Biology Department, Reed College, Portland, Oregon 97202,1 and Departments ofMedicinal ..... Adams, R. L. P., F. I. McKay, L. M. Craig, and R. H. Burdon. 1979 ...
Vol. 169, No. 6
JOURNAL OF BACTERIOLOGY, June 1987, p. 2902-2905
0021-9193/87/062902-04$02.00/0 Copyright X 1987, American Society for Microbiology
Differential DNA Methylation during the Vegetative Life Cycle of Neurospora crassat PETER J. RUSSELL,'* KARIN D. RODLAND,lt ELLIOT M. RACHLIN,2 AND JAMES A. McCLOSKEY2 Biology Department, Reed College, Portland, Oregon 97202,1 and Departments of Medicinal Chemistry and Biochemistry, University of Utah, Salt Lake City, Utah 841122 Received 1 December 1986/Accepted 9 March 1987 Isotope dilution gas chromatography-mass spectrometry analysis of genomic DNAs isolated from the different growth phases of Neurospora crassa revealed significant differences in the amounts of 5methylcytosine; the mol% of 5-methylcytosine was 0.36 in conidia (asexual spores), 0.40 in conidial germlings cultured for 3 h, 0.24 in mycelial cells that had grown exponentially for 6 and 12 h, and 0.40 in stationary-phase mycelial cells. These results indicate an approximate inverse correlation between the level of C methylation in the DNA and the presumed level of developmentally related transcriptional activity.
m5Cyt levels in genomic DNA isolated from various stages in the vegetative life cycle of a wild-type strain of the ascomycetous filamentous fungus N. crassa. (A preliminary report of some of the results of this study was presented at the UCLA Symposium on Molecular Genetics of the Filamentous Fungi, Keystone, Colo., 1985
DNA methylation, a postreplicative modification of DNA, has been reported to occur in both procaryotic and eucaryotic organisms. In eucaryotes, 5-methylcytosine (m5Cyt) in particular has been shown to affect many important biological functions through effects on gene expression (for reviews, see references 9, 10, 15, and 22). Some eucaryotes, particularly vertebrates, contain relatively high levels of m5Cyt, predominantly in the sequence CpG (6, 12, 14, 17). In many of these organisms, there has been reported a negative correlation between m5Cyt levels and gene expression (e.g., 7, 10, 11, 15, 23, and 28). However, in a number of other systems, the correlation between relative m5Cyt content and the level of gene expression is inconsistent (e.g., 5, 7, 9, 19, and 23). While m5Cyt is present in significant amounts in the DNA of higher eucaryotes, including vertebrates and higher plants, it is not present or it is present at relatively low levels in a number of organisms, for example, many insects and arthropods and the protozoan Tetrahymena themophila (1, 7, 21, 31). Among the fungi, diverse patterns of cytosine methylation are seen, with the general aspect being that the fungi contain much lower m5Cyt levels than do higher eucaryotes, making accurate quantification difficult with the usual methods of analysis, namely, restriction enzyme digestion or high-performance liquid chromatography. In Neurospora crassa, the subject of this study, the genomic DNA has been reported to contain very low levels of m5Cyt as judged by restriction enzyme analyses (4, 6), while significant cytosine methylation has been detected in the ribosomal DNA (25). In most studies, quantitative analysis of C methylation in genomic DNA has involved high-performance liquid chromatography. The limitations of this method with respect to its sensitivity have led to the development of a method for the direct analysis of m5Cyt in genomic DNA which exploits the high degree of sensitivity, selectivity, and precision inherent in isotope dilution gas chromatography-mass spectrometry (GC-MS; 8, 13). Using GC-MS, we analyzed the *
[25].)
Conidia (asexual spores) from the wild-type strain 74-OR23-LA were harvested from 7-day-old cultures of the organism on solid Vogel complete medium (27, 32) incubated for 2 days at 35°C and for 5 days at 25°C. Conidia were germinated, and mycelial cultures were produced in liquid Vogel minimal medium in Delong flasks which were incubated with shaking at 25°C. DNA was isolated from the conidia, from germinating conidia, and from exponentially growing mycelial cultures as previously described (24). DNA was isolated from stationary-phase mycelial cultures as described previously (26). The DNA was purified by two successive CsCl equilibrium density gradient centrifugations, dissolved in low-salt buffer (1 mM Tris hydrochloride [pH 7.4], 1 mM NaCl, 0.1 mM EDTA), precipitated with ethanol, and dried by using a Savant Speed-Vac (Savant Instruments, Inc., Hicksville, N.Y.). The procedures followed for isotope dilution GC-MS were improved versions of a method described earlier (8). All reactions were carried out in reaction tubes fabricated from thoroughly cleaned Pyrex (Coming Glass Works, Coming, N.Y.) tubing (outside diameter, 3 mm). A solution containing known concentrations of [2H3]thymine ([3H3]Thy) and [2H3,15N]m5Cyt was added to approximately 1 ,ug of dried DNA. Samples of 20 microliters of 95% formic acid (Aldrich Chemical Co., Inc., Milwaukee, Wis.) were added to the mixture. The tubes were sealed and heated to 180°C for 1 h. After removal of the acid, the samples were treated with pyridine and dried in vacuo to remove any trace quantities of water. Conversion of the free bases to their t-butyldimethylsilyl derivatives was carried out in the original hydrolysis tubes. The reagents, pyridine and N-methyl-N-(t-butyldimethylsilyl)trifluoroacetamide with 1% t-butyldimethylsilylchlorosilane as the catalyst (Regis Chemical Co., Morton Grove, Ill.), were used as supplied by the manufacturer.
Corresponding author.
t This paper is dedicated to the memory of Steven C. Schlitt (1951-1986). t Present address: Department of Cell Biology and Anatomy, Oregon Health Sciences University, Portland, OR 97201. 2902
VOL. 169, 1987
NOTES
2903
B
w
LiJ Un z 0 aU) LiJ
5Cyt*
z
0 IL
V,
0~ cr 0
0:: 0 0
Thy*
w 0
cw
Thy
cL
0
m5Cyt 296 297
5 MINUTES
2
K
3
4
lII6
5
I II I
I
7
8
MINUTES
FIG. 1. Elution profile of spike blank mixture (A) and natural Thy and m5Cyt (B) in DNA from N. crassa conidia by stable-isotope dilution GC-MS. Thy* and m5Cyt* denote the labeled internal standards [2H3]Thy and [2H3,'5N]m5Cyt, respectively. MS responses for channels m/z 296, 297, and 300 are shown for the GC elution period 2 to 8 min. The m5Cyt was quantified relative to Thy and therefore relative to DNA. Detector sensitivity was increased 165-fold at approximately 5.5 min.
All analyses were performed on an LKB-9000S gas chromatograph-mass spectrometer (LKB Instruments, Inc., Rockville, Md.). Samples were introduced through a DB-5 fused-silica capillary column (30 m by 0.25 mm; J & W Scientific, Ranch Cordova, Calif.). The GC column was programmed from 180 to 280°C at 10°C/min. The carrier gas pressure was adjusted to provide for the maximum chromatographic resolution attainable with that column. Other instrumental temperatures were as follows: GC inlet, 250°C; separator, 250°C; and source, 270°C. The ionizing energy was 70 eV, and the accelerating potential was 3 kV. Characteristic M - 57 ions of the derivatized bases were selected by computer-controlled accelerating-voltage switching. The ions monitored were as follows: Thy, m/z 297; [2H3]Thy, m/z 300; m5Cyt, m/z 2%; and [2H3,15N] m5Cyt, m/z 300. Peak heights from DNA-derived bases and from isotopically labeled internal standards were measured from the selected-ion recordings and used in the construction of calibration curves and in the determination of m5Cyt (8). In this manner, the m5Cyt content relative to Thy was measured and related to DNA through the Thy content of DNA (18). The quantities of synthetic standards used for the construction of the curves were within an order of magnitude of the quantity predicted for m5Cyt. All samples were run as replicates to guarantee statistical reliability of the data. Five growth stages were selected in the study of DNA methylation. Each sample was treated independently and run in duplicate to determine the reliability of the results. A spike mixture containing only the internal standard was run as a blank to establish minor background contributions to the m/z 296 and 297 channels (Fig. 1A). A representative selected-ion recording for the quantitative measurement of m5Cyt from N. crassa DNA is shown in Fig. 1B. The level for the signal in the channel used to monitor endogenous m5Cyt was
noise limited, and the contribution of variation of the baseline in the m5Cyt channel to DNA samples was not considered to be significant. The quantities of m5Cyt (in mol%) calculated for the DNAs of conidia, germinating conidia, exponentially growing cells, and stationary-phase cells of N. crassa are shown in Table 1 and clearly indicate a significant variation in the extent of cytosine methylation with growth time. Specifically, the data in the table indicate that the level of cytosine methylation was approximately the same in the conidia, in conidial germlings (3-h cultures), and in stationary-phase cells, while significantly lower levels were seen in the exponentially growing cells from the 6- and 12-h cultures. No difference in cytosine methylation was evident between the cells from the 6- and 12-h cultures. From the data, one can conclude that there was a reduction in the percent cytosine methylation during the more active stages in the vegetative growth cycle. These data contrast with those derived from restriction enzyme analysis which indicated that N. crassa DNA has very low levels of m5Cyt (4, 6). The restriction enzyme TABLE 1. m5Cyt content in N. crassa Growth phase (h)
Trial 1
mol% msCyta Trial 2
Avg
Conidia Conidial germlings (3) Early exponential (6) Mid-exponential (12)
0.37
0.35
0.36
0.42 0.24
0.38 0.23
0.24
0.24
Stationary
0.41
0.39
0.40 0.24 0.24 0.40
The amount of m5Cyt was calculated based on a Thy content for N. crassa DNA of 23.3% (18). a
2904
NOTES
analysis procedure used in the previous studies is far less sensitive than the GC-MS procedure is, however. Moreover, in our work, restriction enzyme analysis of the genes coding for rRNA of N. crassa has indicated significant cytosine methylation in the nontranscribed spacer region (25). Cytosine methylation varies considerably among the fungi. Negligible cytosine methylation has been reported in Aspergillus nidulans and Aspergillus niger DNAs (30), and none has been found in Saccharomyces cerevisiae DNA (20). The results of surveying 20 species of fungi from 15 families indicated that 18 of the 20 species have undetectable levels of cytosines methylated, while Sporotrichum dimorphosporum and Phycomyces blakesleeanus have low levels of m5Cyt (2). In P. blakesleeanus, m5Cyt is present at a level of 2.9% of the total cytosines, and the cytosine methylation pattern has been thoroughly studied (3). The basidiomycete Coprinus cinereus is also reported to contain high levels of m5Cyt (33). Significant cytosine methylation has been found in ribosomal DNA of Schizophyllum commune (29) and N. crassa (25). Our data indicate that the level of cytosine methylation changes during the vegetative life cycle of N. crassa. The highest levels of m5Cyt were found in conidia, conidial germlings (3-h cultures), and stationary-phase cells, and the lowest levels were found in exponentially growing cells from 6- and 12-h cultures. In a number of systems, there is an inverse correlation between the level of cytosine methylation and the level of gene activity, and our results, which are from studies of developmental stages, seem to provide another example of that relationship in that exponentially growing cells were much more transcriptionally active than were either conidia or stationary-phase cells. Similarly, DNA methylation was shown by high-performance liquid chromatography and restriction enzyme analysis to be quantitatively different at different developmental stages in another fungus, the plant pathogen Phymatotrichum omnivorum; approximately threefold more m5Cyt residues are found in the DNA from the dormant sclerotia than in the DNA from the metabolically active mycelia (16). In conclusion, GC-MS is perhaps the most sensitive and selective method available for discerning differences in DNA methylation at low levels, such as is the case in many fungi. More definitive conclusions about the role of methylation in N. crassa development will be possible when more basic information is available about developmentally significant genes and when cloned probes derived from these genes are used to investigate the methylation status of specific sites during development. P.J.R. acknowledges the excellent technical assistance of Sheryl Wagner. This work was supported in part by Public Health Service grants CA 09038 from the National Cancer Institute and GM 21584 from the National Institute of General Medical Sciences to J.A.M. and by grant GM 32922 to P.J.R. from the National Institute of General Medical Sciences. Some equipment used by P.J.R. during this work was purchased from funds from National Science Foundation Special Equipment Program grants PRM-7919955, CDP-8007790, CDP-8007801, and PRM-8114794 and from U.S. Department of the Army grant DAAG29-85-G-0038. LITERATURE CITED 1. Adams, R. L. P., F. I. McKay, L. M. Craig, and R. H. Burdon. 1979. Methylation of mosquito DNA. Biochim. Biophys. Acta
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specific differences in ribosomal DNA from the fungus Schizophyllum commune. Curr. Genet. 8:219-232. Tamame, M., F. Antequera, J. R. Villaneuva, and T. Santos. 1983. High frequency conversion to a "fluffy" developmental phenotype in Aspergillus spp. by 5-azacytidine treatment: evidence for involvement of a single nuclear gene. Mol. Cell. Biol. 3:2287-2297. Urieli-Shoval, S., Y. Gruenbaum, J. Sedat, and A. Razin. 1982. The absence of detectable methylated bases in Drosophila melanogaster DNA. FEBS Lett. 146:148-152. Vogel, H. J. 1964. Distribution of lysine pathways among fungi: evolutionary implications. Am. Nat. 98:435-446. Zolan, M. E., and P. J. Pukkila. 1985. DNA methylation in Coprinus cinereus. UCLA Symp. Mol. Cell. Biol. 34:333-344.
