Analysis of the sulfur-regulated control of the cystathionine γ-lyase gene of Neurospora crassa
© Reveal and Paietta; licensee BioMed Central Ltd. 2012
Received: 16 January 2012
Accepted: 5 June 2012
Published: 2 July 2012
Cystathionine γ-lyase plays a key role in the transsulfuration pathway through its primary reaction of catalyzing the formation of cysteine from cystathionine. The Neurospora crassa cystathionine γ-lyase gene (cys-16 + ) is of particular interest in dissecting the regulation and dynamics of transsulfuration. The aim of this study was to determine the regulatory connection of cys-16 + to the Neurospora sulfur regulatory network. In addition, the cys-16 + promoter was characterized with the goal of developing a strongly expressed and regulatable gene expression tool.
The cystathionine γ-lyase cys-16 + gene was cloned and characterized. The gene, which contains no introns, encodes a protein of 417 amino acids with conserved pyridoxal 5’-phosphate binding site and substrate-cofactor binding pocket. Northern blot analysis using wild type cells showed that cys-16 + transcript levels increased under sulfur limiting (derepressing) conditions and were present only at a low level under sulfur sufficient (repressing) conditions. In contrast, cys-16 + transcript levels in a Δcys-3 regulatory mutant were present at a low level under either derepressing or repressing conditions. Gel mobility shift analysis demonstrated the presence of four CYS3 transcriptional activator binding sites on the cys-16 + promoter, which were close matches to the CYS3 consensus binding sequence.
In this work, we confirm the control of cystathionine γ-lyase gene expression by the CYS3 transcriptional activator through the loss of cys-16 + expression in a Δcys-3 mutant and through the in vitro binding of CYS3 to the cys-16 + promoter at four sites. The highly regulated cys-16 + promoter should be a useful tool for gene expression studies in Neurospora
Cystathionine γ-lyase (E.C. 22.214.171.124; also known as γ-cystathionase) catalyzes the conversion of cystathionine to cysteine and α-ketobutryrate. Cystathionine γ-lyase, therefore, plays a key role in the transsulfuration reactions involved in the interconversion of homocysteine and cysteine routing through the intermediate cystathionine. Secondary reactions catalyzed include elimination reactions of homoserine, cystine, and cysteine; which in the latter case yield hydrogen sulfide (H2S). The proposed role of H2S as a physiologic signaling molecule has led to a number studies with the mammalian cystathionine γ-lyase[1, 2], including gene knock-out constructs in mice. Interestingly, the reverse transsulfuration pathway using cystathionine β-synthase and cystathionine γ-lyase, appears restricted to fungal and mammalian systems among eukaryotes[4, 5]. Thus, the fungi offer an additional model system for cystathionine γ-lyase studies.
The properties and expression of cystathionine γ-lyase in fungi have been examined in several species. In Neurospora crassa the enzyme has been purified and enzyme assays have shown up to a 30x increase in activity upon sulfur starvation[7, 8] as well as low activity under sulfur sufficient conditions. In Aspergillus nidulans, the cystathionine γ-lyase gene (designated mecB) has been cloned; mecB transcript levels show derepression under low sulfate levels; but showed increased transcription (with proportional enzyme activity increases) following growth with methionine supplementation. In contrast, the Acremonium chrysogenum cystathionine γ-lyase gene does not appear to be regulated by the level of methionine. In N. crassa, unlike these other fungi, the sulfur level provided for growth appears to have a straightforward effect on either derepressing or repressing cystathionine γ-lyase enzyme activity level.
The N. crassa sulfur regulatory system is composed of a genetically defined set of trans-acting regulatory genes and a set of structural genes encoding enzymes used in the uptake and assimilation of a variety of sulfur compounds[11–13]. Basically, when N. crassa is cultured under conditions of sulfur limitation (i.e., derepressing conditions) then an entire set of sulfur-related enzymes is coordinately expressed. The available data, therefore, suggests that N. crassa cystathionine γ-lyase is likely under control of this control system; along with other confirmed sulfur-regulated genes encoding arylsulfatase, sulfate permeases I and II, choline sulfatase, and others. The key regulator in the control system is the CYS3 bZIP transcriptional activator which is essential for sulfur structural gene expression. The consensus binding sequence for CYS3 has been determined by in vitro binding-site selection studies.
Besides the cystathionine γ-lyase enzymatic assays done under sulfur-limiting and sulfur –sufficient conditions mentioned above, there is additional data available from microarray studies that have examined gene expression during Neurospora crassa development that include cys-16 + transcript levels. A major increase in cys-16 + transcript level was observed during the initial phase of the conidial germination process. Starting with dormant conidia, there was an approximately 2.5-fold increase in cys-16 + transcript level at 1 hour into the germination process. The cys-16 + transcript level then slowly declined, from the peak level seen at 1 hour, over an incubation time of 16 hours to end up slightly below the starting level (i.e., that seen in dormant conidia). The elevation in cys-16 + transcript (and presumably cystathionine γ-lyase activity) seems likely to represent an accession of the available cystathionine pool in order to supply the cellular demand for cysteine as germination commences and active growth begins. Consistent with this idea, additional microarray studies demonstrated a 3.6-fold higher cys-16 + transcript level in the active growth periphery (i.e., hyphal tips) as compared to a colonies interior region (i.e., 12–15 hours old). A final note regarding available microarray data is that a 2-fold increase in cys-16 + transcript level was observed post-exposure to phytosphingosine (which induces programmed cell death and notable increases in transcript level for many metabolism–related genes). The microarray data provides a useful developmental context for future studies of cys-16 + .
In this report, we present the cloning, transcript and promoter analysis of the N. crassa cystathionine γ-lyase (cys-16 + ) gene. The data extend the role of the CYS3-directed regulatory system to include the transsulfuration pathway through the control of cystathionine γ-lyase and provide an additional model system to study the dynamics of transsulfuration. In addition, the tight regulation and high expression level of the N. crassa cystathionine γ-lyase gene make it’s promoter a potentially valuable tool for studies requiring manipulated control of gene expression.
Results and discussion
Sequence and characterization of the cystathionine γ-lyase gene
Analysis of cys-16 + gene expression
The role of CYS3 in the regulation of cys-16 + was examined using Northern blot analysis of cys-16 + expression in a strain deleted for the cys-3 + gene (Δcys-3). Northern blots of poly(A)+ mRNA isolated from Δcys-3 grown under low- and high-sulfur conditions were probed with the cys-16 + gene (Figure2). The level of cys-16 + transcript detected was equivalently low for both low- and high-sulfur growth conditions; and similar to the repressed level seen in wild-type under high sulfur growth conditions. The Δcys-3 mutant completely blocks the derepression of cys-16 + expression seen under low-sulfur growth conditions in wild-type. In this regard, the cys-16 + gene is showing the typical response of genes previously confirmed to be part N. crassa sulfur control circuit (e.g., arylsulfatase, sulfate permease I and II, choline sulfatase, and others) in that sulfur starvation derepresses transcription in wild-type and mutating the CYS3 regulator abolishes that derepression[11, 13].
Gel mobility shift analysis of CYS3 regulator binding
The cystationine γ-lyase gene, cys-16 + , is tightly regulated and is derepressed upon growth of N. crassa under conditions of sulfur limitation. Taken collectively, the in vivo expression experiments using a Δcys-3 mutant and the in vitro CYS3 binding data strongly supports the CYS3 regulation of cys-16 + expression. These experiments provide a starting point for examining the dynamics of transsulfuration control in a model eukaryotic system. In addition, the cys-16 + gene contains a potentially useful regulatable promoter for gene expression studies.
Strains, plasmids and culture conditions
74OR23-1a was used as the wild-type (WT) for these studies. Δcys-3 (18–4) was constructed and described in a prior study. The λ-J1 N. crassa genomic library and N-EST:Nc3A10 clone were obtained from the Fungal Genetics Stock Center (Kansas City, KS). Vogel minimal medium, with supplements as required, was used. N. crassa cultures were grown at 25o C. Sulfur repression and derepression experiments were done by growth of mycelia on Vogel-minus-sulfur medium plus high sulfur (5.0 mM methionine) and low-sulfur medium (0.25 mM methionine) medium, respectively.
Gene cloning and sequencing
The N. crassa cDNA clone N-EST:Nc3A10 was used to produce a 32P-labeled probe and carry out plaque hybridization as described to a N. crassa λ-J1 genomic library and identify hybridizing clones. A segment containing the cys-16 + gene derived from an isolate designated λ412 was subsequently subcloned into pSPORT and subjected to automated sequencing (Cleveland Genomics; Cleveland, OH) by primer walking. The genomic sequence was submitted to GenBank as AF401238. Following the cloning of the gene, the gene symbol cys-16 + was assigned by A. Radford (University of Leeds) and included in The Neurospora crassa e-Compendium. The gene has also since been given the locus designation NCU09230 in the Broad Institute Neurospora crassa database.
Poly(A)+ mRNA was isolated by phenol extraction and subsequent oligo (dT)-cellulose chromatography as described previously. Briefly, mycelial samples were harvested by filtration, frozen in liquid nitrogen, and homogenized in a 1:1 mixture of phenol-chloroform-isoamyl alcohol (49:49:2) and extraction buffer (1% sarkosyl, 100 mM sodium acetate, 1 mM EDTA [pH 5.0]). After phenol-chloroform extractions, precipitation, and sodium acetate washes, and the poly(A)+ mRNA was isolated by oligo (dT)-cellulose chromatography. 32P-labeled probes were prepared by oligolabeling of DNA fragments. Northern blots were hybridized and washed as outlined elsewhere.
Gel mobility shifts
Purified promoter fragments derived from restriction endonuclease digestion were used in an initial scan for CYS3 binding. Consequently, oligonucleotides representing the binding sites to be tested were synthesized (Applied Biosystems 391EP synthesizer), labeled by T4 polynucleotide kinase with [γ-32P]ATP, annealed and gel purified as described[14, 26]. The following oligonucleotides (and complementary strands) representing the four putative CYS3 binding sites on the cys-16 + promoter were prepared: Site 1 [5′ ACCATGCATTCCGCCATAGAGGTA 3′], Site 2 [5′ GTCGTCGATGGTGTCAGTGGTGCT 3′], Site 3 [5′ AACCTGAATTGCGCCATAGCCAAA 3′], and Site 4 [5′ TCACCAATTGGCGCCATCTCCGTC 3′]. The synthesized mutated versions had a G to T substitution at the sixth position of the 10 bp core of the CYS3 consensus binding site: Site 1 M [5′ ACCATGCATTCCT CCATAGAGGTA 3′], Site 2 M [5′ GTCGTCGATGGGT TCAGTGGTGCT 3′], Site 3 M [5′ AACCTGAATTGCT CCATCGCCAAA 3′], and Site 4 M [5′ TCACCAATTGGCT CCATCTCCGTC 3′]. DNA-binding assays were carried out as we have described previously using Eschericia coli produced CYS3 protein[14, 20]. Specificity of binding was ensured by control experiments using competition by addition of excess unlabeled DNA. Four percent PAGE gels with a 50 mM Tris-80 mM glycine-2 mM EDTA (pH 8.5) running buffer were electrophoresed at 20 mA with the temperature maintained at 4o C. Quantitation of gel shift assays was performed by using a Molecular Dynamics Phosphorimager.
Availability of supporting data
The sequence data supporting the results of this article is available in the GenBank repository [AF401238,http://0-www.ncbi.nlm.nih.gov.brum.beds.ac.uk/nuccore/AF401238].
The work was supported by a Medical Innovations Grant from the Wright State University Boonshoft School of Medicine.
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