Carbohydrates Induce Mono-ubiquitination of H2B in Yeast*

Histone modifications have emerged to be a major regulatory mechanism for gene expression (1–4). However, it is not clear how histone modifications are physiologically regulated. Here, we show that mono-ubiquitinated H2B at lysine 123 (uH2B) in the yeast (Saccharomyces cerevisiae) is present in exponential phase and absent in stationary phase. A wide array of carbohydrates or sugars, including glucose, fructose, mannose, and sucrose, are capable of inducing uH2B in stationary phase yeast. In contrast, non-metabolic glucose analogs are defective in inducing uH2B. Furthermore, uH2B induction is inhibited by iodoacetate, an inhibitor of glyceraldehyde-3-phosphate dehydrogenase in glycolysis. Moreover, uH2B induction is markedly impaired in yeast mutants, in which glycolytic genes are deleted. These data indicate that glycolysis is required for the carbohydrate-induced mono-ubiquitination of H2B at lysine 123. Therefore, our study reveals a novel paradigm of metabolic regulation of histone modifications.

Histone modifications have emerged to be a major regulatory mechanism for gene expression (1-4). However, it is not clear how histone modifications are physiologically regulated. Here, we show that mono-ubiquitinated H2B at lysine 123 (uH2B) in the yeast (Saccharomyces cerevisiae) is present in exponential phase and absent in stationary phase. A wide array of carbohydrates or sugars, including glucose, fructose, mannose, and sucrose, are capable of inducing uH2B in stationary phase yeast. In contrast, non-metabolic glucose analogs are defective in inducing uH2B. Furthermore, uH2B induction is inhibited by iodoacetate, an inhibitor of glyceraldehyde-3-phosphate dehydrogenase in glycolysis. Moreover, uH2B induction is markedly impaired in yeast mutants, in which glycolytic genes are deleted. These data indicate that glycolysis is required for the carbohydrate-induced mono-ubiquitination of H2B at lysine 123. Therefore, our study reveals a novel paradigm of metabolic regulation of histone modifications.
BRE1 with its endogenous promoter region was cloned by PCR from yeast genomic DNA and cloned to SmaI site of pRS414. Subsequently, N-terminal FLAG-FLAG-Bre1 was generated by site-directed mutagenesis (Stratagene) and sequence verified. All the N-terminal FLAGtagged H2B constructs were generated either by site-directed mutagenesis (Stratagene) or generously provided by M. A. Osley.
Western Blot Analysis and Glucose Assay-Whole-cell extracts from normalized number of cells or histones were prepared, analyzed on 12 or 7.5% SDS-polyacrylamide gels, and Western-blotted with anti-FLAG M2 (Sigma) as described previously (9). Glucose analysis was performed with a glucose assay kit (Sigma) according to the manufacturer's instruction.

RESULTS AND DISCUSSION
We made a serendipitous observation that uH2B was not detected in yeast Y96 with FLAG-tagged H2B as the only copy of H2B in water (Fig. 1A, lane 4). We further found that uH2B was also absent in stationary phase Y96 that was cultured in YPD (1% yeast extract, 2% peptone, and 2% glucose) for 7 days (Fig. 1A, lane 3). In striking contrast, significant amount of uH2B was present in exponential phase Y96 (e.g. A 600 ϭ 1) in YPD (Fig. 1A, compare lanes 1, 3, and 4). These initial observations indicated that the induction of uH2B was subjected to environmental and/or nutritional regulation.
Stationary phase yeast is in a non-proliferating or G o state, resulting from nutrient depletion (20). Because uH2B was not detected in stationary phase yeast, we then sought to explore whether addition of nutrients to stationary phase yeast could induce uH2B. To do so, we collected stationary phase Y96, washed it with water, and resuspended it with 5 ml of the following solutions in separation or in combination (A 600 ϭ 1): 2% glucose, nitrogen base, ammonia sulfate, and amino acid mixture. After incubating the yeast at 30°C for 1 h, we prepared whole cell extracts for assaying the presence of uH2B. We found that glucose alone was capable of inducing uH2B (Fig. 1A, lane 11). In contrast, amino acids, nitrogen base, and ammonia sulfate were defective in inducing uH2B (Fig. 1A,  lanes 7 and 12). In addition, uH2B could be readily induced by a range of glucose concentrations (0.05 to 2% tested) within a few minutes of incubation in Y96 (data not shown). The data demonstrate that glucose is an inducer of uH2B.
There are four stages of yeast growth on the glucose-based medium YPD: exponential phase during which glucose is present, diauxic shift when glucose is depleted, post-diauxic shift when yeast growth depends on respiration, and stationary * The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. phase when nutrient is depleted (20). To examine the presence of uH2B in the four stages of yeast growth, we inoculated Y96 in YPD and measured glucose levels in the medium, cell density, and uH2B levels at various time points. We found that uH2B was significantly present in exponential phase (A 600 was less than 12) (Fig. 1B, lanes 3-5; Fig. 1C). However, only less than 4% of the original bulk uH2B was present in post-diauxic shift (A 600 was from about 17 to 41) (Fig. 1B, lanes 6 and 7; Fig.  1C). Moreover, uH2B was not detectable in stationary phase (Fig. 1B, lanes 8 and 9; Fig. 1C). Therefore, the presence of uH2B closely correlated with the presence of glucose (Fig. 1C), further suggesting that glucose is an inducer of uH2B.
Genetic studies indicate that mono-ubiquitination of H2B is catalyzed in vivo by Bre-1, a ubiquitin E3 ligase (12,13). Because uH2B was not detected in stationary phase, we sought to determine whether Bre1 was expressed in stationary phase. To assess Bre1 expression, we first tagged chromosomal BRE1 with green fluorescence protein at its carboxyl terminus. However, presumably because the RING-finger domain of Bre1 is at the extreme carboxyl terminus (12), fusion of green fluorescence protein to the C-terminal of Bre1 rendered Bre1-green fluorescence protein fusion to be catalytically inactive (data not shown). Therefore, we generated a bre1⌬ mutant (Y119, derived from BY4733 strain) and introduced the bre1⌬ mutant with pRS414-(2x) FLAG-Bre1 (CEN TRP) under the control of BRE1 endogenous promoter (Y206) and cultured it in YPD until stationary phase was reached. pRS414-(2x) FLAG-Bre1 directed expression of FLAG-FLAG-Bre1, in which tandem FLAG epitopes were tagged after its start methionine. We found that FLAG-FLAG-Bre1 protein was present throughout the four growth stages, whereas the presence of uH2B was mainly in exponential phase (Fig. 1D) as described earlier.
Glucose is a major carbon and energy source for yeast. There are a number of known glucose sensors monitoring the presence of glucose, which include hexokinase II, Snf3, Snf1, Rgt2, Gpr1, and glucokinase I (21)(22)(23)(24)(25)(26). Therefore, we sought to explore whether individual glucose sensors play an essential role in inducing uH2B. To test this, we generated isogenic strains of Y96, namely, hxk2⌬, snf1⌬, snf3⌬, rgt2⌬, gpr1⌬, and hxk2⌬ glk1⌬ mutants, in which the entire open reading frames of these genes were deleted. We cultured them in YPD until A 600 reached about 2, and prepared whole cell extracts for analyzing the presence of uH2B as described. We found that uH2B was present in all the mutant strains (Fig. 2). These data demonstrate that these sensors do not play an essential role in inducing uH2B.
In addition to glucose, yeast can also utilize a variety of carbohydrates as its carbon and energy sources. Therefore, we sought to test whether other carbohydrates could also induce uH2B. To do so, we collected stationary phase Y96, washed it with water, resuspended it with 2% various carbohydrates (A 600 ϭ 2), and incubated these cultures at 30°C for 6 h. We found that mannose, fructose, and sucrose were also very potent in inducing uH2B when added to stationary phase yeast (Fig. 3A, lanes 5-8). Raffinose and maltose could also induce uH2B (Fig. 3A, lanes 12 and 13). These data indicate that uH2B can be induced by a wide array of carbohydrates.
In contrast, L-glucose, 3-O-methyl-D-glucopyranose, and 2-deoxyglucose, non-metabolic glucose analogs, were defective in inducing uH2B (Fig. 3A, lanes 9 -11). L-Glucose is not transported by the yeast (21). 3-O-Methyl-D-glucopyranose is transported; however, it is not phosphorylated at the C-6 position (21). 2-Deoxyglucose is transported and phosphorylated at the C-6 position but is not further phosphorylated at the C-1 position (21), which is a key step for the entry of glycolysis. Therefore, our data suggest that glucose-mediated mono-ubiquitination of H2B requires glucose metabolism.
These observations led us to explore whether glycolysis, the central carbohydrate metabolic pathway (27), plays a role in inducing uH2B. We first examined whether iodoacetate (IAA), 1 an inhibitor of glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase step of glycolysis (28), could inhibit the induction of uH2B. We collected stationary phase Y96 and resuspended it with fresh YPD containing various amount of IAA (A 600 ϭ 2) and further incubated it at 30°C for 30 min. We found that glucose-induced uH2B levels were markedly reduced in the presence of IAA in a dose-dependent manner (Fig.  3B). In contrast, rotenone, an inhibitor of respiratory chain (29), had no effect on inducing uH2B (Fig. 3C). These data suggest that glycolysis, but not respiration, is required for carbohydrate-induced ubiquitination of H2B.
To further examine the role of glycolysis on carbohydrateinduced uH2B, we examined the presence of uH2B in glycolytic mutants, in which genes for the entry of glycolysis are deleted. The first irreversible step for glycolysis is the conversion of fructose-6-phosphate to fructose-1,6-bisphosphate, catalyzed by 6-phosphofructokinase activity encoded by PFK1 or PFK2 (30). Deletion of both PFK1 and PFK2 genes abolishes glycolysis (31). To test the effect of glycolysis on the induction of uH2B, we cultured isogenic strains Y218 (WT), Y219 (pfk1), and Y223 (pfk1 pfk2) in SC-U/GE (1% glycerol and 3% ethanol) for 3 days. uH2B was not present in the WT, pfk1, and pfk1 pfk2 strains before YPD was added (Fig. 3D, lanes 3, 5, and 7). After resuspending the WT, pfk1, and pfk1 pfk2 strains in YPD (A 600 ϭ 2) and incubating them at 30°C for 3 h, we found that uH2B was induced in the WT strain as expected (Fig. 3D, lane  4). However, the induction of uH2B was impaired in the pfk1 mutant (Fig. 3D, compare lanes 4 and 6). Furthermore, the induction of uH2B was abolished in the pfk1 pfk2 mutant (Fig.  3D, compare lanes 4, 6, and 8). In contrast, deletion of CYT1 or COX9 (Y237 or Y238), which encodes essential respiration protein cytochrome c 1 or cytochrome c oxidase subunit VIIA, respectively, had no effect on uH2B (Fig. 3E). These data further validate our model that glycolysis is required for the carbohydrate-induced mono-ubiquitination of H2B.
Mono-ubiquitination of H2B at Lys 123 is implicated in yeast telomeric silencing (9). Consistent with the gene-silencing role, our comparative genome-wide expression data showed that about 70 metabolic genes, which are normally repressed in exponential phase and up-regulated in post-diauxic shift and stationary phase, were up-regulated in a h2b-K123R strain when it was in exponential phase in YPD. 2 Taken together, our data suggest that uH2B might function as a histone marker for the carbohydrate metabolism or carbon source in yeast.
In summary, we have found that carbohydrates are potent FIG. 3. Glycolysis is required for carbohydrate-induced ubiquitination of H2B. A, ubiquitination of H2B is induced by a variety of carbohydrates except non-metabolic glucose analogs. Stationary phase Y96 was collected, washed with water, and resuspended in 2% indicated carbohydrates (A 600 ϭ 2) and incubated at 30°C for 6 h. B, induction of uH2B is inhibited by IAA. Stationary phase Y96 was diluted with fresh YPD (2% glucose) containing various amount of IAA (A 600 ϭ 2) and further incubated at 30°C for 30 min. C, Stationary phase Y96 was diluted with fresh YPD (2% glucose) (A 600 ϭ 2) containing various amount of rotenone and further incubated at 30°C for 1 h. D, stationary phase isogenic strains Y218(WT), Y219(pfk1), and Y223(pfk1 pfk2) (lanes 3, 5, and 7) were collected, resuspended in YPD, and incubated at 30°C for 3 h (lanes 4, 6, and 8). E, stationary phase isogenic strains Y230 (WT), Y237 (cyt1), and Y238 (cox9) were collected and resuspended in YPD and incubated at 30°C for 3 h. uH2B was analyzed as in Fig. 1. inducers for mono-ubiquitination of histone H2B in yeast. We have further discovered that the induction of mono-ubiquitinated H2B requires glycolysis, the central carbohydrate metabolic pathway. To our knowledge, this study is the first demonstration of novel metabolic regulation of histone modifications by sugars or carbohydrates.