Lecture 7: Eukaryotic Transcriptional Regulation 2
**Lecture 7 begins**
- Today we’re going to start to move forward, going from where we
were, showing the interplay b/w chromatin regulation, the regulation
of transcription, and now finally getting around to making proteins
and the regulation of translation – turning it on and turning it off.
- Gene expression actually just starts with chromatin & actually ends
ultimately with the proteins that interact together.
- Need to consider the multitude of different factors that function to
regulate transcription itself & modify chromatin structure.
- Movie: Modifications can occur at different steps: capping
enzyme, putting 7-methyl-guanine one, we’ve got splicing that can
occurs (introns being removed), poly-adenylation occurs &
eventually have migration out of the nucleus out into the cytoplasm
& now the events involved in translation & the control thereof.
Transcriptional regulation includes insulators/barrier sequences and
gene regulation (initiation of transcription involves) regulatory
- Insulators & barrier sequences are important for defining zones of
gene regulation, making sure that where gene expression occurs is
going to be euchromatin & where gene repression occurs is
heterochromatin. The 2 zones are effectively, to a good extent,
isolated from each other.
- Insulators function to make sure that genes function as discrete
units, that is, they block the activity of DNA motifs known as
enhancers – this is where transcriptional activator proteins would
bind to these enhancer sites. Insulators prevent the info from the
enhancer to spread to the next gene.
- Diagram: we’ve got enhancer that’s going to function to turn on
gene B once the gene regulatory protein is bound – and we don’t
want that binding necessary to influence the expression of gene A.
Consequently, an insulator element, a segment of DNA, occurs b/w the enhancer and gene A so only gene B is regulated by the enhancer
– ensures that each gene is a discrete regulatory element in and of
- Diagram: we’ve got a heterochromatin domain on the DNA near
the end and the barrier sequence is functioning to prevent it from
spreading into gene B, silencing gene B & making it inactive. Saw
this with the example of the white locus – saw barrier sequence
preventing spread of heterochromatin normally except in white
mutants where it gets flipped around.
- Insulators function to ensure (and barrier sequences do the same
actually) that you’ve got regions that are structural (heterochromatin
vs. euchromatin) and functional (active and inactive).
- Diagram: the fact that insulators function to create such domains is
very nicely shown in the diagram. Here we’ve got a chromosome
that has been stained so we can effectively see the compression of
the DNA & those regions of the DNA that are highly condensed are
in a nice bright red. Below what you’re looking at is an anti-body
that has been made that recognizes proteins that bind to the insulator
regions and makes them function as insulators. That anti-body has
been tagged with a fluorescent green dye so wherever the proteins
are that bind to the insulator, the insulator binding proteins are green
showing that they function to divide the genome up into these
different domains of condensation and de-condensation or
heterochromatin or euchromatin or active and inactive.
- It turns out that such info with regards to what regions of the DNA
are condensed or de-condensed can be transmitted from one cell to
its daughter cells. So when mitosis occurs, that info is carried
forward to the daughter cells. How does that actually take place?
There are 3 mechanisms (there is a 4 one that will described in a
1) Histone modification: here we have active chromatin and now we
have inactive or heterochromatin that’s been made and in the
daughter cells, this heterochromatic state is going to be maintained,
that is, that region of heterochromatic DNA in the daughter cells is
also going to be heterochromatic.
2) Positive feedback: a protein is not made in this particular cell but
now in the next cell the protein is made, and b/c the protein is made
and will be present in that cell when it divides, the protein will
function in the daughter cells as well & if that protein itself is active,
to activate its own gene, it will perpetuate the activation of that gene
in daughter cell after daughter cell.
3) DNA methylation: here we have unmethylated DNA regions that
become methylated as shown by these little red dots & this new
methylated state is now effectively transmitted to the daughter cells.
- Note that he’s saying daughter cells, not mother and daughter cells
– talking about mitosis, not talking about the products of sexual
reproduction where something different occurs. This mode of
transmission that is mitotic where daughter cells inherit the gene
expression state that their mother’s have is known as epigenetic
inheritance. Note that it does not involve a change in any of the
nucleotide bases in the DNA but what has been inherited from the
mother cell to the daughter cells is the activity of that DNA region,
either active or inactive. - Inheritance through the somatic cell line: cells that make up
everything but the reproductive tissues in our body – the inheritance
of the info from mother cell to daughter cell.
X chromosome inactivation
- Recall we inherit one set of chromosomes from mom and one set
of chromosomes from dad. Women differ from men in that women
have two X chromosomes and males, their 23 pair of
chromosomes, is an X and Y.
- There’s 2 ways of looking at that: how is it that men only get by
with only one X chromosome? Or how do women contend with
having 2 X chromosomes? From a biological perspective, having 2
X chromosomes is the more difficult thing to deal with so as to
equalize effectively the dosage of X b/w males and females, what
happens in females is one of the X chromosomes, either the paternal
copy or maternal copy is silenced – and it’s silenced by condensing
it into a highly heterochromatic chromosome.
- Dosage compensation, that is, to make sure that women effectively
only have one dose of X is manifest by shutting down one of the X
- At random, either the paternal or the maternal copy is shut down &
depending on whether the paternal or maternal copy that is shut
down, what ends up happening as a consequence is that all the cells
that are derived from that cell, for example where the paternal copy
is shut down inherit that state of the Barr body residing in the
paternal copy of the X chromosome. Whereas the other lineage,
where the maternal copy has been shut down, it’s now highly
condensed, that is transmitted from one cell to the other.
- So the net result of that is that as opposed to men, which are with
their single X chromosome, they are, in terms of their X
chromosome, they are just one great blandness – it’s only the one X
chromosome that they inherited by their mom that is going to be
expressed. Women, by contrast, are a mosaic – they have the X
chromosome from mom and the X chromosome from dad – some
tissues, some organs will only be using the X chromosome from
mom, others the X chromosome from dad – a mosaic.
- How does this shutting down of the X chromosome actually occur?
- Non-translated RNA: This is an RNA that begins and ends its life
as an RNA – never is translated into protein.
- Hypoacetylation – decrease in acetylation, resulting in
heterochromatin formation (condensation of chromatin). C-G (cytosine-guanine)
- Diagram: we have an unmethylated cytosine (see C-G pair in
diagram) and there is a methylated cytosine at another pair. Upon
DNA replication (recall it’s semiconservative), we’ll end up with
one of the methylated strands up here and a non-methylated strand
& the other methylated strand and a newly synthesized, non-
methylated but these are recognized by the maintenance
methyltransferase so that what is transmitted to the daughter cell is
something that is not hemi-methylated (half-methylated) but totally
methylated at that C-G dinucleotide & that’s passed on from cell to
cell (that info).
Epigenetic – not transmitted through genetic mechanisms, it’s “in
addition to” or “over top of” genetic inheritance.
- Diagram: What we have occurring is the transmission of
methylation through reader-writer complex as we’ve seen before –
methylation being transmitted down the length of DNA, such that by
the time that we get to the bottom, we’ve got a purely
heterochromatized region that has been locked in this silent state.
Again, this does not involve any changes at all to A, T, C or G
themselves outside of the fact that methylation occur – no actual
change in the DNA code.
- When gametes are made, these signals are all wiped clean.
- The epigenetic changes can create differences in highly related
individuals – even monozygotic twins (identical twins with the same
genetic material), by the time that they are older, despite the fact that
they may still look identical, have diverged dramatically in terms of
their epigenetic state so that things are very different in the future.
- Monozygotic twins, that is identical twins, twins that were derived
from the same zygote, from the same fertilization event that when
that zygote was cleaved & those genetically identical individuals
began their individual trajectories in life, that despite the fact that
they are genetically identical, there is something different about
them in the molecular sense. **Finishes here at the end of the lecture 7**
**Lecture 8 starts here…**
- Last lecture: we’ve been covering an elaboration of the central dogma of molecular biology, working from
DNA to RNA to protein. We’ve been talking about the organization of genomes, the interplay b/w that &
the transcriptome & finally we’re moving on eventually to proteins, just starting to touch on translation.
Recall: we were talking about the complexities of gene expression & the regulation thereof in eucaryotes –
saw how there are multiple steps at which gene expression can be modified, taking gene expression in the
broadest sense to mean the conversion of DNA info to corresponding protein info – so a # of steps that can
modified along the way, each of them regulated.
- Then we started to work through a list of different types of regulation along this pathway. Finished up with
transcriptional regulation by insulators & barrier sequences & we finally finished on DNA methylation and
this concept of epigenetic inheritance – the idea that what can happen in a mother cell, mitotically speaking
can be transmitted to daughter cells – this does not involve change in DNA sequence but rather the way in
which DNA is used, be it in the euchromatin vs. heterochromatin state and the relationship of that to DNA
- Implications: Epigenetic changes can create differences in highly related individuals.
- Recall 5-methylcytosine occurs at those C-G sites where
methylation is going to occur.
- Acetylation of H3 & H4 is going to bring about differences in the
chromatin state where those histones are localized.
- 2 different experimental approaches that allow them to take a look
at 5-methylcytosine content in the 1 instance and in the 2 instance,
the acetylation states of H3 & H4.
- In the 1 case, there is very little difference b/w 3 year-old twins in
the global 5-methylcytosine levels. By contrast, when we look at 50
year old twins, there is significant difference b/w one twin and the
- Similarly when we take a look at the acetylation of H3 – no
difference effectively b/w young twins, significant difference b/w
old twins. Same case with acetylated histone 4 –