It is well established that acetylation of histone N-termini is necessary to transform or maintain chromatin in a transcriptionally competent state in many organisms 2. Using specific antibodies the presence of H3K9ac (Figure 2A) and H3K14ac (Figure 2B) in macronuclei (M) and the absence of both modifications in micronuclei (m) was demonstrated by in situ antibody staining. Western analyses confirmed that H3K9ac/K14ac was abundant in macronuclei, but could not be detected in micronuclei (Additional file 2A). Notably, using in situ antibody staining we could not observe H3K36ac in macronuclei (M) or micronuclei (m). The existence of this modification has recently been demonstrated in Tetrahymena, Saccharomyces cerevisiae and human 33.

H3K4 methylation has been implicated in gene activation as well as in gene silencing 34. H3K4me1 is a frequent histone modification in macronuclei of Tetrahymena as well as in nuclei of S. cerevisiae and human 2932. This modification has been linked to silenced euchromatin in Chlamydomonas 35. H3K4me3 is found to be associated with 5' ends of euchromatic genes, which contain RNA-Pol II initiation sites 363738. It has been suggested that H3K4me3 is related to the process of active transcription, even though a very recent genome-wide study showed that this modification is found at most promoters of protein-coding genes in human cells, regardless of whether they produce full-length transcripts or not 39. Using immunofluorescence microscopy H3K4me1 (Figure 2C) could not be detected in micronuclei (m) but occurred as foci-like signals in the macronucleus (M). Similar to H3K4me1, H3K4me3 (Figure 2D) was a prominent marker in macronuclei (M), but was also not detected in micronuclei (m). When DNA is stained with To-Pro-3, the macronucleus reveals a sponge-like structure with domains of condensed chromatin separated from regions almost devoid of chromatin 40. It has been shown that nascent Pol I and Pol II transcripts occur at borders between such chromatin-rich and chromatin-poor compartments 41. While the preferential localization of H3K4me1 (Figure 2K) at such compartment borders was not observed in single light optical sections, H3K4me3 (Figure 2L) exhibited a similar distribution as described earlier for nascent RNA. This observation suggests that H3K4me3 is associated with active transcription in macronuclei. The abundance of H3K4me1 as well as of H3K4me3 in macronuclei and the absence of both modifications in micronuclei was confirmed by Western analyses (Additional file 2A).

H3K36me3 has been reported to accumulate at the 3' end of transcriptionally competent genes and is thought to act as a recruiting platform for histone deacetylase (HDAC) activity, preventing internal initiation of mRNA coding sequences in yeast 42. In ciliates the sole methylation observed on H3K36 to date is H3K36me1, in purified Tetrahymena macronuclei 2932. However, in relation to other organisms, for example human, mouse or Arabidopsis, the local sequence motif around H3K36 appears to be less conserved in Tetrahymena than in most H3 variants of Stylonychia 43. Interestingly, in our study H3K36me3 was found in micronuclei (m), whereas this modification was not observed in transcriptionally active macronuclei (M) by immunofluorescence (Figure 2E) and Western analyses (Additional file 2A).

H3K9me3 is implicated in transcriptional repression of euchromatic genes and to the formation of pericentric heterochromatin 24. Previous analyses using antibodies targeted to repressive lysine methylation sites at histone H3 suggested that H3K9me3 occurred in macronuclear anlagen and in micronuclei of Stylonychia 18. It was later confirmed that the antibody directed against H3K9me3 (Abcam, product code: ab8898) used in 18 cross-reacted with H3K27me3, as documented by the manufacturer. Our immunofluorescence analyses have now clarified that H3K9me3 (Figure 2G) is neither present in micronuclei (m) nor in macronuclei (M).

Similar to H3K9me3, H3K27me1 is highly enriched at pericentric heterochromatin in mammalian cell nuclei 44. This modification was reported to be abundant in macronuclei of Tetrahymena 2932. In contrast, in Stylonychia H3K27me1 was neither detected in micronuclei (m) nor in macronuclei (M) (Figure 2H).

H3K27me3 has been related to HOX gene silencing, X chromosome inactivation and genomic imprinting. This silencing system is linked to the function of the polycomb group of proteins (PcG) since complexes composed of variable PcG proteins have been shown to catalyze methylation of H3K27 445. In Tetrahymena, H3K27me3 has been detected in both the micronucleus and the macronucleus 212932. In situ antibody staining in our study revealed an abundance of H3K27me3 in micronuclei (m) of vegetative Stylonychia (Figure 2I), suggesting that the micronucleus-specific H3 variant 'X' 46 carries this modification. In contrast, H3K27me3 was not detected in macronuclei (M).

During mitosis Ser-10 and Ser-28 of H3 become phosphorylated by the kinase Aurora-B 4748. It was shown that H3S10p can occur, even if the neighboring lysine 9 is methylated. H3S10p is sufficient to prevent or disrupt HP1 binding at H3K9me3 4950, similarly to H1S27p preventing HP1 binding at H1K26me3 51. Such binary switches have been preassigned as 'methyl/phospho-switch' 52. The effects of phosphorylation at Ser-28 of histone H3 are far less characterized. Recently, this modification has been associated with transcriptional activity in immature chicken erythrocytes 53. Our immunofluorescence microscopic analyses revealed that H3S28p was abundant in micronuclei (m*) during mitosis (Figure 2Ja), but this modification was also detected in interphase micronuclei (m) of vegetative cells (Figure 2J) as well as in macronuclei (M) as dispersed foci-like signals that were apparently localized at surfaces separating condensed chromatin domains and regions almost devoid of chromatin.

During conjugation the parental macronucleus disintegrates into a variable number of fragments, which are at least partly still detectable in the cell during subsequent stages of macronuclear development. Our immunofluorescence analyses revealed that the histone modification pattern of these fragments does not differ from the pattern of vegetative macronuclei. Strong signals were observed in the parental macronuclei (p) using antibodies directed against H3K9ac (Figure 3A) or H3K14ac (Figure 3B). H3K4me1 (Figure 3C) and H3K4me3 (Figure 3D) also were prominent hallmarks although with weaker signal intensity than that observed in the vegetative macronuclei (M in Figure 2C, D), and H3S28p (Figure 3J) occurred as scattered foci-like signals in the fragmented parental macronuclei (p). These observations suggest that the chromatin of the parental macronuclear fragments still adopts a transcriptional competent or active state in conjugating cells.

Histone modifications in micronuclei of conjugating cells partly deviate from those of vegetative cells. Since H3K36me3 was not detected in micronuclei undergoing meiosis or mitosis (m*) in conjugating cells (Figure 3E), this modification was obviously lost at the onset of sexual reproduction. Remarkably, H3K27me1 was observed transiently during prophase of meiosis or mitosis of micronuclei (m*) during conjugation (Figure 3H).

At early developmental stages following the separation of mating cells, the signal intensity of H3K9ac (Figure 4A; Figure 5A; Figure 6A), H3K14ac (Figure 4B; Figure 5B; Figure 6B), H3K4me1 (Figure 4C; Figure 5C; Figure 6C), H3K4me3 (Figure 4D; Figure 5D; Figure 6D) and H3S28p (Figure 4J; Figure 5J; Figure 6J) in the fragmented parental macronuclei (p) gradually became weaker, or in some cases signals were entirely lost. H3K9me3 (Figure 4G; Figure 5G, Figure 6G) and H3K27me1 (Figure 4H; Figure 5H, Figure 6H) occasionally occurred as sporadic foci-like signals in fragmented parental macronuclei (p) of all macronuclear developmental stages.

H3K27me3 was present in micronuclei (m) of exconjugant cells (Figure 4I; Figure 5I; Figure 6I), as it was during conjugation. After the removal of H3K36me3 from micronuclei in the previous developmental stage this modification accumulated de novo in micronuclei (m) following the separation of conjugating cells (Figure 4E; Figure 5E; Figure 6E). H3K27me1 (Figure 4H; Figure 5H; Figure 6H) as well as H3S28p (Figure 4J; Figure 5J; Figure 6J) were lost from micronuclei (m) after the separation of mating cells as observed by in situ antibody staining, and neither modification was traceable in subsequent developmental stages. Particularly, the loss of H3S28p exhibits a characteristic (epigenetic) feature of micronuclei in the course of macronuclear differentiation. Acetylation of H3K9 or H3K14 was not observed in micronuclei (m) at any developmental stage (Figure 4A, B; Figure 5A, B; Figure 6A, B).

Macronuclear anlagen at early developmental stages (stages a₁/early a₂, compare Figure 1) were morphologically characterized by the decondensation of chromatin, accompanied by the appearance of H3K9ac (Figure 4A) and H3K14ac (Figure 4B). Western analyses confirmed that H3K9ac/K14ac was abundant ~24 hours post conjugation (stage a₂) in macronuclear anlagen stages (Additional file 2A). It appeared that for both modifications signals were restricted to the interior of the early macronuclear anlage, while the condensed chromatin at the nuclear periphery remained unstained. This observation is consistent with earlier microscopic studies, which showed that only a portion of the chromosomes in the early macronuclear anlagen move to the center of the nucleus, are despiralized and develop into polytene chromosomes, while the residual chromosomes become pyknotic and are lost from the macronuclear anlage 1316.

While H3S28p accumulated in the early macronuclear anlagen stages (a₁) (Figure 4J), we could not detect H3K9me3 (Figure 4G), H3K27me1 (Figure 4H), or H3K27me3 (Figure 4I) in this stage (a₁/early a₂) by immunofluorescence. These observations highlight that the fusion of two heterochromatic micronuclei during conjugation leads to a zygote nucleus, in which all repressive micronuclear-specific histone modifications are removed and permissive histone modifications are introduced de novo.

However, repressive methylation hallmarks on histone H3 were shown to be associated with micronucleus-specific sequences during macronuclear differentiation in ciliates such as Tetrahymena 212231 or Stylonychia 18. These modifications are a necessary prerequisite for correct DNA processing. Whereas we did not find repressive methylation markers in early macronuclear anlagen (a₁), our microscopic data show that after transition to the next developmental stage (early a₂) sporadic foci-like signals of these modifications were observed in the anlage (for example, H3K9me3 in Figure 4G and H3K27me3 in Figure 4I). In addition, the de novo introduction of H3K36me3 was detected in macronuclear anlagen at stage a₂ of polytenization (Figure 4E). Western analyses confirmed that this modification occurred in macronuclear anlagen (a₂) ~24 hours post conjugation (Additional file 2A). Due to asynchronous macronuclear differentiation in mass cultures, performance of quantitative normalized Western blot analyses was not possible. We therefore determined the ratios of H3K9ac/K14ac and H3K27me3 to DNA in microscopic sections of macronuclear anlagen in various stages of development (Additional file 3). Although it is generally difficult to compare fluorescence intensities of different antibodies directly, these measurements were a valuable relative approximation to plot the time lapses of H3K9ac/K14ac or H3K27me3 signals, respectively, confirming that H3K9ac/K14ac accumulated early during macronuclear differentiation whereas H3K27me3 signals remained on a threshold level at that time.

As described above, H3K9ac and H3K14ac are introduced de novo in the macronuclear anlage immediately after division of the zygote nucleus. Both modifications were abundant in later stages of macronuclear development, including advanced stages a₂/a₃ with a high degree of polytenization (Figure 5A, B), and stages (e) during DNA-elimination (Figure 6A, B) up to and including the mature macronucleus (Figure 2A, B). Western analysis confirmed that H3K9ac/K14ac was abundant ~48 hours post conjugation (transition stage a₃ → e) in advanced macronuclear anlagen stages (Additional file 2A). While H3K9ac and H3K14ac appeared to be evenly distributed over the entire interior of the macronuclear anlagen in stages a₁ and a₂ (Figure 4A, B), large chromatin domains were unstained in the advanced macronuclear anlagen stages (a₃) (Figure 5A, B). This became even more obvious in single light optical sections (Figure 5K), where large chromatin domains exhibited no H3K9ac/K14ac signals. These domains corresponded to 'heterochromatic blocks' of polytene chromosomes described earlier 13. According to the measurements of the H3K27me3:DNA ratio in microscopic sections it could be roughly estimated that at the late polytene chromosome stage up to 80% of the DNA pixels co-localized with H3K27me3 signals. We performed ChIP experiments using antibodies specific for H3K9ac and H3K14ac respectively. Subsequently DNA fragments from three sequence classes were analyzed by real-time quantitative PCR (Figure 7A–D) using three macronucleus-destined sequences encoding the TEBPα gene (GeneBank accession number: FJ159127, the 1.1 kb and 1.3 kb macronuclear nanochromosome pCE7; GeneBank accession number: X72958; 18 and the actin I gene GeneBank accession number: DQ108616); flanking sequences from the micronuclear 1.1 kb locus and the actin I gene; and the moderate repetitive sequence 'stad5' (Gene Bank accession number: EU687742), a micronuclear genomic sequence, which is specifically eliminated during macronuclear development (Figure 7D; Additional file 2B). Most analyses in the holotrichous ciliate Tetrahymena concentrated on the elimination of IES, which have a length up to several kilo base pairs. In contrast, IES size in stichotrichous ciliates like Stylonychia ranges between 1 to 100 bp 12. Because of this property we did not pursue analyses of IES chromatin structure and elimination in the present study. In the germline TEBPα and actin I genes multiple MDSs, interrupted by micronucleus specific DNA, occur in a scrambled disorder 54 (Figure 7A). To exclude amplification from macronuclear contamination, DNA primers derived from an IES and a MDS (TEBPα), from the 3' end of the 1.1 kb and 5' end of the 1.3 kb locus (pCE7) and a scrambled part of the micronuclear actin I sequence were used for PCR analyses (Figure 7A to 7C). Our ChIP experiments revealed that H3K14ac was associated with all MDSs (Figure 7F). To a significantly lower extent we found these modifications associated with sequences flanking the 1.1 kb locus and the actin I gene. For the micronucleus-specific stad5 no association with H3K14ac (Figure 7F) was observed. Similar results were obtained using the H3K9ac-specific antibody for ChIP (data not shown).

During the first rounds of DNA amplification immunofluorescence signals of H3K4me1 (Figure 5C) and H3K4me3 (Figure 5D) were observed to drastically accumulate de novo in advanced stages (a₃) of macronuclear development. H3K4me1 (Figure 6C) and H3K4me3 (Figure 6D) were also present during the DNA elimination stage (e) and were retained in the mature macronucleus (Figure 2C, D). The presence of both modifications in macronuclear anlagen ~48 hours post conjugation (transition stage a₃ → e) was also demonstrated by Western analyses (Additional file 2A). ChIP experiments using antibodies specific for H3K4me3 demonstrated that this modification was associated with the MDSs and to a lesser extent with their flanking sequences. The association with stad5 was negligible (Figure 7F).

H3K36me3 was detected by immunofluorescence microscopy (Figure 5E) and Western analysis (Additional file 2A) at advanced macronuclear anlagen (a₃) with high level of polyteny. An accumulation of H3K36ac was also observed at this stage using in situ antibody staining (Figure 5F). However, in contrast to H3K4 methylation, H3K36me3 was no longer observed in the subsequent DNA elimination stage (e), indicating the loss of H3K36me3 during DNA elimination (Figure 6E). Similarly, H3K36ac (Figure 6F) exhibited maximum signal intensity at the transition from highly polytene anlagen to the elimination stages (a₃-e), and during the second round of DNA amplification we could not detect this modification. ChIP experiments using antibodies directed against H3K36me3 demonstrated that this modification was associated with MDSs and to lesser extent with sequences in the flanking regions of the MDSs. An association with stad5 was not observed (Figure 7F).

H3S28p persisted during polytenization in macronuclear anlagen (a₃) (Figure 5J) and disappeared during DNA elimination (e) (Figure 6J), and was not retained in mature macronuclei (M) (Figure 2J). ChIP experiments using antibodies specific for H3S28p demonstrated that this modification was associated with the MDS of actin I. To a lesser extent H3S28p was observed to associate with actin I flanking sequence, whereas the association with stad5 was marginal (Additional file 4).

Earlier studies demonstrated that sequences targeted for elimination during macronuclear development are associated with histone modifications typically found within silenced chromatin, such as H3K9me3 and H3K27me3 in Tetrahymena 212231 or H3K9me3 in Stylonychia 18. Our immunofluorescence analyses showed that de novo tri-methylation at H3K9 (Figure 5G) and mono-methylation at H3K27 (Figure 5H) take place at advanced stages of polytenization in macronuclear anlagen (a₂, early stage a₃). Both modifications were still detected with gradually decreasing signal intensity during the DNA elimination stage (e) (Figure 6G; Figure 6H), but were not observed during the second rounds of DNA amplification.

H3K27me3 exhibited the most prominent signals of repressive histone modifications examined in macronuclear anlagen (a₃) late during polytene chromosome formation (Figure 5I). Single light optical sections (Figure 5L) showed that large chromatin domains exhibited H3K27me3 signals exactly where H3K9ac/K14ac signals were obviously omitted (compare Figure 5K). In spread polytene chromosome preparations it became obvious that H3K27me3 was enriched in chromosomal bands as well as in 'heterochromatic blocks' (Figure 5M). H3K27me3 was observed to occur with decreasing signal intensity during stages (e) of DNA elimination (Figure 6I; Additional file 3).

Since it is well established that the selective introduction of H3K9me3 and H3K27me3 is a necessary prerequisite for the programmed elimination of DNA during macronuclear differentiation, we decided to analyze the spatiotemporal occurrence of two chromatin modifying proteins, a putative histone methyltransferase (HMT) and the HP1 homolog Spdd1p, which have been identified previously in ciliates 215556. Initially, we decided to analyze the nuclear localization of a putative E(z)-like HMT (new nomenclature: K-methyltransferase 6/KMT6 57), since we assumed that homologs of this protein could be involved in the establishment of H3K9me3 and H3K27me3 in Stylonychia. The SET-domain-containing PcG protein E(z)/KMT6 or homologous proteins were identified to be core components of all polycomb repressive complexes 2 (PRC2) isolated so far. PRC2 complexes have HMT activity and are capable of transferring methyl groups at H3K27 and/or H3K9 24. An E(z)/KMT6 homolog has recently been identified in the ciliate Tetrahymena 21. We applied an antibody targeted to EZH2 (Abcam; ab3748) for immunofluorescence microscopic analysis (Figure 8A). Although we could not detect any signals at early macronuclear anlagen stages (a_1–2), we observed an accumulation of signals at advanced macronuclear stages (a₃) during polytene chromosome formation. These signals persisted up to and including the DNA elimination stage (e), but were not retained during the second rounds of DNA amplification and in nuclei of vegetative Stylonychia (data not shown). Further immunofluorescence analyses on spread polytene chromosomes revealed that the protein detected with an anti-EZH2 antibody had a similar distribution to H3K27me3 (Figure 8B). However, the specificity of the antibody used still has to be shown in Stylonychia.

Heterochromatin formation in mammalian and Drosophila cell nuclei involves the recruitment of HP1, which specifically binds to H3K9me3 via its chromodomain 585960. The chromodomain-containing proteins Pdd1p in Tetrahymena 2255 and Spdd1p in Stylonychia 56 are homologues of HP1 and both were shown to be involved in programmed DNA elimination. We therefore reinvestigated the occurrence of Spdd1p during Stylonychia macronuclear development. It has been shown that during macronuclear differentiation Spdd1p is present in the macronuclear anlagen as well as in the degenerating parental macronuclei 56. Our immunofluorescence analyses confirmed that Spdd1p occurred in macronuclear anlagen with a similar pattern to H3K9me3 or H3K27me3 (Figure 8). Spdd1p was detected neither in micronuclei nor in macronuclei of vegetative Stylonychia (data not shown). In early macronuclear anlagen (a_1–2) no signals were observed (Figure 8C) before Spdd1p accumulated in macronuclear anlagen (a₃) at later stages of polytenization (Figure 8D) and was present during the DNA elimination stage (e) (data not shown).

In summary, our data show that repressive histone modifications are introduced de novo at later stages during macronuclear differentiation, and they are found in the banded regions and heterochromatic blocks of the polytene chromosomes. While these modifications are lost during the DNA elimination stage, 'open' chromatin markers introduced at very early stages persist throughout macronuclear differentiation up to the vegetative macronucleus.

Direct evidence for the involvement of an RNAi-related machinery in the control of DNA elimination by heterochromatin formation has been recently provided 7891819, and it has been shown that the Piwi-family protein Twi1p involved in the trafficking of small RNA molecules in Tetrahymena is an important component of that pathway 79. In Stylonychia the Piwi-family macronuclear development protein 1 (mdp1, predicted size ~89 kDa) is differentially expressed during macronuclear development 2526. Besides the PIWI-domain, which is thought to be involved in protein-protein interactions, Stylonychia Piwi/mdp1 possesses a putative RNA-binding PAZ domain, suggesting that this protein is involved in the processing of small RNA molecules, which have been shown to be complementary to micronucleus specific sequences 18. We therefore analyzed in detail the spatiotemporal occurrence of Piwi/mdp1 during macronuclear development by immunofluorescence microscopy (Figure 9) as well as Western analyses (Additional file 2A). Using RNAi directed against various regions of the Piwi/mdp1 gene, the intensity of the putative Piwi/mdp1 band was greatly reduced (Additional file 2A), and as reported earlier 27 an arrest of macronuclear development at an early stage was observed. Immunofluorescence analyses showed that Piwi/mdp1 accumulated in a sub-fraction of micronuclei (m) early during conjugation (Figure 9A), whereas the Tetrahymena homologous protein Twi1p was observed in the cytoplasm at comparable stages 7. Coevally we observed the accumulation of scattered foci-like signals in the fragmented parental macronuclei (p). Later during conjugation (Figure 9B) a high signal intensity of Piwi/mdp1 was found in the fragmented parental macronuclei (p), while it was no longer present in micronuclei (m). Piwi/mdp1 was still present in the fragmented parental macronuclei (p) immediately after separation of conjugating cells (Figure 9C). Subsequently Piwi/mdp1 signal density drastically decreased in these parental macronuclear fragments (p) but relocalized to the macronuclear anlagen (a_1–2) early during polytenization (Figure 9D). We detected a maximum Piwi/mdp1 signal intensity in macronuclear anlagen at this stage, and the staining appeared to be homogenously distributed. Piwi/mdp1 signals in macronuclear anlagen (a₂, a₃, e) were observed to become organized as discrete foci, and the signal density continuously decreased in the course of further polytenization (Figure 9E, F) and during DNA elimination (Figure 9G). Subsequently we observed that Piwi/mdp1 was completely lost from developing macronuclei (r) during the second round of DNA amplification (Figure 9H).