SANT proteins modulate gene expression by coordinating histone H3KAc and Khib levels and regulate plant heat tolerance.

Histone post-translational modifications (PTMs), such as acetylation and recently identified lysine 2-hydroxyisobutyrylation (Khib), act as active epigenomic marks in plants. SANT domain-containing proteins SANT1, SANT2, SANT3 and SANT4 (SANT1/2/3/4), derived from PIF/Harbinger transposases, form a complex with HISTONE DEACETYLASE 6 (HDA6) to regulate gene expression via histone deacetylation. However, whether SANT1/2/3/4 coordinate different types of PTMs to regulate transcription and mediate responses to specific stresses in plants remains unclear. Here, in addition to modulating histone deacetylation, we found that SANT1/2/3/4 proteins acted like HDA6 or HDA9 in regulating the removal of histone Khib in Arabidopsis (Arabidopsis thaliana). Histone H3 lysine acetylation (H3KAc) and histone Khib were coordinated by SANT1/2/3/4 to regulate gene expression, with H3KAc playing a predominant role and Khib acting complementarily to H3KAc. SANT1/2/3/4 mutation significantly increased the expression of heat-inducible genes with concurrent change of H3KAc levels under normal and heat stress conditions, resulting in enhanced thermotolerance. This study revealed the critical roles of Harbinger transposon-derived SANT domain-containing proteins in transcriptional regulation by coordinating different types of histone PTMs and in the regulation of plant thermotolerance by mediating histone acetylation modification.

The Arabidopsis epigenetic regulator ICU11 as an accessory protein of Polycomb Repressive Complex 2.

Molecular mechanisms enabling the switching and maintenance of epigenetic states are not fully understood. Distinct histone modifications are often associated with ON/OFF epigenetic states, but how these states are stably maintained through DNA replication, yet in certain situations switch from one to another remains unclear. Here, we address this problem through identification of Arabidopsis INCURVATA11 (ICU11) as a Polycomb Repressive Complex 2 accessory protein. ICU11 robustly immunoprecipitated in vivo with PRC2 core components and the accessory proteins, EMBRYONIC FLOWER 1 (EMF1), LIKE HETEROCHROMATIN PROTEIN1 (LHP1), and TELOMERE_REPEAT_BINDING FACTORS (TRBs). ICU11 encodes a 2-oxoglutarate-dependent dioxygenase, an activity associated with histone demethylation in other organisms, and mutant plants show defects in multiple aspects of the Arabidopsis epigenome. To investigate its primary molecular function we identified the Arabidopsis FLOWERING LOCUS C (FLC) as a direct target and found icu11 disrupted the cold-induced, Polycomb-mediated silencing underlying vernalization. icu11 prevented reduction in H3K36me3 levels normally seen during the early cold phase, supporting a role for ICU11 in H3K36me3 demethylation. This was coincident with an attenuation of H3K27me3 at the internal nucleation site in FLC, and reduction in H3K27me3 levels across the body of the gene after plants were returned to the warm. Thus, ICU11 is required for the cold-induced epigenetic switching between the mutually exclusive chromatin states at FLC, from the active H3K36me3 state to the silenced H3K27me3 state. These data support the importance of physical coupling of histone modification activities to promote epigenetic switching between opposing chromatin states.

The domesticated transposase ALP2 mediates formation of a novel Polycomb protein complex by direct interaction with MSI1, a core subunit of Polycomb Repressive Complex 2 (PRC2).

A large fraction of plant genomes is composed of transposable elements (TE), which provide a potential source of novel genes through "domestication"-the process whereby the proteins encoded by TE diverge in sequence, lose their ability to catalyse transposition and instead acquire novel functions for their hosts. In Arabidopsis, ANTAGONIST OF LIKE HETEROCHROMATIN PROTEIN 1 (ALP1) arose by domestication of the nuclease component of Harbinger class TE and acquired a new function as a component of POLYCOMB REPRESSIVE COMPLEX 2 (PRC2), a histone H3K27me3 methyltransferase involved in regulation of host genes and in some cases TE. It was not clear how ALP1 associated with PRC2, nor what the functional consequence was. Here, we identify ALP2 genetically as a suppressor of Polycomb-group (PcG) mutant phenotypes and show that it arose from the second, DNA binding component of Harbinger transposases. Molecular analysis of PcG compromised backgrounds reveals that ALP genes oppose silencing and H3K27me3 deposition at key PcG target genes. Proteomic analysis reveals that ALP1 and ALP2 are components of a variant PRC2 complex that contains the four core components but lacks plant-specific accessory components such as the H3K27me3 reader LIKE HETEROCHROMATION PROTEIN 1 (LHP1). We show that the N-terminus of ALP2 interacts directly with ALP1, whereas the C-terminus of ALP2 interacts with MULTICOPY SUPPRESSOR OF IRA1 (MSI1), a core component of PRC2. Proteomic analysis reveals that in alp2 mutant backgrounds ALP1 protein no longer associates with PRC2, consistent with a role for ALP2 in recruitment of ALP1. We suggest that the propensity of Harbinger TE to insert in gene-rich regions of the genome, together with the modular two component nature of their transposases, has predisposed them for domestication and incorporation into chromatin modifying complexes.

Polycomb proteins control floral determinacy by H3K27me3-mediated repression of pluripotency genes in Arabidopsis thaliana.

Polycomb group (PcG) protein-mediated histone methylation (H3K27me3) controls the correct spatiotemporal expression of numerous developmental regulators in Arabidopsis. Epigenetic silencing of the stem cell factor gene WUSCHEL (WUS) in floral meristems (FMs) depends on H3K27me3 deposition by PcG proteins. However, the role of H3K27me3 in silencing of other meristematic regulator and pluripotency genes during FM determinacy has not yet been studied. To this end, we report the genome-wide dynamics of H3K27me3 levels during FM arrest and the consequences of strongly depleted PcG activity on early flower morphogenesis including enlarged and indeterminate FMs. Strong depletion of H3K27me3 levels results in misexpression of the FM identity gene AGL24, which partially causes floral reversion leading to ap1-like flowers and indeterminate FMs ectopically expressing WUS and SHOOT MERISTEMLESS (STM). Loss of STM can rescue supernumerary floral organs and FM indeterminacy in H3K27me3-deficient flowers, indicating that the hyperactivity of the FMs is at least partially a result of ectopic STM expression. Nonetheless, WUS remained essential for the FM activity. Our results demonstrate that PcG proteins promote FM determinacy at multiple levels of the floral gene regulatory network, silencing initially floral regulators such as AGL24 that promotes FM indeterminacy and, subsequently, meristematic pluripotency genes such as WUS and STM during FM arrest.

A domesticated Harbinger transposase forms a complex with HDA6 and promotes histone H3 deacetylation at genes but not TEs in Arabidopsis.

In eukaryotes, histone acetylation is a major modification on histone N-terminal tails that is tightly connected to transcriptional activation. HDA6 is a histone deacetylase involved in the transcriptional regulation of genes and transposable elements (TEs) in Arabidopsis thaliana. HDA6 has been shown to participate in several complexes in plants, including a conserved SIN3 complex. Here, we uncover a novel protein complex containing HDA6, several Harbinger transposon-derived proteins (HHP1, SANT1, SANT2, SANT3, and SANT4), and MBD domain-containing proteins (MBD1, MBD2, and MBD4). We show that mutations of all four SANT genes in the sant-null mutant cause increased expression of the flowering repressors FLC, MAF4, and MAF5, resulting in a late flowering phenotype. Transcriptome deep sequencing reveals that while the SANT proteins and HDA6 regulate the expression of largely overlapping sets of genes, TE silencing is unaffected in sant-null mutants. Our global histone H3 acetylation profiling shows that SANT proteins and HDA6 modulate gene expression through deacetylation. Collectively, our findings suggest that Harbinger transposon-derived SANT domain-containing proteins are required for histone deacetylation and flowering time control in plants.

Mechanical stress mediated by both endosperm softening and embryo growth underlies endosperm elimination in Arabidopsis seeds.

Seed development in angiosperms demands the tightly coordinated development of three genetically distinct structures. The embryo is surrounded by the endosperm, which is in turn enclosed within the maternally derived seed coat. In Arabidopsis, final seed size is determined by early expansion of the coenocytic endosperm, which then cellularises and subsequently undergoes developmental programmed cell death, breaking down as the embryo grows. Endosperm breakdown requires the endosperm-specific basic helix-loop-helix transcription factor ZHOUPI. However, to date, the mechanism underlying the Arabidopsis endosperm breakdown process has not been elucidated. Here, we provide evidence that ZHOUPI does not induce the developmental programmed cell death of the endosperm directly. Instead ZHOUPI indirectly triggers cell death by regulating the expression of cell wall-modifying enzymes, thus altering the physical properties of the endosperm to condition a mechanical environment permitting the compression of the cellularised endosperm by the developing embryo.

Kicking against the PRCs - A Domesticated Transposase Antagonises Silencing Mediated by Polycomb Group Proteins and Is an Accessory Component of Polycomb Repressive Complex 2.

The Polycomb group (PcG) and trithorax group (trxG) genes play crucial roles in development by regulating expression of homeotic and other genes controlling cell fate. Both groups catalyse modifications of chromatin, particularly histone methylation, leading to epigenetic changes that affect gene activity. The trxG antagonizes the function of PcG genes by activating PcG target genes, and consequently trxG mutants suppress PcG mutant phenotypes. We previously identified the ANTAGONIST OF LIKE HETEROCHROMATIN PROTEIN1 (ALP1) gene as a genetic suppressor of mutants in the Arabidopsis PcG gene LIKE HETEROCHROMATIN PROTEIN1 (LHP1). Here, we show that ALP1 interacts genetically with several other PcG and trxG components and that it antagonizes PcG silencing. Transcriptional profiling reveals that when PcG activity is compromised numerous target genes are hyper-activated in seedlings and that in most cases this requires ALP1. Furthermore, when PcG activity is present ALP1 is needed for full activation of several floral homeotic genes that are repressed by the PcG. Strikingly, ALP1 does not encode a known chromatin protein but rather a protein related to PIF/Harbinger class transposases. Phylogenetic analysis indicates that ALP1 is broadly conserved in land plants and likely lost transposase activity and acquired a novel function during angiosperm evolution. Consistent with this, immunoprecipitation and mass spectrometry (IP-MS) show that ALP1 associates, in vivo, with core components of POLYCOMB REPRESSIVE COMPLEX 2 (PRC2), a widely conserved PcG protein complex which functions as a H3K27me3 histone methyltransferase. Furthermore, in reciprocal pulldowns using the histone methyltransferase CURLY LEAF (CLF), we identify not only ALP1 and the core PRC2 components but also plant-specific accessory components including EMBRYONIC FLOWER 1 (EMF1), a transcriptional repressor previously associated with PRC1-like complexes. Taken together our data suggest that ALP1 inhibits PcG silencing by blocking the interaction of the core PRC2 with accessory components that promote its HMTase activity or its role in inhibiting transcription. ALP1 is the first example of a domesticated transposase acquiring a novel function as a PcG component. The antagonistic interaction of a modified transposase with the PcG machinery is novel and may have arisen as a means for the cognate transposon to evade host surveillance or for the host to exploit features of the transposition machinery beneficial for epigenetic regulation of gene activity.

Polycomb-Group Proteins and FLOWERING LOCUS T Maintain Commitment to Flowering in Arabidopsis thaliana.

The switch from vegetative to reproductive growth is extremely stable even if plants are only transiently exposed to environmental stimuli that trigger flowering. In the photoperiodic pathway, a mobile signal, florigen, encoded by FLOWERING LOCUS T (FT) in Arabidopsis thaliana, induces flowering. Because FT activity in leaves is not maintained after transient photoperiodic induction, the molecular basis for stable floral commitment is unclear. Here, we show that Polycomb-group (Pc-G) proteins, which mediate epigenetic gene regulation, maintain the identity of inflorescence and floral meristems after floral induction. Thus, plants with reduced Pc-G activity show a remarkable increase of cauline leaves under noninductive conditions and floral reversion when shifted from inductive to noninductive conditions. These phenotypes are almost completely suppressed by loss of FLOWERING LOCUS C (FLC) and SHORT VEGETATIVE PHASE, which both delay flowering and promote vegetative shoot identity. Upregulation of FLC in Pc-G mutants leads to a strong decrease of FT expression in inflorescences. We find that this activity of FT is needed to prevent floral reversion. Collectively, our results reveal that floral meristem identity is at least partially maintained by a daylength-independent role of FT whose expression is indirectly sustained by Pc-G activity.

Reduced function of the RNA-binding protein FPA rescues a T-DNA insertion mutant in the Arabidopsis ZHOUPI gene by promoting transcriptional read-through.

T-DNA insertion mutants have been widely used to investigate plant gene functions. Unexpectedly, in several reported cases, the phenotype of T-DNA insertion mutations can be suppressed because of trans T-DNA interactions associated with epigenetic modification, which indicates that caution is needed when T-DNA mutants are used. In the present study, we characterized a novel process suppressing a T-DNA mutation. The spz2 (suppressor of zou 2) mutant was isolated as a suppressor of the phenotype of the zou-4 mutant caused by a T-DNA insertion in the first intron. The spz2 mutation partially recovered the native ZOU gene expression in the zou-4 background, but not in two other zou alleles, zou-2 and zou-3, with T-DNAs inserted in the exon and intron, respectively. The suppressed phenotype was inherited in a Mendelian fashion and is not associated with epigenetic modification. The recovery of the native ZOU gene expression in the spz2 zou-4 double mutant is caused by transcriptional read-through of the intronic T-DNA as a result of decreased proximal polyadenylation. SPZ2 encodes an RNA-binding protein, FPA, which is known to regulate polyadenylation site selection. This is the first example of FPA rescuing a T-DNA insertion mutation by affecting the polyadenylation site selection.

ZHOUPI controls embryonic cuticle formation via a signalling pathway involving the subtilisin protease ABNORMAL LEAF-SHAPE1 and the receptor kinases GASSHO1 and GASSHO2.

Seed production in angiosperms requires tight coordination of the development of the embryo and the endosperm. The endosperm-specific transcription factor ZHOUPI has previously been shown to play a key role in this process, by regulating both endosperm breakdown and the formation of the embryonic cuticle. To what extent these processes are functionally linked is, however, unclear. In order to address this issue we have concentrated on the subtilisin-like serine protease encoding gene ABNORMAL LEAF-SHAPE1. Expression of ABNORMAL LEAF-SHAPE1 is endosperm specific, and dramatically decreased in zhoupi mutants. We show that, although ABNORMAL LEAF-SHAPE1 is required for normal embryonic cuticle formation, it plays no role in regulating endosperm breakdown. Furthermore, we show that re-introducing ABNORMAL LEAF-SHAPE1 expression in the endosperm of zhoupi mutants partially rescues embryonic cuticle formation without rescuing their persistent endosperm phenotype. Thus, we conclude that ALE1 can normalize cuticle formation in the absence of endosperm breakdown, and that ZHOUPI thus controls two genetically separable developmental processes. Finally, our genetic study shows that ZHOUPI and ABNORMAL LEAF-SHAPE1 promotes formation of embryonic cuticle via a pathway involving embryonically expressed receptor kinases GASSHO1 and GASSHO2. We therefore provide a molecular framework of inter-tissue communication for embryo-specific cuticle formation during embryogenesis.

Arabidopsis guard cell integrity involves the epigenetic stabilization of the FLP and FAMA transcription factor genes.

Arabidopsis guard cell (GC) fate is conferred via a transient pulse of expression of FAMA that encodes a bHLH transcription factor. Stomata often function for years, suggesting that the FAMA expression window stabilizes long-term GC identity or that additional factors operate. Transgenic lines harboring a copy of a FAMA transgene were found to induce the fate resetting of mature GCs to that of lineage-specific stem cells causing new stomata to arise within shells of the old, a Stoma-in-Stoma (SIS) phenotype. These lines disrupt the normal trimethylation on lysine 27 of histone3 (H3K27me3) on stomatal stem cell genes, a phenotype rescued by constitutive expression of the Polycomb Group (PcG) gene CURLY LEAF. Thus the stability of stomatal fate is enforced by a PcG-mediated reduction in the transcriptional accessibility of stem cell genes and by the endogenous FAMA gene itself. Moreover, a transgenic FOUR LIPS gene, which encodes a MYB protein that is not required for GC fate, also induces a SIS phenotype and disrupts H3K27 trimethylation. Thus FLP might indirectly enforce GC fate as well.

Tissue-specific expression of FLOWERING LOCUS T in Arabidopsis is maintained independently of polycomb group protein repression.

The Polycomb Group (PcG) pathway represses transcription through a mechanism conserved among plants and animals. PcG-mediated repression can determine spatial territories of gene expression, but it remains unclear whether PcG-mediated repression is a regulatory requirement for all targets. Here, we show the role of PcG proteins in the spatial regulation of FLOWERING LOCUS T (FT), a main activator of flowering in Arabidopsis thaliana exclusively expressed in the vasculature. Strikingly, the loss of PcG repression causes down-regulation of FT. In addition, our results show how the effect of PcG-mediated regulation differs for target genes and that, for FT expression, it relies primarily on tissue differentiation.

AGAMOUS terminates floral stem cell maintenance in Arabidopsis by directly repressing WUSCHEL through recruitment of Polycomb Group proteins.

Floral stem cells produce a defined number of floral organs before ceasing to be maintained as stem cells. Therefore, floral stem cells offer an ideal model to study the temporal control of stem cell maintenance within a developmental context. AGAMOUS (AG), a MADS domain transcription factor essential for the termination of floral stem cell fate, has long been thought to repress the stem cell maintenance gene WUSCHEL (WUS) indirectly. Here, we uncover a role of Polycomb Group (PcG) genes in the temporally precise repression of WUS expression and termination of floral stem cell fate. We show that AG directly represses WUS expression by binding to the WUS locus and recruiting, directly or indirectly, PcG that methylates histone H3 Lys-27 at WUS. We also show that PcG acts downstream of AG and probably in parallel with the known AG target KNUCKLES to terminate floral stem cell fate. Our studies identify core components of the network governing the temporal program of floral stem cells.

Polycomb repressive complex 2 controls the embryo-to-seedling phase transition.

Polycomb repressive complex 2 (PRC2) is a key regulator of epigenetic states catalyzing histone H3 lysine 27 trimethylation (H3K27me3), a repressive chromatin mark. PRC2 composition is conserved from humans to plants, but the function of PRC2 during the early stage of plant life is unclear beyond the fact that it is required for the development of endosperm, a nutritive tissue that supports embryo growth. Circumventing the requirement of PRC2 in endosperm allowed us to generate viable homozygous null mutants for FERTILIZATION INDEPENDENT ENDOSPERM (FIE), which is the single Arabidopsis homolog of Extra Sex Combs, an indispensable component of Drosophila and mammalian PRC2. Here we show that H3K27me3 deposition is abolished genome-wide in fie mutants demonstrating the essential function of PRC2 in placing this mark in plants as in animals. In contrast to animals, we find that PRC2 function is not required for initial body plan formation in Arabidopsis. Rather, our results show that fie mutant seeds exhibit enhanced dormancy and germination defects, indicating a deficiency in terminating the embryonic phase. After germination, fie mutant seedlings switch to generative development that is not sustained, giving rise to neoplastic, callus-like structures. Further genome-wide studies showed that only a fraction of PRC2 targets are transcriptionally activated in fie seedlings and that this activation is accompanied in only a few cases with deposition of H3K4me3, a mark associated with gene activity and considered to act antagonistically to H3K27me3. Up-regulated PRC2 target genes were found to act at different hierarchical levels from transcriptional master regulators to a wide range of downstream targets. Collectively, our findings demonstrate that PRC2-mediated regulation represents a robust system controlling developmental phase transitions, not only from vegetative phase to flowering but also especially from embryonic phase to the seedling stage.

Convergent evolution of water-conducting cells in Marchantia recruited the ZHOUPI gene promoting cell wall reinforcement and programmed cell death.

A key adaptation of plants to life on land is the formation of water-conducting cells (WCCs) for efficient long-distance water transport. Based on morphological analyses it is thought that WCCs have evolved independently on multiple occasions. For example, WCCs have been lost in all but a few lineages of bryophytes but, strikingly, within the liverworts a derived group, the complex thalloids, has evolved a novel externalized water-conducting tissue composed of reinforced, hollow cells termed pegged rhizoids. Here, we show that pegged rhizoid differentiation in Marchantia polymorpha is controlled by orthologs of the ZHOUPI and ICE bHLH transcription factors required for endosperm cell death in Arabidopsis seeds. By contrast, pegged rhizoid development was not affected by disruption of MpNAC5, the Marchantia ortholog of the VND genes that control WCC formation in flowering plants. We characterize the rapid, genetically controlled programmed cell death process that pegged rhizoids undergo to terminate cellular differentiation and identify a corresponding upregulation of conserved putative plant cell death effector genes. Lastly, we show that ectopic expression of MpZOU1 increases production of pegged rhizoids and enhances drought tolerance. Our results support that pegged rhizoids evolved independently of other WCCs. We suggest that elements of the genetic control of developmental cell death are conserved throughout land plants and that the ZHOUPI/ICE regulatory module has been independently recruited to promote cell wall modification and programmed cell death in liverwort rhizoids and in the endosperm of flowering plant seed.

The CURLY LEAF interacting protein BLISTER controls expression of polycomb-group target genes and cellular differentiation of Arabidopsis thaliana.

Polycomb-group (Pc-G) proteins are important regulators of many developmental processes in plants and animals and repress gene expression by imparting histone H3 lysine 27 trimethylation (H3K27me3). Here, we present the identification of the novel, plant-specific Arabidopsis thaliana protein BLISTER (BLI), which interacts with the Pc-G histone methyltransferase CURLY LEAF (CLF). We map the interaction of BLI with CLF to a predicted coiled-coil domain in BLI that shares similarity with STRUCTURAL MAINTENANCE OF CHROMOSOMES proteins. BLI colocalizes with CLF in the nucleus, shows an overlapping expression pattern with CLF throughout plant development that is strongest in dividing cells, and represses a subset of Pc-G target genes. Loss of BLI results in a pleiotropic developmental mutant phenotype, indicating that BLI prevents premature differentiation. Furthermore, bli mutants exhibit severe epidermal defects, including loss of cell adhesion, outgrowth of cells, and increased cotyledon cell size. As these phenotypes have not been observed in Pc-G mutants, we propose that BLI has functions related to Pc-G proteins but can also act independently in Arabidopsis development.

Stomatal regulators are co-opted for seta development in the astomatous liverwort Marchantia polymorpha.

The evolution of special types of cells requires the acquisition of new gene regulatory networks controlled by transcription factors (TFs). In stomatous plants, a TF module formed by subfamilies Ia and IIIb basic helix-loop-helix TFs (Ia-IIIb bHLH) regulates stomatal formation; however, how this module evolved during land plant diversification remains unclear. Here we show that, in the astomatous liverwort Marchantia polymorpha, a Ia-IIIb bHLH module regulates the development of a unique sporophyte tissue, the seta, which is found in mosses and liverworts. The sole Ia bHLH gene, MpSETA, and a IIIb bHLH gene, MpICE2, regulate the cell division and/or differentiation of seta lineage cells. MpSETA can partially replace the stomatal function of Ia bHLH TFs in Arabidopsis thaliana, suggesting that a common regulatory mechanism underlies setal and stomatal formation. Our findings reveal the co-option of a Ia-IIIb bHLH TF module for regulating cell fate determination and/or cell division of distinct types of cells during land plant evolution.

Polycomb proteins regulate the quantitative induction of VERNALIZATION INSENSITIVE 3 in response to low temperatures.

Vernalization, the promotion of flowering in response to low temperatures, is one of the best characterized examples of epigenetic regulation in plants. The promotion of flowering is proportional to the duration of the cold period, but the mechanism by which plants measure time at low temperatures has been a long-standing mystery. We show that the quantitative induction of the first gene in the Arabidopsis vernalization pathway, VERNALIZATION INSENSITIVE 3 (VIN3), is regulated by the components of Polycomb Response Complex 2, which trimethylates histone H3 lysine 27 (H3K27me3). In differentiated animal cells, H3K27me3 is mostly associated with long-term gene repression, whereas, in pluripotent embyonic stem cells, many cell lineage-specific genes are inactive but exist in bivalent chromatin that carries both active (H3K4me3) and repressive (H3K27me3) marks on the same molecule. During differentiation, bivalent domains are generally resolved to an active or silent state. We found that H3K27me3 maintains VIN3 in a repressed state prior to cold exposure; this mark is not removed during VIN3 induction. Instead, active VIN3 is associated with bivalently marked chromatin. The continued presence of H3K27me3 ensures that induction of VIN3 is proportional to the duration of the cold, and that plants require prolonged cold to promote the transition to flowering. The observation that Polycomb proteins control VIN3 activity defines a new role for Polycomb proteins in regulating the rate of gene induction.