Lecture 3: Transcriptional Regulation 1
- In the last lecture, we took a look at 2 chimps & we talked about the role that the transcriptome plays in giving
rise to the differences b/w those 2 chimps.
- Considered a problem with regards to the generation of that transcriptome, the diversity of RNA transcripts that
one finds in an organism that defines the organism itself, different tissue types, different cell types or different
cells in different points in time or under different conditions.
- Problem that we had to deal with is this: the challenge that the genome has to deal with – not such a big challenge
with procaryotes but big challenge with eucaryotes.
- Last lecture: considered a model that would allow one to accommodate all of those factors so that one could have
a tightly compressed genome within the nucleus but access it at appropriate points in time or in appropriate cells
to give rise to differences in the transcriptome.
- In circumstances where one does not need to access the info contained in the genome, it’s tightly condensed
within the chromosome – heterochromatin. But then when genes need to be accessed for the process of
transcription, they’re de-condensed, made into euchromatin.
Slide 1 - Take a look at the interplay again b/w genome organization & the regulation of
- The idea is that we have to access the 4 letter code in the DNA and the idea
that the chromatin has to be remodeled and exposed to allow for these RNA
polymerase to give them access to the chromosomes.
- Last lecture about transcription of RNA how they were made and now a little
more about regulation.
Slide 3 - How is this orchestrated? How is it controlled? How are specific regions of the
genome regulated in such a way that only those genes are expressed?
- Movie: organization of nucleosomes (octameric core comprising histone
proteins & their interactions with DNA). Wavy bits are histone tails. DNA is
going to interact with the histone proteins. Can see the tails whipping off the
end & waving out – that’s the nucleosome with the histone tails wagging off
around. Important point with regards to those histone tails: the 8 histones, pairs
of 4 different histones, present in the nucleosome core (2A, 2B, 3 & 4) have
tails that are accessible & can be post-translationally modified.
- The chromatin is remodeled for the purposes of gene expression, how is this
- Missing slide showing DNA that is going to be wrapped around histone
proteins. The histones have tails whipping around and what you see is
nucleosome with DNA wrapped around and then the nucleosomes are
assembling into the 30 nm chromatin fibre.
Slide 4 N terminal
Covalent (post-translational modification)
- There are 4 types of nucleosomes, the octamer all have tails.
- The histone tails can be chemically modified and can create their own code.
Slide 5 Amino acids
- The AAs that are susceptible to this modification are Lys and Ser residues.
Specific AAs in the histone tails are These specific AAs are substrates for post-translational modification where they
substrates for post-translational have particular groups added to them by a covalent bond.
modification - Can have acetyl groups added to the end of Lys. 1, 2 (di), 3 (tri) methyl groups
on the Lys as well. The acetylation & the methylation are mutually exclusive
events that occur at this Lys – that is, you have one or the other, or neither.
- To the OH group of Ser, a phosphate can be added to give phosphoserine.
- By these modifications & their specific location within the tail, you establish
the code of sort, a way of flagging things to say ‘here, this is a region that you
want to make a specific chromatin remodeling event’.
- Histones that you remember, they are high in two amino acids which are serine
and lysine. - The lysine can be methylated either mono di or tri or acetylated
- The charge once acetylated becomes neutral and in terms of DNA histone
interaction, it becomes weaker because
- This is a somewhat new idea where before you just thought histones packed
Slide 6 - Histone modifications can occur in either H2A, H2B, H3 or H4 – can see that
they may be comprised of either methylation, phosphorylation, acetylation or
ubiquitylation (generally occurs at carboxyl terminal end). Methylation,
phosphorylation & acetylation activities all occur at the amino terminal tail &
we can see the different Lys & Ser groups are susceptible to that post-
translational modification in the different histone proteins. These together
comprise what is known as the ‘Histone Code’ – a way of tagging histones to
indicate that specific sort of chromatin remodeling event should occur there
(like a flag – ‘make heterochromatin or euchromatin here’) – this is an
important concept in eucaryotic cells as a transcriptional regulatory mechanism.
- There are these specific types of modifications you see there are 4 types of
- Looking at the N terminus of the amino acid for H2A then H2B and you look
at all the modifications that occur on all these amino acids
- You can get methylation, acetylation and phosphorylation or adding a
molecule called ubiquitin
- Just know that all these modifications occur but is there some meaning in
regulation of transcription or structure?
Slide 7 - Example with Histone 3 – can see # of modifications that can occur on the
amino terminal tail. Note that dependent on the modification is taking place so
for example, methylation at K-9 (Lys) – indicates that heterochromatin should
be formed there (condensation of the chromatin) – as a consequence you get
- By contrast acetylation at Lys-9 (K-9) & methylation at Lys-4 results in gene
expression, similarity phosphorylation of Ser-10 & acetylation of Lys-14 gives
rise to gene expression – formation of euchromatin (decondensed DNA that can
be accessed by the transcriptional apparatus). Yet another example: methylation
of Lys-27 silencing a particular set of genes – X chromosome inactivation.
- Different modification will signal different types of things to happen, here are
a few examples and these are the ones known to happen on histone 3
- Below that are specific modification for whom the meaning have been worked
- Its known that on lysine 9, if it gets methylated its known that it makes the
chromatin to become heterochromatin so that it silences or reduces
transcription, heterochromatin formation and gene silencing
- In other things like acetylation on the lysine and phosphorylation makes the
DNA associated with those histones get transcribed whereas methylated K 27,
are silencing hox genes
- They determine your body segment that allow you to have arms coming out of
your torso instead of arms coming out of your head if you play with them then
you get flies with tails coming out of their head
- Gene expression for the phosphorylated S 10 and adenylation of K 14 is just
increased gene expression
Slide 8 - What is it that is reading the histone code? – protein modules that detect
specific modifications on the nucleosome – this is a so-called histone code
reading complex – it detects the covalent modifications that have occurred at the
amino terminal tail & as a consequence, recruit particular protein complexes,
catalytic activities & additional binding sites that are going to end up modifying
the chromatin structure – things like the chromatin remodeling complex, ATP
utilizing catalytic activities that converts one kind of chromatin to another.
- Got proteins that are able to read the code, recruit other proteins & together they function like a machine to either scrunch proteins up here or de-compress
so the gene expression can occur. So depending on the proteins that are binding
to the DNA, these are non-histone proteins, you get the conversion of one
chromatin type to another.
- That’s great you’ve got these modifications but how does this information get
used and also how do you get these modifications in the first place you have two
parts of the problem
- One part is who writes that code and who reads that code
- Notice how important it is that the tails are sticking out of the nucleosome and
there are specific proteins that bind to the histone tails and you see them all
assembled on a scaffold protein and they recognize a very specific grouping of
- Those come in and they are what reads the code and what they do is attract
other factors, maybe the general transcription factors that allow for transcription
to happen or chromatin remodeling factors allowing for less interaction with
- They lead to gene silencing or transcription or other biological function
- The histone code has important information that helps to regulate gene
Slide 9 - In this specific example, can see the spread of the histone code down the
length of the DNA so that now, the length of the DNA scrunches up to become
heterochromatin or become spread out to become euchromatin. So what
happens is a specific protein binds, it in turn recruits a histone modifying
enzyme (so-called “writer enzyme”) – these function then to recruit reader
proteins that then read the code and then recruit other writer proteins and so on
and so forth so that these reader-writer complexes read the code and hit the next
nucleosome & transmit the next code to it – have spreading of one chromatin
type vs. another down the length of the chromosome structure (chromatin).
- What often will happen is that there is a special gene regulatory protein that
will recognize a very specific sequence in the DNA
- The protein will bind to that sequence and what that will do is set up a whole
cascade of events and that is how we get the histone modifications
- We have regulatory protein attracting enzymes that will write information to
- As well there will be a code reader protein that will bind to the code that is
written and that may be linked to another enzyme that will write the code there
- What you have initially is that the reader gets recognized by regulatory
proteins and histone modifying enzyme brought in, then a code reader protein
comes in that modifies the next histone
- You get spreading of the code so that the histones around that area will have
the same information
- You see the reader writer complex so you get regions where you get these
information altering the transcription silencing or promoting transcription
Slide 10 - A protein has been recruited or has found a stretch of DNA – it in turn recruits
a writer protein that then modifies the histone that then you have a read-writer
complex & as you can see, as we go along with the chromatin remodeling
complex, we’re scrunching up the DNA – it started out nice and spread out
euchromatin then on the basis of these histone modifications, reading,
subsequent writing step then at the next nucleotide the signal gets passed along
so eventually the whole region is made into heterochromatin.
- You can spread heterochromatin just as you saw with the fruit fly eye, the
white gene was sometimes expressed or not and what happens in those cells, the
heterochromatin has spread to those genes shutting them down while in other
instances they are still being transcribed
- The reader writer complex and chromatin remodeling complex brought in and
with nucleosomes over to cause chromatin to be very highly condensed - Conceivable this can keep going over and over and shut down the entire
genome and what regulates that?
Slide 11 DNA Barrier
- Of course we don’t want this to proceed all the time – don’t want all of the
chromatin to be made into heterochromatin b/c we need to gain access to some
regions of it. Recall in the last lecture, looked at the white locus from
Drosophilus – the white locus was necessary to make red eye colour in
Drosophilus & recall there was a heterochromatin region right next to the white
locus (the white locus being the white gene that codes the white protein that
makes red pigment) – in b/w that heterochromatin region & the gene was a
barrier that stopped under normal circumstances the spread of heterochromatin
into the gene so the gene could be expressed. If there was a mutation such that
the barrier was now no longer blocking the spread of the heterochromatin, the
gene was made into heterochromatin & expressed, you got a white eye.
- The barrier functions to stop the reading of the histone code & make sure that
there isn’t spread of heterochromatin into regions that you want to retain as
- Here is an example of barrier DNA & an associated barrier protein that
recognizes that barrier DNA, stopping the spread of heterochromatin there on
the left by virtue of its association with the nuclear pore. Recall all of this is
taking place in the nucleus of the eucaryotic cell – the nucleus has pores that
allows transmission of info from the inside to the outside – that is transit of
RNA from the nucleus out into the cytoplasm & conversely, transit of proteins
from the cytoplasm into the nucleus but association of particular proteins with
that nuclear pore where the transit (of RNA from nucleus out into cytoplasm &
proteins from cytoplasm into nucleus) occurs function to stop the spreading of
the heterochromatin into the euchromatin region.
- Other proteins simply recognize a specific sequence & function themselves as
a barrier for this spread of the heterochromatin into euchromatin area – they
function rather passively & here we’ve got a protein that is functioning actively.
- There are actually DNA sequences called barrier DNA that prevent the
spreading from going too far
- There are different mechanisms postulated on how that barrier DNA words
- One example is that a barrier protein binds to the DNA sequence called the
barrier DNA sequence lie the nuclear pore complex (that allows stuff to go in
and out of the nucleus) but it would now act as a physical hindrance of the
- Or a large protein may just protect subsequence DNA
- OR it may acetylate the protein and prevent methylation on lysine 9
Slide 12 Sequence specific DNA binding proteins
- Who/what writes the histone code? – sequence specific transcription factors
that play an absolute crucial role in this chromatin remodeling – sometimes
these are referred to as gene specific regulatory proteins. The proteins bind to
specific sequences & then recruit the chromatin remodeling machinery.
- What are sequence motifs? A motif, when talking about a DNA motif, is a
series of nucleotides, the combination of A, T, C & G (whatever order they are
in). This is what specific sequence transcription factors bind to.
- Many people in the outside world call them transcription factors, they are
different from the general transcription factors that bring RNA polymerase in