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Plant Physiol. (1998) 117: 189-196
Light-Regulated Transcription of Genes Encoding Peridinin
Chlorophyll a Proteins and the Major Intrinsic
Light-Harvesting Complex Proteins in the Dinoflagellate
Amphidinium carterae Hulburt (Dinophycae)1
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ABSTRACT |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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In the dinoflagellate Amphidinium carterae, photoadaptation involves changes in the transcription of genes encoding both of the major classes of light-harvesting proteins, the peridinin chlorophyll a proteins (PCPs) and the major a/c-containing intrinsic light-harvesting proteins (LHCs). PCP and LHC transcript levels were increased up to 86- and 6-fold higher, respectively, under low-light conditions relative to cells grown at high illumination. These increases in transcript abundance were accompanied by decreases in the extent of methylation of CpG and CpNpG motifs within or near PCP- and LHC-coding regions. Cytosine methylation levels in A. carterae are therefore nonstatic and may vary with environmental conditions in a manner suggestive of involvement in the regulation of gene expression. However, chemically induced undermethylation was insufficient in activating transcription, because treatment with two methylation inhibitors had no effect on PCP mRNA or protein levels. Regulation of gene activity through changes in DNA methylation has traditionally been assumed to be restricted to higher eukaryotes (deuterostomes and green plants); however, the atypically large genomes of dinoflagellates may have generated the requirement for systems of this type in a relatively "primitive" organism. Dinoflagellates may therefore provide a unique perspective on the evolution of eukaryotic DNA-methylation systems.
In many respects dinoflagellates are an unusual group of
chromophytic algae, their closest relatives being the apicomplexans (van der Peer et al., 1993). For example, uniquely among eukaryotes, their chromatin appears to be entirely devoid of histones (Rizzo, 1981 In Amphibinium carterae, both PCPs and
LHCs are encoded by nuclear genes (Hiller et al., 1995 At this time, almost nothing is known about the molecular basis of
photoadaptation in dinoflagellates. PCP may account for up to 95% of
the total cellular-soluble protein under low-light conditions
(Prezelin, 1987 In green plants light-regulated transcription of the cab genes is
mediated via phytochrome; response elements have been identified in the
promoters of the cab genes (Terzaghi and Cashmore, 1995 Preliminary studies in our laboratory suggested the presence of
significant levels of 5-MeC at CpG motifs in genomic DNA from the
dinoflagellate A. carterae. Many studies suggest that a
major function of this type of methylation in eukaryotes is in the
regulation of gene expression (for review, see Lewis and Bird, 1991 Although it is not clear whether methylation is a cause or a
consequence of alterations in gene activity, a number of lines of
evidence suggest that the effects of methylation may be mediated via
modifications of chromatin structure. For example, introduced DNAs
generally adopt chromatin structures specified by their methylation status (Keshet et al., 1986 In summary, the evolution and roles of eukaryotic DNA-methylation
systems and the interactions between three processes, methylation, alterations of chromatin structure, and changes in gene activity, are
not well understood. In this respect, dinoflagellates may be highly
informative because, although otherwise typical eukaryotes, they are
unique in posessing permanently condensed chromosomes and in lacking
histones (Rizzo, 1981 To investigate the possible involvement of DNA methylation in the
regulation of gene expression in dinoflagellates, we examined the
methylation status of A. carterae PCP and LHC genes under different light conditions. Photoadaptation involved major changes in
the levels of transcripts encoding both of these classes of light-harvesting proteins. DNA from A. carterae was normally
highly methylated at CpG motifs and significantly methylated at CpNpG motifs. However, when grown under reduced light conditions, elevated mRNA levels were accompanied by decreases in the methylation of CpG and
CpNpG motifs within or near PCP- and LHC-coding regions. Therefore,
cytosine-methylation levels are nonstatic in A. carterae and
may be influenced by environmental factors. However, chemically induced
undermethylation was found to be insufficient for gene activation.
These results provide the first evidence to our knowledge that the
relationship between cytosine methylation and transcriptional activity,
which has been observed for a large number of genes in a wide range of
eukaryotes, does not require histones and nucleosomes.
Source and Maintenance of Cultures
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
,
1991) and is not organized in the normal nucleosomal structure (Herzog
and Soyer, 1981). Light harvesting is mediated in dinoflagellates by
two classes of proteins, the PCPs and the LHCs. PCP, a water-soluble protein containing the carotenoid peridinin, is unique to
dinoflagellates and appears to be unrelated to any other
light-harvesting protein (Norris and Miller, 1994), whereas the LHCs
are related to the cab proteins of higher plants (Hiller et al., 1993)
and their algal homologs (for review, see Grossman et al., 1990). Both
PCPs and LHCs are present in multiple forms in dinoflagellates. The functional significance of this and the nature of the interaction between these two classes of light-harvesting proteins are completely unknown. Chromophyte chloroplasts appear to lack many of the mechanisms known to be central to photoadaptation in chlorophytes, such as lateral mobility of photosynthetic complexes and stacking/unstacking of
thylakoids. One hypothesis is that the multiple forms of
light-harvesting proteins fulfill related roles in dinoflagellates,
i.e. that the synthesis of different PCP and LHC complements
facilitates photoadaptation.
; Sharples et
al., 1996) and synthesized with N-terminal transit peptides directing
chloroplast translocation (Norris and Miller, 1994; Hiller et al.,
1995; Sharples et al., 1996). As in the case of Euglena
gracilis, the A. carterae LHC mRNA encodes a
polyprotein. The PCPs are encoded by discrete mRNAs, but at least some
of the genes appear to be tandemly organized (Hiller et al., 1995).
), and there is some indirect evidence that PCP
synthesis is light regulated at the level of transcription in
Glenodinium sp. (Roman et al., 1988). As stated above, the LHCs are the dinoflagellate homologs of the cab proteins and, by
analogy with higher plants (for review, see Anderson, 1986) and green
algae (Kindle, 1986), light regulation is likely.
). However,
phytochrome has been detected only in chlorophytes, implying that the
regulation of synthesis of light-harvesting proteins may be achieved
via alternative mechanisms in other algal groups. In the chrysophyte
Giraudyopsis stellifer, synthesis of the chlorophyll a/c protein is light regulated at the level of transcription
(Passaquet and Lichtl, 1995), whereas in E. gracilis, LHC
synthesis is apparently regulated posttranscriptionally (for review,
see Houlne and Schanz, 1993). How these effects are mediated in the
apparent absence of the phytochrome system is unknown at this time.
).
Transcriptionally inactive regions of chromatin are generally highly
meth-ylated at CpG motifs (Keshet et al., 1986; for review, see
Lewis and Bird, 1991), whereas active chromatin is often
undermethylated (Antequera et al., 1989; Kochanek et al., 1993).
Moreover, for many genes CpG methylation status reflects
transcriptional activity.
; Buschhausen et al., 1987). Methylation may
affect chromatin structure by modifying nucleosome assembly through the
action of methylcytosine-binding proteins (Nan et al., 1997) or by a
combination of processes. Regions containing 5-MeC may preferentially
bind histone H1 (Ball et al., 1983; Jost and Hofsteenge, 1992) and
associate with histones H3 and H4 mainly in their nonacetylated forms
(Tazi and Bird, 1990), presumably promoting more condensed forms of
chromatin. Methylation-specific binding proteins (Meehan et al., 1989;
Lewis et al., 1992) can also mediate changes in chromatin structure.
, 1991) and nucleosomes (Herzog and Soyer, 1981).
MATERIALS AND METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
1. Cells were normally grown on a 14-h/10-h
light-dark cycle at 80 to 100 µmol m2
s1 (two white, two warm, and two Grow-Lux tubes
[Sylvania]), referred to here as high-light conditions. For low-light
treatments, cells were grown under high-light conditions for 1 week
before being transferred to either 18 to 20 µmol
m2 s1 (moderate-light
cultures; two Grow-Lux fluorescent tubes only) or < 2 µmol
m2 s1 (low-light
cultures) for 14 d.
DNA Extraction, Restriction, and Southern Analysis
To avoid possible interference by intrinsic circadian rhythms, cells were always harvested by centrifugation 6 h into the light phase of growth. Pellets were ground in liquid N2, and genomic DNA was isolated using the standard high-salt method (Dellaporta, 1983). Purified DNA was restricted and fractionated on 0.8 to 1.2% agarose gels. After Southern transfer to Hybond-N membranes (Amersham), blots were hybridized with 32P-labeled (Feinberg and Vogelstein, 1983) DNA probes. Bacteriophage 
DNA was used as an
internal control in restriction digests. Homologous probes for the LHC
(pRGH201; Hiller et al., 1995) or the main form of the PCP genes (see
figure 6 of Sharples et al., 1996) were kindly provided by R. Hiller
(Macquarie University, Sydney, Australia). 16S-Like rDNA probes were
generated from A. carterae DNA using universal PCR primers
(Medlin et al., 1988). Heterologous hybridization probes (from the
coral Acropora millepora) for the highly conserved ubiquitin
CEP52 (ub52; Berghammer et al., 1996) and elongation factor
1
(Ef 1) genes were provided by H. Berghammer (James
Cook University, Townsville, Australia).
RNA Extraction, Transfer, and Hybridization
Poly(A+) RNA was extracted from approximately 107 cells with an mRNA purification kit (QuickPrep, Pharmacia), using the manufacturer's recommended protocol. Precipitated RNA was resuspended in Tris-EDTA buffer, quantified spectrophotometrically, and stored at
70°C. Aliquots of
mRNA (0.25-1.0 µg) were applied to nylon membranes through a slotted
template following the supplier's (Schleicher & Schuell) instructions.
For the preparation of northern blots, 2.0-µg aliquots of mRNA were
subjected to electrophoresis in 1% agarose/formaldehyde gels prior to
transfer to nylon membranes in 20× SSC. Hybridizations (6× SSC, 0.1%
SDS, and 2× Denhardt's solution at 60°C for 24 h) and three
washes (0.5× SSC, and 0.1% SDS at 60°C for 20 min) were conducted
as described by Sambrook et al. (1989).
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RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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PCP and LHC mRNA Levels Increase under Low-Light Conditions
Northern analysis (Fig. 1A) and slot-blotting experiments (Fig. 1B) clearly demonstrated that the abundance of PCP and LHC mRNAs changed substantially in response to light conditions, whereas the level of the mRNAs corresponding to the housekeeping genes ub52 and EF1 did not vary
significantly.
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PCP and LHC Loci Are Normally Hypermethylated at CpG Motifs and Their Methylation Status Is Modified by Light Conditions
Preliminary experiments indicated a substantial level of cytosine methylation in the genome of A. carterae. DNA prepared by Dellaporta's (1983) method was digestible by a range of methylation-insensitive restriction endonucleases, including MvaI and HaeIII. However, the same DNA preparations were highly resistant to a range of CpG-methylation-sensitive restriction endonucleases, including HpaII and HhaI (Fig. 2). The presence of diffusible inhibitors of digestion in the DNA preparations was excluded by the use of bacteriophageDNA as an internal control (see Fig. 2A, right panel).
These results therefore imply that A. carterae DNA is hypermethylated at CpG motifs, as has been reported for a few higher
plants (Gruenbaum et al., 1981) and algae (Jarvis et al. 1992).
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Heterogenous DNA-Methylation Changes at CpNpG Motifs
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Figure 3.
A. carterae PCP and LHC loci are
normally hypermethylated at CpG motifs and their methylation status is
modified by light conditions. A, Southern analysis of PCP, LHC, and
rDNA loci in cells grown under different light conditions using the
restriction enzymes HpaII (H) and MspI
(M); C, controls. The sizes indicated to the left of the figure
correspond to positions of DNA size standards. Note that the apparently
repeating pattern seen for the MspI digest of
high-light-grown cells (A, left panel) is consistent with the
hypothesis that several of the PCP loci are tandemly organized
(Sharples et al., 1996 ). B, Restriction maps of single A. carterae PCP (based on figure 6A of Sharples et al., 1996; bp
35-1110)- and LHC (based on figure 1B of Hiller et al., 1995, bp
10-600)-coding sequences that were used as probes.
Chemically Induced Undermethylation Does Not Alter PCP Transcript
Levels
Light-Regulated Transcription of PCP and LHC Genes
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Figure 4.
CpNpG methylation at LHC loci is variable in
A. carterae. The figure shows Southern analysis of DNA
from cells grown under various light conditions digested with either
EcoRII (E; sensitive to CpNpG methylation) or
MvaI (Mv; an EcoRII isoschizomer
insensitive to CpNpG methylation) hybridized with the corresponding
probe. Lanes C, Controls.
View larger version (73K):
[in a new window]
Figure 5.
Chemically induced undermethylation is not
sufficient to activate transcription of PCP genes in A. carterae. A. carterae cultures in the exponential phase of
growth were transferred to medium supplemented with either
5-azacytidine (200 µm) or ethionine (300 µm), and grown under high-light conditions for 6 weeks,
after which DNA was extracted and digested with HpaII
(H) or MspI (M) prior to electrophoresis (A), and
blotting and hybridization with the PCP probe (B). Lanes C, Controls.
C, Slot blots of mRNA prepared from cells treated in the same manner
and hybridized with the PCP probe. Numbers to the right of the blots
are the corresponding standardized photon equivalents determined by
quantitative phosphor imaging. Note that exposure of A. carterae to these compounds resulted in reductions in growth
rates of around 35 to 50%, but appeared to otherwise have no adverse
effects on morphology or development of the organisms (data not shown).
Transfer of treated cells back into normal medium resulted in an
immediate return to normal growth rates.
). However, although exposure to either
inhibitor reduced the growth rate of A. carterae by around
35 to 50% (data not shown), there appeared to be no adverse effects on
morphology or development. Motility appeared to be unaltered in the
presence of inhibitors, and effects on growth rates were fully
reversible; transfer of azacytidine-treated A. carterae back
into normal medium resulted in an immediate return to normal growth
rates. Treated and untreated cells were indistinguishable in terms of
pigmentation, and levels of both total mRNA and soluble proteins (data
not shown) were shown not to differ significantly between treatments.
In summary, although we cannot completely exclude chemically
induced artifacts, our results strongly suggest that simply reducing
the extent of 5-MeC within or near PCP genes does not appear to affect
transcription.
DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References
).
). The larger (6 kb) form of the A. carterae LHC mRNA detected here corresponds to the previously estimated size (Hiller et al., 1995). Although
lower-Mr forms of the LHC mRNA have previously
been observed (Hiller et al., 1995), these appeared to be very minor
components. The A. carterae LHC-coding sequences are
relatively heterogeneous (Hiller et al., 1993, 1995), and the use of
different LHC probes in the previous and present studies may account
for this difference; the probes have approximately 70% identity,
which, under the conditions employed, may have had a significant effect
on the results of northern analysis. The functional significance of the
qualitative shift in the LHC mRNA population with light intensity is
presently unclear. The LHC mRNA encodes a polyprotein, with the 6.1-kb
form encoding possibly up to 10 LHC protein units (Hiller et al.,
1995). If there is only one LHC locus, we might predict that the
proteins encoded by the 3
end of the mRNA might be required only under reduced illumination. Alternatively, multiple loci under independent regulation may encode the 3- and 6-kb forms of the LHC mRNA. Analyses of the genomic loci will be required to resolve these alternatives.
CpG and CpNpG Methylation Patterns in A. carterae May Be Influenced by Environmental Factors
Although no significant differences were observed between cells grown under moderate- and low-light conditions, when A. carterae cells grown under high-light conditions were shifted to lower light intensities, demethylation occurred at CpG and CpNpG motifs within or proximal to the LHC- and PCP-coding regions. CpG demethylation appears to be partial, affecting a limited number of meth-ylatable sites or loci, a minor proportion of cells, or a combination of both. By contrast, light-induced CpNpG demethylation events appear to be more pronounced and uniform. Consideration of the available sequence data for A. carterae PCP (Sharples et al., 1996) and LHC (Hiller et al., 1995) implies that whereas changes
in the methylation status of CpG motifs may be localized at or near the coding sequence, CpNpG methylation changes are likely to occur over
larger regions, affecting sequences distant to those transcribed or
occurring in tandemly arranged multiple copy loci. The existence of
tandemly organized PCP loci has been recently documented for Gonyaulax polyedra (Le et al., 1997) and has been previously
proposed for A. carterae (Sharples et al., 1996). Our
apparently repeating pattern seen for the MspI digest of
high-light-grown cells (Fig. 3A, left panel) is consistent with this
proposal.
;
Galaud, 1993) or transgenes (e.g. Meyer et al., 1992); for example,
light-induced de-methylation of distant regulatory sites correlated
with increased levels of PEPCase mRNA levels in Zea mays
(Langdale et al., 1991). However, we were unable to find precedents for
methylation changes affecting endogenous genes involved in light
harvesting.
Decreased Methylation Is Not Sufficient to Activate Transcription of PCP and LHC Genes
The question of whether DNA methylation states are required for gene activity or represent consequences of genomic changes involved in the activation of chromatin is controversial. Although treatment with 5-azacytidine or ethionine reduced the extent of methylation of PCP genes, these changes were not accompanied by increased transcript levels, nor did the corresponding protein levels increase (data not shown). This indicates that simply reducing overall levels of methylation is not sufficient to activate PCP transcription in A. carterae. As in our study, 5-azacytidine treatment did not reactivate methylated regions in Z. mays (Brown, 1989); however, there are numerous reports of the opposite effect,
5-azacytidine-treatment-mediated reactivation of T-DNA in plants (Klaas
et al., 1989; Kilby et al., 1992) and of a hepatitis-C cDNA transgene
in mice (Kato et al., 1996) and chemically mediated demethylation
initiating flowering in Arabidopsis thaliana and
Thlaspi arvense (Burn et al., 1993).
).
The Evolution and Possible Roles of Dinoflagellate DNA-Methylation Systems
Our studies establish the presence of significant and variable levels of CpG and CpNpG methylation in dinoflagellates, suggesting the presence of a higher-plant-like methyltransferase(s) (Theiss et al., 1987). Our data are consistent with the hypothesis that methylation systems may have arisen as a means of noise reduction in
organisms with large genomes (Bestor, 1990; Bird, 1995; Jablonka and
Regev, 1995). Dinoflagellates have atypically large genomes, ranging
from 2 to 3 pg in A. carterae to > 200 pg in G. polyedra (Holm-Hansen, 1969; Rizzo, 1987), compared with other
algae at the same apparent level of complexity (e.g. < 0.2 pg for the
green algae Chlamydomonas and Chlorella;
Cattolico and Gibbs, 1975; Holm-Hansen, 1969). This suggests that major
amounts of heterochromatin may be present in dinoflagellates, perhaps
generating a requirement for methylation systems to maintain this in a
transcriptionally inactive state.
), and certainly tens and probably
hundreds of copies of the rbcL gene in Symbiodinium
microadriaticum (R. Rowan, personal communication). None of these
complex multigene families has been fully characterized, and in every
case probes for single genes are likely to cross-hybridize. Thus, it is
not possible to relate demethylation events to active copies of genes.
-regulatory sequences are
also likely to be involved in regulation (e.g. Langdale et al., 1991);
however, with the exception of one copy (of the approximately 1000 copies) of a G. polyedra LBP gene (Lee et al., 1993) and one
copy (of the suggested 5000 copies) of a G. polyedra PCP
gene (Le et al., 1997), sequence data are not available for any
dinoflagellate promoters. Further progress in understanding the
significance of DNA methylation in dinoflagellate gene regulation requires the characterization of genes or gene families and the corresponding promoters to a level that will permit the development of
highly specific probes for single loci.
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FOOTNOTES |
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Received August 28, 1997;
accepted December 29, 1997.
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ABBREVIATIONS |
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Abbreviations: 5-MeC, 5-methylcytosine. LHC(s), chlorophyll a/c-containing intrinsic light-harvesting protein(s). PCP(s), peridinin chlorophyll a protein(s).
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ACKNOWLEDGMENT |
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We thank Dr. R. Hiller for providing cloned A. carterae PCP and LHC cDNAs.
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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