An H3K9 methylation-dependent protein interaction regulates the non-enzymatic functions of a putative histone demethylase

A cell’s identity depends on which of its genes are active. One way for cells to control this process is to change how accessible their genes are to the molecular machinery that switches them on and off. Special proteins called histones determine how accessible genes are by altering how loosely or tightly DNA is packed together.

Histones can be modified by enzymes, which are proteins that add or remove specific chemical ‘tags’. These tags regulate how accessible genes are and provide cells with a memory of gene activity. For example, a protein found in yeast called Epe1 helps reactivate large groups of genes after cell division, effectively ‘re-setting’ the yeast’s genome and eliminating past memories of the genes being inactive.

For a long time, Epe1 was thought to do this by removing methyl groups, a ‘tag’ that indicates a gene is inactive, from histones – that is, by acting like an enzyme. However, no direct evidence to support this hypothesis has been found. Raiymbek et al. therefore set out to determine exactly how Epe1 worked, and whether or not it did indeed behave like an enzyme.

Initial experiments testing mutant versions of Epe1 in yeast cells showed that the changes expected to stop Epe1 from removing methyl groups instead prevented the protein from ‘homing’ to the sections of DNA it normally activates. Detailed microscope imaging, using live yeast cells engineered to produce proteins with fluorescent markers, revealed that this inability to ‘home’ was due to a loss of interaction with Epe1’s main partner, a protein called Swi6. This protein recognizes and binds histones that have methyl tags. Swi6 also acts as a docking site for proteins involved in deactivating genes in close proximity to these histones.

Further biochemical studies revealed how the interaction between Epe1 and Swi6 can help in gene reactivation. The methyl tag on histones in inactive regions of the genome inadvertently helps Epe1 interact more efficiently with Swi6. Then, Epe1 can simply block every other protein that binds to Swi6 from participating in gene deactivation. This observation contrasts with the prevailing view where the active removal of methyl tags by proteins such as Epe1 switches genes from an inactive to an active state.

This work shows for the first time that Epe1 influences the state of the genome through a process that does not involve enzyme activity. In other words, although the protein may ‘moonlight’ as an enzyme, its main job uses a completely different mechanism. More broadly, these results increase the understanding of the many different ways that gene activity, and ultimately cell identity, can be controlled.

The covalent and reversible modification of histones allows cells to establish stable and heritable patterns of gene expression without any changes to their genetic blueprint. Histone H3 lysine nine methylation (H3K9me) is a conserved mark of transcriptional silencing that is associated with the formation of specialized domains called heterochromatin. Heterochromatin formation is critical for centromere and telomere function, silencing of transposons and repetitive DNA elements, and for maintaining lineage-specific patterns of gene expression (Grewal and Jia, 2007; Nicetto et al., 2019). It is widely assumed that a delicate interplay between histone modification readers, writers, and erasers regulates the establishment and maintenance of epigenetic states (Allis and Jenuwein, 2016). In fission yeast, the establishment of H3K9 methylation requires the enzymatic activity of a conserved methyltransferase, Clr4^Suv39h (Rea et al., 2000). H3K9 methylation acts as a platform to recruit chromatin effector proteins with reader domains. Two HP1 homologs, Swi6^HP1 and Chp2^HP1, are involved in H3K9 methylation binding and have distinct, non-overlapping functions during transcriptional silencing (Motamedi et al., 2008). Ultimately, factors such as Epe1, a putative histone demethylase, promote epigenetic erasure, thus reversing H3K9 methylation and transcriptional gene silencing (Ayoub et al., 2003; Trewick et al., 2005).

The sequence-specific recruitment of Clr4^Suv39h restricts heterochromatin establishment to distinct sites in the genome, such as the centromeres, telomeres, and the mating-type locus (Bayne et al., 2010; Hall et al., 2002; Verdel et al., 2004; Volpe et al., 2002). Once established, H3K9 methylation spreads to silence genes that are distant from heterochromatin nucleation centers. H3K9me spreading depends on the read-write activity of Clr4^Suv39h (Al-Sady et al., 2013; Zhang et al., 2008). Two conserved, structural attributes of Clr4^Suv39h mediate this process: a conserved chromodomain that recognizes and binds to H3K9me and an enzymatic SET domain that is involved in catalysis (Ivanova et al., 1998). The ability of Clr4^Suv39h to bind to the product of its enzymatic activity enables H3K9 methylated histones to serve as carriers of epigenetic information (Audergon et al., 2015; Ragunathan et al., 2015). Following DNA replication, H3K9 methylated histones that are partitioned between daughter DNA strands serve as templates to mark newly deposited histones.

H3K9 methylation acts as a multivalent platform that recruits the HP1 homolog, Swi6^HP1 to sites of constitutive heterochromatin (Ekwall et al., 1995; Ekwall et al., 1996). HP1 proteins have a conserved architecture consisting of a chromodomain (CD) that is involved in H3K9 methylation binding and a dimerization domain called the chromoshadow domain (CSD) that mediates protein-protein interactions (Bannister et al., 2001; Cowieson et al., 2000). The oligomerization of HP1 proteins promotes formation of higher-order chromatin complexes that exhibit phase separated, liquid-like properties in vitro and in cells (Larson et al., 2017; Sanulli et al., 2018; Strom et al., 2017). Swi6^HP1 interacts with a broad spectrum of agonists and antagonists that influence epigenetic silencing. Most notably, recruitment of Epe1, a putative histone demethylase that opposes heterochromatin formation, is dependent on Swi6^HP1 (Ayoub et al., 2003; Zofall and Grewal, 2006). Loss of Epe1 leads to increased heterochromatin spreading beyond normal boundary sequences, inheritance of H3K9 methylation via sequence-independent pathways, increased cell to cell variation in H3K9 methylation patterns, and acquisition of adaptive epigenetic traits (Audergon et al., 2015; Ayoub et al., 2003; Ragunathan et al., 2015; Sorida et al., 2019; Trewick et al., 2007; Wang et al., 2015; Zofall and Grewal, 2006; Zofall et al., 2012). Despite its critical role in heterochromatin regulation, how Epe1 exerts its anti-silencing function in cells remains mysterious (Trewick et al., 2007).

Histone demethylases have prominent roles in regulating the reversibility of epigenetic states (Iwase et al., 2007; Whetstine et al., 2006). Changes in their expression lead to widespread chromatin reorganization, which alters both the prognosis and treatment of diseases such as cancer (Liau et al., 2017). Epe1 most closely resembles JumonjiC (JmjC) domain containing proteins that use Fe (II) and α-ketoglutarate as co-factors to catalyze histone demethylation (Tsukada et al., 2006). Unlike active histone demethylases, where an HxD/E….H motif is involved in Fe (II) coordination, Epe1 harbors a non-canonical HXE….Y motif (Tsukada et al., 2006). Although Epe1 shares conserved features with other histone demethylases at the amino acid level, there is no biochemical evidence to support the notion that Epe1 has any in vitro enzymatic activity. Epe1 purified from fission yeast cells, or a recombinant source (insect cells), exhibits no H3K9 demethylase activity in vitro (Tsukada et al., 2006; Zofall and Grewal, 2006). However, point mutations of amino acid residues involved in Fe (II) or α-ketoglutarate binding affect Epe1 activity in cells and lead to heterochromatin spreading beyond normal boundary sequences (Trewick et al., 2007). These conflicting lines of biochemical and genetic data have prompted several alternative explanations for how Epe1 might fulfill its anti-silencing role. These models include the possibility that Epe1 acts as a protein hydroxylase which targets non-histone proteins such as Swi6^HP1, regulates the activity of the multi-subunit H3K9 methyltransferase CLRC complex, or functions as an H3K9 demethylase when in complex with Swi6^HP1 (Aygün et al., 2013; Iglesias et al., 2018; Trewick et al., 2007; Zofall and Grewal, 2006). Although these models represent attractive possibilities for how Epe1 regulates heterochromatin, there is no direct evidence to suggest that any of these proteins represent bonafide enzymatic targets. An alternative hypothesis is that Epe1 has a non-enzymatic function that regulates heterochromatin spreading and epigenetic inheritance. In support of this hypothesis, the overexpression of Fe (II) and α-ketoglutarate binding mutants of Epe1 suppresses heterochromatin spreading defects observed in epe1Δ strains (Trewick et al., 2007; Zofall and Grewal, 2006). Hence, co-factor binding mutants of Epe1 can act as multi-copy suppressors of epigenetic silencing despite the presumptive loss of enzymatic activity.

In this study, we discovered that the putative catalytic JmjC domain of Epe1 is, at least in part, dispensable for its anti-silencing function in cells. The C-terminus of Epe1 directly interacts with Swi6^HP1 and its interaction in the context of full-length Epe1 is regulated by H3K9 methylation. Expressing the Epe1 C-terminus alone is sufficient to reverse heterochromatin establishment and attenuate epigenetic inheritance. We propose that a cis interaction between the Epe1 N- and C-terminus inhibits Swi6^HP1 binding. H3K9 methylation binding attenuates this intramolecular interaction and promotes Swi6^HP1 binding. A requirement for H3K9 methylation to stabilize a complex comprising Epe1 and Swi6^HP1 restricts their interaction to a heterochromatin-specific context. Our work highlights the versatile, non-canonical ways in which histone demethylases can oppose establishment and maintenance of epigenetic states.

The establishment and maintenance of epigenetic states is primarily thought to depend on a balance of enzymatic activities between readers, writers, and erasers of histone modifications (Allis and Jenuwein, 2016). However, the replication-dependent and independent turnover of histones serve as a major mechanism that shapes genome-wide patterns of histone methylation (Chory et al., 2019). In Drosophila, modified histones are turned over more than once during each cell-cycle , which limits their capacity to serve as carriers of epigenetic information (Coleman and Struhl, 2017; Deal et al., 2010; Laprell et al., 2017). In principle, passive genome-wide nucleosome exchange can compete with histone modification-dependent read-write mechanisms to oppose epigenetic inheritance.

Our data support the notion that enzymatic erasure is at least in part, dispensable for regulating the inheritance of transcriptionally silent epigenetic states in fission yeast. Our observations are fully consistent with earlier work demonstrating an antagonistic relationship between Epe1, which enhances nucleosome turnover at sites of heterochromatin formation, and Clr3, which suppresses this process (Aygün et al., 2013). In addition, Epe1 also has newly defined roles in recruiting members of the SAGA complex which mediate histone acetylation, a chromatin feature that is also associated with increased nucleosome turnover (Bao et al., 2019). Recent work demonstrated a surprising and unexpected function for the N-terminus of Epe1 in transcriptional activation, which suppresses stochastic, epigenetic silencing (Sorida et al., 2019). Our findings are complementary to these recent studies and expand the gamut of anti-silencing functions associated with Epe1.

Auto-inhibition is a widely used strategy that proteins use to activate their latent functions in response to specific cellular or environmental signals (Pufall and Graves, 2002). The enzymatic and non-enzymatic properties of proteins can be regulated by internal control mechanisms that act in cis. For example, in the case of the chromatin remodeler ALC1, poly ADP ribosylation in response to DNA damage releases the macrodomain of the protein from an auto-inhibited state and activates its latent ATPase-dependent nucleosome remodeling function (Lehmann et al., 2017; Singh et al., 2017). The non-catalytic heterochromatin associated protein Swi6^HP1 is auto-inhibited by a histone H3 mimic sequence (Canzio et al., 2013). Release from auto-inhibition switches the protein to a spreading competent state.

We discovered that a weak interaction between Epe1 and Swi6^HP1 can be bolstered by H3K9 methylation. We propose that a cis interaction between the N- and C-terminal halves of Epe1 opposes Swi6^HP1 binding. These results suggest that Epe1 may be preserved in an auto-inhibited state until it interacts with H3K9 methylation (Figure 8). In the absence of H3K9 methylation, the two proteins fail to form a stable complex in vivo despite their ability to interact directly with each other in vitro. The ability of Epe1 to bind to H3K9 methylation raises the possibility that it competes with Swi6^HP1 for a shared binding site. However, heterochromatin consists of dense networks of H3K9 methylated nucleosomes. It is also possible that Epe1 while interacting with Swi6^HP1, can also be stimulated via an interaction with an adjacent or a distal nucleosome. Our data do not lend direct support to a structural change that occurs within Epe1 upon H3K9me3 peptide binding but strongly suggest that modified histones can influence the stability of chromatin associated complexes in living cells.

Figure 8

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Swi6^HP1 is dynamic and undergoes rapid exchange on the millisecond timescale between the free and H3K9 methylation-bound state (Cheutin et al., 2004; Cheutin et al., 2003). Given the relative abundance of Swi6^HP1 in cells, a simple protein-protein interaction between Epe1 and Swi6^HP1 would effectively titrate Epe1 away from sites of heterochromatin formation. By enforcing an H3K9 methylation-dependent mode of interaction, Epe1 selectively targets a sub-population of Swi6^HP1 molecules that are bound to sites of H3K9 methylation and have a causal role in heterochromatin formation. Therefore, one outcome of this H3K9 methylation-dependent mode of interaction is that Epe1 selectively interacts with heterochromatin-bound Swi6^HP1 molecules as opposed to freely diffusing Swi6^HP1 proteins. It is likely that this balance of protein-protein interactions is tunable via post-translational modifications. For example, Swi6^HP1 phosphorylation compromises Epe1 binding and promotes histone deacetylase recruitment to sites of heterochromatin formation (Shimada et al., 2009). Our studies demonstrate that altering the balance of Swi6^HP1-dependent protein-protein interactions profoundly affects the stability and heritability of silent epigenetic states.

Tethering Clr3 at an ectopic site renders heterochromatin refractory to the anti-silencing effects of Epe1. Our observations are in part, similar to previous findings relating to the fission yeast mating type locus where DNA binding proteins, Atf1 and Pcr1 recruit Clr3 to maintain epigenetic silencing following an RNAi-dependent initiation mechanism (Wang and Moazed, 2017; Yamada et al., 2005). We also recapitulate a unique requirement for the sequence-dependent recruitment of HDAC proteins in cis to facilitate the inheritance of silent epigenetic states despite the presence of anti-silencing factors such as Epe1. We favor a model where histone deacetylases such as Clr3 create a chromatin environment that promotes the read-write enzymatic function of Clr4^Suv39h. In essence, Epe1 prevents Clr3 mediated histone hypoacetylation by forming an inhibitory complex with Swi6^HP1 within a heterochromatin restricted genomic context.

The question of whether Epe1 harbors any histone demethylase activity remains unanswered. Inspired by earlier studies on the Drosophila dKDM4A protein where HP1 binding stimulates demethylase activity, we attempted to reconstitute the enzymatic function of Epe1 in the presence of Swi6^HP1 (Lin et al., 2008). However, our studies are unable to detect Epe1 mediated enzymatic demethylation in the presence or absence of Swi6^HP1 (Figure 2—figure supplement 2A). It is noteworthy that a histone demethylase like protein in Neurospora, DMM-1 shares many similarities with Epe1 and also surprisingly lacks any in vitro enzymatic activity (Honda et al., 2010). DMM-1 also interacts with the HP1 homolog in Neurospora leading us to speculate that this surprising mode of heterochromatin regulation we uncovered in the case of Epe1 might also extend to other fungal systems. Given that expressing the C-terminus of Epe1 only partially restores wild-type levels of Epe1 activity (Figure 3C,D), we speculate that there could potentially be unique substrates or conditions required to reconstitute the enzymatic activity of Epe1. We favor a model in which heterochromatin associated proteins could act as positive or negative allosteric regulators of Epe1. For example, Bdf2, a BRD4 homolog that localizes to heterochromatin boundaries along with Epe1, is a possible candidate that may activate the latent enzymatic functions of Epe1 as H3K9 methylation levels are negligible at the heterochromatin-euchromatin boundary (Wang et al., 2013).

Our observations add to an expanding list of proteins that mimic histone modifiers but have built-in non-enzymatic functions that regulate the establishment and maintenance of epigenetic states. The Drosophila protein dKDM4A is a prominent example of a histone demethylase with enzymatic and non-enzymatic functions (Colmenares et al., 2017). More extreme examples of histone-modifying enzyme mimicry are observed in the case of JARID2, a subunit of the PRC2 complex that shares several characteristics with JmjC domain-containing proteins but has a structural role in regulating PRC2 complex assembly (Kasinath et al., 2018; Son et al., 2013). Proteins involved in the Arabidopsis RNA-dependent DNA methylation pathway, SUVH2 and SUVH9, resemble SET domain methyltransferases but lack enzymatic activity. Instead, both proteins recognize methylated DNA and are involved in RNA pol V recruitment in plants to establish epigenetic silencing (Johnson et al., 2008; Law et al., 2013). Although Epe1 has a JmjC domain, it is possible that its putative catalytic function has been repurposed to regulate its interaction with Swi6^HP1. Our data suggest that H3K9 methylation promotes the cooperative assembly of complexes between Epe1 and Swi6^HP1 at sites of heterochromatin formation.

We speculate that the enzymatic and non-enzymatic functions of Epe1 are likely to oppose heterochromatin assembly on different timescales. As a first approximation, the stabilization of Epe1 at sites of heterochromatin formation is sufficient to displace histone deacetylases and hasten the loss of transcriptional silencing. Hence, the non-enzymatic function of Epe1 serves as the first line of opposition to heterochromatin establishment. This transient inhibition of heterochromatin assembly is likely to be followed by a slower enzymatic step where Epe1 might demethylate H3K9 methylated histones or activate transcription (Sorida et al., 2019). Our work provides a biochemical basis for how chromatin associated factors that are thought to purely function as enzymes have non-enzymatic properties that regulate heterochromatin assembly and epigenetic inheritance.

All data generated or analysed during this study are included in the manuscript and supporting files.

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Author details

1. Gulzhan Raiymbek

   Department of Biological Chemistry, University of Michigan, Ann Arbor, United States

   Contribution

   Conceptualization, Investigation, Methodology

   Competing interests

   No competing interests declared

2. Sojin An

   Department of Biological Chemistry, University of Michigan, Ann Arbor, United States

   Contribution

   Resources, Investigation, Methodology

   Competing interests

   No competing interests declared

3. Nidhi Khurana

   Department of Biological Chemistry, University of Michigan, Ann Arbor, United States

   Contribution

   Formal analysis, Validation, Investigation

   Competing interests

   No competing interests declared

4. Saarang Gopinath

   Department of Biological Chemistry, University of Michigan, Ann Arbor, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

5. Ajay Larkin

   Department of Biological Chemistry, University of Michigan, Ann Arbor, United States

   Contribution

   Formal analysis, Investigation

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-5967-9535

6. Saikat Biswas

   Department of Biological Chemistry, University of Michigan, Ann Arbor, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

7. Raymond C Trievel

   1. Department of Biological Chemistry, University of Michigan, Ann Arbor, United States
   2. Department of Biophysics, University of Michigan, Ann Arbor, United States

   Contribution

   Resources

   Competing interests

   No competing interests declared

8. Uhn-soo Cho

   1. Department of Biological Chemistry, University of Michigan, Ann Arbor, United States
   2. Department of Biophysics, University of Michigan, Ann Arbor, United States

   Contribution

   Resources, Investigation, Methodology

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-6992-2455

9. Kaushik Ragunathan

   Department of Biological Chemistry, University of Michigan, Ann Arbor, United States

   Contribution

   Conceptualization, Resources, Formal analysis, Supervision, Funding acquisition, Methodology, Project administration

   For correspondence

   ragunath@umich.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-4776-8589

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