The Yeast AMPK Homolog SNF1 Regulates Acetyl Coenzyme A Homeostasis and Histone Acetylation

Abstract

Acetyl coenzyme A (acetyl-CoA) is a key metabolite at the crossroads of metabolism, signaling, chromatin structure, and transcription. Concentration of acetyl-CoA affects histone acetylation and links intermediary metabolism and transcriptional regulation. Here we show that SNF1, the budding yeast ortholog of the mammalian AMP-activated protein kinase (AMPK), plays a role in the regulation of acetyl-CoA homeostasis and global histone acetylation. SNF1 phosphorylates and inhibits acetyl-CoA carboxylase, which catalyzes the carboxylation of acetyl-CoA to malonyl-CoA, the first and rate-limiting reaction in the de novo synthesis of fatty acids. Inactivation of SNF1 results in a reduced pool of cellular acetyl-CoA, globally decreased histone acetylation, and reduced fitness and stress resistance. The histone acetylation and transcriptional defects can be partially suppressed and the overall fitness improved in snf1⌬ mutant cells by increasing the cellular concentration of acetyl-CoA, indicating that the regulation of acetyl-CoA homeostasis represents another mechanism in the SNF1 regulatory repertoire. SNF1 is the budding yeast ortholog of mammalian AMP-activated protein kinase (AMPK). SNF1 and AMPK are highly conserved in eukaryotes and serve as cellular energy sensors and master regulators of metabolism (1-3). The yeast SNF1 complex consists of the catalytic ␣ subunit Snf1p; one of three different regulatory ␤ subunits, Sip1p, Sip2p, or Gal83p; and the stimulatory ␥ subunit Snf4p (4). The kinase activity of Snf1p is activated by upstream kinases Sak1p, Tos3p, and Elm1p, which phosphorylate activation loop residue Thr210 (5, 6). The activity of these upstream kinases is opposed by the activity of protein phosphatases Reg1p-Glc7p and Sit4, which dephosphorylate Thr210 of Snf1p (7-9). SNF1 is regulated at the dephosphorylation step, and ADP activates SNF1 by protecting it against dephosphorylation (10).SNF1 regulates transcription by several mechanisms, including phosphorylation of transcriptional activators and repressors; modification of chromatin; and RNA polymerase II, Srb-mediator, and preinitiation complex assembly. One of the best-studied targets of SNF1 is the repressor Mig1p. Upon glucose depletion, Mig1p is phosphorylated by SNF1, which results in the translocation of Mig1p from the nucleus to the cytoplasm (11-13) or relieves the transcriptional repression imposed by Mig1p by altering its interaction with the corepressor Tup1p/Ssn6p (14). In addition to phosphorylating and regulating the transcriptional factors, SNF1 also regulates directly the RNA polymerase II holoenzyme (15, 16). SNF1 regulates transcription also by a chromatin-based mechanism. SNF1 phosphorylates histone H3 on serine 10 (H3S10), and this modification promotes SAGA and TATA binding protein recruitment at the INO1 gene and acetylation of histone H3 at lysine 14 (H3K14) (17-19). However, since H3S10 phosphorylation is not required for activation of SUC2, GAL1, and HIS3 genes, it is not a general requirement for transcriptional induction o...

Lack of mitochondria-generated acetyl-CoA by pyruvate dehydrogenase complex downregulates gene expression in the hepatic de novo lipogenic pathway

“…Studies in yeast showed that increased availability of AcCoA due to Acc1 gene deletion was sufficient to enhance histone acetylation (13,45). Similarly, activation of ACC1 by inhibition of AMPK in yeast resulted in decreased histone acetylation (44). In budding yeast, AcCoA production by the PDC in the mitochondria was indirectly altered by deletion of mitochondrial pyruvate carrier-1 (MPC1), resulting in accumulation of acetate in the cytosol, triggering upregulation of the cytosolic AceCS1 for AcCoA production, increased histone acetylation, and repression of autophagy genes (10).…”

Reduced Histone Expression or a Defect in Chromatin Assembly Induces Respiration

“…Denatured proteins were separated on a 15% denaturing polyacrylamide gel. Western blotting with anti-histone H3 polyclonal antibody (catalog number ab1791; Abcam) at a dilution of 1:1,000, anti-histone H4 polyclonal antibody (catalog number 2542; Cell Signaling) at a dilution of 1:750, anti-histone H2A polyclonal antibody (catalog number 07-146; Millipore) at a dilution of 1:750, and anti-histone H2B polyclonal antibody (catalog number 07-371; Millipore) at a dilution of 1:750 was carried out as described previously (43,44). To confirm equivalent amounts of loaded proteins, the membranes were also probed with actin polyclonal antibody (catalog number A5060; Sigma) at a dilution of 1:500.…”

Protein Acetylation and Acetyl Coenzyme A Metabolism in Budding Yeast

“…The pools of acetyl-CoA in the nucleocytosolic and mitochondrial compartments are separate; the concentration of acetyl-CoA in the nucleocytosolic compartment affects protein acetylation in the nucleus and cytosol (10,45,106), while the concentration of acetyl-CoA in mitochondrion affects acetylation of mitochondrial proteins (13). In cells exponentially growing on glucose, the mitochondrial pool of acetyl-CoA represents about 30% of the total cellular pool (13).…”

“…The first and rate-limiting reaction in de novo synthesis of fatty acids is carboxylation of acetyl-CoA to form malonyl-CoA, a reaction that is catalyzed by acetyl-CoA carboxylase (Acc1p) (120). Decreased flux of acetyl-CoA through the fatty acid synthesis pathway by reducing ACC1 expression results in an increased cellular level of acetyl-CoA, globally increased histone acetylation, and altered transcriptional regulation (45,106). It is interesting that hypomorphic ACC1 mutations were isolated in a genetic screen for suppressors of inositol auxotrophy caused by low expression of the INO1 gene in snf1⌬ cells (122).…”

“…Global histone acetylation, as opposed to targeted acetylation, functions over large chromosomal regions irrespective of promoters, coding regions, or sequence-specific transcription factors (14). Thus, it is likely that the hypomorphic ACC1 mutations were recovered in both genetic screens because decreased enzymatic activity of Acc1p leads to an increased nucleocytosolic pool of acetyl-CoA and globally increased histone acetylation (45,106).…”