Noncoding RNAs and
Chromatin
Authorship:
Ricardo José Cordeiro Machado Rodrigues (2012)
Student number 3622940
Under the Supervision of René F. Ketting
Noncoding RNAs and Chromatin
Abstract
During embryonic development organisms need to establish different cell types to carry out specific
functions. Gene expression regulation through chromatin changes has more and more been found to
play a major role in cellular differentiation. RNA has also been found to be a player in gene
expression regulation, through mechanisms like RNA interference. Recent studies show a great
connection between RNA and chromatin, though this field is in a very early stage. Here I discuss
these recent findings in the RNA and chromatin fields, focusing in noncoding RNAs. I will go through
the mechanisms by which noncoding RNAs can regulate chromatin and gene expression. Through
examples I will argue in favour that noncoding RNAs are important general players in chromatin
remodelling and gene expression.
Ricardo Rodrigues 2012
2
Introduction
In higher eukaryotes all cells originate
from a single pluripotent cell – the egg. With
rare exceptions, all cells from an individual
share exactly the same genome, though they
show very different phenotypes and play very
different roles, e.g. a neuron versus a muscle
cell. The first performs signal transmission
functions, while the latter has contractile
functions. Through development the identity
of each cell is established by differences in
their gene expression that lead to different
cell organizations and cellular content.
Following the previous example, it is
important that a neuron expresses, for
instance, glutamate receptors so that it may
receive information through its synapses,
while for the muscle cell it is important to
express myosin for proper contraction. These
differences in expression patterns are
acquired mainly through epigenetic means. In
the early embryo, cells have the potency to
differentiate into all cell types, and are for this
reason named pluripotent stem cells. Yet, the
cells need to differentiate to acquire their
specialized functions, which leads to the loss
of their pluripotency. During development the
cells undergo epigenetic changes that ensure
their differentiation into their correct cell
type.
Epigenetic
regulation
of
gene
expression includes several levels where this
regulation can act, from modifications in DNA,
histones and other DNA associated proteins
(chromatin), to nucleosome packaging and
chromosome organization. However in this
thesis we will focus mainly in the chromatin.
Canonically, two main kinds of chromatin have
been distinguished: heterochromatin and
euchromatin (Heitz, 1928). Euchromatin is
typically gene rich, less condensed and more
accessible
for
transcription,
while
heterochromatin is considered gene poor,
highly compacted and inaccessible to the
transcription machinery. Differences between
euchromatin and heterochromatin are mainly
associated modifications in the nucleosome,
the basic unit of the eukaryotic chromosome
that consists of DNA (146 bp) wrapped around
a core of a histone octamer (Luger et al.,
1997). Histone acetylations are positively
correlated with transcription in humans
(Heintzman et al., 2007; Wang et al., 2008).
Euchromatin nucleosomes are generally
enriched in acetylated histones 3 and 4 (H3
and H4), as well as H3 lysine 4 (H3K4me) and
H3 lysine 39 methylation (H3K39me) (Barski et
al., 2007; Heintzman et al., 2007; Noma et al.,
2001). However, histone methylation is a
more complex trait then acetylation, as it may
also be associated with heterochromatin.
Heterochromatin is associated with
histone hypoacetylation, H3 lysine 9
methylation
(H3K9me)
(Lachner
and
Jenuwein, 2002; Nakayama et al., 2001; Rea et
al., 2000; Schotta et al., 2002) , H3 lysine 27
trimethylation (H3K27me3) (Aldiri and Vetter,
2012; Schwartz and Pirrotta, 2007) and the
presence of heterochromatin protein-1 (HP1)
(Bannister et al., 2001; Lachner et al., 2001).
Another characteristic of heterochromatin is
the presence of DNA cytosine methylation
(5mC), but this modification is thought to be a
reinforcement to the histone modifications
(Bird, 2002; Keshet et al., 1986; Suzuki and
Bird, 2008) and not all organisms have this
kind of modification, such as Caenorhabditis
elegans and Drosophila melanogaster (Bird,
2002). Heterochromatin forms mainly in
pericentromeric and telomeric regions that
are enriched in repetitive DNA elements, such
as transposons (Blasco, 2007; Schueler and
Sullivan, 2006).
Noncoding RNAs and Chromatin
Among
other
functions
of
heterochromatin, centromeric heterochromatin has been found to be necessary for correct
chromosome segregation (Folco et al., 2008;
Pidoux and Allshire, 2005). Furthermore,
heterochromatin formation is important for
the silencing of genes. Transposons, once
expressed, may lead to the damage of the
genome of the cell (Kazazian, 2004).
Therefore, silencing transposon-containing
regions of chromosomes, for instance through
chromatin silencing, becomes important for
genome stability. This thesis will mainly focus
on chromatin silencing. The silencing capacity
of heterochromatin was revealed to the
scientific community by the phenomenon of
position-effect variegation (PEV) (Muller and
Altenburg, 1930). In their work, Muller and
Altenburg (1930) mutagenised Drosophila
embryos using X-ray and observed patterns of
variegated gene expression, through the eye
colour of the flies. This was later revealed to
be related to the silencing of the gene
responsible for the eye pigmentation, when it
would be close to heterochromatin (Schotta et
al., 2003). These findings showed not only that
heterochromatinization of a gene has the
capacity to silence it, but also that
heterochromatin can be dynamic, as not all
cells would have their pigmentation gene
silenced.
Heterochromatinization of certain
portions of the genome is known to be
important for cell homeostasis (Aguilo et al.,
2011; Li et al., 2012; Scheen and Junien, 2012;
Yoo and Hennighausen, 2012). A few
mechanisms by which the cells form
heterochromatin are known (Blasco, 2007;
Lachner and Jenuwein, 2002; Lachner et al.,
2001; Nakayama et al., 2001; Schotta et al.,
2002) and some of them are known to be
mediated by RNA (Grewal and Jia, 2007; Tsai
et al., 2010; van Wolfswinkel and Ketting,
2010). Here I will focus on these RNAmediated mechanisms of chromatin silencing,
Ricardo Rodrigues 2012
their variety and cross points. Though I will go
through a variety of these mechanisms, I will
put small noncoding RNAs (sRNAs) and their
relation to chromatin silencing on the
spotlight due to recent findings on this field. I
will then argue that RNA is a key molecule in
chromatin and gene regulation.
RNA as an active molecule
Conventionally, biology’s view on gene
regulation was focused on protein coding
genes that would follow the central dogma of
molecular biology (DNA->RNA->Protein), but
in the last fifteen years the advancements in
genomic tools and the works of noncoding
genes (Mattick, 2004) have changed the view
of the scientific community.
The regulatory potential of the
noncoding parts of the genome has, in fact,
been suggested as the main cause for the
evolution of developmental processes and
organism complexity (Mattick, 2004). Also
there is a positive correlation between the
complexity of an organism and the relative
expansion of the non-protein-coding DNA
regions of the genome (Mattick, 2004). To our
current knowledge, the human genome
portion responsible for protein coding
constitutes only 1,5% of the genome (Lee,
2009; Wang and Chang, 2011). However,
many more elements are known to be
transcribed (Mattick, 2001, 2004; Wang and
Chang, 2011) and the current estimate
suggests that 98% of our transcription output
are noncoding RNAs (ncRNAs) (Mattick, 2001).
These ncRNAs include introns of protein
coding genes and other transcripts that do not
seem to encode proteins. From these
numbers we can infer that either complex
organisms are filled of useless transcription or
that these ncRNAs should have some kind of
function within these organisms (Mattick,
2004). A way to verify if ncRNAs have
functions within the genome is by comparing
ncRNA profiles of different species. If we see
4
Noncoding RNAs and Chromatin
that these elements are conserved between
species, it means that they were selected
upon. This way conservation should be a good
indicator that a certain element has a specific
function within the cell (Bentwich et al., 2005;
Guttman et al., 2009; Guttman et al., 2010).
However, even though there are certain
ncRNAs that are known to have a conserved
sequence (Wutz et al., 2002), certain species
of ncRNA are actually poorly conserved in
sequence and rather have a secondary
structure conservation, such as long
noncoding RNAs. Nevertheless, the hypothesis
that these noncoding elements are important
for cell function and homeostasis has been
supported by several studies throughout the
last decade (Bartel, 2009; Guttman et al.,
2011; Tsai et al., 2010; Wang and Chang,
2011).
Several classes of ncRNAs have been
found since the beginning of the field. Though,
up to now, two main classes of ncRNAs have
emerged as key players in gene regulation and
expression control: long noncoding RNAs
(lncRNAs) and small noncoding RNAs (sRNAs)
associated with RNA interference (RNAi)
(Mercer et al., 2009; Siomi and Siomi, 2009).
These ncRNAs have been found in most
eukaryotes, including plants, fungi and
animals, which shows us the importance of
such elements. These two classes of ncRNAs
have lately been found to influence chromatin
states (Ashe et al., 2012; Gupta et al., 2010;
Luteijn et al., 2012; Tsai et al., 2010) and for
this reason we will discuss these molecules in
more detail in this dissertation.
Long noncoding RNAs and their
function
Long noncoding RNAs (lncRNAs) are
defined as transcribed non protein coding RNA
molecules greater than 200 nucleotides (nt)
(Kapranov et al., 2007; Mercer et al., 2009). In
5
opposite to other ncRNAs, this class is
generally poorly preserved through species
with an apparent lack of conserved motifs
(Wang and Chang, 2011).
Recent high-throughput studies have
shown that, in the mammalian genome,
thousands of sites with low protein coding
potential are transcribed (Guttman et al.,
2009; Guttman et al., 2010). Nonetheless,
most of these transcripts seem to be
transcribed by RNA Polymerase II, as Guttman
et al. (2009) showed that these transcripts
have 5’caps, are polyadenylated and their
genomic regions have RNA Polymerase II
occupancy and transcriptional elongation
associated histone modifications.
The mechanisms by which these
lncRNAs regulate gene expression seem to be
very diverse and generally poorly understood
(Bernstein and Allis, 2005; Mercer et al., 2009;
Wilusz et al., 2009). Probably the most well
studied lncRNA is Xist, a lncRNA involved in
inactivation of the X chromosome in
mammalian females.
Xist, a lncRNA that
chromosomal inactivation
drives
In mammals the sex chromosome
dimorphism leads to an imbalance in gene
dosage between the male and the female. The
strategy adopted by mammals to compensate
for this imbalance was to inactivate one of the
X chromosomes in female cells, so that both
male and female individuals have only one
transcriptionally activate X chromosome
(Lyon, 1961). Early in development, the
inactive
X
chromosome
forms
a
heterochromatic body within the nucleus of
the female cells (Barr and Bertram, 1949). The
process of X chromosome inactivation and
heterochromatinization is regulated by a great
number of elements (Augui et al., 2011),
though they all seem to coincide with the
regulation of X inactivation centre (Xic) and
Cancer Genomics & Developmental Biology MSc Master Thesis
Noncoding RNAs and Chromatin
the X-inactivation specific transcript (Xist)
(Augui et al., 2011; Wutz, 2011).
Several studies culminated in the
discovery of both Xic and Xist (Borsani et al.,
1991; Brockdorff et al., 1991; Brown, 1991).
Early studies of Xist (Clemson et al., 1996),
showed that this RNA accumulated in the
inactive X chromosome site in the nucleus,
which led the authors that found this
localization to suggest that this transcript
should have a function as a noncoding
transcript. Shortly after the discovery of Xist,
this transcript was shown to be required for
initiation of X chromosomal inactivation
(Marahrens et al., 1997; Penny et al., 1996).
Meanwhile, it has also been shown to not be
required for X inactivation maintenance
(Brown and Willard, 1994; Csankovszki et al.,
1999).
The Xist gene is located within the Xic
locus (Borsani et al., 1991; Brockdorff et al.,
1991; Brown, 1991). Xist is known to act in a
cis manner, i.e., once Xist is expressed it acts
upon the chromosome where it is being
expressed, silencing it (Clemson et al., 1996).
Furthermore, ectopic expression of Xist in an
autosome, has been shown to be sufficient for
the inactivation of that same autosome (Wutz
and Jaenisch, 2000). The mechanism by which
Xist localizes within the X chromosome
remains elusive (Wutz, 2011) and a study by
Wutz et al (2002) has shown that several
regions within the Xist transcript are able to
mediate chromosomal localization of this
ncRNA (Wutz et al., 2002).
Interestingly Xist is also regulated by
other lncRNAs. Xist has an antisense
transcription unit (Tsix) that mediates Xist
repression. The exact mechanism by which
Tsix represses Xist remains elusive, but the
ratios of sense/antisense transcription across
this gene are known to be crucial to
determine which Xist locus is upregulated and
therefore, which chromosome will be
silenced. Inducing antisense Xist transcription
Ricardo Rodrigues 2012
is known to prevent its upregulation in cis
(Luikenhuis et al., 2001; Stavropoulos et al.,
2001). Furthermore another lncRNA expressed
at 5’ of Xist, Jpx, has been shown to be
required for female specific Xist activation as
its deletion prevents X inactivation (Tian et al.,
2010). The presence of all these regulating
lncRNAs show how important these molecules
can be in regulating gene expression.
Another mystery associated with Xist
is how it induces the chromosome silencing
(Wutz, 2011). One of the earliest events after
Xist localization is the depletion of
transcription
machinery,
transcription
initiation factors and splicing factors from the
Xist covered domains of the chromosome
(Chaumeil et al., 2006; Okamoto et al., 2004).
This depletion coincides with the depletion of
nascent RNA transcripts, and histone
modifications
associated
with
gene
expression, such as histone acetylation and
H3K4me (Heard et al., 2001). How the
depletion of the transcription machinery is
accomplished is still unknown (Wutz, 2011).
However, the silencing events do not end with
the depletion of the transcription machinery
and
transcription
associated
histone
modifications, as a number of epigenetic
processes involving Polycomb Group Complex
(PcG complex) proteins and DNA methylation
take place in the Xist-covered chromatin
domain (de Napoles et al., 2004; Fang et al.,
2004; Hellman and Chess, 2007; Plath et al.,
2003).
PcG complexes are enriched in the
chromatin within the Xist domain(de Napoles
et al., 2004; Fang et al., 2004; Plath et al.,
2003), namely Polycomb repressive complex 1
(PRC1), which catalyzes histone H2AK119
ubiquitination and Polycomb repressive
complex 2 (PRC2), which catalyzes H3K27
trimethylation (H3K27me3). The recruitment
of these PcG complexes leads to whole
inactivated
X
chromosome
histone
modifications (Wutz, 2011). However, how
6
Noncoding RNAs and Chromatin
these complexes are recruited to the
inactivated chromosome and their importance
in the activation event is still a theme of
discussion within the scientific community
(Wutz, 2011).
Later in development, Xist is no longer
necessary for maintaining X inactivation (Wutz
and Jaenisch, 2000). The transition into Xist no
longer being necessary and to stable
inactivation is also known as “locking-in” of X
chromosome inactivation. This process is
thought to be mediated by DNA methylation
(Hellman and Chess, 2007; Sado et al., 2000).
The inactivated X chromosome is known to be
hypermethylated in gene rich regions,
compared to the activate X chromosome
(Hellman and Chess, 2007; Weber et al.,
2005). This process seems to be dependent of
the DNA methyltransferase DNMT1 (Sado et
al., 2000) and the protein “structural
maintenance of chromosomes hinge domain
1” (SMCHD1) (Blewitt et al., 2008), also
identified in plants as a RNA-mediated DNA
methylation factor (Kanno et al., 2008). Once
the inactivated chromosome is methylated,
gene repression is stable and can be
maintained without the presence of Xist.
This is an example of RNA as a
molecule that signals gene silencing that is
then maintained by epigenetic means.
lncRNAs act
mechanisms
outcomes
through different
towards
similar
Although Xist is a well studied
example of a mechanism by which lncRNAs act
upon chromatin, other examples are known
were lncRNAs influence gene expression (Rinn
et al., 2007; Tsai et al., 2010). Additionally,
even though the function of only a small
number of lncRNAs has been found, they have
been shown to control every level of the gene
expression(Wapinski and Chang, 2011). Our
inability to predict functional motifs in these
7
molecules and the fact that these molecules
are largely diverse and of complex action
makes it difficult to classify them. Wang and
Chang (2011) decided to approach this
problem by establishing archetypes for their
molecular mechanisms, such as guides,
scaffolds and decoys (see Figure 1). These
archetypes will be discussed further bellow in
this thesis.
Like various protein coding genes,
lncRNA gene expression shows tissue and
stimuli specific expression patterns. This kind
of expression patterns shows us that these
molecules are also under fine-tuned
expression control (see Figure 1A). For this
reason it is possible to infer that these
transcripts might work as signals for the cell
Figure 1 – Schematic representation of the lncRNA molecular
mechanisms according to Wang and Chang (2011). (A) lncRNAs act as
signals by being expressed under tight transcription factor control (B)
The different functions of lncRNAs (B1) lncRNAs acting as decoys (B2)
lncRNAs acting as guides (B3) lncRNAs acting as scaffolds. Image
taken from Wang and Chang (2011)
Cancer Genomics & Developmental Biology MSc Master Thesis
Noncoding RNAs and Chromatin
that would help it understand its cellular
context. The use of RNA as a signal would
bypass the need of using the translational
machinery, saving time and energy to the cell.
The signalling properties of these molecules
are then carried on by different mechanisms,
depending on the lncRNA in question. For
instance, Xist is expressed during tight
moments of mammalian female development
(Augui et al., 2011; Wutz, 2011). The
expression of Xist can work as a signal for the
cell that active silencing should take place in
that site of the genome at that time point.
This way this lncRNA works as a signal for both
time and location of action. In the case o Xist,
the localization signal would be subcellular.
Other examples are known where the signal
could be time and space regulated in whole
tissues, such as the lncRNA HOTAIR (Rinn et
al., 2007).
HOTAIR is a lncRNA that is associated
with the HOXC cluster (Rinn et al., 2007). In
mammals the HOX loci are organized in four
clusters (HOXA, B, C and D) and these clusters
have an expression pattern that is both
collinear between the gene position within the
cluster and their spatial position in the
anterior-posterior axis during development
(Wang et al., 2009), e.g. the gene HOXA1 is
expressed in earlier time points and on the
anterior part of the embryo, while HOXA13 is
expressed at later time points in the posterior
part of the embryo. HOTAIR was found to
have a similar expression pattern (Rinn et al.,
2007). Particularly, it is expressed in more
distal posterior areas of the embryo. The tight
expression pattern of HOTAIR works as a
signal for cell positioning. The positional
information is translated to the cell through
the actions of the lncRNA itself, i.e., the fact
that this RNA is expressed in a particular cell
means that this RNA may only change the
expression programme of that same cell,
informing it of what differentiation path it
should follow.
Ricardo Rodrigues 2012
The mechanisms by which lncRNAs
can act upon a cell are largely variable.
Though, I will now discuss some general
functional archetypes of these molecules, by
which they may exert their signalling
properties in the cell. Here I will focus on
examples where lncRNAs act upon chromatin.
lncRNAs acting as Guides
A lncRNA acts as a guide when the
RNA molecule binds proteins, forming a
ribonucleoprotein (RNP) complex, and directs
them to their specific targets (see Figure 1 B2).
The RNP complexes can then have an effect
on the site where they are transcribed, i.e.
they act in cis or if they guided towards other
sites by targeting specific DNA sequences or
by RNA recognition of certain chromatin
structures, i.e. they act in trans. The effects on
gene expression will differ on the charge of
which proteins are recruited to that site by the
lncRNA.
The lncRNA Xist can be included in this
kind of function as it acts as a guide in a cis
manner. The 5’end of Xist has a highly
conserved region that interacts with PRC2, the
A repeat region (Wutz et al., 2002), or RepA.
The current thought is that, once express, Xist
recruits PRC2, which catalyzes H3K27me3
modification associated to the silencing of
chromatin. The spreading of Xist in the X
chromosome would lead to a spreading of the
PRC2 induced modification and the
chromosome silencing.
Another example of a lncRNA that acts
as a guide is HOTAIR, though this lncRNA
exerts its functions across chromosomes. So in
opposition to Xist, HOTAIR acts in trans.
HOTAIR is known to be able to alter and
regulate epigenetic states in several genome
sites (Gupta et al., 2010; Tsai et al., 2010). The
overexpression of this lncRNA has been
recently associated with cancer metastasis
and the depletion of HOTAIR from cancer cells
leads o a decrease in invasiveness (Gupta et
8
Noncoding RNAs and Chromatin
al., 2010). HOTAIR in cancer cells was found to
interact with PRC2 and lead to an altered
H3K27me3 pattern in the genome and its
depletion to lead to the loss of the PRC2
excessive activity in the same cells (Gupta et
al., 2010).
These two examples show us how
lncRNAs can act as guides and tethers to
chromatin remodelling machinery and show
us a way by which these molecules can
influence gene expression.
lncRNAs acting as Scaffolds
Scaffolding complexes is important for
the cell as in various cases coordinating
different cell complexes towards a certain
function is necessary (Good et al., 2011;
Spitale et al., 2011). For long, proteins have
been regarded as the key players in this kind
of function (Good et al., 2011), though, in
these recent years, several studies showed
that lncRNAs can also play this kind of function
in the cell (Spitale et al., 2011; Tsai et al.,
2010). These kind of lncRNAs have the
capacity of binding multiple effectors in order
to coordinate their activity (see Figure 1 B3).
HOTAIR is a lncRNA that is known to have this
kind of activity.
Recently HOTAIR has been shown to
bind two different histone modification
complexes: PRC2 complex and the
LSD1/CoREST/REST complex (Tsai et al., 2010).
The PRC2 complex, as mentioned before, has
a chromatin silencing activity by catalyzing
H3K27me3 (Aldiri and Vetter, 2012). The
LSD1/CoREST/ REST complex has a H3K4me2
demethylation activity by LSD1 that also leads
to gene repression (Shi et al., 2004). Using a
series of HOTAIR deletion mutants Tsai et al.
(2010) show that the PRC2 complex binds to
HOTAIR in the first 300 nt, while the LSD1
complex binds to HOTAIR in the nucleotides
1500 – 2146. The co-precipitation of these
two complexes was found to be positively
dependent of HOTAIR expression levels (Tsai
9
et al., 2010). Furthermore ChIP-chip analysis
of these complexes revealed a significant
overlap (one third) in promoter occupancy
between them (Tsai et al., 2010).
This example shows how a lncRNA can
be used to coordinate different complexes
with the same goal. Genes targeted by this
macro complex are suppressed not only by
adding the H3K27me3 modification by PRC2
but also by the removal of the gene
expression associated modification H3K4me2
by LSD1. The use of lncRNAs as scaffolds might
be a general mechanism by which the cell is
able to coordinate different and specific
histone modifications to target genes.
lncRNAs acting as Decoys
In the same way that lncRNAs can
interact with proteins to help carry on their
function, lncRNAs have the potentiality to
interact with RNA binding proteins in a
negative manner. A way for these molecules
to have a negative effect on their interactors is
to act as molecular decoys, i.e., these lncRNAs
can interact with proteins in such a way that it
will avoid the binding of proteins to their real
target, where they would exert their activity
(see Figure 1 B1). This way the lncRNA will
titrate the protein with whom it is interacting,
limiting its function. Telomeric repeat
containing RNA (TERRA) is a lncRNA known to
act in such way.
TERRA a lncRNA that is part of the
telomeric heterochromatin (Azzalin et al.,
2007). The telomerase template sequence is
complementary to a repeat sequence in
TERRA. This lncRNA is then though to inhibit
telomerase activity and telomere extension by
binding to the telomerase template sequence
at the same time that it sequesters the
telomerase at the telomeric 3’end (Redon et
al., 2010).
From the given examples, we can see
that lncRNAs can have several effects upon
chromatin silencing and should be regarded as
Cancer Genomics & Developmental Biology MSc Master Thesis
Noncoding RNAs and Chromatin
Figure 2 – Different RNA interference pathway effects. Adapted from Ketting,R.F. (2011) and Maartje Luteijn
active molecules. Further studies will surely
give us new insights on this type of gene- and
chromatin regulation. Nevertheless, these are
not the only ncRNAs that are known to
influence gene expression and chromatin. In
the next section of this thesis I will discuss
how sRNAs can influence gene expression at
various levels.
RNA interference as a RNAmediated silencing mechanism
RNA interference (RNAi) is a process
by which small noncoding RNAs typically
reduce the expression of target genes (Ender
and Meister, 2010; Siomi et al., 2011). Since
the discovery of RNAi (Fire et al., 1998) a large
number of sRNA species has been described in
most classes of eukaryotes (Hutvagner and
Simard, 2008; Ketting, 2011). A common
feature of RNAi pathways is the partaking of
an argonaute (AGO) protein (Ender and
Meister, 2010). Small RNAs are loaded in into
AGO proteins and guide target inhibition in a
sequence specific manner (Tolia and JoshuaTor, 2007). The targets of the AGO protein are
identified through base pairing between the
loaded sRNA and the target RNA. Once the
Ricardo Rodrigues 2012
loaded AGO pairs with a target, silencing can
be induced by different processes: target
cleavage by the AGO or cleavage induction by
the recruitment of additional factors, affecting
translation or RNA stability, or even by
altering the chromatin of a target gene (see
Figure 2) (Siomi and Siomi, 2009; van
Wolfswinkel and Ketting, 2010). Here I will
briefly introduce some of the known sRNA
classes.
Probably the most famous class of
sRNAs are the microRNAs (21-25nt long
depending on organism). microRNAs (miRNAs)
are created from transcripts that form a
hairpin (pri-miRNA) that is recognized and
cleaved by Drosha, creating small double
stranded RNAs (dsRNAs) called pre-miRNAs
(Siomi and Siomi, 2009). These pre-miRNAs
are then recognize and cleaved by Dicer and
only then one of its strands is loaded into an
AGO that carries out its silencing function (see
Figure 3A) (Siomi and Siomi, 2009). This
process is very similar to the formation of
another class of sRNAs, the endogenous
interfering small RNAs (endo-siRNAs). In
mammals and Drosophila, endo-siRNAs are
formed from dsRNAs that originated from the
pairing of larger transcripts (see Figure 3A), in
opposition to the single transcript hairpin
formation of miRNAs (Siomi and Siomi, 2009).
10
Noncoding RNAs and Chromatin
Figure 3 – Different RNA interference pathways examples. Pathways are indicated in figure. For description see text. Adapted from
Ketting,R.F. (2011)
Still, just like miRNAs, endo-siRNAs are
dependent on Dicer. This protein cleaves the
large dsRNA transcripts that are then loaded
into an AGO (Ender and Meister, 2010; Siomi
and Siomi, 2009).
The
siRNA
pathway
in
Schizosaccharomyces pombe and the 26G
pathway in C. elegans are two other RNAi
pathways in which sRNAs also originate from
dsRNA molecules (Ketting, 2011). The 26G
pathway has earned its name for the size of its
sRNAs (26nt) and a sequence bias of a
guanosine in their 5’end. The source of these
dsRNAs is from RNA-dependent RNA
polymerases (RdRPs) (Gent et al., 2009;
Martienssen et al., 2005; Pavelec et al., 2009).
In these two pathways RdRPs synthesize
dsRNAs from single strand RNAs (ssRNAs) that
are then cleaved by Dicer and loaded into an
AGO (see Figure 3B)(Ketting, 2011). The siRNA
pathway of S. pombe is known to be
important in heterochromatin formation in
this organism and will be further discussed
bellow in this thesis.
Common between the mentioned
RNAi pathways is that they all use Dicer to
process the sRNAs. The absence of Dicer leads
to the inactivation of these pathways,
therefore they qualify as Dicer-dependent
11
pathways (Ketting, 2011). However not all
RNAi pathways are Dicer-dependent. In C.
elegans secondary siRNAs, also known as 22G
RNAs for their size (22nt) and 5’end sequence
bias (guanosine), are derived directly from
RdRP activity and then loaded into AGOs,
without Dicer processing (see Figure 3C) (Pak
and Fire, 2007; Sijen et al., 2007). The
presence of a triphosphate group in their
5’ends serves as evidence that there are newly
synthesized and not digested from a longer
transcript (Ketting, 2011). 22G RNAs are
known to be loaded into a multitude of AGOs
and are not believed to induce target
cleavage. Though their precise function is still
under discussion, recent findings suggest a
strict relationship between this class of RNAs
and chromatin (Ashe et al., 2012; Buckley et
al., 2012; Gu et al., 2012; Lee et al., 2012;
Luteijn et al., 2012; Shirayama et al., 2012),
which we will discuss further ahead in this
thesis.
Another Dicer-independent pathway is
the Piwi-pathway, which seems to be animal
specific and restricted to the germ line. In this
pathway, endogenous ~26-32nt long sRNAs
are derived from genomic elements
(Brennecke et al., 2007; Houwing et al., 2007;
Ketting, 2011; Siomi et al., 2011). These sRNAs
Cancer Genomics & Developmental Biology MSc Master Thesis
Noncoding RNAs and Chromatin
associate with Piwi proteins, a subclass of the
AGO protein family and are for this reason,
called Piwi-interacting RNAs (piRNAs). Piwi
proteins are germline specific AGOs and
together with piRNAs they are thought to be
key elements in the silencing of transposable
elements (TEs), whose activity may cause
damages in the genome. It has been shown
that deficiencies in the Piwi pathway lead to
the upregulation of TEs in the germline (Aravin
et al., 2006), and similar effects have been
found in zebrafish (Houwing et al., 2007). The
Piwi proteins are known to have the capacity
of cleaving the transcripts that they are
targeting (Hutvagner and Simard, 2008; Siomi
et al., 2011), though they have also been
implicated in other silencing mechanisms,
such as epigenetic modifications (Aravin et al.,
2008; Klattenhoff et al., 2009). In mice the
piRNA pathway has been implicated as a
specificity determinant of de novo DNA
methylation in germ cells (Aravin et al., 2008).
The Piwi-pathway can actually be
divided into a primary and a secondary
pathway. The first is responsible for the
processing of long single-stranded RNAs
(ssRNAs), derived from genomic piRNA
clusters in an AGO dependent manner. The
latter corresponds to a loading of a secondary
AGO protein that carries out further silencing
functions, which are slightly different
depending on the species (see Figure 3D)
(Aravin et al., 2006; Houwing et al., 2007;
Ketting, 2011; Siomi et al., 2011). In the
primary pathway it is believed that an
unknown endonuclease cleaves the long
ssRNAs, into small single stranded piRNAs that
are then loaded into a Piwi protein. The
primary pathway is then believed to induce
the secondary pathway (Ketting, 2011). In the
secondary pathway two or more Piwi-protein
paralogues silence target sequences or
transcripts (Aravin et al., 2006; Brennecke et
al., 2007; De Fazio et al., 2011; Houwing et al.,
2008; Houwing et al., 2007). Characteristic
Ricardo Rodrigues 2012
signatures of primary piRNAs are a sequence
bias to have a uracil in their 5’end and to be
2’-O-methylated in their 3’ends (Aravin et al.,
2006; Brennecke et al., 2007; Houwing et al.,
2007; Kamminga et al., 2010; Kamminga et al.,
2012).
In C. elegans piRNAs are known as 21U
RNAs and they differ significantly from piRNAs
in other animals (Batista et al., 2008; Das et
al., 2008). Though the same characteristic
signatures of piRNAs (5’end U bias and 3’end
2’-O-methylation) are present in the 21U
RNAs, they do not seem to target the majority
of TEs in the genome (Batista et al., 2008). By
sequence comparison most 21U RNAs have no
close sequence matches beyond their own
locus (Batista et al., 2008; Das et al., 2008),
though recent studies have shown that
perfect pairing between the 21U RNA and the
target RNA is not necessary to induce target
silencing (Bagijn et al., 2012). Furthermore, it
has also been shown that the cleaving activity
of PRG-1, a Piwi protein that drives 21U RNAs,
is not necessary to silence a target (Bagijn et
al., 2012). Work made in the last six months
suggests that 21U RNA silencing activity is also
associated with silencing of chromatin (Ashe
et al., 2012; Luteijn et al., 2012; Shirayama et
al., 2012). However, proof of a direct
connection between 21U RNAs or PRG-1 and
chromatin remains to be found.
A few known examples show a clear
bridge between sRNAs and chromatin
silencing (Ashe et al., 2012; Gu et al., 2012;
Klattenhoff et al., 2009; Luteijn et al., 2012;
Shirayama et al., 2012; Sienski et al., 2012;
Volpe et al., 2002). Here I will discuss some of
these examples and show sRNAs as a common
mechanism for gene specific silencing.
siRNA directed heterochromatin
assembly in S. pombe
In
fission
yeast
centromeric
heterochromatin assembly is RNAi dependent.
12
Noncoding RNAs and Chromatin
This heterochromatic area is enriched in
H3K9me and the heterochromatic protein1
(HP1) homolog Swi6, which has important
functions
in
cohesin
assembly
on
chromosomes (Bernard et al., 2001; Volpe et
al., 2003). In this organism a RNA-induced
transcriptional silencing (RITS) complex that
targets chromosome regions for inactivation
through siRNA has been identified (Verdel et
al., 2004). This complex includes the only AGO
protein (Ago1) of S. pombe. This protein is
loaded with a siRNA and directs the RITS
complex to the silencing regions. Proper
centromeric heterochromatin assembly is
necessary
for
proper
chromosome
segregation, revealing the importance of this
process (Bernard et al., 2001; Volpe et al.,
2003).
Centromeres
of
fission
yeast
chromosomes are flanked by repetitive DNA.
These repetitive sequences are transcribed in
both strands (Verdel and Moazed, 2005). The
transcripts of the repetitive elements then
form dsRNAs that are recognize and cleaved
by Dicer turning them into siRNAs that are
then loaded into Ago1 (Volpe et al., 2002). The
loaded AGO directs the RITS complex to the
locations where the repetitive elements are
being transcribed (Motamedi et al., 2004). The
RITS complex includes the protein Chp1, a
chromodomain containing protein that is
known to be part of the heterochromatic
structure (Verdel et al., 2004; Verdel and
Moazed,
2005).
Chp1
binds
to
heterochromatin by recognizing H3K9me
through its chromodomain (Verdel et al.,
2004). This binding allows the RITS complex to
be kept near the heterochromatin.
Furthermore, the RITS complex is known to
recruit Clr4, a histone modifier that catalyzes
H3K9me (Noma et al., 2004), through the LIM
domain protein Stc1 (Bayne et al., 2010). The
H3K9me histone modification also allows for
the protein Swi6 to bind to the
heterochromatin (Noma et al., 2004). Studies
13
have shown that the presence of Swi6 at
centromeric chromatin is necessary for the
recruitment of the RNA-directed RNA
polymerase complex (RDRC) which includes a
RdRP (Rdp1) (Motamedi et al., 2004). RDRC
uses the local repetitive DNA transcripts as a
template, generating more dsRNA and
therefore amplifying the siRNA response
(Motamedi et al., 2004).
The heterochromatic assembly at the
S. pombe centromeres is therefore a loop that
is self reinforcing (see Figure 4A). The
recognition of nascent transcripts by the RITS
complex, leads to H3K9me of the local
histones by recruitments of Clr4. This
modification then reinforces the presence of
the RITS complex, allowing it to improve
target recognition, through the binding of
Chp1 to H3K9me. The presence of H3K9me
also reinforces the siRNA response through
the binding of Swi6 to heterochromatic sites,
which recruits the RDRC complex. This way
the cell manages to silence and repress those
sites locally, i.e. in cis. Though we would think
that these mechanisms would be sufficient for
the silencing of this heterochromatin, recent
work by Keller et al. (2012) showed that these
processes are more complex than once
anticipated.
Heterochromatin is usually viewed as
a static chromatin compartment, inaccessible
to the transcriptional machinery, though it has
recently been shown that HP1 is a highly
dynamic protein in heterochromatin (Cheutin
et al., 2004; Festenstein et al., 2003).
Furthermore, RNAi-independent RNA turnover
mechanisms have been found to be necessary
for the complete silencing of heterochromatic
genes (Buhler et al., 2007). Without disturbing
heterochromatin structure, the silencing of
heterochromatic genes is impaired in the
absence of the protein Cid14 (Buhler et al.,
2007), a noncanonical poly(A) polymerase that
is thought to target heterochromatic
transcripts for degradation (Keller et al.,
Cancer Genomics & Developmental Biology MSc Master Thesis
Noncoding RNAs and Chromatin
2010). In addition, Swi6 was also known to be
necessary for this kind of silencing (Buhler et
al., 2007). Through a series of elegant
experiments Keller et al. (2012) were able to
demonstrate a key function of Swi6 in the
silencing of these transcripts.
Swi6 had been associated with
heterochromatic transcripts (Motamedi et al.,
2008). Keller et al. (2012) were able to
demonstrate that this HP1 homolog is able to
bind to RNA through its hinge region. In the
same study they demonstrate that disturbing
the RNA binding capacity of Swi6 does not
influence the H3K9me binding capacity of
Swi6 chromodomain. This would show that
the recruitment of Swi6 to chromatin is
independent of its RNA binding capacity
(Keller et al., 2012). However the binding of
Swi6 to the RNA causes a conformation
change in the protein that leads to the
decrease in Swi6 affinity to H3K9me and its
dissociation from heterochromatin (Keller et
al., 2012). These findings suggest that Swi6
binds to heterochromatic transcripts and
leaves the heterochromatin sites.
Heterochromatic
transcripts
are
targeted for degradation, as they are not
translated (Buhler et al., 2007; Keller et al.,
2012). Ablation of Cid14 has been associated
with the accumulation of this kind of
transcripts (Buhler et al., 2007). For this
reason Keller et al. (2012) hypothesized that
Cid14 might be involved in the degradation of
these transcripts. The authors then use DNA
adenine methyltransferase identification
method (DamID) combined with tiling arrays
to show that both Swi6 and Cid14 localize to
the heterochromatic sites of S. pombe.
Furthermore they show that the localization
of Cid14 to heterochromatin is Swi6
dependent (Keller et al., 2012). These findings
lead the authors to suggest a model where
Swi6 prevents the production of proteins from
these transcripts by sequestering the mRNAs.
Swi6 then escorts the transcripts to the
Ricardo Rodrigues 2012
degradation machinery that is located next to
the heterochromatin and also associated with
Cid14 (Keller et al., 2012)(see Figure 4B).
In this example siRNAs employ a
mechanism of silencing that is translated into
heterochromatinization of the target area. The
formation of closed heterochromatin then
decreases the chance of transcription by the
transcription machinery. Still, the Swi6-Cid14
heterochromatic RNA checkpoint reassures
that the few transcribed RNAs are still not
translated, leading to a complete silencing of
these genes.
The crosstalk between HP1 (Swi6) and
the silencing of RNAs has not only been
described in yeast. Recent work in Drosophila
has also shown crosstalk between the
Drosophila HP1 homologue Rhino and RNA
silencing through the piRNA pathway
(Klattenhoff et al., 2009), suggesting that this
might be a common mechanism among
eukaryotes.
RNAi and chromatin crosstalk
leads to heterochromatin silencing
in Drosophila
In Drosophila the piRNA pathway
protects the genome against TEs (Brennecke
et al., 2007; Siomi et al., 2011). Mutations in
this pathway lead to DNA damage and
genome instability (Brennecke et al., 2007).
In this organism piRNAs are derived
from transposon rich clusters that localize at
the pericentromeric and subtelomeric
heterochromatin. Most of these clusters are
transcribed in both sense and antisense,
though some of these clusters are mostly
transcribed in a single strand (Brennecke et
al., 2007; Brennecke et al., 2008). This
suggests that the piRNAs that target these
sequences are generated in different sites of
the genome and therefore act in trans
(Brennecke et al., 2007), though this
mechanism
is
poorly
understood.
Furthermore, piRNA pathway mutations are
14
Noncoding RNAs and Chromatin
known to modify PEV and Piwi proteins have
been found to interact with HP1 (BrowerToland et al., 2007), showing an association
between
the
piRNA
pathway
and
heterochromatin.
In the work by Klattenhoff et al.
(2009), the authors show that mutating Rhino
(rhi), an HP1-like homologue of Drosophila,
triggers both transposon upregulation and
DNA
damage,
without
affecting
heterochromatin formation or the expression
levels of non-TE heterochromatic genes.
Furthermore they demonstrate that the
localization of Drosophila piwi proteins to
nuage (Aub, Ago3), a perinuclear structure
implicated in RNA processing, and to the
nucleus (Piwi) is affected in these mutants
(Klattenhoff et al., 2009). These effects are
very similar to known mutations in the piRNA
pathway (Chuma et al., 2006; Huang et al.,
2011a; Huang et al., 2011b) and are a good
indicator of the involvement of Rhino protein
in this RNAi pathway.
To search for defects in the Piwipathway the authors look into the piRNA
abundance in rhi mutants. They find an 80%
decrease in piRNA abundance with the loss of
the characteristic ping-pong amplification loop
signature. Interestingly, the decrease in piRNA
abundance was mainly in antisense piRNAs
from dual transcribed clusters (Klattenhoff et
al., 2009). These antisense piRNAs are thought
to be originated from primary antisense
transcripts, which lead the authors to believe
that Rhino might be associated with the
transcription of these elements. Through ChIPqPCR experiments the authors show an
enrichment of Rhino in dual-stranded clusters
and later they verify that the absence of Rhino
leads to a decrease of primary transcript
expression (Klattenhoff et al., 2009). This
would explain the decrease in piRNAs, as the
absence of the primary pathway leads to an
inefficient secondary pathway.
Interestingly,
Drosophila
piRNA
biogenesis has been also associated with the
germline
specific
H3K9me
histone
methyltransferase dSETDB1 (Rangan et al.,
2011). Mutations in this histone modifier
showed similar effects to those cause by the
Figure 4 – RNAi mediated heterochromatin assembly in S. pombe and Drosophila. (A) Schematic representation of RNAi mediated
heterochromatin formation in fission yeast, notice that it is a feedback loop. (B) Swi6-mediated heterochromatic RNA decay model by Keller
et al. (2012). (C) Piwi-related heterochromatin formation in Drosophila. Notice that compared to (A) this is not a closed loop, but the
components are very similar. (D) Model by Zhang et al. for the function of Rhino and UAP56 in the Piwi pathway and relation to
heterochromatin in Drosophila. For detailed description see text. Adapted from (Olovnikov et al., 2012), Keller et al. (2012) and Zhang et al.
(2012)
15
Cancer Genomics & Developmental Biology MSc Master Thesis
Noncoding RNAs and Chromatin
rhi mutation (Klattenhoff et al., 2009). In the
same work, Rangan et al. (2011), show that
methylation by dSETB1 allows Rhino to bind to
heterochromatin and that H3K9me is enriched
in piRNA clusters. The components of this
pathway seem to be very similar to the ones
mentioned before in the S. pombe
heterochromatin assembly process. We can
then infer that in Drosophila a similar adapted
mechanism exists, for chromatin silencing (see
Figure 4C). dSETB1 catalyzes H3K9me in the
piRNA clusters in the germline nucleosomes
and allows for Rhino to associate with
heterochromatin. In a recent study, UAP56, a
DEAD Box protein, has been suggested to
escort heterochromatic transcripts from the
nuclei to the nuage (Zhang et al., 2012)(see
Figure 4D). These escorted transcripts are
linked to Rhino-associated piRNA clusters.
Once in the nuage, the transcripts would be
processed into piRNAs. Drosophila Piwi
protein is known to localize to the nucleus and
has been suggested to recruit HP1a to
heterochromatin (Brower-Toland et al., 2007).
Furthermore, it has also been shown that the
presence of heterochromatin-associated
histone modifications in piRNA clusters is
dependent of Piwi and the existence of TE
transcripts (Sienski et al., 2012). These findings
suggest that Piwi may recognize TE transcripts
and recruit chromatin silencers, such as
dSETDB1. Nevertheless, it is not known if Piwi
protein interacts with dSETDB1, as RITS
interacts with Clr4 through Stc1. If this is the
case
the
self
reinforced
heterochromatinization loop in S. pombe
could also be generated in Drosophila. Further
research in this field might elucidate the
mechanisms of heterochromatin formation in
Drosophila and, as other Piwi proteins are
known to have nuclear localization (Aravin et
al., 2008; Houwing et al., 2008), establish a
general mechanism for RNAi-directed
heterochromatin assembly in eukaryotes.
Ricardo Rodrigues 2012
Furthermore, in recent publications
(Ashe et al., 2012; Lee, 2009; Luteijn et al.,
2012; Shirayama et al., 2012), a bridge
between piRNAs (21U RNAs in C. elegans) and
heterochromatin formation has been shown
in C. elegans.
This process has been
denominated as RNA-induced epigenetic
silencing (RNAe) and shows a new link
between RNAi and epigenetic silencing.
RNAe an inheritable epigenetic
memory induced by Piwis
The short generation time (~3days),
their easy maintenance and the large amount
of progeny have turned C. elegans into a great
tool for forward genetic screens and
multigenerational effect studies. In the past
decade, this nematode has been extensively
used for the study of RNAi mechanisms.
During these studies inheritance of the
silencing effect caused by RNAi has been
reported (Burton et al., 2011), and during the
last year this inheritance has been linked to
chromatin silencing (Ashe et al., 2012; Gu et
al., 2012; Lee et al., 2012; Luteijn et al., 2012;
Shirayama et al., 2012).
Work by Gu et al. (2012) has shown
that in C. elegans sequence specific chromatin
silencing can be induced by exogenous dsRNA.
In this study, the authors show that targeting
the endogenous gene lin-15B with exogenous
RNAi triggered H3K9me modification in the
nucleosomes of this locus (Gu et al., 2012).
Furthermore, the authors show that
secondary siRNAs associated AGOs and are
required to obtain this kind of response. Also
the H3K9me modification revealed itself to be
dependent of NRDE-2 protein (Gu et al.,
2012), a RNAi nuclear factor that is known to
inhibit transcript elongation (Guang et al.,
2010). Gu et al. (2012) then suggest this
mechanism to be analogous to the S. pombe
siRNA-chromatin relation, where tethering
RITS to nascent transcripts leads to their
heterochromatin based silencing (Buhler et
16
Noncoding RNAs and Chromatin
al., 2006). Interestingly, during this process
the RNAi-induced heterochromatic response
in C. elegans seems to be inheritable up to
two generations (Gu et al., 2012).
In nematodes, exogenous siRNA
response is known to be mediated by RDE-1
argonaute (Gu et al., 2009), which then
induces the secondary 22G RNA response. In
this pathway, 22G RNAs are loaded into worm
specific AGOs (WAGOs) that amplify the
silencing response (Gu et al., 2009). WAGOs
are also known to silence endogenous
elements in the germline, such as transposons
and pseudogenes (Gu et al., 2009). However,
RDE-1 is not required for the silencing of these
endogenous elements, suggesting that
WAGOs may have alternative primary triggers
besides RDE-1. Mutations in PRG-1, a Piwi
homologue of C. elegans, are known to reduce
22G RNAs associated with WAGOs that target
the transposon Tc3 (Bagijn et al., 2012; Batista
et al., 2008; Das et al., 2008). These findings
lead to the suggestion that piRNAs may recruit
a RdRP that generates 22G RNAs and initiate
the WAGO pathway (Bagijn et al., 2012).
Furthermore, Bagijn et al. (2012) have shown
that PRG-1-21U complexes are able to trigger
an endogenous RNAi pathway that is
mediated by WAGO-9 and other endogenous
RNAi factors such as MUT-7 and RDE-3.
In the studies by Bagijn et al (2012),
piRNA-mediated silencing is shown to require
a subset of siRNA pathway genes: the putative
helicases MUT7, DRH-3 and MUT-14 as well as
the RdRPs EGO-1 and RRF-1. The authors are
able to show the targeting capacity of the
pathway by using a synthesised 21U RNA
sensor (Bagijn et al., 2012). This sensor
consisted of a H2B-GFP fusion protein, which
mRNA had a 3’UTR with a known 21U RNA
target sequence. Using a stable transgene
worm with this 21U sensor, the authors
manage to show that PRG-1 cleaving activity
or target mRNA-21U perfect pairing are not
required for target silencing (Bagijn et al.,
17
2012). Silencing induced by PRG-1-21U
complex seemed rather dependent on the
generation of 22G RNAs. Though, in the same
study, the authors show that the amplitude of
the 22G response declines with the increase of
mismatches between the complex and the
target mRNA.
Since the mentioned sensor was
generated with a single-copy genome locus
directed insertion technique (Frokjaer-Jensen
et al., 2008) the authors were also able to
demonstrate the trans silencing activity of the
Piwi-pathway in C. elegans (Bagijn et al.,
2012). Meanwhile, follow up studies (Ashe et
al., 2012; Lee et al., 2012; Luteijn et al., 2012;
Shirayama et al., 2012) where able to
demonstrate that the effects of 21U RNA
silencing where not only an effect of RNAi
silencing but that this phenomenon was
actually associated with chromatin silencing.
In the follow up work by Ashe et al.
(2012), these authors find that the silencing of
the previously described 21U Sensor by the
piRNA pathway is not only stable, but it is also
transmitted through generations (>F20). Using
the presence or absence of H2B-GFP, the
authors develop an essay where they can
observe
the
phenomenon
of
transgenerational silencing, i.e. the absence of
H2B-GFP indicates active silencing of the
sensor. In this work, the authors postulate and
are able to demonstrate that this silencing
mechanism is dependent on the continuous
generation of RNAi. Furthermore, using this
sensor for a forward genetic screen, Ashe et
al. (2012) find that this inheritance is
dependent on the nuclear factors WAGO-9 (or
HRDE1), NRDE-2, previously shown by Gu et
al. (2012) to be important for silencing
inheritance, and SET-25, a histone H3K9
methyltransferase. In the same screen the
authors identify NRDE-1, NRDE-4 (proteins of
unknown function), SET-32 and an HP1
ortholog HPL-2 to be necessary for this effect.
As these are all nuclear factors and some of
Cancer Genomics & Developmental Biology MSc Master Thesis
Noncoding RNAs and Chromatin
them have been previously implied in other
RNAi based silencing inheritance, the authors
conclude that there is a common
RNAi/Chromatin pathway in the germline that
is both required for exogenous siRNA induced
and piRNA induced silencing inheritance (Ashe
et al., 2012).
Ashe et al. (2012) identified both RNAi
pathways and nuclear factors as key elements
in this inherited silencing process. Though a
question arises: Are the RNAi pathways
upstream or downstream of the nuclear
factors activity? The authors reply to this
question by looking into the 22G RNA
populations. In the nuclear proteins hpl-2 and
nrde-2 mutants, 22G RNAs that map to the
sensor are not affected, while they are
significantly diminished in prg-1 mutants
(Ashe et al., 2012). These data strongly
suggest that the nuclear response is
downstream from the RNAi response.
Furthermore, the authors then test if the
piRNA pathway could be the maintainer of this
silencing by constantly inducing the silencing
of the target gene. Interestingly, introducing
prg-1 mutation, which leads to absence of
piRNAs (Batista et al., 2008), in sensorsilenced strains would not recover H2B-GFP
expression. Though, introducing nrde-1 or 2
mutations in these strains caused the sensor
to be once more expressed (Ashe et al., 2012).
From these observations the authors conclude
that the nuclear factors are necessary for the
maintenance of silencing, whereas the piRNA
pathway is only necessary to trigger the
silencing of the gene.
This silencing phenomenon was also
observed by others (Lee et al., 2012; Luteijn et
al., 2012; Shirayama et al., 2012). Multicopy
transgene in C. elegans generally leads to
transgene silencing in the germline. The
technique of Mos1-mediated single-copy
insertion (MosCI) created by Frokjaer-Jensen
et al. (2008) had as one of their intents to
avoid the silencing of these genes and
Ricardo Rodrigues 2012
decrease co-suppression (Ketting and Plasterk,
2000). However, Shirayama et al. (2012), find
that, even with this technique, transgene
silencing in the germline still occurs. These
authors take these findings further and show
that silenced transgenes are targeted by PRG1 and 21U RNAs and that this targeting
strongly correlates with H3K9me modification
in the 21U RNA targeted sequences
(Shirayama et al., 2012). For this reason they
baptise this mechanism as RNA-induced
epigenetic silencing or simply RNAe. In this
study Shirayama et al. (2012) show that 22G
RNAs are generated against exogenous
sequences within the transgene fusions. Using
gfp::csr-1 and gfp::cdk1 transgene lines the
authors find that the silencing of these lines
correlates with the accumulation of 22G RNAs
against the GFP portion of the sequence and
the H3K9me modification of the transgenic
loci. This silencing was partially recovered by
introducing wago-9 mutation in these worms,
in accordance with the findings of Ashe et al.
(2012). Interestingly, in this study, the authors
also find that mutations in cytoplasmic
WAGO-1, and nuclear WAGO-10 and 11
partially recover the transgene expression as
well (Shirayama et al., 2012). These findings
suggest redundancy between WAGOs, but
also suggest that cytoplasmic WAGOs also
have a function in RNAe. In the same study, by
performing similar experiments to Ashe et al.
(2012), Shirayama et al. (2012) also find that
PRG-1 is necessary to initiate RNAe response
but not to maintain it. Though these authors
also suggest a second pathway induced by
PRG-1.
Shirayama et al. (2012) observe that
crossing silenced transgenes into several years
old GFP expressing strains caused the
reactivation of the transgene (transactivation),
which sometimes would be maintained after
genetically isolating the transgene once more.
For this reason they suggest that a third
pathway might be associated with insuring self
18
Noncoding RNAs and Chromatin
memory, i.e. PRG-1 recognizes a target and
initiates a nonself and a self recognition
response. The first would be associated with
22G RNA biogenesis that are loaded into
WAGOs and the RNAe chromatin silencing
pathway, while the latter, which remains to be
found, would prevent the recruitment of
WAGOs by PRG-1, protecting endogenous
expressed genes that might be recognized by
this protein and targeted for silencing.
Nevertheless, the authors suggest the CSR-1
RNAi pathway as a possible antisilencing
pathway (Shirayama et al., 2012). There is a
known 22G RNA population that recognizes
native germline expressed genes and are
known to be loaded into CSR-1 (Claycomb et
al., 2009). However they do not seem to cause
target silencing (Claycomb et al., 2009). In
opposition to the upregulation of WAGO
associated 22G RNAs, this population of 22G
RNAs was not altered in the MosCI transgenic
lines (Shirayama et al., 2012). The authors
propose that CSR-1 may inhibit the WAGO
pathway, for instance by cleaving the
transcripts to which RdRPs are bound to,
decreasing the 22G RNA response. It would
then be interesting to see if CSR-1 is bound to
GFP associated 22G RNAs in the several year
old GFP expressing transgenic lines that
reactivated the newly synthesized transgenics.
Finding such 22G RNAs would work as further
support of CSR-1 dependent self memory.
A study from the same group further
supports the idea of an antisilencing capacity
of CSR-1 (Lee et al., 2012). In this study the
authors show that WAGO associated 22G
RNAs are depleted in prg-1 mutants, as well as
there is an upregulation of WAGO-22G-RNA
mRNA targets. On the other hand, CSR-1
bound 22G RNAs or their targets did not suffer
significant changes (Lee et al., 2012).
Furthermore, they show that predicted targets
of CSR-1 bound 22G RNAs that overlap with
predicted targets of 21U RNAs are not
significantly altered in the prg-1 mutant
19
background, whereas WAGO targets that
overlap with 21U RNA targets are upregulated
in these mutants. Since CSR-1 is known not to
silence its germline targets (Claycomb et al.,
2009), the authors once more suggest that
they may have a protective function of
germline endogenous transcripts. However,
further insights in the works of CSR-1 are
needed to confirm this hypothesis.
In their work, Ashe et al. (2012) and
Shirayama et al. (2012) found various
components of this new pathway, though,
using genetic crosses Luteijn et al. (2012) gave
us new insights into this pathway. Using the
same sensor by Bagijn et al. (2012) and Ashe
et al. (2012) these authors find that the
silencing state of the transgene can be
imposed in trans (Luteijn et al., 2012), in
accordance with Shirayama et al. (2012),
though this is only imposed by the female
germline. These authors perform different
crossings in a prg-1 mutant background to
study the inheritance of this silencing
mechanism. The use of the prg-1 mutant
background eliminates the chance of de novo
silencing, as PRG-1 is the initiator of
endogenous RNAe. By crossing a silenced
transgene of a male worm with a
hermaphrodite worm with an active sensor
these authors observe that all descendants
are GFP positive. On the other hand if the
female worm has a silenced sensor and is
crossed with a GFP positive male all
descendants are GFP negative. These findings
indicate that RNAe can work in trans and that
this activity is dependent of a diffusible agent
in the female germline (Luteijn et al., 2012).
Interestingly, in the same study,
Luteijn et al. (2012) register different
observations from the work of Shirayama et
al. (2012). Investigating the involvement of
MUT-7 and WAGOs in this pathway, they do
observe that MUT-7 and WAGO-9 proteins are
necessary for RNAe maintenance through
generations, though these proteins reveal a
Cancer Genomics & Developmental Biology MSc Master Thesis
Noncoding RNAs and Chromatin
maternal effect in their descendents. Luteijn
et al. (2012) observe that the activation of a
silenced sensor only occurs in the second
generation of homozygous mutants for these
genes. These findings suggest that RNAe is
established in the maternal germline. Also the
observations of Shirayama et al. (2012) where
they suggest that other WAGOs besides
WAGO-9 are redundant in the RNAe function
are not observed by Luteijn et al. (2012). In
their studies the wago-9 mutation partially
activates the 21U sensor as in Shirayama et al.
(2012), but in opposition to that study the
same is not verified for wago-10, wago-11 and
nrde-3 mutants. Still, the fact that wago-9 only
partially rescues the silencing of the
transgene, suggests that the RNAe
maintenance function of WAGO-9 has
redundancy with other proteins. Furthermore,
the authors show that wago-9 and nrde-1
mutations, but not mut-7 have enhanced
sensor reactivation ability in the prg-1
background, which reveals some redundant
silencing ability mediated by PRG-1.
Nevertheless, the H3K9me mark of the sensor
was lost in the wago-9 and nrde-1 mutants,
revealing the importance of these two
proteins in the pathway.
Integrating their findings Luteijn et al.
(2012) suggest that RNAe can be separated
into two phases: initiation and maintenance.
Initiation of this pathway can be triggered by
21U RNAs or dsRNAs that will lead to the
generation of 22G RNAs (See Figure 5).
In the maintenance phase 22G RNAs
are still generated, but in a PRG-1/dsRNA
independent manner. Though the RdRP that
generates these 22G RNAs is still unknown,
the authors suggest that their biogenesis
might be associated with known RdRPs like
RRF-1 or EGO-1. The biogenesis of these 22G
RNAs seems to be dependent of MUT-7 and
WAGO-9. The latter is probably the AGO
protein in which the 22G RNAs are loaded. The
inheritance of the 22G RNAs is also dependent
on nuclear factors like NRDE-1, which has
Figure 5 – Schematic representation of the RNAe functional model suggested by Luteijn et al. (2012). PRG-1 or dsRNA initiate a response that
will induce 22G siRNA generation and heterochromatin silencing. On the other hand PRG-1 may also initiate an anti-silencing response. Taken
and adapted from Luteijn et al. (2012)
Ricardo Rodrigues 2012
20
Noncoding RNAs and Chromatin
previously been propose to have a role on this
kind of inheritance (Gu et al., 2012). From the
observations that mut-7 and wago-9 mutants
maintain the silencing activity for one
generation, the authors also deduce two steps
of RNAe silencing maintenance: MUT7/WAGO-9-independent and MUT-7/WAGO9-dependent steps. The first might be
associated with the chromatin changes in the
transgene locus that may be sufficient to keep
it silenced, while the latter may reflect the
necessity of re-initializing heterochromatin
formation, such as in S. pombe siRNAdependent heterochromatin assembly. The
absence of the RNAi nuclear machinery, such
as WAGO-9 and NRDE-1 (Guang et al., 2008)
would lead to the absence of these
heterochromatin formation sRNAs.
From these findings we can see that
RNAi, particularly the piRNA pathway, can be
used as a mechanism to protect the genome
against invasive sequences. Not only does this
pathway recognizes exogenous sequences and
silences them through RNAi mechanisms, it
also adds a second layer of regulation by
assembling heterochromatin in the exogenous
locus.
Concluding
perspectives
remarks
and
Cells need a fine tuned regulation of
gene expression and genetic programs, so that
they are able to maintain their homeostasis.
Multicellular
organisms
have
evolved
specialized cell types to carry out specific
functions, which gives them the need for a
greater tuning of their genetic programs. Gene
expression regulation through epigenetic
mechanisms opens a door of opportunities for
these cells to add layers of regulation to their
expression programs. Furthermore, epigenetic
regulation of gene expression has been found
21
in all layers of the genome part of gene
expression. Modifications in DNA and
chromatin have been found to be of great
importance for program fine-tuning. It is
therefore not surprising that cells evolved
upon using RNA, a highly dynamic molecule,
as a tool for a further improvement of
regulation networks. In this thesis we give a
few examples of how this potent molecule can
regulate chromatin and therefore gene
expression.
Long noncoding RNAs are a great
example of how in various ways RNA can
regulate gene expression. Here we emphasize
the silencing capacities of RNA-mediated
mechanisms, though lncRNAs have also been
found to activate genes (Wang and Chang,
2011). These opposing functions show us the
potential of ncRNAs in the cell. Furthermore,
crosstalk between the ncRNA pathways might
be possible. Here we describe the works of
HOTAIR as a scaffold for both LSD1 and PRC2
(Tsai et al., 2010). We also describe the
function of sRNAs in heterochromatin
formation in Drosophila and S. pombe
(Klattenhoff et al., 2009; Sienski et al., 2012;
Volpe et al., 2003). LSD1 and PRC2 are known
to interact with more lncRNAs in different cell
types (Khalil et al., 2009) and LSD1 is known to
regulate heterochromatin boundary formation
in both Drosophila and S. pombe. Therefore it
is possible that LSD1 may interact with
different lncRNAs to regulate heterochromatin
boundaries in these organisms and that it may
have a regulatory effect in the sRNA pathways
to define these boundaries. However further
studies have to be done to assess this
hypothesis.
Though lncRNAs are been shown to be
important in cells, the problems associated
with the studying these molecules, like finding
predictive functional motifs, limit the field.
This problem might be overcome through
more detailed studies of different lncRNAs.
Now that we know that high throughput
Cancer Genomics & Developmental Biology MSc Master Thesis
Noncoding RNAs and Chromatin
studies showed that these molecules are
highly abundant in cells, follow up functional
detailed studies of these elements should be
eminent. The further study of these molecules
not only will give us more insight of their
detailed work in the cell, but might also give
us a broader perspective of their common
features. The increase in lncRNA knowledge
might give the scientific community the data it
needs to create computerized tools that will
help identify lncRNA functions. As this is a new
field, a positive feedback loop of knowledge,
where the works in lncRNAs will help create
bioinformatic tools and the latter will help to
better understand lncRNA features, is still in
early stages.
Since the discovery of RNAi, small
regulatory RNAs have been found to be
important expression regulators. The cells
need to silence specific genes and the
capability of the RNAi machinery to identify
specific sequences makes these pathways
great tools for the cell to target specific loci.
Therefore the described examples of RNAi
directed heterochromatin silencing should
come as no surprise, as living organisms
usually evolve using the tools they have in
their “tool box”, rather than creating new
ones. Here we go through examples that
include single cell and multicellular organisms,
of RNAi pathways that can regulate
heterochromatin assembly and therefore gene
expression. In S. pombe a feedback loop of
heterochromatin formation directed by siRNA
has been described (Verdel and Moazed,
2005). In this organism, heterochromatin also
has a function in silencing heterochromatic
RNAs, through the HP1 ortologue Swi6 (Keller
et al., 2012). This shows us that there are
multiple layers of silencing mechanisms, as all
the potential for silencing by both RNAi and
chromatin is used when silencing a certain
gene. Interestingly, a similar pathway has
been found in Drosophila. In this organism a
similar set of proteins is used to silence TEs in
Ricardo Rodrigues 2012
the germline. It would then be interesting to
see if a similar mechanism of feedback loop
and
heterochromatin
functions
in
heterochromatic RNA silencing. Though some
steps in the pathway still need to be
identified, like how Piwi manages to lead to
H3K9me modifications in the targeted loci. If
this is the case this suggests that this
mechanism may be a common feature in
eukaryotes, though it may be both a
conserved mechanism that has evolved
differently and specialized by using the Piwi
pathway or rather a convergent evolution
where both organisms have “discovered” the
advantages of using RNAi as a tool for gene
specific heterochromatinization. Furthermore,
nuage, a subcellular perinuclear structure
associated with RNAi, seems to be affected
when heterochromatin components are
depleted (Klattenhoff et al., 2009; Rangan et
al., 2011; Sienski et al., 2012; Zhang et al.,
2012). Here we discussed that Rhino and
UAP56 are involved in shuttling transcripts to
the nuage, for piRNA biogenesis. These
transcripts might be needed to maintain
nuage integrity. In zebrafish, deletion of
Tdrd1, a protein that scaffolds Piwi proteins
and transcripts together for transcript
degradation, leads to abnormal nuage (Huang
et al., 2011b). In this sense the nuage
disturbances in rhi mutants and UAP65
hypomorphs might be associated with the
absence of transcripts that might be needed
for proper Piwi scaffolding. Under the same
hypothesis it would be interesting to see if
disturbing the nuage would lead to chromatin
alterations to see if the structure itself is
needed for chromatin silencing, or if this is
just a side-effect of Piwi defects. For instance,
disturbing dynein, previously described to be
important in proper nuage morphology
maintenance (Strasser et al., 2008), and
checking for altered H3K9me patterns in the
genome, might be an approach.
22
Noncoding RNAs and Chromatin
Interestingly, the usage of the Piwi
pathway as a tool for gene specific
heterochromatinization has also been found in
C. elegans (RNAe). This finding also suggests
that the usage of this pathway for chromatin
silencing is a common feature among
multicellular eukaryotes. Interestingly, HP1 C.
elegans ortholog HPL-2 deletion has been
found to be required for RNAe (see Table 1).
RNAe identified
component
Required for RNAe
maintenance
Gene Function
Ashe Shirayama Luteijn
Set-25
Set Domain
+
hpl-2
HP1 homologue
+
mes-3
Polycomb complex
+
mes-4
Trithorax complex
+
mut-7
3' to 5' exonuclease
+
+
nrde-1
+
+
nrde-2
+
+
nrde-3
Nuclear WAGO
-
+
-
nrde-4
prg-1
Piwi homologue
-
-
-
rde-3
Poly(A) polymerase
wago-1
Cytoplasmic WAGO
wago-9
Nuclear WAGO
wago-10
wago-11
+
+
+
+
+
Nuclear WAGO
+
-
Nuclear WAGO
+
-
Table 1 – Summary of known RNAe components and their requirement
for silencing maintenance. Since different studies have different
observations, they are discriminated in the table. (+) means required (-)
means not required.
The requirement of this protein might be
associated with similar mechanisms to the
ones mentioned before and/or for proper
heterochromatin assembly. If this protein
would have a similar function to the one of
Rhino, verifying the integrity of nuage in hpl-2
mutants might give us clues about if this
protein is also Piwi-pathway related.
Nevertheless, it would be interesting to see
how this protein is required for RNAe and
further testing is needed to understand this
mechanism.
In recent studies a large set of genes
have been found to be required for RNAe (see
Table 1). Though since this is a recent finding,
some players are still missing, such as which
23
specific RdRPs are involved in this mechanism,
or which proteins cause redundancy in the
wago-9 mutant phenotype. As C. elegans is
known to be an easy forward genetics model
organism, finding new players of this pathway
should probably go through this kind of
approach. Also to decrease false negative hits
due to redundancy, making these genetic
screens in other mutant backgrounds could
improve the screen sensitivity. For instance,
using the 21U sensor, mentioned before, for
screening in a wago-9 mutant background
could increase the number of obtained hits. As
the wago-9 mutant has an intermediary GFP
signal, lower than prg-1 mutation, a second
mutation might increase the levels of GFP,
which would be considered a hit. A problem of
this approach is that it can lead to missing
elements whose mutation is lethal or causes
sterility. A possible solution is to search for
hypomorphs mutations of RNAe components.
For instance, looking for temperature sensitive
mutants could increase the number of RNAe
elements found. Another RNAe element
searching approach could be isolating the
known
pathway
elements
through
immunoprecipitation followed by mass
spectrometry. Though, this approach has also
its disadvantages, such as raising antibodies
against these proteins or missing fast
interactions between proteins that will not be
isolated in precipitated complexes. For this
reason the use of the BioID technique (Roux et
al., 2012) might be a good option. In this
technique a BirA mutant protein is tethered to
a protein of interest. The mutant BirA has the
ability of biotynilating proteins in its proximity
without sequence specificity. This way, by
tethering this protein to a protein of interest,
it will be able to biotynilate proteins in
proximity of our protein of interest (Roux et
al., 2012), which may later be isolated through
streptavidin affinity purification and identified
through mass spectrometry. Applying this
technique to components the RNAe pathway
Cancer Genomics & Developmental Biology MSc Master Thesis
Noncoding RNAs and Chromatin
may further elucidate which is the machinery
used in this process. Furthermore, it would
also be interesting to see where the actions of
the argonaute components take place, the
cytoplasm or specific locations in the
nucleoplasm. To approach this, the technique
known as DamID (van Steensel and Henikoff,
2000) might be a good option. Through DamID
analysis of targeted proteins we would see if
they are all recruited to the same location in
the genome. For instance, by applying this
technique to PRG-1, WAGO-9 and CSR-1 we
could see if all these argonautes are recruited
to the same locations in the genome. If they
are recruited together, it is a good indication
that PRG-1 is recruiting WAGO-9 and CSR-1.
For the latter it would be a good indicator that
this protein is involved in the antisilencing
response.
In this thesis we can see that RNA is a
common chromatin influencing molecule and
that molecule should be seen as a key
chromatin regulator. Even though plenty of
work still has to be done in the field, we can
already unforeseen the implications of these
studies in biology and disease, as more and
more findings come up with new implications
of RNA in chromatin and gene regulation.
Acknowledgements
I would like to thank René F. Ketting
for accepting and supervising this thesis.
Furthermore, I would like to thank him for his
understanding and patience towards obstacles
in the making of this dissertation.
I would also like to thank Patrick
Wijchers for accepting to be the second
reviewer of this work.
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