A permissive chromatin structure is adopted prior to site-specific DNA demethylation of developmentally expressed genes involved in macronuclear differentiation
© Bulic et al.; licensee BioMed Central Ltd. 2013
Received: 12 November 2012
Accepted: 17 January 2013
Published: 5 March 2013
DNA methylation and demethylation are important epigenetic regulatory mechanisms in eukaryotic cells and, so far, only partially understood. We exploit the minimalistic biological ciliate system to understand the crosstalk between DNA modification and chromatin structure. In the macronucleus of these cells, the DNA is fragmented into individual short DNA molecules, each representing a functional expression and replication unit. Therefore, long range epigenomic interaction can be excluded in this system.
In the stichotrichous ciliate Stylonychia lemnae, cytosine methylation occurs in a small subset of macronuclear nanochromosomes expressed only during sexual reproduction. Methylation pattern shows similarity to that observed in fungi and Drosophila. Cytosine methylation correlates with gene activity and chromatin structure. Upon gene activation, cytosines become demethylated and a redistribution of histone post-translational modifications (PTMs) takes place. Evidence is presented that the formation of a permissive chromatin structure in the vicinity of the 5meCs precedes cytosine methylation and is probably a necessary prerequisite for their demethylation. Shortly after demethylation of cytosines occurs, the parental macronucleus degenerates, a new macronucleus is formed from a micronuclear derivative and the specific methylation pattern is transmitted from the germline micronucleus to the new macronucleus.
We show that very few, or even only one, discrete methylated cytosines are required to assign regulatory functions at a specific locus. Furthermore, evidence is provided that a permissive chromatin structure is probably a necessary prerequisite for the demethylation of specific cytosines. Our results allow us to propose a mechanistic model for the biological function of cytosine methylation in the ciliate cell and its regulation during the cell cycle.
KeywordsCytosine methylation Chromatin structure Demethylation Ciliates Macronucleus
There is general agreement that differential molecular signatures of both the DNA and the proteinous contents of chromatin, such as histones, above the primary DNA sequence encode epigenetic information that are prerequisites for the spatiotemporal control of gene expression in a potentially heritable way. On the molecular level these signatures include DNA methylation at carbon number five in the pyrimidine ring of cytosines (5meC) , covalent post-translational modifications (PTMs) of all types of histone proteins, mostly at their N-terminal tails , and very probably the incorporation of specific histone variants into nucleosomal arrays . DNA methylation has been frequently observed at symmetric CpG motifs in humans and many other organisms [4, 5] as well as at asymmetric motifs (CpNpG and CpHpH), depending on the species and the developmental stage of an organism [6, 7]. Cytosine methylation is mostly associated with transcriptional repression, possibly by direct blocking of transcription factor binding or by recruitment of histone deacetylases, thus impeding the decondensation of higher order conformations . But it is now evident that cytosine methylation is more dynamic than previously thought .
The next level of epigenomic regulation of gene expression above the level of DNA modifications is the compaction of the 10 nm chromatin fiber, in which the DNA is wrapped around nucleosomes. This 10 nm fiber becomes further compacted by the interaction with linker histone H1 and other proteins . Through altering the degree of chromatin compaction, PTMs of histone tails create chromatin structures favorable either for activation or repression of genes, depending on the genomic context and the combination of modifications at a given site . Modifications found at distinct residues of the histone protein N-termini include - among others - lysine acetylation, lysine and arginine methylation as well as serine and threonine phosphorylation.
A crosstalk between DNA methylation and demethylation and chromatin structure has been assumed for a long time. In fact, histone modifications and DNA methylation could be mechanistically interconnected in various organisms. There are descriptions of DNA methylation directing histone modifications at specific loci in mammals [10–12] but only a limited number of examples have been reported in which histone modifications direct DNA methylation [13–17]. Demethylation processes are even more intricate as only one recent report shows that in Arabidopsis, a histone acetyltransferase regulates active DNA demethylation .
Although cytosine methylation in the ciliate genome has been described as being potentially involved in DNA processing during macronuclear differentiation [24, 25], a potential role in the regulation of gene expression has so far not been demonstrated. To address this issue, we analyzed the occurrence of 5-methyl cytosine in either constitutively expressed macronuclear genes or genes only activated during sexual reproduction. We provide strong evidence for a pivotal role of the DNA methylation status of specific cytosines for the regulation of differential gene expression and propose that site-specific cytosine methylation may be involved in long-term silencing of a sub-fraction of developmentally regulated genes. This methylation pattern seems to be transmitted from the micronucleus to the macronucleus during macronuclear differentiation. Moreover, we demonstrate that the creation of a permissive chromatin structure above the methylated cytosines precedes cytosine demethylation and may be a necessary prerequisite for this process.
The majority of macronuclear nanochromosomes are continuously expressed during vegetative growth of the cell. However, recently we showed that at the onset of sexual reproduction, ten hours post-conjugation a sub-fraction of nanochromosomes becomes developmentally expressed during early macronuclear development (Figure 1A, stage a1) while they are silent in the vegetative macronucleus. According to a recent microarray analysis, less than 1% of the nanochromosomes (approximately 100 nanochromosomes, most of which are not yet characterized) present in the macronucleus are developmentally expressed , for example, nanochromosomes encoding some histone variants or proteins involved in programmed DNA elimination show developmentally regulated expression.
In the macronucleus, 5meC is associated with some genes in their silent state and becomes removed upon activation
To find out whether there is also a possible link between cytosine methylation and gene expression, we tried to determine whether any cytosine methylation occurs in macronuclear DNA and whether this DNA modification differs between genes permanently expressed in the macronucleus from those which are transcriptionally repressed during vegetative growth and only activated during sexual reproduction.
Analyses of bisulfite-modified macronuclear DNA revealed that in the genes constitutively expressed (actin I, β tubulin, histone H4) no cytosine methylation can be detected in either vegetative cells or in exconjugant cells. In contrast, in DNA isolated from vegetative cells, cytosine methylation was observed in the 5′-non coding region of both mdp1 and mdp2. In mdp1, three methylated cytosines were found at positions 28, 35 and 38 in the sequence context CAG and CG. In mdp2, only one methylated cytosine could be found at position 44 in the sequence context CTG (for statistical significance see Additional file 1). In both cases the methylated cytosines are upstream of the putative TATA boxes. No sequence homology between mdp1 and mdp2 in the regions in which these methylated cytosines were located could be found except that both sequences are very AT rich. In both cases the first cytosine(s) downstream, the telomeric sequence was methylated while no cytosines further downstream were modified (Figure 3B, C). Remarkably, although the enormous multiplication of both mdp1 and mdp2 expression in exconjugant cells strongly indicate that both genes are silent during vegetative growth (Figure 1G), we could find a significant subset of mdp2 nanochromosomes unmethylated (Figure 3C; Additional file 1), suggesting that at least for some genes, DNA methylation alone is not sufficient to induce repression of their expression.
In DNA isolated from exconjugants at a time point where mdp1 and mdp2 are expressed, no cytosine DNA methylation could be observed, suggesting that they are actively demethylated (Figure 3B, C). Thus, demethylation of very few, or in the case of mdp2, even only one methyl group at a specific cytosine correlates with activation of gene expression. Interestingly, it has been reported that induced CD4+ T-cell activation leads to demethylation of a single CpG site in the promoter-enhancer of the human IL2 gene, and that this change is necessary and sufficient to enhance transcription of a reporter plasmid .
DNA methylation signatures of mdp1 and mdp2 nanochromosomes are reminiscent of their micronuclear patterns
We made an attempt to understand how this specific methylation pattern is introduced into these developmentally expressed nanochromosomes. For this, we isolated DNA from micronuclei and early macronuclear anlagen in the precursor sequences of mdp1 and mdp2. As shown in micronuclear DNA as well as in DNA from the differentiating macronucleus, the same cytosines are methylated as in the vegetative macronucleus. Our data therefore suggest that the methylation pattern of the germline micronucleus is preserved and transmitted to the new macronucleus. In contrast, in the nanochromosomal precursor sequences, macronuclear-destined sequences (MDSs) of either the micronucleus or the developing macronucleus of the constitutively expressed β tubulin, no methylation was found in either the micronuclei or macronuclear anlage (see Additional file 2).
Repressive chromatin markers become relocalized in a subset of genes which are activated at the onset of sexual reproduction only
We have recently shown that activation of mdp1 and mdp2 correlates with a redistribution of histone modifications typical for active genes. While in the silenced status, these PTMs accumulate at the 3′-end of the gene they are enriched at the 5′-end upon activation . In this former study the distribution of modifications typical for repressed chromatin was not included. In fact, no signals are obtained when macronuclei of vegetative cells (data not shown) or fragments of parental macronuclei are stained in situ with an antibody directed against H3K9me3/K27me3 (Figure 2A), suggesting that these PTM do not occur or only in minor concentrations in the macronucleus; a very similar situation to that of the methylated cytosines. Notably, unlike in many animals studied so far, a functional discrimination between H3K9me3 and H3K27me3 has not been demonstrated in ciliated protozoa to date [20, 29]. Using ChIP experiments we now analyzed the distribution of these repressive markers in combination with PTMs typical for active chromatin (H3K4me in Figure 2B, H3K9ac/K14ac in Figure 2C) on the mdp1 and mdp2 nanochromosomes either in their silenced or active state (Figure 2D, E). In the silenced status we observe an enrichment of the active marker at the 3′- end similar to those observations reported for other PTMs typical for active chromatin , but interestingly, enrichment of H3K9me3/K27me3 could be detected on the 5′-end of both mdp1 and mdp2 nanochromosomes at a similar position to that where we also find methylated cytosines (Figure 2D and E, vegetative growth phase). Upon activation, the concentration of H3K9me3/K27me3 at the 5′-end is greatly reduced while H3K4me and H3K9ac/K14ac relocalize from the 3′- to the 5′-end. No H3K9me3/K27me3 associated with constitutively expressed genes could be found, while the distribution of active PTMs was similar to those expressed in mdp1 and mdp2 (data not shown) . Thus we conclude that PTMs typical for repressive chromatin are involved in the regulation of a small sub-fraction of macronuclear genes which exhibit short-term expression during sexual reproduction but are repressed during most of the cell’s life cycle.
Inhibition of HAT activity impedes DNA demethylation as well as activation of mdp1 and mdp2 expression
Until very recently it was believed that cytosine methylation does not occur in ciliated protozoa, or only at a defined stage of development . Here we show that this DNA modification occurs also in a small subset of macronuclear nanochromosomes developmentally expressed during sexual reproduction (Figure 3), while no such DNA modification is observed in constitutively expressed nanochromosomes. Therefore, as in other organisms, cytosine methylation also correlates with transcriptional activity in the ciliate cell. Surprisingly, only very few, or in the case of mdp2, only one methylated cytosine are found in the 5′-subtelomeric regions of the nanochromosomes upstream the TATA box, and removal of these singular methyl groups correlates with activation of expression. This indicates that only very few and discrete methylated cytosines are required to assign regulatory functions to a specific locus. Such a low level of methylated cytosines is below the detection limit of the standard RP-HPLC used in this study which could explain the failure of cytosine methylation detection in vegetative cells in former studies . While in mammals methylated cytosines are preferentially found in the CpG context, we find them also in the CAG or CTG context, somehow similar to what has been described in Neurospora, plants or Drosophila[8, 33, 34]. The presence of methylated cytosines correlates with the distribution of histone PTMs on the nanochromosomes. While in nanochromosomes active throughout the cell’s life PTMs typical for active chromatin accumulate at the 5′-end and no PTMs typical for repressed chromatin are found, active PTMs accumulate at the 3′-end of the nanochromosomes in the presence of 5meC and a PTM typical for repressed chromatin is present at the 5′-end above the methylated cytosines (Figure 2). Upon activation of expression, not only cytosines become demethylated but PTMs also redistribute on the nanochromosome. H3K9me3/K27me3, typical for repressed chromatin becomes removed and the active PTMs, H3K4me and H3K9ac/K14ac, are removed from the 3′-end and accumulate at the 5′-end or at least are equally distributed along the entire nanochromosome as in the case of mdp2 (Figure 2) . The combined presence of 5meC as well as H3K9me3/H3K27me3 at the 5′-end of nanochromosomes could be relevant regarding the effectiveness of gene silencing. This could explain our observation that a significant subset of mdp2 nanochromosomes without 5meC was present in vegetative cells, whereby no expression of this gene above threshold could be detected. To understand the relationship between cytosine demethylation and chromatin context, we either inhibited histone acetylation or histone deacetylation. Until very recently, such a relationship remained unclear but now it has been shown in Arabidopsis that H3 acetylation creates a chromatin environment permissible for 5-methyl cytosine DNA glycolysis . Our results suggest a similar mechanism, that is, after inhibition of acetyl transferase, which prevents the accumulation and redistribution of H3K9ac/14ac at the 5′-end, thereby creating a permissive chromatin structure, demethylation cannot be observed. But these observations do not completely exclude the possibility that the drugs applied may affect other pathways involved in DNA methylation or demethylation. A relevant question is how a specific methylation pattern is established in a small subset of nanochromosomes. We show that a similar methylation pattern has already been observed in the macronuclear precursor sequences in the micronucleus and the developing macronucleus. Shortly after activation of the genes developmentally expressed during sexual reproduction, the old macronucleus degenerates and a new macronucleus is formed by a micronuclear derivative and it seems reasonable to assume that the specific methylation pattern is transmitted from the germline micronucleus through macronuclear development towards the mature, vegetative macronucleus. As such, no specific de novo methylation has to occur. Taking all presented results together allows us to propose a mechanistic model for the biological function of cytosine methylation in the ciliate cell and its regulation throughout the life cycle of these single cell eukaryotes. The presence of the methylated cytosines correlates with gene activity and suggests that very few, or even a single methylated cytosine mark, is sufficient for long-term repression of gene expression, thus adding one further example of a specific cytosine correlating with gene expression . Upon gene activation, a relocalization of histone PTMs takes place and assembly of a permissive chromatin structure above the methylated cytosines, a necessary prerequisite for subsequent demethylation takes place. This mechanism has also recently been suggested for Arabidopsis. Shortly after this demethylation event, the old macronucleus disintegrates and the specific methylation pattern is transmitted from the micronucleus to the vegetative macronucleus during nuclear differentiation.
We introduce a biological model system, ciliated protozoa, whose minimalistic nature of macronuclear genome organization seems to be especially suited to analyze the crosstalk between different levels of epigenomic regulation. Macronucleus DNA is fragmented into small nanochromosomes, each representing a functional, transcriptional and replicational unit. We show that a low level of cytosine methylation occurs in the subtelomeric region of a small subset of macronuclear nanochromosomes only expressed during sexual reproduction, and this specific methylation pattern seems to be passed on from the germline nucleus to the vegetative macronucleus. Upon gene activation these cytosines become demethylated, demonstrating that only very few and discrete methylated cytosines are required to assign regulatory functions at a given locus. Cytosine methylation and demethylation also correlate with chromatin structure. Upon gene activation, a permissive chromatin structure is formed prior to cytosine demethylation and may be a prerequisite for this process. Results obtained in this study may not only be relevant for ciliated protozoa but for eukaryotes in general.
Growth of Stylonychia, synchronization, administration of drugs
Growth of Stylonychia lemnae and isolation of macronuclei, micronuclei or macronuclear anlagen were performed as described previously . DNA contamination of other nuclear types was avoided by purification of macronuclear, micronuclear or macronuclear anlagen DNA by electroelution from agarose gels. Purification was always repeated twice. To set up conjugation, cells of different mating types were mixed. Conjugation efficiency was between 90 and 95%. Cells from various stages of macronuclear development as well as vegetative cells were used to isolate total RNA, DNA and chromatin.
Inhibition of histone deacetylase and histone acetyl transferase
In some experiments, ciliates were treated with histone deacetylase (HDAC) inhibitor trichostatin A (TSA) (final concentration 0.4 μM, administered during conjugation), or histone acetyl transferase (HAT) inhibitor C646 (Calbiochem, Merck Millipore, Darmstadt, Germany) (final concentration 3 μM, added two hours prior to conjugation set up). RNA and DNA from such treated cells were isolated 30 hours post conjugation.
Purification of nucleic acids and cDNA synthesis
Isolation of DNA was performed as described . RNA was isolated from exconjugant cells harvested 30 hours post conjugation. Total RNA was isolated using InnuSOLV RNA reagent (Analytik Jena, Jena, Germany) according to the manufacturer’s recommendations. From obtained RNA, genomic DNA was removed using RNase-free DNase I (Fermentas, Thermo Fisher Scientific, Waltham, MA, USA). 2 μg of RNA were used to synthesize cDNA with InnuSCRIPT reverse transcriptase (Analytik Jena, Jena, Germany).
Analyses of gene expression
Macronucleus specific primers for the N-ChIP analyses
5′- CTTGTCTGGTGTATCACCGATACCATC- 3′
Macronucleus specific primers for bisulfite analysis
Actin 1 for
Actin 1 rev
Actin 2 for
Actin 2 rev
Actin 3 for
Actin 3 rev
Tubulin 1 for
Tubulin 1 rev
Tubulin 2 for
Tubulin 2 rev
Histone 1 for
Histone 1 rev
Histone 2 for
Histone 2 rev
Mdp1 1 for
Mdp1 1 rev
Mdp1 2 for
Mdp1 2 rev
Mdp1 3 for
Mdp1 3 rev
Mdp1 4 for
Mdp1 4 rev
Mdp1 5 for
Mdp1 5 rev
Mdp2 1 for
Mdp2 1 rev
Mdp2 2 for
Mdp2 2 rev
Chromatin isolation and ChIP
Chromatin isolation and ChIP analyses were carried out as described . Antibodies used for ChIP were directed against H3K9me3/K27me3, H3K4me or H3K9ac/K14ac (Abcam, Cambridge, UK). The specificities of these polyclonal antibodies on ciliate histone modifications are well documented .
Confocal laser scanning microscopy
Sample treatment for immunofluorescence and subsequent analyses by confocal laser scanning microscopy (CLSM) was performed using a protocol, antibodies and dyes, which are described in detail in . Images were assembled using ImageJ (Rasband, WS, ImageJ, National Institutes of Health, Bethesda, MD, USA, http://rsb.info.nih.gov/ij/, 1997–2004) and Adobe Photoshop CS5 software (Adobe Systems, San Jose, CA, USA).
Confocal laser scanning microscopy
Internal eliminated sequences
Polymerase chain reaction
Reverse phase-high performance liquid chromatography
This work was supported by grants of the Deutsche Forschungsgemeinschaft to HJL and GR.
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