Questa Julia (Orcid ID: 0000-0002-0462-8423)


Mechanisms of epigenetic regulation of transcription by lncRNAs in plants

Benjamin J.M. Tremblay, Julia I. Qüesta

Centre for Research in Agricultural Genomics, Barcelona, Spain.


Abstract

Long noncoding RNAs (lncRNAs) are a ubiquitous feature of eukaryotic genomes, and in
recent decades have been shown to be highly abundant and varied. Many prominent
examples have been described as having essential roles in regulating the expression of
genes in different developmental and environmental contexts. As a result, much work has
been done on elucidating the mechanisms by which they modulate the expression of
protein coding genes. In this review, we focus on those which have been characterized
in plants. We specifically examine common epigenetic mechanisms which regulate gene
expression at the level of transcription. In this regard, we focus on the lncRNAs in plants
that have primarily been associated with controlling the chromatin environment of genes
at the level of modifications, RNA POLYMERASE II (RNAPII) processivity and efficiency
of transcription, and mediating the formation of transcriptionally activating and repressive
chromatin loops. We discuss open questions in plant lncRNA epigenetic regulation and
opportunities for future study of functionally significant lncRNAs with yet-unknown
epigenetic mechanisms.

Introduction

Long noncoding RNAs (lncRNAs) are a class of RNAs longer than 200 nucleotides
generally lacking in coding potential. LncRNAs are not universally noncoding however,
as a small number may produce peptides or have dual functions as lncRNA and peptide
(1-5). LncRNAs can share many structural similarities with mRNAs, including being
transcribed by RNAPII and containing spliced introns, a 5’ cap, and a poly-A tail. LncRNAs
are a common genetic feature of eukaryotes, for example numbering in the thousands in
yeast (6) and the tens of thousands in human and mouse genomes (7). Efforts at
identifying lncRNAs genome-wide in plants suggest they are similarly widespread (8, 9).
LncRNAs are classified based on their relative position to nearby protein coding genes.
Further classifying them based on their functional potential remains a challenging task,
and thus far only a small subset has been experimentally determined to have regulatory
importance (10). Already in plants (and primarily in the model system Arabidopsis thaliana
(Arabidopsis)) lncRNAs have been implicated in having contributing roles to stress
responses (11-15), nutrient homeostasis (16-19), and developmental processes (20-46).

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Detection and characterization of lncRNAs still remains a challenge in many species
including plants, partly due to their low expression levels compared to protein coding
genes. Given their regulatory function, lncRNA molecules may be subject to fast turnover.
As a result, lncRNAs may escape detection by standard steady-state RNA sequencing
(RNA-seq) methods, requiring more sophisticated nascent RNA profiling methods to
achieve de novo annotation of novel long non-coding transcripts. In addition, lncRNAs
are remarkably cell- and tissue-specific (47) and thus significantly diluted when sampling
organs or whole individuals, a common practice in plant research. With regards to
subcellular localization, as shown in animals lncRNAs tend to be retained in the nuclear
fraction (48, 49), however they can also be exported to the cytoplasm and other
organelles (50). Association of lncRNAs to ribosomes has been observed in Arabidopsis
(51), although their potential function in regulating translation requires further exploration
in plants.

Unlike mRNA genes, which are under a strong selective pressure to maintain their coding
reading frames, lncRNA sequences are generally not conserved across different species
(52). lncRNAs do not seem to depend on their sequence to perform their function, but
rather conservation of their secondary structure or position in the genome may prevail for
propagation of their biological roles as has been demonstrated for the well described
Arabidopsis COLD INDUCED LONG ANTISENSE INTRAGENIC RNA (COOLAIR) (53).
The systematic evaluation of secondary and higher-level structures of non-coding RNAs
remains a promising field to provide answers on lncRNA function. In vivo lncRNA
structure dynamics may be a key factor contributing to the formation of biomolecular
condensates. In recent years, the formation of membraneless subcellular compartments
with distinct biological roles have gained a lot of attention. Many nuclear and cytoplasmic
condensates are rich in RNA and RNA-binding proteins (54). Some examples are nucleoli
and Cajal bodies in the nucleus, as well as stress granules in the cytoplasm. Recent work
depicts lncRNA molecules as facilitators of phase separation and formation of biologically
relevant RNA-protein aggregates across cell compartments and species (55), a feature
likely explaining functionality of lncRNAs at very low, substoichiometric expression levels
in cellular environments (56, 57).

Epigenetic mechanisms are an integral part of transcriptional regulation in eukaryotes.
Chromatin structure and dynamics determine the accessibility of gene regulatory regions
for the transcriptional machinery, fine tuning gene expression in responses to stress and
development (58). Epigenetic regulation relies on a plethora of proteins and complexes
that function as readers, writers, and erasers of different chromatin marks (59), such as
posttranslational modifications of histones and DNA methylation. Furthermore, the
expanding field of epitranscriptomics (60) has added RNA editing and non-coding RNA
regulation in the list of epigenetic regulators.

Increasing evidence from different eukaryotic systems demonstrate the ability of lncRNAs
to act as epigenetic modulators. Seminal work on mammalian Xist has provided an
excellent example of the role of a lncRNA in developmentally regulated epigenetic
silencing. Xist lncRNA spreads over the entire length of the X chromosome from which it
is transcribed, recruiting chromatin modifiers to induce epigenetic silencing of X-linked
genes in cis (61). One remarkable feature of Xist is its ability to direct major 3D
reorganization of the genome. Over the last decade, multiple efforts to understand the
biological function of plant lncRNAs are shedding light into the role of these non-coding
molecules in the regulation of epigenetic processes. Recent reviews have covered the
challenges of lncRNA classification, the computational tools to detect them, as well as
their roles in plant development and stress responses (62-67). In the current work, we
plan to highlight examples of plant lncRNAs with demonstrated impact in epigenetic
regulation (Table 1), such as mediating the deposition of chromatin marks, modulation of
RNAPII dynamics and facilitating the formation and dissociation of chromatin loops.

Influence on histone modifications

LncRNAs have been proposed to function as scaffolds facilitating recruitment of
chromatin modifiers. In the case of lncRNAs overlapping a coding gene (intragenic,
intronic and antisense lncRNAs) that remain locally associated to the site of transcription,
this “recruiter” function of the lncRNA could affect the local chromatin status either
promoting or inhibiting transcription at the locus. Several examples of lncRNAs inducing
epigenetic silencing by bringing Polycomb Repressive Complexes (PRC) to chromatin
have been reported in eukaryotes, including plant species (68). Originally characterized
in the fly Drosophila melanogaster and later in multiple eukaryotic species including
plants, PRC1 and PRC2 complexes carry out silencing of developmental gene programs
in a tissue specific and temporally regulated manner. While PRC1 catalyzes mono
ubiquitination of histone H2A (H2Aub), PRC2 mediates tri-methylation of the lysine 27 on
histone H3 (H3K27me3). These roles can also be overlapping, as for example LHP1
promotes both the spread PRC1 and PRC2 activity in Arabidopsis (69). Deposition of
both PRC1 and PRC2 repressive marks has been associated with chromatin compaction
and long-term epigenetic silencing (70). Despite their high spatio-temporal target
selectivity, PRC expression is ubiquitous, thus relying on “recruiters” for target chromatin
binding and gene silencing. The ability of sequence-specific DNA-binding proteins as well
as of lncRNAs to guide PRC to chromatin have been documented in several species. In
mammals, lncRNAs such as Xist, HOTAIR, Braveheart and Kcnq1ot1 were shown to
recruit PRC2 to chromatin to regulate developmental processes (reviewed in (71)). In line
with this, the first example of PRC2 recruitment by a lncRNA in plants was Arabidopsis
COLDAIR (28). However, work over the last decade has demonstrated that PRC2
complex components may bind promiscuously to RNA molecules, challenging the
“specific recruiter” model, and suggesting that lncRNA-PRC2 interactions would not be
as widespread as originally expected. In fact, current models of lncRNA-PRC interactions
propose that lncRNAs modulate PRC complex function rather than its recruitment (72).

In Arabidopsis, recruitment of the PRC2 H3K27me3 methyltransferase CURLY LEAF
(CLF) by lncRNAs have been proposed for different developmental processes, including
the floral transition and flower development (28, 30, 73). This is reminiscent of the
recruitment of the mammalian H3K27me3 methyltransferase EZH2 by Xist (74). To
promote spring flowering in Arabidopsis, the master floral repressor gene FLOWERING
LOCUS C (FLC) needs to be stably repressed during winter. The acceleration of flowering
by winter cold temperatures is known as vernalization, which is key to Brassicaceae such
as Arabidopsis, as well as for winter cereals. A vernalization specific Plant Homeodomain
(PHD) PRC2 (PHD-PRC2) complex assembles at the 5´ end of FLC gene during winter
to deposit H3K27me3 for long-term gene silencing (75). A series of lncRNA transcripts
are upregulated from the FLC locus upon exposure to cold, which coincides with
transcriptional shutdown of FLC gene: the antisense lncRNA COOLAIR (22, 25, 32, 34,
37, 38, 45) and two sense lncRNAs, COLD-ASSISTED INTRONIC NONCODING RNA
(COLDAIR) (28) and COLDWRAP (30), transcribed from the FLC intron 1 and promoter
region, respectively. Both sense lncRNAs have been linked to recruitment of CLF to FLC
during vernalization thus promoting accumulation of H3K27me3 (28, 30). In contrast,
COOLAIR antisense promotes FLC transcriptional downregulation in response to cold but
seems not to be required for guiding PHD-PRC2 to FLC chromatin.

Parallel and synergistic mechanisms have been proposed for COOLAIR transcriptional
shutdown of sense FLC. First, transcription from FLC sense and antisense strands is
mutually exclusive, suggesting that the act of COOLAIR transcription prevents activation
of FLC sense promoter (34). Additionally, COOLAIR coordinates the switch from active
(H3K36me3) to inactive (H3K27me3) chromatin states possibly through the stabilization
of the H3K36me3 demethylation activity (22). More recent work has shown that COOLAIR
promotes cold induction of nuclear condensates that contain the FLC transcriptional
activator FRIGIDA (FRI) (76). Thus, sequestering away the FRI activator from the FLC
locus during cold appears as an interesting mechanism for COOLAIR-induced FLC
repression. In a cold-independent context, FLC transcription is quantitatively regulated by
the co-transcriptional processing of COOLAIR antisense transcript which is linked to
chromatin regulation. The proximal polyadenylation of COOLAIR results in a repressive
chromatin environment that reduces FLC transcriptional initiation and elongation rates
(77). This process requires the action of multiple components including the RNA-binding
protein FCA and the H3K4 demethylase LSD1 FLOWERING LOCUS D (FLD) (25, 78).
Interestingly, COOLAIR 3′-end processing localize to nuclear bodies (79) suggesting that
specialized subnuclear compartments may provide a platform for joint co-transcriptional
RNA 3′ processing and histone modification mechanisms.

One additional lncRNA that has been shown to interact with CLF to regulate the plant
reproductive transition is the sense intronic lncRNA AG-incRNA4 transcribed from the
second intron of the AGAMOUS (AG) gene (73). AG is a MADS BOX transcription factor
required for specification of stamens and carpels in Arabidopsis flowers. Given its unique
function in development, AG expression is highly tissue specific and restricted to
reproductive organs. In this context, AG-incRNA4 lncRNA tightly regulates AG
expression, securing repression of this floral gene in vegetative tissues.

Apart from the control of master developmental genes, PRC2 has been linked to the
regulation of gene clusters in plants (80). In Arabidopsis, biosynthesis of marneral
triterpene is carried out by enzymes encoded by genes organized in a small cluster (81).
In aerial tissues, marneral cluster is immersed in a repressive chromatin environment
covered by H3K27me3 and the PRC2-accessory protein LHP1. The phytohormone
Abscisic Acid (ABA) triggers epigenetic reprogramming of the marneral cluster resulting
in transcriptional activation of all loci in the cluster. ABA-induced local reduction of
H3K27me3 accumulation and binding of LIKE HETEROCHROMATIN PROTEIN 1
(LHP1) protein results in transcriptional activation of the genes in the marneral locus, a
process that depends on the intergenic lncRNA MARneral Silencing (MARS) that maps
the marneral cluster (35). Apart from its very well documented function as “reader” of
H3K27me3 acting downstream from PRC2 (82), LHP1 associates with RNA molecules in
vitro through its RNA-binding hinge domain, a region that is also important in vivo to
repress Polycomb target genes (83). In line with this, MARS lncRNA has been shown to
physically interact with LHP1 to modulate chromatin conformation of the marneral cluster
(35). Interestingly, the analysis of MARS-LHP1 interaction has revealed a dosage-
dependent mechanism, with the levels of MARS accumulation affecting LHP1 association
to the marneral locus. While basal levels of MARS help recruitment of LHP1, low or high
levels of MARS transcripts may titrate away LHP1 (35). Unlike previously expected,
lncRNA activity is not limited to guiding silencing complexes to target regions but likely
comprises a much more complex and dynamic mechanism of transient stabilization of
chromatin modifiers over a specific site that depends on the local concentration of lncRNA
molecules.

Regarding recruitment of chromatin activation complexes, the MADS AFFECTING
FLOWERING 4 (MAF4) ANTISENSE (MAS) lncRNA has been shown to bind and recruit
to chromatin the WD REPEAT DOMAIN 5 (WDR5) component of the Arabidopsis
COMPASS-like complex (44). Both in animals and plants the RNA-binding protein WDR5
promotes H3K4me3 activity. Remarkably, mammalian WDR5 homolog interacts with
lncRNAs including HOTTIP, NeST and Evx1 (84-86). In Arabidopsis, MAS lncRNA
localize to the nucleus and is produced in antisense position to the FLC homolog MAF4.
Similar to antisense COOLAIR mapping to the FLC locus, MAS expression peaks in
increasing weeks of winter cold. A key difference to COOLAIR is that MAS induces
concordant activation of MAF4 during cold. MAS remains attached to MAF4 chromatin
positively influencing transcriptional dynamics of MAF4 locus likely by bringing the
H3K4me3 activity associated to WDR5 (Figure 1A) (44). In rice, the ncRNA LRK
ANTISENSE INTERGENIC RNA (LAIR) not only binds and recruits to chromatin the rice
homolog OsWRD5, but also interacts directly with MALES ABSENT ON THE FIRST
(OsMOF) protein, which is involved in H4K16 acetylation (40). The antisense lncRNA
LAIR is relatively long (1.8 kb, 5´-capped and polyadenylated transcript with 6 exons) and
maps at the 5´end of the LRK gene cluster in rice chromosome 2. Interestingly, the first
gene in the cluster (LRK1) is fully immersed in LAIR intron 1. Overexpression of LAIR
leads to transcriptional activation of LRK1 and LRK4 possibly through recruitment of
OsMOF and OsWDR5. Thus, LAIR provides another example of an antisense lncRNA
promoting a permissive chromatin environment thereby activating transcription of its
sense coding gene. Moreover, LAIR represents another lncRNA capable of coordinating
gene expression within a cluster.

In summary, plant lncRNAs are versatile molecules with the ability to recruit and induce
local stabilization of different chromatin modifiers resulting in either gene activation or
repression. What features of the lncRNAs at the sequence or structural level determine
either function remains a very exciting and open question.

Modulation of RNAPII transcriptional activity

Besides modulation of the activity of chromatin modifiers, increasing evidence suggests
that the act of lncRNA transcription per ser may exert a regulatory function directly
impacting RNAPII activity. RNAPII transcription in eukaryotes proceeds through distinct
stages (87-89). The first of these, initiation, involves recognition and opening of promoters
by the preinitiation complex (PIC). This is followed by phosphorylation of the Ser5 residue
of the carboxy-terminal domain (CTD) of RNAPII by the TFIIH subunit of the PIC. To
promote transcriptional elongation, P-TEFb phosphorylates the Ser2 residue of the
RNAPII CTD, as well as other factors involved in maintaining RNAPII promoter-proximal
pausing. Finally, termination occurs either by its eviction by XRN2 after cleavage of the
nascent transcript, or after a conformational change in RNAPII induced by the poly-A
signal at the end of the gene. Various lncRNAs in non-plant models have been shown to
regulate different aspects of this process, such as by suppressing transcription initiation
of genes (90, 91) or interfering with elongation (92, 93).

Transcriptional interference is a widely prevalent biological phenomenon whereby active
transcription from one promoter can suppress in cis the transcription from a neighboring
promoter. Efficient termination of RNAPII transcription of an upstream gene is one of the
key processes to avoid transcription read-through, and hence transcriptional interference
of a downstream locus (94). Intergenic lncRNAs can be a source of transcriptional
interference when located upstream of a coding gene (95). An example of such a lncRNA
in plants has yet been described, though examples of transcription interference derived
from T-DNA insertions have been observed. In the last decades, Arabidopsis reverse
genetic analyses have heavily relied on T-DNA insertion mutants. While T-DNA insertions
over gene bodies often result in reduction of gene expression, promoter T-DNA insertions
can either ectopically induce or disrupt expression of coding genes. One way of gene
inactivation by T-DNA insertions is transcriptional interference: aberrant transcripts
generated from T-DNA insertions upstream of the Arabidopsis RFD1 gene (T-DNA-RFD
lncRNA) and QUA1 gene (T-DNA-QUA1 lncRNA) inhibit transcription initiation from the
RFD1 and QUA1 TSSs, respectively, likely through transcriptional interference (96, 97).
These T-DNA driven lncRNAs are chimeras of T-DNA plus promoter (intergenic)
sequences followed by the downstream mRNA. Interestingly, in the case of RFD1 gene,
T-DNA insertions located over the 5´UTR do not inhibit transcription. Instead, transcription
from the upstream T-DNA inactivates the RFD1 TSS (96). This mechanism of T-DNA-
lncRNA-dependent downstream promoter repression may be driven by co-transcriptional
histone modifications (97). Each RNAPII stage is characterized by the co-transcriptional
recruitment of factors involved in nascent RNA processing and specific chromatin
modifications (98). For example, methylation of H3K4 exhibits a distinct profile that
correlates with the different phases of transcription, with enrichment of H3K4me3 at the
TSS (initiation), H3K4me2 over the gene body (elongation) and H3K4me1 towards the
3´end (termination). The proposed mechanism for silencing of QUA1 and RFD1 by
upstream lncRNAs is the switch from promoter- to elongation-type co-transcriptional
histone marks (97). RNAPII initiates transcription from the upstream T-DNA insertions,
elongating through the promoter and coding sequences of QUA1 and RFD1, thus
suppressing the TSSs of both genes at least partly via the activity of the FACT complex.

Convergent antisense transcription can likewise interfere with RNAPII transcription of
genes. A prominent case is the light-dependent transcription of the antisense lncRNA qrf
leads to early transcriptional termination of the clock gene rfq in the fungus Neurospora
(93). Specific examples of lncRNAs involved in such processes have also been
characterized in Arabidopsis, including the lncRNA SVALKA (SVK). Exposure to cold
triggers rapid activation of the C-REPEAT/DEHYDRATION-RESPONSIVE ELEMENT
BINDING FACTORS (CBFs) transcription factors which promote tolerance to cold
temperatures by activating downstream cold-responsive genes (99). SVK is a cold-
inducible intergenic lncRNA located downstream and head-to-head to the CBF1 gene
(Figure 1B) (11). CBF transcripts are only transiently activated, reaching high levels of
expression a few hours after start of cold treatment and then returning to basal, almost
undetectable levels. This time-dependent regulation of CBF expression is partly due to
the production of lncRNAs. Transcriptional read-through of SVK results in the expression
of a cryptic antisense CBF1 lncRNA (asCBF1) from the 3´UTR of CBF1. asCBF1
transcription results in RNAPII collision thereby repressing the expression of full-length
CBF1. Thus, production of two antisense lncRNAs during cold limit the production of
maximal CBF1 mRNA levels by an RNAPII collision mechanism over the CBF1 locus.

Several other plant antisense lncRNAs with functional importance (but for which their
mechanisms remain to be fully understood) share similar patterns of anticorrelation with
their sense genes which may in part be as a result of RNAPII interference. Examples
include, CDF5 LONG NONCODING RNA (FLORE), which is antisense to the circadian
clock gene CYCLING DOF FACTOR 5 (CDF5) and regulates its phasic expression
pattern (27); the seed-maturation DELAY OF GERMINATION 1 (DOG1) gene antisense
1GOD, which represses DOG1 expression during seed maturation (26, 43); a convergent
isoform of COOLAIR, transcribed from the first intron of FLC (45); and SUF, an antisense
lncRNA expressed in male tissues to suppress the female sexual differentiation regulator
MpFGMYB in Marchantia polymorpha (29). Additional work is needed to clarify their
modes of regulation, as well as to characterize the large numbers of discovered antisense
lncRNAs in plants (44, 100)

Mediating chromosomal looping dynamics

3D genome organization plays a significant role in regulating gene expression. In
metazoan genomes, chromosomal loci tens of kilobases apart come into contact and form
topologically associated domains (TADs) and constrain epigenetic marks to specific parts
of the genome (101). TAD looping (and additionally sub-TAD chromatin looping) events
driven by pioneer transcription factors also bring distal regulatory elements near
promoters and subsequently activate gene expression (102, 103). Seminal studies in
human disease also demonstrated the importance of lncRNAs in this area, revealing them
to be essential for the formation and maintenance of specific chromatin loops involved in
activating gene expression (104, 105). In plants, HiC analyses of crop species including
maize, tomato, sorghum, foxtail millet, rice and wheat confirmed the presence of TAD-
like features within their genomes, though sub-TAD chromatin loops could only be
confirmed in the plants with the largest genomes: maize, rice, tomato and wheat (106-
109). Currently little is known about the possible role of lncRNAs in regulating chromatin
loops in these plants, in part owing to the limited availability of molecular techniques.

Comparatively, the plant Arabidopsis has been much more extensively studied. Likely
owing to its smaller genome, early studies utilizing 4C and HiC techniques revealed that
the bulk of chromatin interactions occur primarily at pericentromeric heterochromatin and
telomeres and failed to identify clear TAD-like structures (110-112). Regardless,
chromatin loops were found to be abundant between heterochromatin islands containing
H3K27me3 or H3K9me2 within euchromatin, which was supported by subsequent HiC
experiments in Arabidopsis (113-115). H3K27me3-HiChIP experiments later revealed in
greater resolution how this mark plays a significant role in mediating chromatin loop
formation and primarily producing tissue-specific Polycomb-dependent compartments
leading to gene repression (116). This class of chromatin interaction was also observed
in maize, rice, and wheat (106, 108, 117). In parallel with this research, several studies
were published which revealed the role of lncRNAs in mediating the formation and
maintenance of individual instances of these H3K27me3-dependent loops in Arabidopsis.

The first of these was the lncRNA APOLO. After observing that it was coregulated with
the approximately 5 kilobase upstream auxin transport regulator gene PINOID (PID)
during auxin treatment, 3C experiments revealed the existence of a chromatin loop
between the APOLO locus and the 5’ region of PID (20). These contact regions were
marked with H3K27me3 and DNA methylation, the former of which decreased alongside
contact frequency in lhp1 mutants. ChIRP-qPCR experiments revealed that APOLO was
bound to both contact sites, and RIP-qPCR experiments further showed it interacted with
LHP1. Studying loop dynamics during auxin treatment and in various mutant and
transgenic lines revealed a mechanism whereby an initial repressive loop was quickly
relaxed via loss of DNA methylation and LHP1 binding in a RNAPII-transcribed APOLO-
dependent manner, leading to elevated RNAPII levels at the PID locus and increased
transcription, followed by APOLO-dependent closing of the loop once again at 24h by an
increase in DNA methylation (via RNA POLYMERASE V (RNAPV)-transcribed APOLO)
and LHP1 binding (Figure 1C) (20). A follow-up study provided further details on the
mechanism by showing that an essential TTCTTC element within the APOLO sequence
allowed for the formation of R-loops at contact sites via complementary binding to
GAAGAA sequences in the genome, and furthermore revealed using overexpression
lines of APOLO that increases in APOLO levels led to a decrease in LHP1 binding by
decoying it away from its targets (118). In addition, the authors found several additional
auxin-related putative target genes and confirmed this for WAVY ROOT GROWTH
(WAG2) and AZA-GUANINE RESISTANT 2 (AZG2), suggesting a wider role for APOLO
in regulating auxin response (118). This role was further expanded to root hair elongation,
as some of its putative targets included root hair development related genes (118).
Additional studies confirmed that two such genes had similar APOLO target dynamics
(this time in cold-treated plants): ROOT HAIR DEFECTIVE 6 (RHD6) and EXTENSIN 3
(EXT3), though now it involved an interaction with the transcription factor WRKY42,
suggesting these two may be working together alongside the target transcription factor
RHD6 to regulate many root hair elongation processes (119, 120). APOLO functional
characterization was also extended to show that in the case of the auxin biosynthesis
gene YUCCA2, it can interact with the DNA methylation regulator VARIANT IN
METHYLATION 1 (VIM1) in addition to LHP1 and modulate both DNA methylation and
H3K27me3 deposition at the YUCCA2 promoter (121).

Another important example of a lncRNA mediating a repressive chromatin loop was
discovered by (30), who characterized COLDWRAP as being transcribed in the sense
direction 225 base pairs upstream of FLC using RT-qPCR and interacting with CLF using
RIP-RT-PCR during vernalization. RNA binding assays demonstrated its interaction with
CLF occurred at the 5’ end and was dependent on its secondary structure, and decreased
H3K27me3 accumulation over FLC in COLDWRAP RNAi lines suggested this interaction
was necessary for proper CLF activity (30). Importantly, the authors found a
COLDWRAP-dependent chromatin loop occurred the FLC promoter and the 3’ end of its
first intron, and that this loop (as well as its interaction with CLF) could be maintained by
COLDWRAP in trans using transgenic COLDWRAP and FLC lines (30). This suggests a
similar mechanism to the loop reforming dynamics of APOLO, whereby the loop is closed
in part by APOLO interacting with LHP1 and increasing H3K27me3 levels at the contact
loci (20, 118).

Finally, in an example of mediating a chromatin loop instead associated with gene
activation, the lncRNA MARS was recently characterized as facilitating the formation of a
loop between the MRN1 gene and a distal enhancer (35). As mentioned above, MRN1
is part of the marneral gene cluster including CYP705A12 and MRO; the MARS locus is
also found within it. Plant biosynthetic gene clusters generally share a common
transcriptionally active or inactive environment via 3D compartmentalization (122).
Accordingly, (35) found that upon ABA treatment, a loop formed between MRN1 and an
upstream intergenic region in the middle of the gene cluster, specifically between MRO
and MARS. MARS RNAi lines demonstrated that the formation of this loop was dependent
on increased transcription of MARS which then decoys away LHP1, leading to loss of
H3K27me3, decompaction of the chromatin and loop formation (35). The formation of this
loop is accompanied with increased transcription of the cluster genes, suggesting the
intergenic is acting as an enhancer; indeed, cloning this region into a minimal 35S
promoter system demonstrated it was sufficient to increase expression of the associated
GUS reporter (35). Interestingly, the authors also showed that under normal conditions
basal levels of MARS instead promote LHP1 binding to the gene cluster and subsequent
H3K27me3 deposition, thus compacting the chromatin and preventing loop formation,
suggesting a dual mechanism of loop control similar to what was described for APOLO
(20, 35, 118).

As our knowledge of RNA-DNA interactions and chromatin loops increases, opportunities
for future studies to explore specific lncRNA-chromatin loop dynamics increase. Many
recent large-scale studies in plants of R-loops (123, 124), RNA-DNA interactions (125)
chromatin looping as well as PRC2-mediated chromatin looping (69, 106, 116) are
revealing many new possible candidate lncRNA-loop regulatory dynamics for
investigation.

Conclusions

While the number of mechanistic studies on plant lncRNAs have significantly increased
over recent years, we are still at the early days of fully understanding the potential of
these non-coding transcripts in regulating biological processes. Some clear examples as
the ones described above argue against the idea of lncRNAs being just transcriptional
noise. Yet, the many thousands of natural antisense lncRNAs detected in plant genomes
(44, 100) open the question of how many actually affect the expression of their sense
genes. Additional lncRNAs will have to be characterized in depth to define conserved
mechanisms of epigenetic regulation of transcription by lncRNAs in plants.

The capacity of lncRNAs to modulate chromatin structure has been recognized for several
years. However, the original view of lncRNAs as recruiters of chromatin modifiers could
now be upgraded to more dynamic mechanisms that involve the transient stabilization of
epigenetic modulators over specific regions in the genome. Strikingly, this role as
stabilizers of chromatin complexes depends not only on the local accumulation of
lncRNAs but also on the concentration of lncRNA molecules. While small amounts of
lncRNA transcripts may transiently retain protein complexes at specific sites, over-
production of these molecules act as molecular barriers sequestering away chromatin
modifiers. This dual regulatory role characterized for MARS lncRNA in Arabidopsis (35)
had previously been proposed for mammalian lncRNAs interacting with PRC2 complexes
(126). RNA invasion of DNA via formation of R-loops both locally (37) or even at distant
loci in the genome (118) appears as another common mechanism that may help to
stabilize or decoy away epigenetic regulators. As research on biomolecular condensates
continues to expand, we will be learning about new examples of lncRNAs participating in
formation of membraneless subcellular components. In plants, COOLAIR antisense RNA
stands out in this field by being able to induce formation of nuclear condensates that
sequester FLC sense positive regulators thus triggering transcriptional silencing (76).
Additionally, our understanding of 3D chromatin organization in plant genomes is steadily
growing and the role of lncRNAs in mediating chromatin looping remains an area of
research with great potential for additional discovery. Prominent examples such as
APOLO and MARS have demonstrated the essential role of lncRNAs in controlling the
formation of repressive and activating chromatin loops (35, 118).

Regardless of the mechanism, it has become clear that lncRNAs are key factors in plant
transcriptional modulation with significant impacts in both development and stress. This
presents many opportunities to exploit these modes of regulation for fine-tuning plant
transcription in agronomic contexts. Future work is needed in this area to help overcome
the challenges associated in translating all this fundamental research on lncRNAs into
agronomical applications.


Acknowledgements

We apologise for not citing all the relevant primary research articles and reviews due to
space limitation. This work was supported by the Junior Leader Fellowship
[LCF/BQ/PI19/11690003], from “laCaixa” Foundation [ID100010434] awarded to J.I.Q,
the Severo Ochoa Excellence Programme for Centres 2016-2019 (SEV-2015-0533)
awarded to CRAG. B.J.M.T. PhD work is funded by an FPI predoctoral fellowship
(PRE2019-090156) from the Spanish Ministry of Science.

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Figure legends

Figure 1: Overview of example epigenetic mechanisms of actions of plant lncRNAs. A.
Prior to vernalization, the flowering gene MAF4 is only expressed at low basal levels.
Upon cold treatment and vernalization, its antisense lncRNA MAS increases in
expression. This leads to the recruitment of WDR5A and the COMPASS-like complex to
the MAF4 locus and subsequently increases H3K4me3 deposition. Accumulation of
H3K4me3 at the MAF4 TSS leads to increased transcriptional activation of the gene. B.
Upon cold exposure, the freezing tolerance gene CBF1 is activated. After 4 hours,
expression of the downstream convergently-oriented lncRNA SVALKA is activated.
Read-through transcription into the CBF1 locus leads to transcription of the cryptic
antisense RNA asCBF1. Transcription on both the sense and antisense strand leads to
collision of RNAPII. Stalling of the RNAPII transcribing CBF1 leads to rapidly-degraded
truncated transcripts and a reduction in gene expression. C. The promoter of the auxin
transport regulator gene PID forms a repressive loop with the locus of the lncRNA
APOLO. This loop is maintained by recruitment of LHP1 directed to these sites by APOLO
forming R-loops with target regions, promoting H3K27me3 deposition. Additional RNAPV
transcription of APOLO also leads to DNA methylation of the APOLO locus. Upon auxin
treatment, transcription of APOLO increases and it decoys LHP1 away from the loop
contact regions. This leads to a loss in H3K27me3 and DNA methylation, relaxing the
loop and leading to increased expression of PID. APOLO also acts in trans, relaxing
repressive loops inhibiting the transcription of other auxin-responsive genes.
IUB_2681_Tremblay_IUBMB_Fig1_600dpi_FINAL.jpg
Table 1: List of plant lncRNAs with characterized epigenetic mechanisms mentioned in
this review


    lncRNA            Epigenetic                  Target(s)      Reference(s)
                      mechanism

AG-incRNA4       Increase H3K27me3          AG                (73)
                 via recruitment of CLF

APOLO            Repressive loop            PID, WAG2, AZG2, (20, 118-121)
                 formation and              RHD6, EXT3
                 dissociation via
                 recruitment and
                 decoying of LHP1,
                 respectively



COLDAIR          Increase H3K27me3          FLC               (28)
                 via recruitment of CLF

COLDWRAP         Repressive loop            FLC               (30)
                 formation, increase
                 H3K27me3 via
                 recruitment of CLF

COOLAIR          Switch to H3K27me3         FLC               (22, 25, 32, 34, 37,
                 via stabilization of the                     38, 45)
                 H3K36me3
                 demethylation complex
LAIR         Increase H3K4me3 and LRK1, LRK4             (40)
             H4K16ac via
             recruitment of OsMOF
             and OsWDR5

MARS         Activating loop            MRN1,            (35)
             formation via decoying     CYP705A12, MRO
             of LHP1

MAS          Increase H3K4me3 via MAF4                   (44)
             recruitment of WDR5A
             and the COMPASS-like
             complex

T-DNA-QUA1   Transcriptional            QUA1             (97)
             interference of RNAPII
             via elongation over
             QUA1 TSS

T-DNA-RFD1   Transcriptional            RFD1             (96, 97)
             interference of RNAPII
             via elongation over
             RFD1 TSS

SVALKA       Transcriptional            CBF1             (11)
             interference of RNAPII
             via read-through
             transcription of cryptic
             asCFB1