anti-YY1 antibodies (alpha YY1). In A and B, samples are as follows: 50% of input (IN) (lanes 1); not bound (NB) (lanes 2); bound and eluted by boiling in buffer containing 1 M GlcNAc and 1% SDS (B) (lanes 3). The relative amount of YY1 in the fractions is shown in the diagrams on the right side of the panels. The amount of YY1 in the input was used as a base for comparison. The calculations were conducted as described in the legend to Fig. 2.

O-GlcNAc on YY1 Inhibits Binding with Rb-- The O-linked GlcNAc moiety has been found in several viral, cytoplasmic, and nucleoplasmic proteins, but its function in the regulation of protein behavior is still subject to speculation.

YY1 interacts with several proteins with important functions in transcription. Here we tested how the O-GlcNAcylation of YY1 would affect YY1-Rb complex formation. Previously, we have shown that Rb binds YY1 in vitro and in growth-arrested CASMC (9). Here, we compared the content of YY1-Rb complexes in glucose-stimulated and glucose-deprived growth-arrested CASMC. We mixed nuclear extracts from both glucose-stimulated and glucose-deprived growth-arrested cells with Rb-immunoaffinity beads and measured the amount of captured YY1 together with Rb by Western blot (Fig. 5, A and B). As expected, in extracts from glucose-deprived and growth-arrested cells (NE-NG) Rb-antibody captured ~40% of the YY1 together with Rb (Fig. 5A, lane 3). However, in extracts from growth-arrested CASMC cultures in high glucose (NE-HG) where most of YY1 was found to be O-GlcNAc-modified, the Rb antibody captured <15% of the YY1 (Fig. 5B, lane 3), suggesting that the O-glycosylated YY1 was not predominantly in a complex with Rb. Therefore, the glycosylated YY1 should be mainly in the unbound fraction. To confirm that the glycosylated YY1 was not bound to the Rb matrix, we mixed the Rb-unbound fraction with a WGA matrix. We observed that most of the YY1 in this fraction was captured on the WGA matrix (Fig. 5C, lane 3). Importantly, the Rb-captured YY1 was not retained on the WGA affinity matrix (Fig. 5D, lane 3).


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Fig. 5.   The YY1 heterocomplex with Rb is sensitive to the O-GlcN acylation of YY1. The Rb-YY1complexes were isolated from growth-arrested CASMC in high glucose (NE-HG) and from growth arrested CASMC in the absence of glucose (NE-HG). NE-NG (250 µg of protein) (A) or NE-HG (250 µg of protein) (B) were mixed and incubated with an affinity matrix containing covalently attached monoclonal antibody to Rb (+Rb affi). Captured proteins were analyzed by Western blot (antibody specificity is identified to the left of the panels). Samples are as follows: 100% of input (IN) (lanes1); not bound (NB) (lanes 2); bound and eluted by boiling in buffer containing 1% SDS (B) (lanes 3); and captured with pre-immune mouse polyclonal antibody (C) (lanes 4). The amount of YY1 in the fractions is shown in the diagrams on the right side of the panels. It was estimated (relative (rel.) units) using as a measurement the intensity of the immunological ECL YY1 staining on the Western blots. The calculations were conducted in NIH Image 1.31 and Photoshop programs. Data was collected from three independent experiments. The presence of O-GlcNAcylated YY1 in the Rb-affinity matrix fractions was determined by WGA affinity bead-capturing. C, the Rb-unbound NE-HG fraction was mixed with the WGA affinity beads (WGA-affi). D, the Rb-bound NE-HG fraction was mixed with WGA affinity beads. Captured proteins in C and D were analyzed for YY1 content by Western blot with YY1-specific antibody (alpha YY1). Lanes 1, the input (IN); lanes 2, the WGA unbound protein; lanes, bound and eluted by boiling in buffer containing 1 M GlcNAc and 1% SDS (B). E, interaction between glycosylated YY1 and unphosphorylated Rb in solution. Glycosylated YY1 (purified from HeLa cells) or recombinant YY1 (rYY1) (purified from E. coli) and recombinant Rb (rRb) alone or after mixing were subjected to size exclusion chromatography on a Superose 6 matrix. Row 1, rRb alone; row 2, rYY1 alone; rows 3, rRb plus rYY1; rows 4, rRb plus glycosylated HeLa YY1. Fractions (numbers at the top of the gels) were analyzed by Western blot with antibodies (alpha ) to YY1 or Rb as indicated on the right-hand side of the gels.

Consistent with the above experiments, when purified recombinant Rb and partially purified O-GlcNAcylated YY1 from HeLa cells (Fig. 1A) were mixed in solution and analyzed for complex formation by gel filtration chromatography, both proteins eluted alone, and complex formation was not observed (Fig. 5E, rows 4). When E. coli-produced, unmodified YY1 and Rb were mixed before chromatography, a substantial portion of both proteins co-eluted as a large complex (Fig. 5E, rows 3).

Activated YY1 O-- GlcN Acylation Coincides with YY1-CArG DNA Box Interaction in CASMC---We have previously demonstrated that, in complex with Rb, YY1 will no longer bind DNA. Therefore, we reasoned that the inability of the glycosylated YY1 to interact with Rb should allow O-GlcNAcYY1-DNA interaction. To test this idea, we used the YY1 binding site on the smooth muscle alpha -actin promoter. When bound to the CArG DNA box of the promoter, YY1 acts as a strong transcriptional repressor. We used a gel shift assay to analyze the YY1 interaction with a double-stranded oligonucleotide corresponding to the CArG DNA box of the smooth muscle alpha -actin promoter, which contains a binding site for YY1 and SRF. As a positive control, we conducted gel shift reactions with the P5 adeno-associated viral promoter element, which is known to present a strong YY1 binding element. Reactions were assembled with equal protein amounts from NE-HG and NE-NG cell extracts. The obtained results suggest that more YY1 was indeed available for interaction with DNA in NE-HG than in NE-NG. YY1 glycosylation alone did not affect the thermodynamics of the DNA complex formation (Fig. 6B) as was found to be the case for the glycosylated forms of Sp1 and SRF.


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Fig. 6.   Gel shift reactions in nuclear extracts from cells with activated and with suppressed O-GlcNAcylation on YY1. A, effect of the YY1 glycosylation-inhibited interaction with Rb on complex formation between YY1 and the CArG box element. Band shift reactions received a 32P-labeled, double-stranded oligonucleotide containing the CArG box sequence (0.12 nM). Samples are as follows: lane 1, bovine serum albumin (BSA); lanes 2-5 received amounts (in micrograms) of nuclear extract from growth arrested CASMC in high glucose (NE-HG) as indicated above the lanes; lanes 6-9 received amounts (in micrograms) of nuclear extract from growth arrested CASMC in the absence of glucose as indicated above the lanes. The reactions in lanes 4 and 9 received 20 nM of unrelated cold oligonucleotide as a competitor. The reactions in lanes 5 and 10 received 3 nM of homologous cold oligonucleotide as a competitor. The presence (+) or absence (-) of competitor oligo DNA in the reactions is indicated above the lanes. The positions of the gel shift start (S), the free DNA (F), and YY1complexes (YY1) are indicated. B, band shift reactions received a 32P-labeled, double-stranded oligonucleotide containing the P5 initiator sequence (0.12 nM). Samples are as follows: lane 1, bovine serum albumin (BSA); lanes 2-5 received amounts (in micrograms) of nuclear extract from growth-arrested CASMC in high glucose (NE-HG) as indicated above the lanes; lanes 6-9 received amounts (in micrograms) of nuclear extract from growth-arrested CASMC in the absence of glucose as indicated above the lanes. The reactions in lanes 2 and 6 received 3 nM unrelated cold oligonucleotide as a competitor. The reactions in lanes 5 and 10 received 3 nM homologous cold oligonucleotide as a competitor. The presence (+) or the absence (-) of competitor oligo DNA in the reactions is indicated above the lanes. The positions of the gel shift start (S), the free DNA (F), and YY1 complexes (B) are indicated at the right side of the panel. C, reactions received a 32P-labeled, double-stranded oligonucleotide containing the CArG box sequence (0.12 nM). Samples are as follows: lane 1, bovine serum albumin (BSA); lanes 2-5 received different amounts of YY1 purified from HeLa cells (HeLa YY1) as indicated above the lanes; lanes 6-9 received amounts of E. coli purified recombinant YY1 (rYY1) as indicated above the lanes. The reactions in lanes 2 and 6 received 3 nM unrelated cold oligonucleotide as a competitor; the reactions in lanes 5 and 9 received 3 nM CArG DNA box oligo. The presence (+) or absence (-) of competitor oligo DNA in the reactions is indicated above the lanes. The positions of the gel shift start (S), the free DNA (F), and YY1 complexes (B) are indicated at the left side of the panel.

These results illustrate how O-glycosylation may alter YY1 function. Preventing YY1 from interacting with Rb will restore its ability to bind to DNA resulting in the repression or activation of YY1-regulated transcription.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have shown that a subset of the nuclear human YY1 exists as a glycoprotein carrying GlcNAc moieties. Together with beta -elimination, we used PNGase F treatment, in vitro labeling with galactosyl transferase, and WGA binding to demonstrate that the carbohydrate is attached by O-glycosidic linkage to serine and/or threonine on YY1. All of the transcription factors that have been reported to undergo glycosylation are O-linked and not N-linked. Our data also fit these observations.

YY1 typically migrates as a 69-kDa band in SDS-PAGE, though its average peptide mass is only 44.7 kDa. Careful removal of the glycosidic moieties under mild alkaline conditions did not significantly change the migration of YY1. Therefore, the altered electrophoretic migration presumably occurs because of modifications other than glycosylation. A similar observation was reported for the major 95-105 kDa variants of transcription factor Sp1, a peptide with an average mass of 72 kDa and multiple O-linked monosaccharides per single peptide chain.

The observed high affinity of YY1 for WGA binding together with the level of [3H]galactose incorporation suggest that YY1 may carry more than one GlcNAc residue, as has been found for other transcription factors such as Sp1 (22), Pax-6 (26), SRF (25), and the CTD domain of the catalytic subunit of polymerase II (28). The exact stoichiometry of GlcNAcylation on YY1, however, remains to be determined. It is likely that the nuclear YY1 represents a heterogeneous population of molecules with respect to the attached GlcNAc residues.

YY1 is ubiquitously expressed regardless of the differentiation status of the cell. We observed the presence of O-GlcNAcylation on YY1 in nuclear extracts from both actively proliferating and quiescent cell types, suggesting that this modification occurs independently of the cell type. It would not be surprising, however, if we were to find that the level of O-GlcNAc on YY1 is dependent on cell type and cell cycle position, thereby adjusting the YY1 functions in gene expression regulation according to the specific cellular requirements.

The levels of O-GlcNAcylation of proteins are regulated, at least in part, by the levels of cytoplasmic UDP-GlcNAc (38) produced by the hexosamine biosynthetic pathway, which is directly responsive to intracellular glucose concentrations. Therefore, it is not surprising that we found the GlcNAcylation on YY1 to be directly responsive to the glucose level in the cellular growth medium. How the external glucose level is translated to GlcNAcylation on YY1 remains to be determined.

At present, the function of the O-linked GlcNAcyl moieties in the regulation of protein behavior are not well understood. Though sugars are hydrophilic in nature, specific orientations of their non-polar CH-groups can create a hydrophobic patch that could interact with a hydrophobic pocket on a neighboring protein. Highly hydrophilic clusters of carbohydrate may alter the polarity of the proteins with which they are conjugated and prevent specific interactions with other proteins. The bulkiness and amphipathic nature of the sugar moieties may alter protein-protein interactions. Therefore, it is not surprising that the activated GlcNAcylation on YY1 in response to high glucose results in dramatically different functional outcomes. First, when YY1 is glycosylated it no longer binds to Rb. Upon dissociation from Rb, the glycosylated YY1 becomes an effective repressor of the alpha -actin promoter by binding efficiently to the CArG DNA box. The repression of this promoter results from the competitive displacement by YY1 of SRF, which was also found to be O-GlcNAcylated (25). O-GlcNAcylation on both YY1 and SRF does not inhibit their DNA binding capability. It appears likely that the O-GlcNAcyl moieties on both YY1 and SRF play a role in the competitive interaction for regulation, but their relative contribution is unknown. Nevertheless, our findings suggest that glycosylation of YY1 represents a novel means of regulating the activity of YY1.

Finally, the YY1-Rb interaction is cell cycle-dependent (9), with complex formation occurring to a greater extent in the G0/G1 compartment than in S, where Rb is hypophosphorylated. The ability of O-glycosylation on YY1 to block interaction with hypophosphorylated Rb in vitro leads us to speculate that O-glycosylation might have a profound effect on cell cycle transitions regulating the YY1-Rb heterodimerization and promoting activity in YY1. It appears likely that the heterodimer formation is the culmination of several signaling cascades that regulate both the O-GlcNAcylation on YY1 and differential phosphorylation of Rb.

The glycosylation of YY1 represents a novel means of regulating the activity of YY1. Our observations provide strong evidence that YY1-regulated transcription may be directly connected to glucose metabolism via O-GlcNAcylation on YY1.

    ACKNOWLEDGEMENTS

We thank E. Harlow for the monoclonal antibody to Rb and V. Gelev for suggestions.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant HL62458 (to A. U.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 617-632-0522; Fax: 617-667-2927; E-mail: ausheva@bidmc.harvard.edu.

Published, JBC Papers in Press, February 13, 2003, DOI 10.1074/jbc.M300789200

    ABBREVIATIONS

The abbreviations used are: Rb, retinoblastoma protein; GlcNAc, N-acetylglucosamine; O-GlcNAc, O-linked N-acetylglucosamine; SRF, serum-response factor; CTD, carboxyl-terminal domain; CASMC, coronary artery smooth muscle cells; WGA, wheat germ agglutinin; PNGase F, peptide N-glycosidase F; NE, nuclear extract; NE-HG, NE from CASMC cultures in high glucose; NE-NG, NE from CASMC cultures deprived of glucose; ConA, concanavalin A.

    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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