Snf1 dependence of peroxisomal gene expression is mediated by Adr1

Abstract

Eukaryotes utilize fatty acids by beta-oxidation, which occurs in the mitochondria and peroxisomes in higher organisms and in the peroxisomes in yeast. The AMP-activated protein kinase regulates this process in mammalian cells, and its homolog Snf1, together with the transcription factors Adr1, Oaf1, and Pip2, regulates peroxisome proliferation and beta-oxidation in yeast. A constitutive allele of Adr1 (Adr1(c)) lacking the glucose- and Snf1-regulated phosphorylation substrate Ser-230 was found to be Snf1-independent for regulation of peroxisomal genes. In addition, it could compensate for and even suppress the requirement for Oaf1 or Pip2 for gene induction. Peroxisomal genes were found to be regulated by oleate in the presence of glucose, as long as Adr1(c) was expressed, suggesting that the Oaf1/Pip2 heterodimer is Snf1-independent. Consistent with this observation, Oaf1 binding to promoters in the presence of oleate was not reduced in a snf1Delta strain. Exploring the mechanism by which Adr1(c) permits Snf1-independent peroxisomal gene induction, we found that strength of promoter binding did not correlate with transcription, suggesting that stable binding is not a prerequisite for enhanced transcription. Instead, enhanced transcriptional activation and suppression of Oaf1, Pip2, and Snf1 by Adr1(c) may be related to the ability of Adr1(c) to suppress the requirement for and enhance the recruitment of transcriptional coactivators in a promoter- and growth medium-dependent manner.

FIGURE 1.

Adr1^c and Adr1 overexpression can regulate peroxisomal genes upon oleate induction in oaf1Δ, pip2Δ, or snf1Δ. A and B, gene expression was measured by qPCR in adr1Δoaf1Δ or adr1Δpip2Δ carrying plasmids for Adr1 (pKD16), Adr1^c (S230A, pKD14) or Adr1 overexpression (pKD17) after subjecting to glucose-derepressing, oleate-inducing conditions (0.1% oleate + 3% glycerol (derepressing-inducing, DRI)) for 1 h. C, adr1Δsnf1Δ strain carrying plasmids for either WT Adr1 (pKD16) or one of three Adr1^c alleles (S230A, pKD14; Δ226–233, pKD26; and R228K, pKD27) was assayed for gene expression by qPCR after subjecting to glucose-derepressing, oleate-inducing conditions (0.1% oleate + 3% glycerol (DRI)) for 1 h. Values are expressed as percent wild type expression; error bars indicate standard deviation of three biological samples assayed in duplicate.

FIGURE 2.

Adr1^c binds some gene promoters better than WT Adr1 under repressing conditions, and Ser-230-phosphorylated Adr1 binds less well in derepressing conditions. Adr1 binding at promoters was detected by ChIP under repressing (A, 5% glucose) and derepressing (B, 3% glycerol) conditions using adr1Δ transformed with plasmids pKD14HA (Adr1^c-HA) or pKD16HA (wtAdr1-HA). C, binding of total Adr1 and Adr1 phosphorylated at Ser-230 was determined under derepressing (3% glycerol) conditions using anti-HA and anti-Ser(P)-230Adr1 antibodies in an adr1Δ strain carrying pKD16HA (wtAdr1-HA). Values were normalized to input and binding at telomere (TEL); error bars represent standard deviations of two biological samples. IP, immunoprecipitation.

FIGURE 3.

Adr1^c partially suppresses coactivator defects in derepressing conditions. mRNA levels were measured after 4 h of incubation in 3% glycerol by qPCR in strains deleted for specific coactivator components and ADR1, carrying either plasmid pKD14 for Adr1^c or pKD16 for Adr1. A, data for individual mutants of the Mediator complex (med2Δ, med3Δ, med15Δ, and med16Δ) were combined and averaged; B, snf2Δ; and C, snf5Δ; D and E, SAGA mutants gcn5Δ and ada1Δ. Values are expressed as percentage of WT expression; error bars indicate standard deviations of three biological samples. DR, derepressing.

FIGURE 4.

Adr1^c partially suppresses coactivator defects during oleate induction. mRNA levels were measured after 1 h of incubation in 0.1% oleate + 3% glycerol by qPCR in strains deleted for ADR1 and specific coactivator components as well as carrying either plasmid pKD14 for Adr1^c or pKD16 for Adr1. A, average expression in individual mutants of the Mediator complex (med2Δ, med3Δ, med15Δ, and med16Δ); in Swi/Snf mutants: snf2Δ (B), and snf5Δ (C); and in SAGA mutants: gcn5Δ (D) and ada1Δ (E). Values are expressed as percentage of WT expression; error bars indicate standard deviations of three biological samples. DRI, derepressing-inducing.

FIGURE 5.

Adr1^c recruits some coactivator components better than WT Adr1. Recruitment of coactivator components was measured by ChIP-qPCR in strains deleted for ADR1 but carrying either plasmid pKD14 for Adr1^c or pKD16 for Adr1 and with Myc-tagged coactivator components. Samples were collected under repressing (R; 5% glucose), derepressing (DR; 3% glycerol) and inducing (DRI; 0.1% oleate + 3% glycerol) conditions. The recruitment of SAGA components (Gcn5 and Ada1) was determined upon repression (A) and derepression (B). C and D show Snf2 recruitment under derepression and oleate induction, respectively, compared with repressed conditions. Error bars indicate standard deviations of two biological samples. IP, immunoprecipitation.

FIGURE 6.

Model for the interaction of Snf1, Adr1, Oaf1, and Pip2 at co-dependent promoters. In this model, Snf1 controls the activity of Adr1 by indirectly stimulating its dephosphorylation (23). The inactive transcription factors are shown in rectangles with dashed borders and are denoted with an i; the active transcription factors are enclosed in solid rectangles and are denoted with an a. Adr1^c does not require Snf1 for activation because it lacks that phosphorylatable Ser-230. Swi/Snf1 was recruited to Adr1^c-activated peroxisomal genes, but it is shown with a dashed border because their transcription was less sensitive to its loss when Adr1^c was the activator (Table 5; Figs. 3 and 4). Mediator was not assessed at peroxisomal genes because their transcription was relatively insensitive to its loss when Adr1^c was the activator. Adr1^c-activated transcription of peroxisomal genes was dependent on SAGA but was less sensitive to the loss of the histone acetyltransferase component Gcn5 than to the loss of the structural component Ada1 (Table 5; Figs. 3 and 4).