SREBP controls oxygen-dependent mobilization of retrotransposons in fission yeast.
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ABSTRACT:
SUBMITTER: Sehgal A
PROVIDER: S-EPMC1941750 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Sre1, the fission yeast sterol regulatory element binding protein, is an endoplasmic reticulum membrane-bound transcription factor that responds to changes in oxygen-dependent sterol synthesis as an indirect measure of oxygen availability. Under low oxygen, Sre1 is proteolytically cleaved and the released N-terminal transcription factor (Sre1N) activates gene expression essential for hypoxic growth. Here, we describe an oxygen-dependent mechanism for regulation of Sre1 that is independent of sterol-regulated proteolysis. Using yeast expressing only Sre1N, we show that Sre1N turnover is regulated by oxygen. Ofd1, an uncharacterized prolyl 4-hydroxylase-like 2-oxoglutarate-Fe(II) dioxygenase, accelerates Sre1N degradation in the presence of oxygen. However, unlike the prolyl 4-hydroxylases that regulate mammalian hypoxia-inducible factor, Ofd1 uses multiple domains to regulate Sre1N degradation by oxygen; the Ofd1 N-terminal dioxygenase domain is required for oxygen sensing and the Ofd1 C-terminal domain accelerates Sre1N degradation. Our data support a model whereby the Ofd1 N-terminal dioxygenase domain is an oxygen sensor that regulates the activity of the C-terminal degradation domain.
| S-EPMC2396400 | biostudies-literature
Project description:In fission yeast, orthologs of mammalian SREBP and Scap, called Sre1 and Scp1, monitor oxygen-dependent sterol synthesis as a measure of cellular oxygen supply. Under low oxygen conditions, sterol synthesis is inhibited, and Sre1 cleavage is activated. However, the sterol signal for Sre1 activation is unknown. In this study, we characterized the sterol signal for Sre1 activation using a combination of Sre1 cleavage assays and gas chromatography sterol analysis. We find that Sre1 activation is regulated by levels of the 4-methyl sterols 24-methylene lanosterol and 4,4-dimethylfecosterol under conditions of low oxygen and cell stress. Both increases and decreases in the level of these ergosterol pathway intermediates induce Sre1 proteolysis in a Scp1-dependent manner. The SREBP ortholog in the pathogenic fungus Cryptococcus neoformans is also activated by high levels of 4-methyl sterols, suggesting that this signal for SREBP activation is conserved among unicellular eukaryotes. Finally, we provide evidence that the sterol-sensing domain of Scp1 is important for regulating Sre1 proteolysis. The conserved mutations Y247C, L264F, and D392N in Scp1 that render Scap insensitive to sterols cause constitutive Sre1 activation. These findings indicate that unlike Scap, fission yeast Scp1 responds to 4-methyl sterols and thus shares properties with mammalian HMG-CoA reductase, a sterol-sensing domain protein whose degradation is regulated by the 4-methyl sterol lanosterol.
| S-EPMC2262954 | biostudies-literature
Project description:Sre1, the fission yeast sterol regulatory element-binding protein, is an ER membrane-bound transcription factor that controls adaptation to low oxygen growth. Under low oxygen, Sre1 is proteolytically cleaved and the N-terminal transcription factor domain (Sre1N) is released from the membrane and enters the nucleus to activate hypoxic gene expression. Ofd1, a prolyl 4-hydroxylase-like 2-oxoglutarate dioxygenase, controls the oxygen-dependent stability of Sre1N. In the presence of oxygen, Ofd1 accelerates the degradation of Sre1N, but under low oxygen Ofd1 is inhibited and Sre1N accumulates. To identify the regulators of Sre1N, we performed a plasmid-based screen for genes that increased Sre1N transcriptional activity. Here, we identify Nro1 (SPCC4B3.07) as a positive regulator of Sre1N stability and a direct inhibitor of Ofd1. In the absence of oxygen, Nro1 binds to the Ofd1 C-terminal degradation domain and inhibits Sre1N degradation. In the presence of oxygen, Nro1 binding to Ofd1 is disrupted, leading to rapid degradation of Sre1N. We conclude that the Ofd1 dioxygenase domain functions as an oxygen sensor that regulates binding of Nro1 to Ofd1 to control oxygen-dependent Sre1N stability.
| S-EPMC2634736 | biostudies-literature
Project description:Proteins of the Argonaute family have been identified as key components of RNA interference (RNAi) pathway. RNAi-related mechanisms are implicated in the regulation of gene expression and repression of transposable elements in eukaryotes. The piwi gene encoding protein of the Drosophila Argonaute family was shown to be required for the germ stem cells maintenance. Here, we show that piwi is involved in silencing of LTR retrotransposons in testes. piwi mutations led to derepression of endogenous retrotransposon copia as well as to upregulation of the reporter gene driven by copia LTR. piwi mutation causes accumulation of retrotransposon mdg1 transcripts at the apical tip of testes, including germinal proliferative center where PIWI protein was shown to be expressed. We applied inverse PCR approach to detect the newly arisen insertions of the mdg1 retrotransposon in the progeny of individual piwi mutant males. Owing to piwi mutation a high rate of mdg1 transpositions was revealed. Thus, piwi is involved in the silencing of retrotransposons in the precursors of male gametes. Our results provide the first evidence that protein of the Argonaute family prevents retrotranspositions. It is supposed that the disturbance of RNA silencing system in germinal cells might cause transposition burst.
| S-EPMC1074743 | biostudies-literature
Project description:In fission yeast, the endoplasmic reticulum membrane-bound proteins Sre1 and Scp1, orthologs of mammalian sterol regulatory element binding protein (SREBP) and Scap, monitor sterol synthesis as an indirect measure of oxygen supply. When cellular oxygen levels are low, sterol synthesis is inhibited, and the Sre1-Scp1 complex responds by increasing transcription of genes required for adaptation to hypoxia. Sre1 and Scp1 are believed to detect a blockage in sterol synthesis by monitoring levels of particular sterols, but the evidence concerning which sterol signals this condition is unclear. Here, we demonstrate that Sre1-Scp1 senses ergosterol. Processing experimental data with a mathematical model of Sre1 and Scp1 function reveals a clear quantitative relationship between ergosterol concentration in the endoplasmic reticulum and Sre1 activation. Based on this relationship, we predict that the Sre1-Scp1 complex exists under "active" and "inactive" states and that the transition between these states is cooperatively mediated by ergosterol.
| S-EPMC3003404 | biostudies-literature
Project description:BackgroundRetrotransposons are transposable elements that proliferate within eukaryotic genomes through a process involving reverse transcription. The numbers of retrotransposons within genomes and differences between closely related species may yield insight into the evolutionary history of the elements. Less is known about the ongoing dynamics of retrotransposons, as analysis of genome sequences will only reveal insertions of retrotransposons that are fixed--or near fixation--in the population or strain from which genetic material has been extracted for sequencing. One pre-requisite for retrotransposition is transcription of the elements. Given their intrinsic sequence redundancy, transcriptome-level analyses of transposable elements are scarce. We have used recently published transcriptome data from the fission yeast Schizosaccharomyces pombe to assess the ability to detect and describe transcriptional activity from Long Terminal Repeat (LTR) retrotransposons. LTR retrotransposons are normally flanked by two LTR sequences. However, the majority of LTR sequences in S. pombe exist as solitary LTRs, i.e. as single terminal repeat sequences not flanking a retrotransposon. Transcriptional activity was analysed for both full-length LTR retrotransposons and solitary LTRs.ResultsTwo independent sets of transcriptome data reveal the presence of full-length, polyadenylated transcripts from LTR retrotransposons in S. pombe during growth phase in rich medium. The redundancy of retrotransposon sequences makes it difficult to assess which elements are transcriptionally active, but data strongly indicates that only a subset of the LTR retrotransposons contribute significantly to the detected transcription. A considerable level of reverse strand transcription is also detected. Equal levels of transcriptional activity are observed from both strands of solitary LTR sequences. Transcriptome data collected during meiosis suggests that transcription of solitary LTRs is correlated with the transcription of nearby protein-coding genes.ConclusionsPresumably, the host organism negatively regulates proliferation of LTR retrotransposons. The finding of considerable transcriptional activity of retrotransposons suggests that part of this regulation is likely to take place at a post-transcriptional level. Alternatively, the transcriptional activity may signify a hitherto unrecognized activity level of retrotransposon proliferation. Our findings underline the usefulness of transcriptome data in elucidating dynamics in retrotransposon transcription.
| S-EPMC2848245 | biostudies-literature
Project description:Sterol regulatory element-binding proteins (SREBPs) are central regulators of cellular lipid synthesis and homeostasis. Mammalian SREBPs are proteolytically activated and liberated from the membrane by Golgi Site-1 and Site-2 proteases. Fission yeast SREBPs, Sre1 and Sre2, employ a different mechanism that genetically requires the Golgi Dsc E3 ligase complex for cleavage activation. Here, we established Sre2 as a model to define structural requirements for SREBP cleavage. We showed that Sre2 cleavage does not require the N-terminal basic helix-loop-helix zipper transcription factor domain, thus separating cleavage of Sre2 from its transcription factor function. From a mutagenesis screen of 94 C-terminal residues of Sre2, we isolated 15 residues required for cleavage and further identified a glycine-leucine sequence required for Sre2 cleavage. Importantly, the glycine-leucine sequence is located at a conserved distance before the first transmembrane segment of both Sre1 and Sre2 and cleavage occurs in between this sequence and the membrane. Bioinformatic analysis revealed a broad conservation of this novel glycine-leucine motif in SREBP homologs of ascomycete fungi, including the opportunistic human pathogen Aspergillus fumigatus where SREBP is required for virulence. Consistent with this, the sequence was also required for cleavage of the oxygen-responsive transcription factor Sre1 and adaptation to hypoxia, demonstrating functional conservation of this cleavage recognition motif. These cleavage mutants will aid identification of the fungal SREBP protease and facilitate functional dissection of the Dsc E3 ligase required for SREBP activation and fungal pathogenesis.
| S-EPMC3711301 | biostudies-literature
Project description:Sterol regulatory element-binding protein (SREBP) transcription factors are master regulators of cellular lipid homeostasis in mammals and oxygen-responsive regulators of hypoxic adaptation in fungi. SREBP C-terminus binds to the WD40 domain of SREBP cleavage-activating protein (SCAP), which confers sterol regulation by controlling the ER-to-Golgi transport of the SREBP-SCAP complex and access to the activating proteases in the Golgi. Here, we biochemically and structurally show that the carboxyl terminal domains (CTD) of Sre1 and Scp1, the fission yeast SREBP and SCAP, form a functional 4:4 oligomer and Sre1-CTD forms a dimer of dimers. The crystal structure of Sre1-CTD at 3.5 Å and cryo-EM structure of the complex at 5.4 Å together with in vitro biochemical evidence elucidate three distinct regions in Sre1-CTD required for Scp1 binding, Sre1-CTD dimerization and tetrameric formation. Finally, these structurally identified domains are validated in a cellular context, demonstrating that the proper 4:4 oligomeric complex formation is required for Sre1 activation.
| S-EPMC5099872 | biostudies-literature
Project description:The sterol regulatory element-binding protein (SREBP) and SREBP cleavage-activating protein (SCAP) are central players in the SREBP pathway, which control the cellular lipid homeostasis. SCAP binds to SREBP through their carboxyl (C) domains and escorts SREBP from the endoplasmic reticulum to the Golgi upon sterol depletion. A conserved pathway, with the homologues of SREBP and SCAP being Sre1 and Scp1, was identified in fission yeast Schizosaccharomyces pombe. Here we report the in vitro reconstitution of the complex between the C domains of Sre1 and Scp1 as well as the crystal structure of the WD40 domain of Scp1 at 2.1 Å resolution. The structure reveals an eight-bladed β-propeller that exhibits several distinctive features from a canonical WD40 repeat domain. Structural and biochemical characterization led to the identification of two Scp1 elements that are involved in Sre1 recognition, an Arg/Lys-enriched surface patch on the top face of the WD40 propeller and a 30-residue C-terminal tail. The structural and biochemical findings were corroborated by in vivo examinations. These studies serve as a framework for the mechanistic understanding and further functional characterization of the SREBP and SCAP proteins in fission yeast and higher organisms.
| S-EPMC4387560 | biostudies-literature
Project description:In fission yeast, meiosis-specific transcripts are selectively eliminated during vegetative growth by the combined action of the YTH-family RNA-binding protein Mmi1 and the nuclear exosome. Upon nutritional starvation, the master regulator of meiosis Mei2 inactivates Mmi1, thereby allowing expression of the meiotic program. Here, we show that the E3 ubiquitin ligase subunit Not4/Mot2 of the evolutionarily conserved Ccr4-Not complex, which associates with Mmi1, promotes suppression of meiotic transcripts expression in mitotic cells. Our analyses suggest that Mot2 directs ubiquitination of Mei2 to preserve the activity of Mmi1 during vegetative growth. Importantly, Mot2 is not involved in the constitutive pathway of Mei2 turnover, but rather plays a regulatory role to limit its accumulation or inhibit its function. We propose that Mmi1 recruits the Ccr4-Not complex to counteract its own inhibitor Mei2, thereby locking the system in a stable state that ensures the repression of the meiotic program by Mmi1.
| S-EPMC5614563 | biostudies-literature