Sterol regulatory element binding protein is a principal regulator of anaerobic gene expression in fission yeast

Abstract

Fission yeast sterol regulatory element binding protein (SREBP), called Sre1p, functions in an oxygen-sensing pathway to allow adaptation to fluctuating oxygen concentrations. The Sre1p-Scp1p complex responds to oxygen-dependent sterol synthesis as an indirect measure of oxygen availability. To examine the role of Sre1p in anaerobic gene expression in Schizosaccharomyces pombe, we performed transcriptional profiling experiments after a shift to anaerobic conditions for 1.5 h. Of the 4,940 genes analyzed, expression levels of 521 (10.5%) and 686 (13.9%) genes were significantly increased and decreased, respectively, under anaerobic conditions. Sre1p controlled 68% of genes induced > or = 2-fold. Oxygen-requiring biosynthetic pathways for ergosterol, heme, sphingolipid, and ubiquinone were primary targets of Sre1p. Induction of glycolytic genes and repression of mitochondrial oxidative phosphorylation genes largely did not require Sre1p. Using chromatin immunoprecipitation, we demonstrated that Sre1p acts directly at target gene promoters and stimulates its own transcription under anaerobic conditions. sre1+ promoter analysis identified two DNA elements that are both necessary and sufficient for oxygen-dependent, Sre1p-dependent transcription. Interestingly, these elements are homologous to sterol regulatory elements bound by mammalian SREBP, highlighting the evolutionary conservation between Sre1p and SREBP. We conclude that Sre1p is a principal activator of anaerobic gene expression, upregulating genes required for nonrespiratory oxygen consumption.

FIG. 1.

Sre1p is required at early times for anaerobic growth. (A) Wild-type and sre1Δ yeast cells were cultured in rich medium under aerobic (+O₂) or anaerobic (−O₂) conditions for 16 h. Cell density was measured using a hemacytometer at the indicated time points. Data are the means from two replicates. Error bars equal 1 standard deviation. (B) Log-phase wild-type yeast cells were cultured in rich medium and shifted to anaerobic conditions at time zero. At the indicated time points, cell extracts and total RNA samples were prepared and analyzed. (Top) Cell extracts were subjected to immunoblot analysis using anti-Sre1p IgG before and after treatment with alkaline phosphatase (+AP). P and N denote the precursor and cleaved nuclear forms of Sre1p, respectively. (Bottom) Total RNA (5 μg) was subjected to Northern blot analysis with the indicated ³²P-labeled probes.

FIG. 2.

Oxygen-regulated and Sre1p-dependent genes involved in ergosterol biosynthesis. Genes predicted to be involved in ergosterol biosynthesis are shown with key intermediates in black boxes. The average change (n-fold) in expression of each gene in wild-type cells in the absence of oxygen is given on the right. Significant anaerobically induced genes (boldface type), significant anaerobically repressed genes (underlined), and significant Sre1p-dependent genes (boxed) are shown. The S. pombe ergosterol synthesis pathway is modeled after S. cerevisiae using the S. pombe ortholog table and has not been determined experimentally (22, 37).

FIG. 3.

Oxygen-regulated and Sre1p-dependent genes in heme biosynthesis. Genes predicted to be involved in heme biosynthesis are shown with intermediates in black boxes. The average change (n-fold) in expression of each gene in wild-type cells in the absence of oxygen is given on the right. Significant anaerobically induced genes (boldface type) and significant Sre1p-dependent genes (boxed) are shown. The S. pombe heme synthesis pathway is modeled after S. cerevisiae using the S. pombe ortholog table and has not been determined experimentally (30, 37). Abbreviations: δ-ALA, 5-aminolevulinate; PBG, porphobilinogen; Preurogen, hydroxymethylbilane; Urogen, uroporphyrinogen; Coprogen, coproporphryinogen; Protogen, protoporphyrinogen; Proto, protoporphyrin.

FIG. 4.

Sre1p is recruited to target gene promoters in vivo. (A) Wild-type and sre1Δ yeast cells were cultured in rich medium under aerobic (lanes 1 and 3) (+) or anaerobic (lanes 2 and 4) (−) conditions for 4 h. Total RNA (5 μg) was subjected to Northern blot analysis with the indicated ³²P-labeled probes. (B) Chromatin immunoprecipitation was performed in parallel using formaldehyde-treated, sonicated cell extracts and anti-Sre1p IgG. Binding of Sre1p to upstream regions was assayed by real-time PCR, and bound DNA is expressed as the change normalized to the wild-type aerobic (+O₂) sample. Data are means of three replicates from one experiment, and error bars indicate 1 standard deviation. erg3⁺ (SPAC1687.16c) and osm1⁺ (SPAC17A2.05) were assigned names of their closest S. cerevisiae sequence homologs.

FIG. 5.

The region −550 to −450 bp upstream of the sre1⁺ start codon is required for Sre1p-dependent transcription. (A) Wild-type and sre1Δ cells carrying a reporter plasmid containing the 850 bp upstream of sre1⁺ fused to lacZ were cultured in rich medium under aerobic conditions (+O₂) or conditions of 0.2% oxygen for 12 h. Cells were assayed for β-galactosidase activity as described in Materials and Methods. (B) Wild-type and sre1Δ yeast cells containing the indicated reporter plasmids were cultured in rich medium under conditions of 0.2% oxygen and assayed for β-galactosidase (β-gal) activity. Data are the means of six replicates from two independent experiments, and error bars indicate 1 standard deviation. The region required for Sre1p-dependent lacZ expression is denoted by SRE.

FIG. 6.

Identification of a functionally conserved S. pombe SRE element. (A) Sequence alignments of the human LDL receptor SRE sequence and putative sre1⁺ SRE sequences. Shaded boxes indicate sequence conservation with human LDL receptor SRE. (B) Purified, recombinant Sre1p(aa 256-366) (3 μg) was subjected to SDS-polyacrylamide gel electrophoresis, and the gel was stained with Gelcode blue (Pierce). (C) Purified Sre1p(aa 256-366) (lanes 2 to 11) was mixed with the indicated ³²P-labeled DNA probes in the presence (lanes 5 to 7 and 9 to 11) or absence (lanes 1 to 4 and 8) of unlabeled competitor DNA probes. The human LDL receptor wild-type probe sequence is shown with the SRE element in boldface type. The mutant (mut) probe contains the indicated mutations in the SRE element. Competitor probes were used at a 10-fold excess (lanes 5 and 9), a 100-fold excess (lanes 6 and 10), and a 1,000-fold excess (lanes 7 and 11). Underlined bases were included to label the probe. (D) Purified Sre1p(aa 256-366) (lanes 2 to 13) was mixed with the indicated ³²P-labeled DNA probes in the presence (lanes 7 to 9 and 11 to 13) or absence (lanes 1 to 6 and 10) of unlabeled competitor DNA probes. The wild-type probe sequence is shown, and putative SRE elements 1 and 2 are in boldface type. Mutant probe sequences are as follows: 1* indicates mutations in SRE1 with the wild-type SRE2 sequence, 2* indicates the wild-type SRE1 sequence with indicated mutations in SRE2, and 1*2* indicates mutations in both SRE1 and SRE2. Competitor probes were used at a 10-fold excess (lanes 7 and 11), a 100-fold excess (lanes 8 and 12), and a 1,000-fold excess (lanes 9 and 13). (E) Wild-type yeast containing the indicated reporter plasmids were cultured in rich medium under aerobic (+O₂) or anaerobic (−O₂) conditions for 12 h. Mutated SRE sequences are the same as those described above (D). Cells were assayed for β-galactosidase activity. Data are the means from six replicates of two independent experiments, and error bars indicate 1 standard deviation.

FIG. 7.

SRE2 and SRE3 are each necessary and sufficient for anaerobic regulation of the sre1⁺ promoter. (A) Identification of SRE3. Purified Sre1p(aa 256-366) was mixed with the indicated overlapping ³²P-labeled DNA probes that scan across the region at bp −570 to −430 of the sre1⁺ promoter in 10-bp increments. Probes including SRE1, SRE2, and SRE3 are indicated. The SRE3 sequence identified by the sequence overlap between probes 8 and 9 is shown below. (B and C) Wild-type yeast cells containing the indicated reporter plasmids were cultured in rich medium under aerobic (+O₂) or anaerobic (−O₂) conditions for 12 h. Cells were assayed for β-galactosidase (β-gal) activity. (B) Mutations in SRE2 are the same as those shown in Fig. 6D. For SRE3, the same nucleotide positions were changed to ACT and ACA. Data are the means of three replicates, and error bars indicate 1 standard deviation. (C) Data are the means of six replicates from two independent experiments, and error bars indicate 1 standard deviation.