Chromo Domain Proteins in the Maintenance of Cell Fate
In metazoan development, coordinated regulation of transcription and chromatin architecture is important for the transition from cell fate plasticity to commitment [reviewed in Yadav et al. (2018)]. During C. elegans embryogenesis, the transition to a more differentiated state is accompanied by a progressive increase in chromatin compaction (Mutlu et al., 2018; Costello and Petrella, 2019). Furthermore, multiple chromatin-based mechanisms, including both repressive and activating chromatin-modification and chromatin remodeling activities, ensure proper cell-type- and developmental-stage-specific gene expression in the germline and soma (Cui et al., 2006; Petrella et al., 2011; Wu et al., 2012; Rechtsteiner et al., 2019) [reviewed in Robert et al. (2015)]. Thus, a network of chromatin-associated factors governs the maintenance of cell fate in C. elegans.
Cell fate maintenance can be countered by both naturally-occurring cell fate conversions (transdifferentiation) and experimentally-induced reprogramming. Ectopic expression of cell fate-determining transcription factors in C. elegans has revealed an important role for histone modification pathways [reviewed in Rothman and Jarriault (2019)]. Chromo domain proteins can modulate the susceptibility of embryonic and differentiated cells to induced reprogramming (see below). These findings highlight the roles of chromo domain proteins in linking chromatin organization to transcriptional regulation and cell fate.
In early development, the blastomeres of the C. elegans embryo are susceptible to cell fate conversion by forced expression of the transcription factor HLH-1, the homolog of the master regulator of myogenesis, MyoD [reviewed in Rothman and Jarriault (2019)]. This assay revealed that cec-4 mutant embryos were less susceptible than wild-type to ectopic cell fate reprogramming (Gonzalez-Sandoval et al., 2015). Whereas all wild-type embryos were reprogrammed to muscle, ∼25% of cec-4 mutant embryos hatched. However, this “escape” from induced muscle fate was incomplete, as these hatched embryos were fragile, expressed muscle markers ectopically, and did not continue to develop further. As discussed above, CEC-4 facilitates H3K9me-dependent anchoring of heterochromatin at the nuclear lamina and influences chromatin compartmentalization. These findings suggest that CEC-4-dependent spatial regulation is important for repression of non-induced developmental programs, and therefore that the cells in cec-4 mutant embryos did not fully commit to the induced muscle fate (Gonzalez-Sandoval et al., 2015).
In contrast to CEC-4, the histone methyltransferases MES-2 (H3K27me) (Yuzyuk et al., 2009) and MET-2 (H3K9me2) (Mutlu et al., 2019) promoted the loss of cell fate plasticity, as the mutant embryos were more susceptible than wild-type to reprogramming. The contrasting mutant phenotypes of cec-4 and met-2 suggest that a CEC-4-independent function, such as impaired heterochromatin compaction (Mutlu et al., 2018), is relevant for the increased plasticity in met-2 mutant embryos. However, it is difficult to directly compare the effects of cec-4 and met-2 mutations, as different embryonic timepoints and readouts for plasticity were examined. Since CEC-4, MET-2, and MES-2 all affect genome organization during embryogenesis, analysis of combinations of mutants in parallel will help to decipher whether they also regulate plasticity through similar pathways.
In contrast to the early embryo, differentiated cells lose plasticity and become more resistant to induced reprogramming. In mitotic germ cells and cholinergic motor neurons, this barrier can be overcome following loss of mrg-1 or the HP1 homologs, respectively, indicating roles for these chromo domain proteins in protecting cell identity. When the gustatory neuron fate-inducing transcription factor CHE-1 is ectopically expressed from a heat shock responsive promoter, knockdown of mrg-1 results in ∼25% of animals exhibiting “converted” germ cells, whereas control animals did not have converted germ cells. The conversion was assayed by expression of a fluorescent reporter for a neuronal CHE-1 target (the chemoreceptor GCY-5) and converted germ cells also developed axon-like projections (Hajduskova et al., 2019). In contrast to other factors which sensitize germ cells to CHE-1-mediated neuronal reprogramming, such as Polycomb Repressive Complex 2 (PRC2) components which regulate H3K27 methylation (Patel et al., 2012), the genomic binding sites of MRG-1 are enriched for marks of active chromatin, and MRG-1 appears to function independently of PRC2 in reprogramming (Hajduskova et al., 2019). Interestingly, MRG-1 physically interacts with the SET domain protein SET-26, which has in vitro H3K9 methyltransferase activity (Greer et al., 2014) and mutation of set-26 increases the efficiency of MRG-1-mediated reprogramming. Thus, MRG-1 and SET-26 might work together through a histone methylation read-write crosstalk mechanism [reviewed in Zhang et al. (2015)] to protect germ cell fate and fertility.
Similar to the effects of mrg-1 in the germline, hpl-1, hpl-2 and heterochromatin pathways restrict the plasticity of post-mitotic cholinergic motor neurons (Patel and Hobert, 2017). When CHE-1 is induced at the last larval stage (L4), loss of both hpl-1 and hpl-2 led to a more robust increase in reprogramming than either alone, as measured by the number of neurons reprogrammed per animal by expression of a gcy-5 reporter. Interestingly, the effects of hpl-1 and hpl-2 were partly H3K9 methylation-independent, as the efficiency of reprogramming was higher in hpl-1;hpl-2 double mutants than in met-2;set-25 mutants. Notably, loss of the cholinergic cell fate-determining transcription factor, unc-3, also sensitized these neurons to reprogramming. Combinatorial mutations indicated that unc-3 acts in the same pathway as met-2 but in parallel to mes-2 and H3K27 methylation (Patel and Hobert, 2017). Collectively, these data highlight the interplay between heterochromatin-associated factors and transcription factors in specifying cell fate.
These experimental reprogramming studies reveal the roles of chromo domain proteins in connecting chromatin landscape with developmental plasticity. While studies discussed earlier focused on global chromatin reorganization, local effects on gene regulation likely also contribute to the roles of chromo domain proteins in cell fate maintenance. Indeed, fluorescent reporter assays revealed roles for H3K9 methylation readers in restricting the expression patterns of key transcription factors. For example, hpl-1 and hpl-2 prevent ectopic expression of reporters for homeodomain transcription factors important for male tail, vulval and gonad development (Coustham et al., 2006; Schott et al., 2006; Studencka et al., 2012b). In addition, loss of either cec-3 or hpl-2 leads to ectopic expression of the homeodomain transcription factor unc-4 in non-vulval ventral nerve cord neurons and disrupted egg laying behavior (Zheng et al., 2013). These reporter assays do not reveal direct effects at the genomic loci of interest. However, the correspondence between the reporter assays and phenotypic readouts suggests that the reporters effectively model the chromo domain-dependent regulation of loci encoding transcription factors with key roles in cell fate.
Together, the cell fate induction experiments described above have revealed roles for both heterochromatin and euchromatin-associated factors in the regulation of cell fate plasticity in several developmental contexts and cell types. Looking beyond C. elegans, chromatin-based mechanisms have also been identified as key barriers to the reprogramming of mammalian cells [reviewed in Brumbaugh et al. (2019)]. Robust characterization of the epigenetic mechanisms governing cell fate therefore holds promise to influence advancements in regenerative medicine. One fruitful avenue will be to take advantage of the screening capabilities of C. elegans to identify modifiers of chromatin factor-mediated reprogramming. Such efforts have already identified connections between H3K27 methylation, the highly conserved Notch signaling pathway, and control of cell proliferation (Seelk et al., 2016; Coraggio et al., 2019). Another challenge will be to determine the mechanisms underlying cell type-specific reprogramming, and to connect the cell fate phenotypes to broad disruption of chromatin organization, or to misregulation of specific target genes. These approaches will provide a more complete understanding of the molecular networks governing cell fate plasticity.
Germline Immortality and Transgenerational Epigenetic Inheritance
Given the importance of chromo domain proteins in cell fate, it is not surprising that they also play key roles in germ cells and fertility. Chromatin regulation affects several of the inter-related mechanisms that jointly contribute to the maintenance of the germ lineage, including the preservation of germ cell fate, repression of transposable/repetitive elements, and genome stability [reviewed in Smelick and Ahmed (2005), Kelly (2014)]. In addition, the interplay between chromatin architecture and small RNA pathways exerts a significant role in the characteristic “immortality” of the germline, or its capacity to indefinitely give rise to gametes transgenerationally. The short generation time and genetic tractability of C. elegans have made it a powerful model to study the mechanisms of germline immortality as well as the related phenomenon of transgenerational epigenetic inheritance (TEI), or the retention of epigenetic information across multiple generations. Here we highlight recent studies that connect chromo domain proteins to the network of mechanisms linking fertility, germline immortality and TEI.
A key model for understanding TEI in C. elegans is the inheritance of RNA interference (RNAi). Gene silencing initiated by RNAi can be inherited for several or many generations in the absence of the initial RNA trigger, with the duration depending on the specific pathway of silencing initiation and the nature of the genetic target [reviewed in Minkina and Hunter (2018)]. The maintenance of this silencing depends on nuclear RNAi which involves small RNA-mediated recruitment of nuclear Argonaute proteins to target loci to effect transcriptional silencing and deposition of repressive histone methylation (Figure 1) [reviewed in Weiser and Kim (2019)]. Further emphasizing the importance of TEI pathways in fertility, loss of factors essential for RNAi inheritance, including the nuclear Argonaute HRDE-1, also have a “mortal germline” phenotype (Buckley et al., 2012; Spracklin et al., 2017).
In a genetic screen for factors that prolong the transgenerational retention of RNAi inheritance, a recent study characterized the chromo domain protein HERI-1 (heritable enhancer of RNAi; formerly known as CEC-9) (Perales et al., 2018). Interestingly, ChIP assays revealed recruitment of HERI-1 to genes undergoing nuclear RNAi; this recruitment is dependent on HRDE-1 and SET-32 (also known as HRDE-3), a methyltransferase contributing to H3K9 methylation and nuclear RNAi inheritance. Together with evidence that HERI-1 inhibits nuclear RNAi, these data suggest that the silencing machinery itself recruits HERI-1 as an inhibitor, potentially forming a negative feedback loop to prevent runaway heritable epigenetic silencing. This “braking” activity may be crucial for sperm development, as heri-1 mutants exhibit impaired spermatogenesis, which was suppressed by mutation of hrde-1. It will be of great interest to identify the endogenous targets of HERI-1. Additional intriguing mechanistic questions include whether its chromo domain directly interacts with methylated histones, and the potential function of its serine-threonine kinase-like domain as an allosteric regulator or scaffold (Perales et al., 2018). While much attention has been directed to the factors required for RNAi inheritance, HERI-1 joins a handful of genes or environmental perturbations identified so far that restrict TEI (Houri-Ze’evi et al., 2016; Lev et al., 2017).
The maintenance of germline immortality requires the concerted activity of multiple histone methyltransferases and demethylases [reviewed in Kelly (2014)], and genetic interaction approaches have uncovered contributions of chromo domain proteins CEC-3 and CEC-6 to this network (Greer et al., 2014; Saltzman et al., 2018). Loss of the H3K9 methylation reader cec-3 has distinct effects in different backgrounds with compromised fertility. Strains with a mutation of the H3K4me2 demethylase spr-5 have a mortal germline phenotype (Katz et al., 2009) which can be suppressed by a cec-3 deletion (Greer et al., 2014). In stark contrast, cec-6 mutants have a comparatively mild fertility defect that is sharply exacerbated in combination with loss of cec-3 (Saltzman et al., 2018). One attractive model to account for these progressive fertility defects posits that disruption of these chromatin factors permits the aberrant spreading of transcriptionally-active euchromatin into transcriptionally-silenced heterochromatin, or vice versa, consequently disrupting germline-specific programming. Indeed, spr-5 mutants exhibit a global increase in H3K4 methylation and a decrease in H3K9 methylation, which are associated with a progressive loss of CEC-3 association with the heterochromatin-enriched chromosome arms. In a similar manner, the ATPase MORC-1, which is required for germline immortality and RNAi inheritance (Spracklin et al., 2017; Weiser et al., 2017), also prevents the spread of H3K36 methylation into heterochromatin, and this effect can be counteracted by loss of the H3K36 methyltransferase met-1 (Weiser et al., 2017). In the case of the cec-3;cec-6 double mutants, the loss of both of these H3K9me and H3K27me readers may eliminate the capacity for compensatory heterochromatin recognition. However, this hypothesis remains to be tested. Further identification of both physical and genetic interactors of chromo domain proteins will help to reveal the molecular details of these models.
In addition to transgenerational effects, chromo domain proteins directly influence the development of the germline and gametes through several mechanisms. Loss of hpl-2 results in temperature-sensitive sterility, abnormal oocyte accumulation (Couteau et al., 2002) and upregulation of repetitive elements such as transposons (McMurchy et al., 2017). The germlines of hpl-2 mutants also exhibit hypersensitivity to DNA damage and increased apoptosis (McMurchy et al., 2017). Brood sizes of hpl-2 mutants are further reduced by loss of additional heterochromatin factors that exhibit significant overlap in their genomic binding patterns with HPL-2 (including the synMuv factors LIN-61, LIN-13, MET-2, and LET-418), particularly at H3K9me2-marked heterochromatin and repetitive elements (McMurchy et al., 2017). These findings suggest that HPL-2 is part of a network of heterochromatin-associated proteins, including the H3K9me2 methyltransferase MET-2, that safeguard genome integrity in the germline (Zeller et al., 2016; McMurchy et al., 2017). The fertility-associated role of chromo domains in genome stability also extends to the euchromatin-associated MRG-1, which is implicated in DNA repair during meiosis and in the primordial germ cells (Xu et al., 2012; Miwa et al., 2019). Together, these studies emphasize the importance of chromo domain proteins in repetitive element and transposon repression and the response to genotoxic stress and DNA damage, in addition to their roles in gene expression regulation.
The mechanisms through which chromo domain proteins maintain germline immortality continue to be investigated. Several mortal germline phenotypes described here are reversible or temperature-sensitive (Spracklin et al., 2017; Saltzman et al., 2018), implicating epigenetic mechanisms such as the remodeling of chromatin states between generations or small RNA-based inheritance. However, given the importance of heterochromatin maintenance in genome stability [described above and reviewed in Janssen et al. (2018)], the contribution of genetic changes to this loss of fertility remains an open question.
Overall, these studies highlight the roles of multiple C. elegans chromo domain proteins at the intersection of chromatin architecture, small RNA pathways, and transgenerational epigenetic inheritance. C. elegans has also become an important model system for the transgenerational influences of environmental factors [reviewed in Perez and Lehner (2019)]. Given the associations among epigenetic mechanisms, environmental effects, aging and cancer [reviewed in Cavalli and Heard (2019)], studies in accessible model systems such as C. elegans are a crucial step toward a mechanistic understanding of epigenetic regulation in health and disease.