While the human genome sequence has transformed our understanding of human biology, it isn’t just the sequence of your DNA that matters, but also how you use it! How are some genes activated and others are silenced? How is this controlled? The answer is epigenetics. Epigenetics has been a hot topic for research over the past decade as it has become clear that aberrant epigenetic control contributes to disease (particularly to cancer). Epigenetic alterations are heritable through cell division, and in some instances are able to behave similarly to mutations in terms of their stability. Importantly, unlike genetic mutations, epigenetic modifications are reversible and therefore have the potential to be manipulated therapeutically. It has also become clear in recent years that epigenetic modifications are sensitive to the environment (for example diet), which has sparked a large amount of public debate and research. This course will give an introduction to the fundamentals of epigenetic control. We will examine epigenetic phenomena that are manifestations of epigenetic control in several organisms, with a focus on mammals. We will examine the interplay between epigenetic control and the environment and finally the role of aberrant epigenetic control in disease. All necessary information will be covered in the lectures, and recommended and required readings will be provided. There are no additional required texts for this course. For those interested, additional information can be obtained in the following textbook. Epigenetics. Allis, Jenuwein, Reinberg and Caparros. Cold Spring Harbour Laboratory Press. ISBN-13: 978-0879697242 | Edition: 1
2.7 Noncoding RNAs - long noncoding RNAs introduction
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Do curso por The University of Melbourne
Epigenetic Control of Gene Expression
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Week 2 - Epigenetic Modifications and Organisation of the Nucleus
We’ll discuss the molecular mechanisms for regulating gene expression in some detail, from how the DNA is packaged at a local level, right up to how the chromatin is positioned within the nucleus. We’ll learn about the chromatin modifications implicated in gene silencing and activation, the role of non-coding RNA, and higher order chromatin structures. This week will provide you with a good understanding of the basic mechanisms that will help you understand the processes we discuss throughout the rest of the course.
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Dr. Marnie BlewittHead, Molecular Medicine Laboratory
Walter and Eliza Hall Institute of Medical Research
So, the final class of non-coding RNAs we want to think about are the long
non-coding RNAs. So, as you'd suggests what, from the
name, these are much longer than those short RNAs we're talking about before the
piRNA's or the microRNAs. And in this case, they're now more than
200 nucleotides in length. In addition, according to their
definition they are usually spliced. So, they undergo splicing like a regular
messenger RNA would. They are capped so they have a five prime
cap like a messenger RNA would. And then have polyA tail so they're
polyadenylated. They also have the same sort of promoter
as a regular gene would have. And so they're subject to expression in a
developmentally controlled manner. But what's interesting about them is they
tend to be or they should be constrained to the nucleus, at least predominantly.
And this is looked at by using a method known as RNA fluorescence in situ
hybridisation. So you look at where the RNA is within a
cell. Using the homology of a probe that
can bind to RNA in particular. And you want to see for long noncoding
RNA that the vast majority of the RNA is staying with the DNA in the nucleus.
So the numbers of RNAs that long noncoding RNAs that are found in the gene
and the estimates vary dramatically. And so perhaps there is 10,000 to 200,000 that
are found in mammalian genome. The vast majority of these found at very,
very low copy numbers. So there may be only a few copies
of these, these long noncoding RNAs in each nucleus.
So the question now becomes what are these long noncoding RNAs doing?
And are the ones that are found at such low copy really doing anything, or are
they perhaps just the result of transcriptional read-through, or
transcriptional noise in the genome. What's interesting about the ones that
have been characterised is that these long noncoding RNAs appear to regulate
epigenetic processes. And this is what's interesting for them
in relation to epigenetic control in this course.
So these long noncoding RNAs can regulate many different processes. And as I said,
there are a very large number of them. But there's a smaller set of them that
have been characterised in any detail. We're going to talk about Xist as well
as HOTAIR, and these two long noncoding RNAs have roles in X inactivation for
Xist and Hox gene silencing for HOTAIR. You'll recall X inactivation, the dosage
compensation mechanism in female mammals. It actually involves a very large number
of long noncoding RNAs. But, the prime example is Xist.
And this was indeed, the first long noncoding RNA to be discovered back in
1991. There are many more that have been
discovered since that have a role in X inactivation, including Tsix.
Genomic imprinting, which I introduced in the last lecture just with a couple of
sentences, this mono allelic expression of genes between that differs, or is based
on, the parent of origin of the gene. Also can involve long noncoding RNAs that have
been very well studied. And Hox gene silencing involved HOTAIR
which we'll talk about. This long noncoding RNA called HOTAIR.
So, Hox gene silencing we have a large number of Hox genes in our genome and these
are involved in setting up the segmentation through the body axis.
So, for example if you think of your vertebrae.
So each Hox gene needs to be on at a particular region, and then is switched
off in other regions. And this switching off involves this long
noncoding RNA called HOTAIR. Finally there are other, many other
processes that are regulated in, with long noncoding RNAs including DNA damage
response. So, what do these long noncoding RNAs
actually do? Well, you'll remember one of these
longstanding questions in the field of epigenetics that I brought up in the
previous lecture was, how are the epigenetic complexes
so, the complexes, the laying down of DNA methylation or histone modifications, or
that are moving around the nucleosomes, the chromatin remodellers, how are these
actually targeted to specific sites in the DNA?
We know that most sites actually don't have much DNA binding specificity.
so your most complexes don't have the DNA binding specificity all of their own.
So we also know there are transcription factors, these factors that that would
bind with RNA polymerase. And they can provide some sequence
specificity. However, the sequence specificity that's
provided by a transcription factor is, tends to not be, particularly high.
So they may recognise maybe a 6 base recognition sequence or even a 12 base
recognition sequence. But if you calculate the number of times
that these 6 or 12 base recognition sequences can be found in the mammalian
genome. It doesn't allow for unique targeting of
any complex but rather there'll be thousands of these sites spread
throughout the genome. So finally we also said that we can, the
chromatin remodelling complexes, for example, will be targeted based on
previous histone marks or histone, they will bind to histone, other histone
marks. And we know also there are some
epigenetic complexes that lay down a histone modification that also bind a
previously made histone modification. But again this doesn't allow for that
specificity. We don't get to come down to just a few
loci, or one allele in by using just these mechanisms.
So, the features of long noncoding RNAs somehow allow them to direct these
epigenetic complexes, at least in some instances that we know about at the
moment. And they appear able to do this in cis,
so in other words. On the chromosome from which they are
transcribed. Or in trans, so acting somewhere else.
They do this amongst other functions. There are many functions that we'll come
to, and we're just going to go through these ones that are concentrating on them
directing the epigenetic machinery. So, what are the features that allow them
to be involved in this targeting, and especially to allow this
allele-specificity? Well, because they are transcribed.
While they are being transcribed from a particular locus from a particular
chromosome they are still tethered there. They're still tethered to the place from
which they're being transcribed. So this allows for that allele
specificity. And it also shows how they might act in cis.
Now the reason they can have provide specificity that's greater than that
of a transcription factor is simply because of their length. Because of the
length of the molecule, the length being over 200 nucleotides in
length and in fact most long noncoding RNA is about which we know something at
the moment a several kilo-bases or tens of kilo-bases in length.
So this means thousands of bases, of course.
This really gives unparalleled site-specificity to these long noncoding
RNAs. But it's important to note that this
specificity and to the allele or specificity based on the
sequence is not necessarily always required for their function.
So what are some of those functions? What are some of the mechanisms by
which they work? The two that we'll focus on are long
noncoding RNAs acting as guides, and the example that we like to talk about here
is Xist. We'll also talk about how HOTAIR acts as
a guide. The difference being, that Xist acts in
cis, whereas HOTAIR acts in trans. And I'll go through this in more detail.
We know that these long noncoding RNAs can act as scaffolds.
And again HOTAIR is the example here. But in addition to these two mechanisms,
there are many others that long noncoding RNAs many other mechanisms by which long
noncoding RNAs can function. They can act as decoys.
They can act as signals, and what this means is that it's actually the act of
transcription itself running through a locus, which has the function rather
than the product, rather than the long noncoding RNA that's produced.
They can act as enhancers and this is has always been shown to work in cis.
So, you can have transcription at one point which alters transcription nearby.
And they can even act as reservoirs. And in this context, H19, which is one of
the best characterised long non coding RNAs, acts as a reservoir of a micro RNA.
So it can sit around being a stable long noncoding RNA, that can then be
processed to release the microRNA that it contains.
So, while there are so many different ways that these long noncoding RNA's can
actually work, there is a common feature. And that is that there's the formation of
an RNA protein complex. And an, this RNA protein complex is able
to influence gene expression. So I'll just briefly describe by what I
mean when things act as a guide versus a scaffold and then in the
next lecture we'll go through an example of each of these.
So as a guide what I mean is that this epigenetic complex which I've drawn here
will bind to the noncoding RNA. The noncoding RNA is drawn in red.
And so we have this long non-coding RNA coming along.
And it is tethering an epigenetic complex to, a particular region.
The reason that it's site specific is that there is, through here, this
sequence identity, which is allowing this RNA to bind to the
DNA in a sequence specific manner. In other words, the long noncoding RNA is
guiding the epigenetic machinery. This epigenetic machinery can then
participate in whatever it normally does. In this case, histone methylation.
So in this way one complex is being guided to a specific site in the DNA.
However when these long noncoding RNAs act as a scaffold now, rather than just
recruiting one complex, they tend to recruit more than one, in this case two.
So different regions of the long noncoding RNA. So, this region here, or
the region down here, combine to two different epigenetic complexes.
And so in this way they act as a scaffold.
They unite two complexes so that they are tethered to one another, and act as a
molecular scaffold to bring epigenetic complexes somewhere else.
This scaffolding may not necessarily require that their
long noncoding RNAs provide sequence specificity.
But, you have then got tethered together these two different complexes which are
then able to act on chromatin and at the same location tethered by these
scaffolding long noncoding RNAs. So in the next lecture we'll talk about
examples of each of these cases and go through their particular functions.
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