Epe1 contributes to activation of AMPK by promoting phosphorylation of AMPK alpha subunit, Ssp2

AMP-activated protein kinase (AMPK) is a pivotal cellular energy sensor. It is activated by stresses that cause depletion of energy and initiates adaptive responses by regulating metabolism balance. AMPK forms αβγ heterotrimer. In fission yeast, activation of AMPK mainly depends on the phosphorylation of AMPKα subunit Ssp2 at Thr189 by upstream kinase Ssp1. However, not much is known about the regulation of this process. In this study, we identified Epe1 as a novel positive regulator of AMPK. Epe1, a jmjC-domain-containing protein, is best-known as a negative regulator of heterochromatin spreading. Although the novel role of Epe1 in regulation of AMPK relies on predicted iron- and 2-oxyglutarate-binding residues inside jmjC domain, it seems to be irrelevant to inhibition of heterochromatin spreading. Epe1 is associated with Ssp2 directly and promotes phosphorylation of Ssp2 upon various environmental stresses, including low-glucose, high-sodium, high-pH and oxidative conditions. Similar to Epe1, Jmj1 and Msc1 also contribute to phosphorylation of Ssp2. Deletion of epe1 + impairs downstream events following phosphorylation of Ssp2, including nuclear translocation of Ssp2, sexual differentiation and inhibition of fatty acid synthesis. Our study reveals a novel way in which a jmjC-domain-containing protein regulates adaptive response by directly binding to a principal sensor.

In this study, we revealed that Epe1 is a novel positive regulator of AMPK. Epe1 is a jmjC domain-containing protein. Epe1 prevents heterochromatin from spreading beyond its natural borders and limits ectopic heterochromatin formation. The novel role of Epe1 in regulation of AMPK relies on its predicted catalytic residues inside the jmjC domain, but seems to be irrelevant to inhibition of heterochromatin spreading. Epe1 directly associates with the AMPK catalytic subunit Ssp2 and promotes its phosphorylation upon various environmental stresses. Deletion of epe1 + abolishes nuclear accumulation of Ssp2 and downstream events, including sexual differentiation and inhibition of fatting acid synthesis. Our results highlight the role of a jmjC domain-containing protein in adaptive responses upon stresses.

Epe1 is involved in adaptive responses to environmental stresses.
In a genome wide screen, epe1Δ mutant displayed decreased growth in low-glucose medium. Epe1 is best-known as a negative regulator of heterochromatin spreading 10,11 . However, its role in response to limited glucose was unclear. Thus, the growth of an epe1Δ mutant in medium containing different concentrations of glucose was investigated. As shown in Fig. 1a, Epe1 displayed normal growth on high-glucose medium (5% or 3%). The growth of epe1Δ mutant was reduced slightly on medium containing 0.1% glucose, and the growth defect became more apparent in even lower concentrations of glucose (0.03% and 0.02%). More severe growth defects were observed in mutants that abolish AMPK activity, including deletion of ssp2 + (encoding the catalytic α subunit), and deletion of ssp1 + (encoding a CaMKK homolog that phosphorylates Ssp2 at Thr 189 ). This is consistent with an essential role of AMPK in the adaptation to glucose limitation 7,12 . Intriguingly, the growth of the ssp1Δ mutant was poorer than that of the ssp2Δ mutant, suggesting that Ssp1 regulates factor(s) other than AMPK in the response to limited glucose. Impaired growth of ssp1Δ, ssp2Δ and epe1Δ mutants in low-glucose conditions was further confirmed by monitoring growth curves in liquid medium. The results were consistent with those on solid medium (Fig. 1b).
Besides low-glucose condition, budding yeast AMPK is required for tolerance to a broad range of stresses, including salt, alkaline, and oxidative stress 12 . The growth of epe1Δ, ssp2Δ and ssp1Δ cells in the presence of high sodium was investigated. Unexpectedly, ssp2Δ and ssp1Δ cells displayed better growth than WT cells, on solid medium containing 0.1 M NaCl, or in liquid medium containing 0.05 M NaCl (Fig. 1c). This result contradicts the defective growth reported for an ssp1Δ mutant in 1 M KCl 8 . It suggests Ssp1, along with AMPK, play different roles in responding to high-sodium and high-potassium stresses. It is possible that AMPK induces mitotic delay upon high-sodium stress. Disruption of AMPK activity by deletion of ssp2 + or ssp1 + might bypass the mitotic delay and result in improved growth. Similarly, epe1Δ cells grew better in medium containing high concentrations of NaCl, but improvement of growth of epe1Δ cells was weaker than that observed in ssp2Δ and ssp1Δ mutants.
The growth of epe1Δ, ssp2Δ and ssp1Δ cells at high pH was also examined. ssp2Δ and ssp1Δ mutants showed a marked reduction of growth at pH 10.5. The epe1Δ mutant also displayed defective growth, but to a lesser extent (Fig. 1d). Since the epe1Δ mutant shares similar phenotypes with ssp1Δ or ssp2Δ mutants in responding to various environmental stresses, it suggests Epe1 is involved in the AMPK pathway. Epe1 is more likely a regulatory factor, owing to its relative weaker phenotype.

Epe1 promotes phosphorylation of Ssp2 upon various stresses.
In fission yeast, activation of AMPK depends on the phosphorylation of Ssp2 Thr 189 by upstream kinase Ssp1 5 . To verify a potential role of Epe1 in AMPK activation, the phosphorylation of Ssp2 upon glucose limitation was investigated by using a Phospho-AMPKα (Thr 172 ) monoclonal antibody. As shown in Fig. 2a, levels of Ssp2 phosphorylation in high-glucose condition (5%) was kept low in both WT and epe1Δ cells. In the presence of 0.1% glucose, phosphorylation in WT was induced, and the induction was milder in epe1Δ mutant. In the presence of 0.07% glucose, phosphorylation of Ssp2 was induced markedly in WT cells, but was induced slightly in epe1Δ cell, which resulted in a substantial difference between WT and epe1Δ cells. As a control, phosphorylation of Ssp2 was totally abolished in an ssp1Δ mutant, indicating the antibody specifically recognizes phosphorylated Thr 189 in fission yeast. The result suggests Epe1 is required for complete induction of Ssp2 Thr 189 phosphorylation in low-glucose condition.
The effect of Epe1 on the phosphorylation of Ssp2 upon other stresses was also investigated. The level of Ssp2 Thr 189 in oxidative (0.03% H 2 O 2 ), high-sodium (1 M NaCl), or high-pH (pH 11) stresses was dramatically decreased in an epe1Δ mutant compared to that in WT cells, suggesting the induction of phosphorylation was impaired by the deletion of epe1 + (Fig. 2c-e). In standard YES medium, phosphorylation of Ssp2 in WT cells was induced rapidly over time upon the depletion of nutrients, including glucose and nitrogen. In contrast, no apparent induction was observed in epe1Δ mutant (Fig. 2b). Thus, the results indicate Epe1 promotes the induction of Ssp2 Thr 189 phosphorylation upon various environmental stresses.

Epe1 is associated with Ssp2 in vivo.
The mechanism underlying Epe1-promoted phosphorylation of Ssp2 was investigated. Deletion of epe1 + causes formation of ectopic heterochromatin and results in silencing of genes in euchromatin 13 . To examine whether the deletion of epe1 + represses the transcription of AMPK subunits and upstream kinase, an RT-PCR assay was performed. As shown in Fig. 3a, there was no significant difference between the mRNA level of ssp2 + , cbs2 + , amk2 + or ssp1 + in WT and epe1Δ cells. Accordingly, the protein level of Ssp2 in an epe1Δ mutant was the same as that in WT cells in regular culture or in environmental stress (Fig. 2). It suggests Epe1 does not regulate phosphorylation of Ssp2 by affecting the transcription of components of AMPK. In a previous study, fragments of Ssp2 were found by mass spectrometry in affinity purification of Epe1-FLAG 14 .

Role of Epe1 in regulation of AMPK requires its predicted iron-and 2-oxyglutarate-binding residues in jmjC domain. Epe1 blocks the expansion of heterochromatin into neighboring euchromatin
and prevents formation of ectopic heterochromatin 10,11 . Although no demethylase activity has been detected for Epe1, the predicted iron-binding residue (H297) and 2-oxyglutarate-binding residue (K314) in the jmjC domain are essential for function of Epe1 in preventing heterochromatin spreading 11 . To investigate the contribution of both residues to the role of Epe1 in AMPK regulation, H297A and K314A point mutants were constructed. Epe1-H297A and Epe1-K314A were expressed from native promoters and their levels are similar to that of wild-type Epe1 (Fig. 3d). Intriguingly, the phenotypes of Epe1-H297A and Epe1-K314A were indistinguishable from epe1Δ mutant. Epe1-H297A and Epe1-K314A displayed a growth defect in low-glucose condition (Fig. 3c). Accordingly, the induction of Ssp2 phosphorylation upon the depletion of nutrients was impaired in either mutant (Fig. 3e). Thus, the predicted iron-and 2-oxyglutarate-binding residues are essential for the role of Epe1 in the AMPK pathway. The role of Epe1 in preventing heterochromatin spreading is redundant with Mst2, a H3K14 acetyltransferase 13 . Deletion of mst2 + did not cause a growth defect on low-glucose plate or delayed induction of Ssp2 phosphorylation (Fig. 3c,f). Thus, the role of Epe1 in the AMPK pathway seems unrelated to its role in limiting heterochromatin spreading. Jmj1 and Msc1 contribute to phosphorylation of Ssp2 and activation of AMPK pathway. Besides Epe1, Jmj1, Jmj2, Jmj3, Jmj4, Lid2 and Msc1 also contain a JmjC domain. Because jmj3Δ and lid2Δ mutants were both inviable, neither mutant was investigated for growth in this study. As shown in Fig. 4a,  jmj2Δ and jmj4Δ mutants both grew as well as WT cells in low concentration of glucose. Similar to epe1Δ mutant, the growth of jmj1Δ and msc1Δ mutants was reduced slightly in low-glucose condition. The growth  defect of epe1Δjmj1Δ or epe1Δmsc1Δ mutant was about the same as epe1Δ mutant. Accordinly, deletion of jmj1 + and msc1 + reduced phosphorylation of Ssp2 upon depletion of nutrients, to a similar extent as deletion of epe1 + (Fig. 4b). The results suggest Jmj1 and Msc1 also contribute to the activation of AMPK, and they probably function in the same pathway as Epe1.

Epe1 is required for sexual differentiation and inhibition of fatty acid synthesis upon depletion of nutrients.
Next, we examined the effect of Epe1 on the downstream events following phosphorylation of Ssp2. Under nutritional stress, phosphorylated AMPK is translocated into the nucleus and promotes sexual differentiation 5 . Whole cell extracts, cytoplasmic and nuclear fractions were prepared from cells after 10-hour growth. Amount of Ssp2-8xHA in whole cell lysate were about the same in WT, epe1Δ and ssp1Δ cells. In WT cells, a strong signal of Ssp2-8HA was detected in the nuclear fraction, compared to a weak signal in the cytoplasmic fraction. Nuclear accumulation of phosphorylated Ssp2 (P-Ssp2) was also observed in WT cells (Fig. 5a). In epe1Δ cells, the signals of Ssp2-8xHA and P-Ssp2 in the cytoplasmic fraction were much stronger than those in the nuclear fraction, indicating the nuclear translocation of Ssp2 is impaired by the deletion of epe1 + (Fig. 5a). As a control, nuclear accumulation of Ssp2-8xHA was totally abolished in ssp1Δ cell, indicating the phosphorylation is essential for nuclear translocation of Ssp2 (Fig. 5a). To evaluate the role of Epe1 in sexual differentiation, mating efficiency was measured in the cells after growing on SPAS medium for 24 hours. As expected, the percentage of zygotes in ssp2Δ (7%) or ssp1Δ cells (10%) was significantly lower than that in WT cells (18%) (Fig. 5b). Deletion of epe1 + caused a relative mild, but still significant decrease in the percentage of zygotes (15%). Consistently, epe1Δ cells exhibited a reduction of iodine staining comparing to WT cells, suggesting Epe1 is required for the sexual differentiation upon depletion of nutrients (Fig. 5c).
To maintain energy balance, AMPK conserves ATP by switching off anabolic pathways, including fatty acid synthesis 15 . In mammals and budding yeast, AMPK inhibits fatty acid synthesis by inactivating acetyl-coA carboxylase (Acc1), which catalyzes the irreversible carboxylation of acetyl-CoA to produce malonyl-CoA 16,17 . To examine whether fission yeast AMPK functions in a conserved way, levels of acetyl-CoA and malonyl-CoA in WT, epe1Δ, ssp1Δ and ssp2Δ cells were measured after growing in the YES for 10 hours. The level of acetyl-CoA decreased and that of malonyl-CoA increased significantly in epe1Δ, ssp1Δ and ssp2Δ cells, compared to that in WT cells (Fig. 5d,e). The results suggest inhibition of acetyl-CoA carboxylase is impaired in the mutants. Malonyl-CoA serves as a precursor for fatty acid synthesis 18 . Accordingly, the amount of total fatty acid was significantly increased in epe1Δ, ssp1Δ and ssp2Δ cells (Fig. 5f). The results suggest fission yeast AMPK shares functional homology with its counterparts in mammals and budding yeast in inhibiting fatty acid synthesis. Fission yeast AMPK inactivates acetyl-coA carboxylase under nutritional stress, and this process might be positively regulated by Epe1.

Discussion
JmjC-domain-containing proteins are implicated in chromatin modification and transcriptional regulation, where in most case their roles are dependent on the demethylase activity of the JmjC domain. Ndy1, a jmjC-domain-containing demethylase, is required for the transcription of antioxidant enzymes. Overexpression of Ndy1 relieves oxidative stress and inhibits the phosphorylation of AMPK 19 . In this study, Epe1 was identified as a novel positive regulator of AMPK. Epe1 does not promote activity of AMPK through transcriptional regulation. Instead, Epe1 is associated with Ssp2 directly and promotes the phosphorylation of Ssp2 at Thr 189 by Ssp1. Our results reveal a novel way in which a JmjC domain-containing protein regulates adaptive responses by directly binding with a principal sensor. Whether a similar mechansim was applied in Jmj1 and Msc1-mediated regulation of AMPK is worth futher investigation.
Although no demethylase activity has been reported for the Epe1, Epe1-promoted Ssp2 phosphorylation requires the predicted iron-and 2-oxyglutarate binding residues in the jmjC domain of Epe1. Thus, it is tempting to speculate that Epe1 demethylates AMPK subunits through its jmjC domain and thus demethylation facilitates the phosphorylation of Ssp2. Activity of AMPK is regulated by various modifications. Besides phosphorylation inside the activation loop, AMPKα subunits are regulated by ubiquitination, sumoylation and oxidation/reduction. AMPKβ is subjected to acetylation, myristoylation, ubiquitination, sumoylation and glycogen binding 20,21 . It is unknown whether Epe1 is also involved in the regulation of these modification. Mass spectrometry analysis of AMPK subunits in epe1Δ and WT cells during stress responses is required to test this hypothesis.

Methods
Yeast strains and media. All the strains used in this study are listed in Supplementary Table S1. Gene deletion and tagging were performed by homologous recombination using a plasmid-based method 22 . Regular yeast extract medium with supplements (YES) containing 3% glucose and SPAS medium was prepared as describe before 23 .
Fivefold serial dilution assay and growth curve analysis. Overnight culture was diluted into fresh YES to an OD 600 of 0.2. Cells were grown at 32 °C and were collected till OD 600 reached 0.8. For serial dilution assay, cells were collected and adjusted to an OD 600 of 1.0. Samples were diluted by fivefold for five times. 5 μl dilutions were spotted onto YES medium containing indicated concentrations of glucose, 0.1 M NaCl, or at pH 10.5. Plates were incubated for 2 or 3d at 32 °C before imaging. For growth curve analysis, cells were washed and resuspended in YES liquid medium containing indicated concentrations of glucose, 0.05 M NaCl, or at pH 10.5, to start at an OD 600 of 0.08. Cells were grown at 32 °C and OD 600 of the culture was measured at indicated time points. Experiments were performed in triplicates.

RT-PCR.
Overnight culture was diluted into fresh YES to an OD 600 of 0.2. 1 × 10 8 cells were collected after growing for 10 h. Total RNA were extracted by using the RiboPure Yeast (AM1926, Life Technologies, Carlsbad, CA, USA) and reverse transcribed into cDNA by using PrimeScript RT (RR037A, Takara, Dalian, China). qPCR was performed using SYBR Premix Ex TaqII (RR820A, Takara) in a LightCycler 480 II Real-Time PCR System (Roche Applied Science, Penzberg, Upper Bavaria, Germany). Primers used are listed in Supplementary Table S2. Whole cell extract and fraction preparation. Cells were grown in YES overnight. Cells were harvested and washed by PBS for three times. Cells were then resuspended to an OD 600 of 0.2 in fresh YES liquid medium containing indicated concentrations of glucose, 0.1 M NaCl, 0.03% H 2 O 2 , or at pH 11. Cells were grown at 32 °C and were collected till OD 600 reached 0.6~0.8. To induce depletion of nutrients, cells were collected after growing in standard YES liquid medium for 4, 10 and 15 h. 5 × 10 8 cells were pelleted and washed with lysis buffer (1 M Tris-HCl (pH 8.0), 167 mM NaCl, 1.2 mM EDTA, 1% TritonX-100, 0.1% Na-deoxycholate, 20 mM NaF, 2 mM phosphoglycerol). Cells were resuspended in lysis buffer containing protease inhibitors cocktail (05892970001, Roche Applied Science, Penzberg, Upper Bavaria, Germany) and homogenized with a bead-beater (FastPrep-24, MP, California, USA) by glass beads. Lysate was centrifuged. Supernatant was supplemented with 4xSDS-PAGE loading buffer and then boiled. Cytoplasmic and nuclear fractions were prepared from 5 × 10 8 cells by using Minute Cytoplasmic & Nuclear Extraction Kits (SC-003, Invent Biotechnologies, Inc, Plymouth, MN, USA). Elution from different fractions was boiled with 4xSDS-PAGE loading buffer. Samples were subjected to Western blot by using following antibodies: anti-HA (M20003L, Abmart, Shanghai, China), anti-Phospho-AMPKα (Thr172) (2531, Cell Signaling Technology, Boston, MA, USA), anti-Tubulin (T8203, Sigma-Aldrich, St Louis, MO, USA). The blots were scanned by GeneGnome HR system (Syngene, Cambridge, UK).

Co-immunoprecipitation.
Overnight culture was dilute into fresh YES to an OD 600 of 0.2. 5 × 10 10 cells were harvested after growing for 10 h. Cells were washed and then resuspended in Buffer 1 supplemented with protease inhibitors as described above. Cells were broken by a high-pressure cell homogenizer (JN-02C, JNBIO, Guangzhou, China) and supernatant were collected after centrifugation. Supernatant was incubated with anti-HA antibody and then protein A/G PLUS-agarose beads (sc-2003, Santa Cruz, Dallas, TX, USA) for 6 h. Bead-conjugated complexes were washed with lysis buffer, boiled in 1xSDS-PAGE loading buffer and subjected to Western blot.