Sequential histone-modifying activities determine the robustness of transdifferentiation

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Science  15 Aug 2014:
Vol. 345, Issue 6198, pp. 826-829
DOI: 10.1126/science.1255885

Epigenetics direct transdifferentiation

To make an entire animal, many and varied cell types form and interact. Some of these differentiated cells take a U-turn and can de-differentiate or transdifferentiate to another cell fate. Although relatively rare in nature, Zuryn et al. followed such a program in the tiny roundworm Caenorhabditis elegans, where a rectal cell–to–motor neuron conversion is seen. Transcription factors with conserved roles in cell plasticity and terminal fate selection partner up with specific histone-modifying enzymes in discrete steps to specify separate sequential phases of cell identity.

Science, this issue p. 826


Natural interconversions between distinct somatic cell types have been reported in species as diverse as jellyfish and mice. The efficiency and reproducibility of some reprogramming events represent unexploited avenues in which to probe mechanisms that ensure robust cell conversion. We report that a conserved H3K27me3/me2 demethylase, JMJD-3.1, and the H3K4 methyltransferase Set1 complex cooperate to ensure invariant transdifferentiation (Td) of postmitotic Caenorhabditis elegans hindgut cells into motor neurons. At single-cell resolution, robust conversion requires stepwise histone-modifying activities, functionally partitioned into discrete phases of Td through nuclear degradation of JMJD-3.1 and phase-specific interactions with transcription factors that have conserved roles in cell plasticity and terminal fate selection. Our results draw parallels between epigenetic mechanisms underlying robust Td in nature and efficient cell reprogramming in vitro.

Tissue and organ regeneration occurs in multiple animal species and can originate from transdifferentiation (Td) of surrounding cell populations (1, 2). Understanding how individual cells interconvert efficiently and precisely to contribute to or result in the robust regeneration of whole tissues is a major goal of regenerative medicine. To characterize these mechanisms in a physiological setting, we investigated a traceable and predictable Td event in Caenorhabditis elegans. The Y cell is a postmitotic hindgut cell that constitutes the half section of the third ring of the rectal tube. This cell exhibits a remarkable behavior by which it disengages from the tube and changes into a motor neuron called PDA during normal development (Fig. 1A) (3). We found that conversion between the two specialized cell types occurred with virtually invariant precision in each animal (100%, n = 2209 animals). To identify those factors that endowed such precision, we chemically mutagenized worms, screened for low-penetrance mutants in which Td was no longer invariant (Td), and isolated several mutant alleles (fp11, fp13, fp15, and fp25) (Fig. 1B and fig. S1, A and B). Using deep-sequencing–based mapping (4), we cloned and confirmed that the causal genetic lesions of all mutants affected jmjd-3.1 (fig. S1, C and D).

Fig. 1 jmjd-3.1 and the Set1 complex determine invariant Td.

(A) Schematic of Y-to-PDA Td. (B) jmjd-3.1 mutations cause defects in Td. (C) Schematic of the JMJD-3.1 protein. NLS, nuclear localization signal; JmjC, Jumonji catalytic domain; ZnB, zinc-binding, H3 tail recognition domain. Amino acids highlighted in gray boxes were mutated in order to disrupt either lysine demethylation (blue) or H3 tail recognition (red). (D) Effect of deficiencies in Set1 complex subunits on Td.

jmjd-3.1 encodes an ortholog of human Jmjd3, which displays specificity for demethylating tri- and dimethylated histone H3 lysine 27 (H3K27) via a highly conserved Jumonji C terminus (JmjC) domain (5, 6). In each jmjd-3.1 mutant, the JmjC domain is either mutated, truncated, or absent, suggesting that H3K27me3/me2 demethylase activity mediates its Td role (Fig. 1C). Generating jmjd-3.1(fp15) strains harboring Cherry-tagged JMJD-3.1 forms, we found that nuclear JMJD-3.1 acted cell-autonomously [as well as nonredundantly (fig. S2)] and that disrupting its ability to demethylate lysine residues or recognize histone H3 tails abolished its activity during Td (Fig. 1C and figs. S3 and S4). Moreover, expressed and purified JMJD-3.1 mutant proteins mimicking fp11, fp15, and fp25 had completely lost their capacity to demethylate H3K27me3 (fig. S5). This correlated with the Td phenotypes and suggested that each allele encoded enzymatically null products. Last, deficiencies in the worm Polycomb Repressive Complex 1 component SPAT-3 (Ring1B) (7), which acts to fortify gene repression upon recruitment to trimethylated H3K27 residues (8), partially restored Td in jmjd-3.1(fp15) mutants (fig. S6). These data indicated that jmjd-3.1 acts antagonistically to H3K27me3-mediated transcriptional repression during Td by demethylating H3K27me3.

Alongside H3K27, other histone tail residues can exist in several states of methylation, each of which can be modulated by specific enzymes. We performed RNA interference (RNAi) screens to determine whether other histone methyltransferases or demethylases, and hence other specific histone modifications, performed a role similar to jmjd-3.1 in ensuring precise Td (fig. S7 and table S1). Only RNAi of the mammalian SET1A/SET1B ortholog, set-2, disrupted invariant Td. SET-2 is the catalytic subunit of the Set1 complex, a highly conserved multi-subunit ensemble that methylates H3K4 (9). Null deficiencies in set-2, as well as other Set1 complex subunits (wdr-5.1/WDR5, ash-2/ASH2L, rbbp-5/RbBP5, dpy-30/DPY30, and cfp-1/CFP1), resulted in a low-penetrance Td phenotype, which again is not attributable to hypomorphism or redundancy (Fig. 1D and fig. S8). Set1 deficiencies could be phenocopied by overexpressing several functionally antagonistic enzymes (H3K4me3/me2 demethylases) in the Y cell in a demethylase-activity–dependent manner (fig. S9). The same effect was observed by overexpressing a K4 unmethylable replication-independent H3.3K4A in Y (fig. S9). Further implicating Set1 H3K4 methyltransferase activity in Td, only a catalytically active set-2 transgene expressed in Y could rescue the defect in set-2 mutants (discussed below). Thus, multiple specific histone-modifying activities acting on distinct lysines of histone H3 mediate invariant cell conversion in a natural setting.

How do specific histone methylation states elicit these effects? To answer this question, we investigated the progression of Td at the single-cell level in live mutant animals. Td cells in Set1 mutants, and in transgenic animals overexpressing Set1 antagonists or H3K4A in Y, could be broken into two distinct classes: Approximately half displayed a characteristic, hindgut fried-egg–shaped nuclear morphology, whereas the rest displayed speckled nuclear morphology (Fig. 2A and fig. S10A). In contrast, Td cells in jmjd-3.1 mutants had exclusively speckled nuclei. These initial observations hinted that specific K4 and K27 methylation states mediated different transitions through successive stages of cell conversion, which became blocked in the absence of their respective modifiers. We have previously shown that Y-to-PDA proceeds through discrete phases: dedifferentiation and then redifferentiation into the new cell type (10), as observed in vertebrate examples of Td, such as newt lens regeneration (1). We thus tested whether jmjd-3.1 and Set1 mediated distinct Td phases. Indeed, those Td cells with hindgut morphology in Set1 mutants persistently expressed hindgut molecular markers and remained attached to the rectal tube via intercellular adherens junctions (fig. S10, B and C), indicating a failure in dedifferentiation. In Td cells with speckled morphology in either jmjd-3.1 or Set1 backgrounds, hindgut markers were correctly extinguished, and the cells disengaged from the rectal tube, but they did not properly activate a range of PDA neural markers (fig. S10B), indicating a failure to properly redifferentiate. In addition, timely rescue of wdr-5.1 and jmjd-3.1 mutant strains by heat shock–inducible rescue constructs demonstrated that the temporal requirements of each factor correlated precisely with their successive cellular roles (Fig. 2B). Therefore, Set1 acts dichotomously, mediating both dedifferentiation and redifferentiation, whereas jmjd-3.1 mediates only redifferentiation. This suggests that stepwise modifications to distinct lysine residues on H3 mediate precise transition through distinct cellular phases of Td (Fig. 2C).

Fig. 2 Set1 and jmjd-3.1 mediate different Td phases.

(A) Fluorescent micrographs of cell fate markers in Td cells in live animals. Asterisk indicates signal from adjacent cell. (B) Timed heat shock pulses show that wdr-5.1 and jmjd-3.1 have distinct temporal requirements matching their successive cellular roles. Ø, no heat-shock. (C) Schematic showing requirements for histone-modifying activities. (D) Photoconversion of Dendra2 fused to JMJD-3.1 indicates that photoconverted protein is degraded during dedifferentiation and resynthesized during redifferentiation. (E) Modulation of JMJD-3.1 nuclear localization indicates that degradation is nuclear-dependent and that removal from the nucleus is sufficient for eliminating its activity. **P < 0.01, *P < 0.05; n > 100 animals.

The model above implies a mechanism for efficient partitioning (temporally and functionally) of K4- and K27-modifying activities within a postmitotic cell. To determine the mechanisms at play, we first addressed how each distinct requirement was established. Consistent with Set1’s action at different steps of the process, functional, green fluorescent protein (GFP)–tagged Set1 subunits were nuclear-localized in Y/PDA throughout Td (fig. S11A). Contrastingly, Cherry-tagged JMJD-3.1 and a jmjd-3.1 fosmid-based GFP construct were dynamically localized during Td (fig. S11, A to C). Both JMJD-3.1 reporters were detectable in the nucleus at all stages during, before, and after Td, except for a precise window when Y dedifferentiated. This pattern matched jmjd-3.1’s temporal requirements and exclusive redifferentiation role, suggesting that regulation of JMJD-3.1 levels may provide a mechanism to ensure stepwise K4 and K27 modification. Through the use of a photoconvertible JMJD-3.1::Dendra2 fusion protein, as well as artificial modulation of JMJD-3.1’s nuclear localization, we determined that JMJD-3.1 levels were dynamically regulated through nuclear-dependent degradation during dedifferentiation (Fig. 2, D and E, and figs. S12). Moreover, precise temporal regulation of JMJD-3.1 levels, and hence stepwise histone modification, was critical for precise Y-to-PDA conversion because forced ectopic JMJD-3.1 expression during Y dedifferentiation induced a Td phenotype and resulted in cells displaying a mixture of abnormal identities (fig. S13). Catalytic inactivation of JMJD-3.1 largely mitigated these effects (fig. S13D). We conclude that regulation of nuclear JMJD-3.1 protein levels ensures partitioning of K27 demethylase activity into the redifferentiation phase of Td and that such control is critical for invariant Td.

We next turned our attention to the mechanism (or mechanisms) that partitioned Set1’s K4 methylase activity into temporally separable, seemingly opposite cellular functions (dedifferentiation and redifferentiation). We reasoned that each distinct role might be segregated functionally through discrete temporal associations with coregulators. Using coimmunoprecipitation, we found that the core Set1 component WDR-5.1 associates with several subunits of the Nanog and Oct4 Deacetylase (NODE)–like complex (Fig. 3A) (11). We have previously found that the NODE-like complex is required for Y dedifferentiation (12) and is composed of factors that have conserved roles in the maintenance and induction of cell plasticity in a variety of species (13). Using Set1;NODE double mutants carrying catalytically active or inactive set-2 rescue constructs, we found that the two complexes functionally cooperated—in a Set1-H3K4 methyltransferase–dependent manner—to specifically mediate Y dedifferentiation (Fig. 3B). This suggested that the NODE-like complex, which can bind DNA targets directly (11), provides dedifferentiation functional specificity to Set1.

Fig. 3 Phase-specific interactions specify Set1 and JMJD-3.1’s Td roles.

(A) WDR-5.1 immunoprecipitates members of the C. elegans NODE complex, CEH-6(OCT), SOX-2(SOX2), SEM-4(SALL), and EGL-27(MTA). (B) Set1 functionally cooperates with the NODE-like complex to mediate hindgut dedifferentiation. m, mutant; wt, wild type. (C and D) JMJD-3.1 immunoprecipitates WDR-5.1 and UNC-3 via an N-terminal domain. (E) unc-3 cooperates with jmjd-3.1 and Set1 to mediate PDA redifferentiation. Red and blue P values correspond to dedifferentiation and redifferentiation, respectively. Error bars indicate SEM. ND, not determined; ns, not significant. Statistical significance was determined by using two-way analysis of variance.

Next, we uncovered an alternative functional interaction network between WDR-5.1, JMJD-3.1, and the phylogenetically conserved COE (Collier, Olf, EBF)–type transcription factor (TF) UNC-3 that appeared to specify Set1’s redifferentiation role. UNC-3 transcriptionally regulates batteries of genes that specify terminal features of motor neurons in C. elegans as well as chordates (14). We showed that UNC-3, which is required for PDA redifferentiation (10), interacts with a functionally important N-terminal region of JMJD-3.1. JMJD-3.1 in turn associates with WDR-5.1 (Fig. 3, C and D, and fig. S14), providing a mechanistic rationale for the precise regulation of JMJD-3.1 protein levels that we unraveled, one that may serve to prevent interaction with Set1 until redifferentiation, for which both activities are needed. Moreover, double unc-3;jmjd-3.1 and unc-3;wdr-5.1 mutants suggested that these three factors functionally cooperate specifically during the redifferentiation phase of Td and not during dedifferentiation (Fig. 3E). Thus, UNC-3 appears to act as an important regulatory hub for the redifferentiation phase of Td onto which chromatin modifiers may log their activities.

To gain deeper insight into SET1 and JMJD-3.1’s mode of action, we analyzed double Set1;jmjd-3.1 mutant strains carrying active or inactive set-2 or jmjd-3.1 transgenes, or jmjd-3.1(fp15) mutants overexpressing H3.3K4A (Fig. 4A). Our results suggested that K4 methylation and K27 demethylation (i) acted in parallel, rather than in a mutually dependent manner, and (ii) ultimately converged on a common downstream target (or targets) to specifically promote redifferentiation. One possibility—suggested by their physical association as well as data presented earlier indicating that Set1 and JMJD-3.1 cellular localizations and activities intersected precisely during redifferentiation—is that both activities act in a bifunctional K4/K27–modifying complex. Such complexes have been shown in other species (6, 1517) and have been predicted to resolve bivalent domains: nucleosomes containing both H3K4me3 and H3K27me3 signatures (18). Evidence suggests that such domains act to poise developmental loci for timely and efficient activation, representing an attractive mechanism for ensuring precise PDA redifferentiation (19). However, bivalency has only been reported thus far in vertebrates (20). Here, we demonstrate the co-occurrence of H3K4me3 and H3K27me3 marks on mononucleosomes purified from worms, pointing to the existence of bivalent domains in C. elegans (fig. S15) and opening the possibility that targets of Set1 and JMJD-3.1 may be bivalently marked.

Fig. 4 jmjd-3.1 and Set1 cooperate to ensure robust Td against stress.

(A) jmjd-3.1 and Set1 components exhibit synergistic genetic interactions in a H3K27 demethylase and H3K4 methyltransferase activity–dependent manner during redifferentiation. m, null mutant; wt, wild type. (B to E) Stressful environmental conditions enhance Td defects in jmjd-3.1 and Set1 null mutants. Error bars indicate SEM. ND, not determined; ns, not significant. n > 100 animals; ***P < 0.001, **P < 0.01, *P < 0.05; n > 100 animals.

Our results suggest a model in which Set1 and jmjd-3.1’s role is to ensure precise Td, whereas key TFs, which exhibit complete penetrance in null mutants (3, 10, 12), act as drivers. We found that histone modifications additionally are critically needed when the worm is exposed to adverse conditions. In absence of jmjd-3.1 or Set1, Y-to-PDA Td was hypersensitive to a variety of stressful treatments, whereas Td remained unaffected in wild-type animals (Fig. 4, B to E). This suggests that both Set1-dependant H3K4 methylation and jmjd-3.1–dependent H3K27me3/me2 demethylation provide protection against variations that may be encountered in the worm’s natural environment. These roles in ensuring the robustness of cell conversion in stressful circumstances may underlie invariant Td under normal conditions. Last, our in vivo results suggest that Set1 and jmjd-3.1 perform highly conserved roles across phyla and cell types to reinforce TF-driven changes in identity. Indeed, modulation of H3K4 and H3K27 methylation state has been shown to regulate somatic reprogramming (2123). Specifically, the core Set1 subunit, Wdr5, was recently shown to interact with and potentiate Oct4’s roles in the initial phases of mammalian induced pluripotent stem (iPS) cell induction (22). Furthermore, Jmjd3 was found to specifically inhibit the initial stages of iPS reprogramming (23), drawing a distinct parallel to its disruption of the initial stages of Td we observed.

Supplementary Materials

Materials and Methods

Figs. S1 to S16

Table S1

References (2442)

References and Notes

  1. Acknowledgments: We are grateful to S. Gasser, R. Klose, M. Labouesse, B. Prud’homme, and R. Schneider for their comments on the manuscript. Work in R.M.’s laboratory is funded by a European Research Council–Stg (REPODDID) grant, an ATIP-Avenir grant, and a Fondation pour la Recherche Medicale (FRM) grant. Work in S.J.’s laboratory is funded by grants from FRM, the Fondation ARC pour la recherche sur le cancer, the Association Française contre les Myopathies (AFM), the Fondation Schlumberger pour l’Enseignement et la Recherche (FSER), and the European Molecular Biology Organization Young Investigator program (EMBO YIP). S.Z. is a FRM and ARC postdoctoral fellow, and S.J. is a Centre National de la Recherche Scientifique research director.
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