Our recent progress in epigenetic research using the model ciliate, Tetrahymena thermophila
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Epigenetic research focuses on heritable changes beyond the DNA sequence, which has led to a revolution in biological studies and benefits in many other fields. The well-known model ciliate, Tetrahymena thermophila offers a unique system for epigenetic studies due to its nuclear dimorphism and special mode of sexual reproduction (conjugation), as well as abundant genomic resources and genetic tools. In this paper, we summarize recent progress made by our research team and collaborators in understanding epigenetic mechanisms using Tetrahymena. This includes: (1) providing the first genome-wide base pair-resolution map of DNA N6-methyladenine (6mA) and revealed it as an integral part of the chromatin landscape; (2) dissecting the relative contribution of cis- and trans- elements to nucleosome distribution by exploring the unique nuclear dimorphism of Tetrahymena; (3) demonstrating the epigenetic controls of RNAi-dependent Polycomb repression pathways on transposable elements, and (4) identifying a new histone monomethyltransferase, TXR1 (Tetrahymena Trithorax 1), that facilitates replication elongation through its substrate histone H3 lysine 27 monomethylation (H3K27me1).
KeywordsEpigenetics Tetrahymena N6-methyladenine (6mA) Nucleosome Transposable elements Histone methyltransferase TXR1
Eukaryotic genomes have multiple layers. Beyond genetic basis, DNA methylation, noncoding RNA and histone modifications/variants serve as a platform of epigenetic signals which regulate the gene expression leading to profound impact on cell development (Badeaux and Shi 2013; Bird 1992; Jeffares et al. 1998; Poole et al. 1998; Reisenauer et al. 1999; Zacharias 1993).
Our group is dedicated to understanding epigenetic regulation mechanisms using Tetrahymena thermophila as a model system (Fig. 1b–e). A summary of our recent progress is presented here.
The genomic distribution and determinants of DNA N6-methyladenine (6mA) in Tetrahymena
We generated the first genome-wide, base pair-resolution map of 6mA in Tetrahymena, by employing single-molecule real-time (SMRT) sequencing (Fig. 2b) (Wang et al. 2017a) 6mA preferentially located in the consensus sequence of 5′-AT-3′ (Fig. 2c), indicating that DNA sequence per se provides necessary information to locate 6mA. However, in a genome as AT rich as Tetrahymena, only a small percentage of adenines (0.66%) are methylated, suggesting that other factors beyond sequence also contribute to 6mA distribution. Further analysis revealed that 6mA accumulated in linker DNA regions between well-positioned nucleosomes (Fig. 2d, g), but the direction of the causal effect remains to be determined (Luo et al. 2018). Moreover, nucleosomes adjacent to 6mA were enriched with the conserved histone variant H2A.Z (Fig. 2e) which is dynamically regulated by ATP-dependent chromatin remodelers (Havas et al. 2001; Jaskelioff et al. 2000). Given that 6mA accumulated right downstream of the transcription start sites (TSS of RNA polymerase II (Pol II) transcribed genes) (Fig. 2f), we posit that 6mA was recruited to the promoter region by a similar mechanism to H2A.Z. It was previously reported that 6mA is either positively or negatively correlated with transcription (Fu et al. 2015; Liang et al. 2018; Wang et al. 2018; Wu et al. 2016; Zhou et al. 2018), and usually enriched in specific pathways in metazoa such as in mouse brain (Yao et al. 2017) and in human cancer cells (Xiao et al. 2018; Xie et al. 2018). In Tetrahymena, however, the correlation between gene expression level and the level/amount of 6mA was rather weak (Fig. 2h). Given that most Tetrahymena genes (> 90%) are decorated with 6mA, it is not surprising that 6mA in unicellular eukaryotes may have not evolved to regulate specific pathways. Collectively, the evidence suggests that distribution of 6mA in Tetrahymena is determined by both DNA sequence and the chromatin environment, and its special pattern is probably achieved by specific methyltransferase(s) deposition.
To better decipher the function and biological significance of 6mA, it is essential to perturb 6mA levels in vivo by manipulating its catalyzing enzymes. Our recent work identified a candidate 6mA methyltransferase (unpublished data), the deletion of which impaired cell growth and development. Systematic investigations are underway to find out how this methyltransferase regulates 6mA level and how the change in 6mA level affects cell fitness. It will also be interesting to look into other factors involved in the regulation of 6mA such as demethylase(s), reader(s), and chromatin modifications and how 6mA interacts with them to jointly shape the chromatin environment.
Epigenetic regulation of nucleosome distribution
How cis- and trans- determinants coordinate with each other and shape nucleosome distribution has long been debated. For example, an early study focusing on in vitro reconstituted nucleosomes showed that nucleosome distribution might be dependent largely on intrinsic DNA sequence rather than trans-acting factors (Sekinger et al. 2005). However, comprehensive in vivo mapping of nucleosomes in budding yeast, using high-throughput sequencing following micrococcal nuclease (MNase) digestion, showed that nucleosome distribution was affected by its chromatin contexts in which gene regulatory elements function on a genomic scale (Lee et al. 2007).
Recently, N6-methyladenine (6mA) DNA methylation, as one of the potential cis-determinants, has been reported to play a role in nucleosome positioning (Fu et al. 2015). The result from our work showed that, in Tetrahymena, linker DNA regions with 6mA are usually flanked by well-positioned nucleosomes (Fig. 2e, 3f) (Wang et al. 2017a). By in vitro nucleosome assembly in Tetrahymena, a recent study revealed that the distribution pattern of nucleosomes could be recapitulated in native genomic DNA but not in DNA without 6mA (Luo et al. 2018). A study of another ciliate, Oxytricha trifallax, demonstrated that nucleosome fuzziness increased after loss of 6mA in the linker DNA (Beh et al. 2019). Overall, our studies and those of others have revealed the intrinsic repulsion between 6mA and nucleosomes which contributes to determining the nucleosome position. However, the causal functions of related factors and the precise mechanisms of how they coordinate with each other remain to be elucidated.
RNAi-dependent Polycomb repression controls transposable elements in Tetrahymena
Transposable elements (TEs) are DNA sequences which can induce mutations and affect the organism’s genome structure by changing their positions within a genome (Bourque et al. 2018). They are drivers of host genome evolution as well as threats to host genome integrity (Bennetzen and Wang 2014; Freeling et al. 2015; Lynch 2007). Therefore, different hosts have developed a wide variety of defense mechanisms to control TE expression, including small RNA, sequence-specific repressors, DNA, and chromatin modification pathways (Berrens et al. 2017; Ecco et al. 2017; Goodier 2016; Liu et al. 2017; Molaro and Malik 2016). In flies and mammals, Piwi-interacting RNAs (piRNAs), which are produced from piRNA clusters genomic loci, mediate TE silencing (Guzzardo et al. 2013; Siomi et al. 2011). In nematodes, Argonaute–small RNA complexes are used as transgenerational binary signals and program the ON/OFF expression state for TEs (Seth et al. 2013). Plants and yeasts identify and repress TEs by recognizing their intrinsic features (Lee et al. 2013; Slotkin et al. 2009). Yet little is known about how TEs are controlled in the single cell organism Tetrahymena thermophila.
In our recent work, we and our collaborators analyzed the polyadenylated RNA transcripts from ~ 10,000 IESs in knockout strains of three key players (DCL1, EZL1 and PDD1) in the RNAi-dependent Polycomb repression pathway (Zhao et al. 2019). We found a significantly higher level of polyadenylated RNA in the mutants than that in the wild type (Fig. 4c). These IES-specific polyadenylated transcripts (many containing TE-related sequences) have mRNA features, such as poly-A tailing, strand specificity, splice sites and a similar codon usage pattern with that of MDS (Fig. 4d), demonstrating the capacity of protein coding, and probably TE coding. As evidence, we detected a dramatic increase in germline mobilization of a recently active TE in the mutants, this mobilization being achieved via a “cut-and-paste” manner, i.e., TE is excised from the genomic locus, leaving an AT footprint in the original locus (Fig. 4e). Both mRNA and ncRNA (scnRNA) were detected in the same IES locus, but mRNA was only produced in the developing MAC where key components in Pol II-driven mRNA biogenesis are present. More intriguingly, mRNA levels of TE-containing IESs are positively correlated with late-scnRNA, while are negatively correlated with early-scnRNA (Fig. 4f, g) (Noto et al. 2015). Together, these results indicate that the balance between ncRNA and mRNA production was probably mediated by RNAi-dependent Polycomb repression and co-transcriptional processing, which is essential for TE control. Considering the conservation of key components in this RNAi-dependent Polycomb repression pathway and the wide distribution of similar pathways throughout eukaryotes (Fig. 4h), we posit that interplay between RNAi and Polycomb repression may be a universal way for TE silencing and transcriptional repression of developmental genes.
Previous studies have revealed the recent transposition of TEs in Tetrahymena based on TE insertion polymorphisms in some IES (Huvos 2004) and purifying selection in predicted coding sequences of some potentially active TEs (Fillingham et al. 2004; Gershan and Karrer 2000; Hamilton et al. 2006, 2016). Our results demonstrate that Polycomb repression defects result in not only the activation of TE transcription but also the germline mobilization of TE. Future studies will focus on the mechanism underlying the molecular nature of TEs, such as where the mobilized TEs reassemble into the genome and which factors determine their insertion sites.
Histone methylation H3K27me1 is involved in the regulation of DNA replication
Residues of histones, especially those in N-terminal tails, can be post-translationally modified, which has been proved to play important roles in many chromatin-templated processes such as DNA replication, RNA transcription, DNA repair, chromatin condensation, and segregation (Allis and Jenuwein 2016; Jenuwein and Allis 2001; Lantermann et al. 2010; Lee et al. 2007; Strahl and Allis 2000). Histone 3 lysine 27 (H3K27) methylation is one of the most well-studied histone post-translational modifications (PTMs) (Cyrus and Yi 2005; Gao et al. 2013; Jacob et al. 2009; Jamieson et al. 2017; Klose and Yi 2007; Liu et al. 2007; Ru and Yi 2004; Zhang et al. 2018). In Drosophila, enhancer of zeste (E(z)) is the histone methyltransferase (HMT) for H3K27 methylation (Cao et al. 2002; Czermin et al. 2002; Jürg et al. 2002). In Arabidopsis, however, besides three homologs to E(z), MEDEA (Langmead and Salzberg 2012), CURLY LEAF (CLF), and SWINGER (SWN), two SET domain-containing proteins, i.e., ARABIDOPSIS TRITHORAX-RELATED PROTEIN5 (ATXR5) and ATXR6, which are phylogenetically divergent from E(z), possess the ability to specifically add one methyl group to H3K27 (Baumbusch et al. 2001; Jacob et al. 2009). The hypomorphic mutant, atxr5/atxr6 showed a moderate yet incomplete reduction of H3K27me1 level (Raynaud et al. 2006), and, more dramatically, re-replication in heterochromatin regions enriched with transposons and repetitive elements, indicating that this clade of HMTs is involved in replication regulation (Jacob et al. 2009, 2010; Yannick et al. 2010).
Our work revealed a new scenario that deletion of a histone methyltransferase could severely impair the replication elongation instead of replication license, but the regulatory mechanism of lysine monomethylation in DNA methylation has yet to be elucidated. Focusing on the modulating mechanism of TXR1, we recently discovered that, in addition to its important role in replication, TXR1 may also be involved in transcription regulation (unpublished data). This is consistent with the finding that H3K27me1 in Arabidopsis exhibits two distinct distribution patterns in constitutive heterochromatin and genic regions, suggesting its role in transcription regulation (Roudier et al. 2011). These bring us to one of the most basic questions in biology: with the same DNA template, how can cells solve the conflicts of replication and transcription? (García-Muse and Aguilera 2016; Hamperl et al. 2017; Lin and Pasero 2017). It will be interesting to dissect roles played by TXR1 in replication and transcription, and how it contributes to coordinating conflicts between these two essential processes.
Summary and perspectives
Epigenetic studies have kept yielding cutting-edge knowledge in different eukaryotic models including Tetrahymena. Benefiting from the unique biological properties such as nuclear dimorphism, Tetrahymena has become a powerful model for epigenetic studies. In this paper, we review recent progress made by our group using Tetrahymena to elucidate the distribution and mechanism of DNA 6mA methylation, nucleosome occupancy driven by both cis- and trans-determinants, transposable elements regulated by RNAi-dependent Polycomb repression and H3K27me1-related regulation of DNA replication.
To improve the understanding of epigenetics using Tetrahymena, future studies will focus on: (1) the regulation mechanisms of 6mA together with its co-factors; (2) the relationship of 6mA and nucleosomes in the chromatin environment; (3) the molecular nature of TEs; and (4) roles of TXR1 in replication and transcription.
The authors would like to thank the following people for assistance with this study: Ms. Yalan Sheng, Mr. Bo Pan, Ms. Yuan Li, Ms. Lili Duan for their help in draft preparation. Our special thanks are due to our collaborators, in particular Dr. Yifan Liu (University of Michigan) and Dr. Wei Miao (Institute of Hydrobiology of Chinese Academy of Sciences).
All authors wrote the paper and approved the final manuscript.
This work was supported by Natural Science Foundation of Shandong Province (JQ201706), The Marine S&T Fund of Shandong Province for Pilot National Laboratory for Marine Science and Technology (Qingdao) (2018SDKJ0406-2), Fundamental Research Funds for the Central Universities (201841005), and the Blue Life Breakthrough Program of LMBB of Qingdao National Laboratory for Marine Science and Technology (MS2018NO04).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no competing interests.
Animal and human rights statement
This article does not contain human participants or animals.
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