Phase separation of Arabidopsis EMB1579 controls transcription, mRNA splicing, and development

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Open Access
Peer-reviewed

Research Article

Yiling Zhang, 

Zhankun Li, 

Naizhi Chen, 

Yao Huang, 

Shanjin Huang

x

Published: July 21, 2020

https://doi.org/10.1371/journal.pbio.3000782

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AbstractTight regulation of gene transcription and mRNA splicing is essential for plant growth and development. Here we demonstrate that a plant-specific protein, EMBRYO DEFECTIVE 1579 (EMB1579), controls multiple growth and developmental processes in Arabidopsis. We demonstrate that EMB1579 forms liquid-like condensates both in vitro and in vivo, and the formation of normal-sized EMB1579 condensates is crucial for its cellular functions. We found that some chromosomal and RNA-related proteins interact with EMB1579 compartments, and loss of function of EMB1579 affects global gene transcription and mRNA splicing. Using floral transition as a physiological process, we demonstrate that EMB1579 is involved in FLOWERING LOCUS C (FLC)-mediated repression of flowering. Interestingly, we found that EMB1579 physically interacts with a homologue of Drosophila nucleosome remodeling factor 55-kDa (p55) called MULTIPLE SUPPRESSOR OF IRA 4 (MSI4), which has been implicated in repressing the expression of FLC by forming a complex with DNA Damage Binding Protein 1 (DDB1) and Cullin 4 (CUL4). This complex, named CUL4-DDB1MSI4, physically associates with a CURLY LEAF (CLF)-containing Polycomb Repressive Complex 2 (CLF-PRC2). We further demonstrate that EMB1579 interacts with CUL4 and DDB1, and EMB1579 condensates can recruit and condense MSI4 and DDB1. Furthermore, emb1579 phenocopies msi4 in terms of the level of H3K27 trimethylation on FLC. This allows us to propose that EMB1579 condensates recruit and condense CUL4-DDB1MSI4 complex, which facilitates the interaction of CUL4-DDB1MSI4 with CLF-PRC2 and promotes the role of CLF-PRC2 in establishing and/or maintaining the level of H3K27 trimethylation on FLC. Thus, we report a new mechanism for regulating plant gene transcription, mRNA splicing, and growth and development.

Citation: Zhang Y, Li Z, Chen N, Huang Y, Huang S (2020) Phase separation of Arabidopsis EMB1579 controls transcription, mRNA splicing, and development. PLoS Biol 18(7):
e3000782.

https://doi.org/10.1371/journal.pbio.3000782Academic Editor: Xuemei Chen, University of California Riverside, UNITED STATESReceived: October 23, 2019; Accepted: July 6, 2020; Published: July 21, 2020Copyright: © 2020 Zhang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Data Availability: All relevant data are within the paper and its Supporting Information files.Funding: This work was supported by a grant from the National Natural Science Foundation of China (31471266). The research in the Huang Lab is also supported by the funding from Beijing Advanced Innovation Center for Structural Biology. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Competing interests: The authors have declared that no competing interests exist.Abbreviations:
A3SS,
alternative 3′ splice site; A5SS,
alternative 5′ splice site; CLF,
CURLY LEAF; COP1,
CONSTITUTIVELY PHOTOMORPHOGENIC1; CUL4,
Cullin 4; DAG,
day after germination; DDB1,
DNA Damage Binding Protein 1; E(z),
enhancer of zeste; EMB1579,
EMBRYO DEFECTIVE 1579; EMF2,
EMBRYONIC FLOWER 2; ESC,
extra sex combs; FIE,
FERTILIZATION INDEPENDENT ENDOSPERM; FIS2,
FERTILIZATION INDEPENDENT SEED; FLC,
FLOWERING LOCUS C; FRAP,
fluorescence recovery after photobleaching; H3K27me3,
trimethylation of lysine 27 of histone H3; hnRNP,
heterogeneous nuclear ribonucleoprotein; IDR,
intrinsically disordered protein region; LLPS,
liquid-liquid phase separation; MEA/FIS1,
MEDEA; MSI1–5,
MULTIPLE SUPPRESSOR OF IRA 1–5; MXE,
mutually exclusive exon; NLS,
nuclear localization signal; NPM1,
nucleophosmin; p55,
nucleosome remodeling factor 55-kDa; PcG,
Polycomb group; PRC1,
Polycomb Repressive Complex 1; qRT-PCR,
quantitative reverse transcription PCR; RBP,
RNA binding protein; RFP,
red fluorescent protein; RNA-seq,
RNA sequencing; RS,
serine/arginine; SIM,
structured illumination microscopy; snRNP,
small nuclear ribonucleoprotein; SPA,
SUPPRESSOR OF PHYA; Su(z)12,
suppressor of zeste 12; SWN,
SWINGER; TGFP,
tandem copies of enhanced green fluorescent protein; U snRNP,
uridine-rich snRNP; VRN2,
VERNALIZATION 2; WT,
wild type

IntroductionPlant growth and development are tightly regulated in response to many endogenous and environmental signals by genetic and cellular programs that determine plant form. As sessile organisms, plants need to efficiently organize various cellular events to cope with the ever-changing surrounding environment [1–3]. Among various cellular events, gene transcription and mRNA splicing play essential roles during plant growth and development as well as during the interaction of the plant with its surrounding environment [4–6]. Dysfunction in gene transcription and mRNA splicing causes dramatic defects in development and environmental adaptation in plants [7–11]. Therefore, it is important to understand how plants tightly and efficiently control gene transcription and mRNA splicing in response to various internal and external cues.
The nucleus contains a dynamic mix of nonmembranous subcompartments, including the nucleolus, nuclear speckles, paraspeckles, Cajal bodies, nuclear stress bodies, histone locus bodies, and the perinuclear compartment [12–14]. The absence of a surrounding membrane enables those subcompartments to assemble or disassemble rapidly following alterations in the cell’s environment and in response to intracellular signals [15–20]. Subnuclear compartmentalization might be especially important in mediating rapid changes in gene transcription and mRNA splicing in response to intrinsic and environmental variations, as proteins associated with transcription and mRNA splicing are often localized to nuclear speckles or dots [21,22]. Those proteins often exhibit multivalent features that are contributed by repetitive folded domains and/or disordered regions (also referred to as intrinsically disordered protein regions [IDRs]) [23], and they are able to undergo liquid-liquid phase separation (LLPS). Indeed, such proteins have been implicated in the regulation of gene transcription and mRNA splicing in different organisms [24–29]. However, it remains largely unknown how and to what extent LLPS of these proteins is linked to gene transcription and mRNA splicing.
Polycomb group (PcG) proteins have been implicated in the regulation of transcription to establish and maintain specific gene expression patterns to drive organismal development. PcG proteins form multisubunit protein complexes, such as Polycomb Repressive Complex 1 (PRC1) and PRC2. PRC2 is recruited to target genes and catalyzes the trimethylation of lysine 27 of histone H3 (H3K27me3) [30]. There are four core components of the PRC2 complex, first identified in Drosophila: enhancer of zeste (E[z]); extra sex combs (ESC); suppressor of zeste 12 (Su[z]12); and nucleosome remodeling factor 55-kDa (p55) [31]. Homologues of these four core subunits exist in plants. Specifically, Arabidopsis has three E(z) homologues, CURLY LEAF (CLF), MEDEA (MEA/FIS1), and SWINGER (SWN), which catalyze H3K27me3. In addition, Arabidopsis has three Su(z)12 homologues, EMBRYONIC FLOWER 2 (EMF2), VERNALIZATION 2 (VRN2), and FERTILIZATION INDEPENDENT SEED (FIS2); one Esc homologue, FERTILIZATION INDEPENDENT ENDOSPERM (FIE); and five p55 homologues, MULTIPLE SUPPRESSOR OF IRA 1–5 (MSI1–5). Based on their different subunit compositions, at least three different PRC2-like complexes with distinct functions exist in Arabidopsis: the EMF, VRN, and FIS complexes [32]. Among them, the vegetative EMF complex, which comprises EMF2, FIE, CLF, or SWN and one p55 homologue, has been implicated in the regulation of vegetative development and the transition to flowering in Arabidopsis [33,34]. Biochemical purification of the EMF complex showed that MSI1, but not MSI4, was a core subunit [35]. Nevertheless, MSI4 plays a key role in the regulation of floral transition, which has been linked to the function of the CLF-containing EMF complex (CLF-PRC2) in repressing the expression of FLOWERING LOCUS C (FLC) [36]. MSI4 has also been suggested to play a role in histone deacetylation [37]. Molecular characterization showed that MSI4 is linked to the epigenetic regulation of the FLC locus through its interaction with Cullin 4 (CUL4)–DNA Damage Binding Protein 1 (DDB1) and a CLF-PRC2 complex [36]. Specifically, MSI4 is a WD40 repeat-containing protein with a conserved WDxR motif, which is a typical feature of the previously identified WD40-containing DDB1 and CUL4-associated factors [38]. MSI4 forms a complex with DDB1 and CUL4, named CUL4-DDB1MSI4 [36]. Although CUL4-DDB1 acts in the photoperiod flowering pathway by interacting with the CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1)–SUPPRESSOR OF PHYA (SPA) complex [39] to control the abundance of CONSTANS protein [40,41], both CUL4 and MSI4 are required to maintain the level of H3K27me3 on FLC chromatin [36]. It was also demonstrated that CUL4–DDB1MSI4 physically associates with a CLF-PRC2 complex [36].Therefore, the emerging scenario is that MSI4 forms the CUL4-DDB1MSI4 complex, which physically interacts with CLF-PRC2 to establish and/or maintain the level of H3K27me3 on the FLC locus to control the expression of FLC [36].
Here we report that the plant-specific protein EMBRYO DEFECTIVE 1579 (EMB1579), which was uncovered during the systematic identification of genes required for normal embryo development in Arabidopsis [42], is able to undergo LLPS in vitro and in vivo. EMB1579 condensates exhibit liquid-like properties and turn over extremely rapidly within the nucleus. We found that many nuclear proteins crucial for chromosomal function and RNA biology interact with EMB1579 condensates and some of them colocalize with EMB1579 condensates in the nucleus. Loss of function of EMB1579 alters global gene transcription and mRNA splicing, which provides an explanation for why emb1579 mutants exhibit pleiotropic developmental defects. Using floral transition as the representative physiological process, we demonstrate that EMB1579 is involved in regulating the level of H3K27me3 on FLC and the expression of FLC. EMB1579 likely controls the function of CLF-PRC2 via direct interaction with CUL4-DDB1MSI4. We propose that EMB1579 condensates condense important biomolecules in the Arabidopsis nucleus to regulate their functions in controlling key nuclear events, such as gene transcription and mRNA splicing. Our study thus reveals a new mechanism for the regulation of plant growth and development through LLPS of EMB1579.

Results
Loss of function of EMB1579 induces pleiotropic growth and developmental defects in Arabidopsis
EMB1579 initially caught our attention because it encodes a protein that is homologous to the tobacco protein MAP190, which interacts with both actin filaments and microtubules [43]. It was also proposed to be involved in nuclear calcium signaling during the salt response, as it contains an EF-hand motif [44]. To gain insights into the developmental functions of EMB1579, we examined its tissue expression pattern and found that it is widely expressed, especially in highly proliferative tissues, including embryos, the root meristematic region, the basal region of shoots, and the base of the cauline-leaf branch (S1 Fig). To examine the function of EMB1579, we characterized two T-DNA insertion lines that were shown to be knockout alleles (Fig 1A). We observed embryonic developmental defects at different stages, resulting from distorted cell division and cell expansion in emb1579 embryos compared to wild type (WT) (Fig 1B). Specifically, we found that the division plane of cells was mispositioned and cells were swollen in emb1579 mutants (Fig 1B). Consequently, the development of seeds and seedlings was defective in emb1579 mutants (Fig 1C–1E). Consistent with the distorted cell phenotypes in embryos, we found that the cell files were altered in the roots of emb1579 mutants (Fig 1F). Notably, we found that the number of meristematic cells was decreased significantly in emb1579 mutants compared to WT (Fig 1G). We also found that the floral transition was delayed in emb1579 mutant plants (Fig 1H and 1I). Thus, our study suggests that EMB1579 is crucial for Arabidopsis growth and development.
Fig 1. Loss of function of EMB1579 induces pleiotropic growth and developmental defects in Arabidopsis.(A) Structure of the EMB1579 gene and identification of T-DNA insertion mutants of EMB1579. Two T-DNA insertion lines, CS16026 and Salk_007142, were designated as emb1579-1 and emb1579-3, respectively. The positions of the T-DNA insertions are indicated by inverted triangles. Three independent pairs of primers were used to identify truncated EMB1579 transcripts in emb1579-1 and emb1579-3. The positions of the primers are indicated under the gene. The expression of EMB1579 in WT and emb1579 mutants was also confirmed by qRT-PCR analysis with primer pairs EMB1579-qRT-F1/EMB1579-qRT-R2 (S4 Table). Data are presented as mean ± s.e.m, n=3. Numerical data underlying the graph are available in S1 Data. The original pictures are available in S1 Raw Images. (B) Micrographs of embryos at different stages. Embryos at the 8-cell stage, 16-cell stage, and globular stage were revealed by whole-mount clearing methods, and embryos at the triangular stage, heart stage, and torpedo stage were revealed by staining with PI as described previously [45]. In emb1579 mutants, the swollen cells are outlined with green lines, and white arrowheads indicate the formation of abnormal cell plates. Bars=50 μm. (C) Images of Arabidopsis seeds. White arrowheads indicate dry wrinkled seeds. Bar=1 mm. (D) Images of Arabidopsis seedlings. Bar=0.5 cm. (E) Quantification of primary root length of 7-day-old seedlings in WT, emb1579-1, and emb1579-3. Data are presented as mean ± s.e.m. ***P Read More