[Instructor] Hello and welcome to module three,
this is the first lecture
and we're going to be talking about
gene expression and regulation.
This is definitely going to build on
what we learned in module two.
So if at any point you feel you might wanna go back
and read through the materials from module two again,
just revisit, especially if it's been a few days,
you might just want to refresh yourself there.
Maybe even just go back and look at the
assignment, the definitions that you did for module two,
or even have that sitting right next to you so that
if there's a word that pops up
that I start to use more regularly
that you just learned about in module two,
you can take a peek and know what it is I'm talking about.
So let's start with just a quick review
of what we learned in the last module.
DNA is located in the nucleus and a segment of DNA
that gives the instructions for making
one particular protein is called a gene.
Genes are transcribed in the nucleus
into complementary mRNA sequence
and that mRNA sequence moves out of the nucleus
to the cytoplasm, where ribosomes read the sequence
and assemble amino acids
in the correct order to create the protein.
And this is called, the process is called translation.
And if you remember this whole process of going from
DNA to being copied into RNA,
which then gets read and translated into protein,
that process was worked out as the central dogma,
which we talked about in module two.
Well, now we're going to be talking about
how the cell actually regulates on many different levels,
how it can regulate that process.
In particular, the process of transcription.
So let's first talk about a few important definitions
that I'd like to make sure we all have clear
before we move forward.
When I say gene expression,
this is the process by which
that information encode in a gene
is used to directly assemble a protein.
So the expression of a gene is basically going from
the gene sequence to having a protein
at the end that is needed.
And gene regulation is the actual process of modulating
gene expression.
So the extent to which a gene is expressed
as in turning it on or off or up or down.
And that usually occurs at the level of the mRNA.
So either how much of it is swarmed
or what version of it is actually used for translation,
which we'll talk about in this lecture.
Constitutive gene,
so these are genes that are transcribed all the time.
Facultative genes,
these are genes that are transcribed only when needed.
So let's pick up a metaphor here, right?
You know, I love my metaphors. (chuckles)
So what I'd like to use for this metaphor,
let's think about the cell as your house, let's say, okay.
And in your house,
there are basic functions that always need to occur,
always need to occur.
These would be like the
functions that constitutive genes that
their proteins would address if this were a cell.
Let's say in your house, you always need electricity,
you always need plumbing, you need water supply,
you need the basic roof over your head,
walls, floor, that kind of thing.
The proteins in a cell,
which perform the functions that are needed
for just every day run of the mill,
keeping the cell up and running,
those are constitutive,
those are encoded by constitutive genes,
which means they are always being expressed
because they're always needed.
No matter what's going on in the cell,
it always needs to maintain its membrane,
so basically keeping its roof over its head.
It needs to maintain its basic metabolism,
so kinda like the electricity going on.
It needs to maintain the proper influx and efflux
of materials like basically getting rid of waste,
sort of like in plumbing,
you always need the water coming in and water going out.
Those are things you need all the time,
no matter what's going on, really.
Let's think about specific functions that might happen
or might be needed in your house at certain times
but not in others.
Let's take today, for example,
it's really cold outside, right?
It's freezing cold outside,
this is January and it's Vermont, so it's gonna be cold.
But as a result, I have my heat turned all the way up
in order to keep it warm enough in my house
because it's really cold outside.
So in response to the environmental condition
of it being cold outside, I've cranked my heat way on up.
Let's assume it's actually August now
and it's 95 degrees and a hundred percent humidity,
which is really hard for me to imagine
what that feels like right now 'cause it's so cold.
But let's say, let's just think of a time
when that actually does happen
and it will happen again in August, let's say.
Instead of having my heat cranked all the way up,
I want my heat off and I want my air condition
cranked all the way up.
So in my house, I don't always have my heat on,
I don't always have my air conditioning on.
In fact, it's very important sometimes,
like if I need, like on a day like today,
it's very important I have my heat on
and my air conditioning off.
And when it's really hot outside,
it's important I have my A/C on and my heat off.
This is very similar to facultative genes.
So in the case of facultative genes,
these would be genes that encode proteins
that are only needed in response to certain things going on
in and around the cell.
In this case, in this particular metaphor,
turning the heat on would be like turning on
genes that would be responsive
to those particular environmental conditions.
And you want to be able to turn them on
and turn them off when you need them.
Coding sequence, these are segments of DNA or RNA
which give instructions to ribosomes
for proper formation of a protein.
So these are the regions that contain those codons,
remember what a codon is, a triplet of bases,
so three bases in a row
that encodes, that provides instructions
for one specific amino acid.
Coding sequence would be
like a long stretch of these codons,
all of which provide instructions for in total,
all of which provide instructions for making one protein.
Non-coding sequences,
these are segments of DNA or RNA sequence
which do not give instruction to ribosomes.
Some of these sequences have other functions
like regulating expression of genes.
And I mentioned before that
something only like 2% of our genome,
of all of our DNA sequences is genes,
so the remaining 98%,
you might hear some people calling that junk DNA,
it's not actually junk. (laughs)
It's really, if you think about it,
why would we as a species keep that much junk around
and go to a lot of trouble to replicate that junk
every time our cell divides and every time
we produce a new offspring.
We wouldn't and we don't.
That 98% is not just junk.
While it doesn't code for a specific protein necessarily,
what it does do is it regulates
how, when, and in response to what the 2%
and so the coding sequence, the 2% of the genome,
those genes are expressed.
So the 98%, I mean most of it,
we don't actually know what all of that 98% does,
but a good portion of it, we have come to find out,
a good portion of it is involved in regulating
the expression of that 2% of the genome,
which actually encodes for proteins.
So cells, as I talked about in that metaphor,
cells must respond to changing environment,
growth, signals from other cells and tissues,
developmental timing, anything that's going on
in and around it.
And not all proteins are needed all the time
or even good to have around all the time.
So what in the end, can we boil this down to one problem?
Yep, the problem is that cells
must be flexible and responsive to their environment,
yet receive no new genes after the time of conception.
So at the time that the egg and the sperm fuse,
you have your fertilized egg,
you have all the DNA that you're ever going to have.
And basically, I mean
all the different DNA that you're going to have,
the cells will replicate and divide
and will duplicate that DNA
but won't make exact copies of it.
So it's not as though in say a kidney cell
compared to a heart cell
you have different genes or different DNA, no,
all of it is still all there,
it's just that some of those genes
are expressed specifically in heart cells
and some genes are expressed
specifically in say kidney cells
or liver cells or skin cells,
which give them their specific qualities and functions.
So what is the solution the cell has come up with
to address this problem?
Certain genes will be turned on
and certain genes will be turned off.
And the genes that are turned on
are those that are needed at the time
and those that are off or not expressed
or not transcribed are those genes that are not necessary,
they're proteins that they code for are not needed,
those functions are not needed in the cell,
are not desirable in the cell at that particular time.
They can be turned on later if they're needed,
but for now, for the particular situation,
they can be turned off and the other genes that are needed,
their proteins are needed, are turned on.
So in which stages is this actually occurring?
Well, if we look back at our DNA to mRNA to protein,
we have transcription,
which is the process of copying DNA to mRNA,
translation being the process of reading mRNA
and assembling the correct amino acids for protein.
Most of what's actually occurring
in gene regulation is actually,
most gene regulation is actually occurring
at the transcriptional level.
So this is at the level of
forming mRNA or what basically with the end result
of that mRNA going into being translated.
Let me make my disclaimer again.
So this is a very complicated,
complex subject of gene regulation,
which can and does take
entire lifetimes of careers contributing to this,
including a lot of my own,
so I'm just really gonna give you the highlights here.
There are many other types of gene regulation
that we're not going to cover,
which may be specific to a certain subset of genes
or this or that, but I'm really just gonna again,
majority rule, just gonna give you
the main ways in which this occurs
and if there's something specifically
that we don't talk about
that comes up for you in your practice
or something you came across and you want to discuss it,
I'd be more than happy to talk with you about it.
But with the goal of really just focusing
on what I think will be most useful to you,
I'm really just going to give you
the sort of the primary ways in which genes are regulated.
And that would include at the transcriptional level
transcription factors,
which we'll talk about momentarily,
in the presence or absence
of these transcriptional activators or repressors
and epigenetic modifications,
which include DNA methylation and histone modification,
which affect early access to particular genes.
And then it also occurs at the level
of post-transcriptional regulation.
And that means once you formed your mRNA,
so transcriptional regulation is
in the process of actually forming mRNA,
post-transcriptional regulation
would be what happens to mRNA after it's transcribed,
but before it's translated.
So this would be called post-transcriptional.
And what we'll really focus on here are splicing variants,
which I'll tell you what that means shortly,
but there's also another way that it can be regulated
through stability of mRNA,
but we won't really focus on that here
because I feel like that gets into
a little too into the weeds for what we need to focus on.
But if ever you want to talk about
or you hear about something called microRNAs
and you want to talk about that more,
I'm more than happy to do it, but for the purpose of this,
let's just kind of glaze over that for now.
All right, a little more detail on transcription.
A promoter is a region of sequence upstream
from the start of a gene
used to align the transcriptional machinery
to start transcription.
This is sort of like a staging area.
So when I say a promoter is a region of sequence upstream,
what do I mean by that?
Well, if you recall the specific order in which bases align
is actually very, very important
and that gives the specific codons which codes for
the amino acids which are used
and this occurs linearly, right?
So it's sort of like basically like letters forming a word
that would be a linear arrangement of letters.
So you have one letter,
then the next, then the next, then the next.
Similarly with bases you have one base,
then the next, then the next, then the next,
left to right, you can think of it that way.
So upstream, when we say upstream, that means
in that same strand of DNA, it's basically
kind of before the start of that sequence.
So along the line of that sequence,
you can think of it as
before you reach the first codon, right?
What's the start codon,
if you remember it is ATG, right that,
that codon is ATG, which encodes for which amino acid?
Methionine, that's right.
So ATG, which is the start.
So upstream of that, so before that sequence,
along the same strand of DNA, you have other sequence
which would be part of that non-coding, non-coding sequence.
It's all attached and all part of the same strand of DNA,
but before you reach that
is a region of DNA called a promoter.
This is not code for a protein at all,
nothing in there codes for protein, so it's non-coding,
but it is necessary for the proper alignment of the
transcriptional machinery,
which is used to transcribe that RNA polymerase
and other factors which are used to
actually make the mRNA molecule off of
one of the template strand.
In facultative gene specific transcription factors,
which are highly specialized proteins,
are needed to bind to the promoter
to enable proper alignment of transcriptional machinery
and for transcription to start,
we'll look at that in just a moment.
If those transcription factors are not present,
the gene is not expressed or not expressed as much,
and genes use different transcription factors
for the regulation.
For example, if we were to take, say
the gene which encodes for the protein insulin,
the transcription factors which are used,
which are needed for it to bind to its promoter
and therefore initiate its transcription,
are different than the transcription factors
which bind to the promoter for say
a gene which encodes a protein
that is involved in say, cell proliferation.
So something like the CD44 gene,
which encodes for the CD44 protein,
which is necessary for regulating cell proliferation.
Its transcription factors are very different
than the transcription fact,
it use a different set of transcription factors
than the transcription factors which are needed
for the expression of say the insulin gene.
And that's important because you need the insulin gene
in response to very different external stimuli
than something like the CD44 gene,
which is only going to be needed
when the cell needs to proliferate rapidly.
All right, let's take a look
at what it is I'm talking about.
So here what we're actually looking at is a
sort of a pictographic representation of a gene,
which you might see quite often.
It's really just for simplicity's sake that I am
indicating all these different components in this way.
You don't think of anything
as actually being shaped this way
with the exception of a DNA strand is
kind of linear like this.
But basically we're looking at a DNA strand.
So it would be a sequence,
you would have a sequence of bases,
C-T-G-A-G-G-T-C-A, whatever.
You know you have some combination of A's, T's, C's, and G's
and a single line along here, so let's say then
if this is your gene, if this is the start of your gene,
it would read ATG as your first codon,
then it would have whatever your next.
whatever your next bases are in your gene,
C-T-A-G-T-A-A-A-C-T, it goes along long, long, long.
And this would be the coding portion,
so this is a gene, so it encodes for protein.
But it is, again, it's a part of a very long
continuous DNA strand.
So you have sequences which would be upstream.
So when I say upstream,
it's usually depicted to the left of the start of a gene,
if this is where we're showing the gene starts here
and the gene we're depicting in this rose color,
then upstream of that, you still have sequence,
I mean it's one long continuous DNA strand.
It's just that this particular part of it starts,
this is the start of a region
and it would continue on through to the end
or a stop codon would be in here somewhere.
And the DNA sequence continues on past the end of that gene,
right, it continues on past,
it's just not a part of the gene, okay.
So when we say upstream,
this would be upstream of a gene group,
this would be downstream of the gene.
So kinda like left is upstream, right is downstream
as is sort of how it's commonly drawn.
All right, this yellow region here
is what we would consider to be say the promoter sequence
of this gene and it's just, again,
it's just a part of the sequence.
So if this is where we start with ATG,
then you can think of this part of it, just again,
it's just some, it's just some sequence.
So C-T-G-A-G-G-C-T-A-G-G, whatever, you know?
It's some combination of bases upstream
of the start of the gene.
And this is considered,
would you call this coding or non-coding?
It's non-coding, right?
Because it's not actually a part of a gene,
it doesn't give the instructions for a specific protein
or amino acids for a protein.
What its function is to align the machinery
needed to transcribe this gene here.
And what do I mean by that?
Well, what's needed is, if you recall,
I mentioned transcription factors,
these are proteins which bind to specific sequences
along the genome
and these sequences are oftentimes found
in promoter regions,
so just upstream of the start of a gene.
And why does that matter?
So what happens is transcription factors will come in
and just through sort of diffusion,
they'll find their way into these sequences
and they'll bind to it, so they'll stick through
chemical bonding, they'll actually bond to, stick to
the DNA but in very specific sequences, so let's say
I'm depicting two different transcription factors here.
Let's say that this transcription factor in white
binds to C-C-A-G-G sequence,
oh, it found a C-C-A-G-G here, it binds there.
Let's say this transcription factor binds to T-T-A-G-T-C,
it found a T-T-A-G-T-C sequence here, and it binds here.
Once those two have bound in this particular arrangement,
then that allows something called a scaffolding protein
to come in, which is depicted with this pentagon here,
not actually what it looks like, but you know,
just for simplicity's sake drawing it that way.
Scaffolding protein is another kind of protein
that will bind to transcription factors
that are bound to the DNA.
So when the transcription factors are bound
in their specific correct arrangement
onto a promoter sequence,
a scaffolding protein can come in and bind.
And why is that needed?
Well, it helps to align RNA polymerase in the right location
at the very beginning or start of the gene.
So that way the RNA polymerase can go along
and can actually transcribe the gene into mRNA, right?
So this is another common depiction
of transcription is and it's not particularly creative,
but it is efficient in looking at it.
Transcription is usually depicted from the start of the gene
as a line going up
and then a line going to the right to demonstrate
the direction in which transcription occurs.
Transcription always occurs from the start of the gene
to the end of the gene.
And if transcription is occurring in this gene,
it will be demonstrated by an arrow going left to right,
coming up from the start of the gene
and continuing to the end of it.
All right, so that's what happens
if the transcription factors are present
then everything else, which we'll talk about
what everything else means momentarily,
everything else is conducive to the gene being transcribed,
you will get the mRNA being formed
and that mRNA can then go on to be translated into protein.
Well, let's say the transcription factors are not present,
what happens then?
So if the transcription factors are not present,
if they're not binding,
then the scaffolding protein
has no idea where it's supposed to go, it can't bind.
So while it's diffusing around in the nucleus,
it doesn't bind there because it doesn't have those
transcription factors to stick onto.
So as a result, RNA polymerase comes in
and woo, I don't know where I'm supposed to go.
So it doesn't actually transcribe the gene.
This means the gene is what we would call off, it is off.
mRNA is not being formed, it is not transcribed,
it's protein is not being made.
All right, so let's think about
the second form of transcriptional regulation,
which I mentioned are epigenetic modifications.
So this is a real up and coming field
epigenetic modifications
and I wanted to at least scratch the surface of this
so you have an idea of what people are talking about
when they talk about this because this,
I'd say over the next five to 10 years is going to
really start to dramatically change the way that
drug development occurs, diagnostics occur,
it's going to be really the next Human Genome Project.
So we know now where really all the genes are,
we understand the sequence of the DNA.
Epigenetic modifications is really like
a sequence on top of our DNA sequence.
And I can talk about that in more detail with you
if you are interested in more on epigenetics,
but really just to give you the basics of it
so you know what we're talking about.
When we say epigenetic modifications,
these are heritable and changeable modifications
and affect gene expression,
but not the actual sequence of DNA.
When I say the sequence of DNA, that's the order of
A's, T's, C's, and G's that are found.
So it doesn't actually change that,
but it does change how genes are expressed
and certainly can have a huge impact on that.
There's two main classes of epigenetic modifications
that we'll talk about in humans at least.
Those include DNA methylation and histone modifications.
So if we start with DNA methylation, what is that?
It's basically adding a methyl group onto cytosine.
So just cytosine,
not the A, not the T, not the G, just the C.
So only the C can be methylated, again, in humans.
So, which is really, that's kind of what we care about here.
So in DNA methylation, what occurs,
again, just showing you the chemical structure
of the cytosine base, remember as a nucleotide
it's attached to the sugar phosphate backbone,
but here we're just looking at the base of cytosine.
A methyl group is attached into the carbon ring,
just sort of on the outside of the carbon ring here
to form 5-methylcytosine.
So it still base pairs with guanines,
so C still binds with G across the two strands of DNA
even if the cytosine is methylated,
it can still bind to its other strand.
What can't happen,
what's different about it when it's methylated
is that transcription factors that would bind
to a specific sequence that contains a cytosine.
If that cytosine is methylated
most of the time, pretty much all of the time,
that transcription factor can no longer bind.
And you can start to imagine
what the implications of that would be.
Basically means that if a promoter region,
if the cytosines in a promoter region for a gene
are methylated, that shuts off
the transcription of that gene
because you can no longer bind transcription factors
to that promoter region.
The protein or enzyme that's responsible
for this process of DNA methylation
is called DNA methyltransferase.
It's actually has a very nice and descriptive name,
many of proteins do.
So if we look at this, if we break down this word,
transfers methyl onto DNA.
So it's a DNA methyltransferase or DNMT for short.
And I bring this up because
we will probably touch on this when we are talking about
certain genetic diseases which involve
changes in DNA methylation, specifically cancer is a big one
that involves DNA methylation changes many times.
The other modification is histone modifications
and here I'm going to just show you
a picture of sort of the end result of that.
We're not going to talk about
the specific histone modifications that can occur.
It's a long list and it gets kind of confusing
and you just really don't need to know that at this point,
again, unless you are interested
in something specifically there,
then please feel free to ask, but otherwise
we'll just kind of glaze over it
and say that there are different histone modifications
and the end result of those histone modifications is
either tightening DNA around histones, if you remember,
what are histones?
Histones are those proteins around which DNA is wrapped
like the spool and DNA is like
the thread that winds around it.
So what actually happens is
depending upon how those histones are modified,
they can actually contract into one another
and bring all the spools really close together
and really, really tight, tight, tight kind of ball of
thread or DNA and the end result of that,
what that structure is actually called
when it's all tightened and packed up together
is called heterochromatin.
And you can have regions along a single strand of DNA
that are tightly packed like heterochromatin
and those that are loosely organized,
which we'll talk about momentarily.
But within a region of heterochromatin,
what happens is that,
let's say that this particular part of the DNA
actually is the gene that you're interested in
and this gene needs to be transcribed,
so its promoter would be, let's say right about here,
what's the problem?
Like what could you anticipate being a problem here?
Well the problem is it's all tightly mushed up together,
wound up together with all these other proteins
and all this other DNA, there's no access,
you can't get into it.
So transcription factors can't actually
get into where the promoter is
to bind into initiate transcription.
So it's basically there's no access, it's shut down,
that means the gene is off.
Even if the transcription factors are present
and are around in the area, it can't turn on
because it is currently in this tight
heterochromatin formation
because these histones are modified in such a way
to bring it all to kind of like shrink it all together
and really make it a tight structure.
On the other hand, there are different modifications
to histones which can occur that result in
a structure called euchromatin
or stretch or region of the DNA, which is loose.
And so there's very few histones say in the area
and that gives the DNA space to breathe a little bit.
You know, let's say that this is where your gene is
and let's say this is where your promoter is,
well now you have lots of space,
the transcription factors can come in and bind
and they can initiate transcription
and so all can be good and happy
with transcribing your gene there.
Again, let's just reinforce what what I just covered.
So DNA methylation can block
transcription factor binding to promoters,
thus shutting off transcription
even when transcription factors are present.
So they're present, they just can't bind the DNA
because they don't recognize it anymore
'cause they don't recognize
when there's a methyl group on the cytosine.
Histone modifications that constrict DNA structure
block access of transcriptional machinery.
And DNA methylation, I also want to mention,
so these are not just completely separate processes,
they can actually compliment one another.
DNA methylation being commonly associated
with histone modifications
that constrict DNA structure further
reinforcing the repression of transcription
for that particular gene.
So you can imagine
you can have any combination of transcription factors
being present or not.
You can have, whether the promoter is methylated or not,
and you can have whether the gene
is in a region of heterochromatin or euchromatin,
all of those together can kind of,
you can have really any versions of those
coming together to determine the overall
level of transcription for a gene.
So this regulation is happening
on a lot of different levels.
Let's take a quick peek at pulling this all together.
So again, if you have a chromosome
and you stretch it out.
Chromatin, you can just kind of think of it
as another word for chromosome, so chromosome,
this is basically like a chromosome stretched out
looking at chromatin.
And again that's a double stranded DNA molecule
wrapped around histones.
These are the histones again.
And if we zoom in on
some of the epigenetic modifications
that can be occurring here,
what we can see is that
histones themselves can be modified with different groups,
so like a phospho group, a methyl group, acetyl group,
you don't need to know these specific modifications
just telling you for your own information.
And the combination of different modifications here
can determine whether or not those histones
really tighten up with one another
or they say really loosey goosey
and allow those genes to be
in a euchromatin state
where they can actually be transcribed.
You can also have methylation on cytosines
and this really matters a lot
if this methylation on cytosine is occurring
in promoter region of a gene
because that can block the binding of what?
Of transcription factors, that's right.
So those transcription factors are necessary
for initiating transcription of the gene.
Okay, let's put this all together.
And in this depiction they're showing white circles,
well they're showing the circles,
let's say these small circles as cytosine bases
along the stretch of DNA, these are your histones,
and let's say in this particular state
in order for transcription to occur,
so there's a lot of hurdles that need to be crossed,
barriers that need to be crossed
before a gene is transcribed.
So first of all, you need to have, what?
You need to have your transcription factors present
that are needed for that particular gene, okay?
You need to have those there.
Then you also need to make sure,
if you want to transcribe this gene,
you need to make sure that the cytosine residues
or that the cytosine bases
in the promoter region for this gene are
unmethylated, right?
Okay, so if they're unmethylated
that allows the transcription factors to be able to bind
into their promoter.
Then what's the third thing that needs to be there?
Well, you need to have your gene in a euchromatin region
or a region where the histones are more relaxed
and are more loosely spaced.
And this is what is showing here on transcription.
Possible, possible.
Remember that it's possible
for a gene to be transcribed
if it's in a region where there's no
methylation on the cytosines
and the histones are modified in such a way
that the DNA is more loosely stretched out.
What else do you need to have
before transcription would happen here?
That's right, you need to have the correct transcription
factors being present.
So this is saying transcription is possible.
If the transcription factors are present,
then transcription would actually occur here.
On the other side, there are a lot of ways
in which transcription can be shut down.
Any one of which
can either completely shut down transcription
or really, really slow it down.
And that would be either/or
methylating the cytosines
specifically in the region of the promoter,
which would prevent transcription factors binding
or condensing the DNA down into heterochromatin
because of histone modifications that cause tightening.
And the other way that you can impede transcription,
not shown here, is just that the transcription factors
needed for that particular gene are not present.
There are lots of ways that can shut it down,
any one of which can shut down transcription,
but you need all of them to be in the on state
before you actually get transcription.
Let's take a quick peek at,
let's go back to our depiction of the gene and its promoter
and sort of its surrounding sequence
and look at what happens with methylation.
The first epigenetic modification that we're talking about.
So let's say you have, I'm depicting,
let's say these are where your cytosines,
there are a couple of cytosines located,
and let's say that they're methylated,
it's depicted by these red M's in the promoter.
So you do have your transcription factors around,
but guess what?
They can't bind, they don't know what's going on
because they no longer recognize their normal sites here
because they're methylated,
those cytosines are methylated, so they can't bind.
So they just go on about their business, they leave,
they don't bind to the promoter.
And what does that mean?
Well that means the scaffolding protein has nowhere to land
and so it doesn't bind.
And then subsequently that also means
RNA polymerase cannot bind.
If you don't have RNA polymerase binding,
you cannot have transcription of the gene.
So even though transcription factors were present,
if the promoter was methylated,
you're not getting transcription.
Okay, the big red X across the line depicting transcription.
So this would be considered the gene being off
or the mRNA is not transcribed.
Let's take a look at what this means in heterochromatin
or when you have those histone modifications that
result in a tightening around the histones
and basically constriction of access to the DNA sequences.
So this is the promoter
and this is basically the DNA
that's kinda wrapping itself around,
it's no longer like nicely stretched out,
it's all smushed together.
So here's your promoter,
but it's all bound up to these histones.
It doesn't have space to breathe, which means
that even though the transcription factors are there,
they don't know where to land
so they can't bind to the promoter,
which means scaffolding protein can't bind,
which means RNA polymerase cannot bind.
And the result of that is no transcription.
So the gene is off.
And even if there's no DNA methylation,
even if the transcription factors are present,
if this is in a heterochromatin formation,
you're not getting transcription.
Cell types have variable patterns
of epigenetic modifications
which contribute significantly
to the differences in which genes are expressed.
So while, let's take for example a kidney cell
and a heart cell.
So they have very different functions,
they look quite different from one another,
they have exactly identical genetic material,
so their genomes are 100% the same,
they all came from the same original cell
that had the same DNA, right?
They all have the same looking DNA,
but they have different proteins being expressed
and that's because they have different
epigenetic modifications.
And those epigenetic modifications
can be inherited from one cell division to the next.
So as a cell progresses down a particular lineage,
say the kidney cell lineage,
it's accumulating epigenetic modifications
that result in the proper genes being expressed
for kidney cell function.
On the other hand, heart cells are accumulating
epigenetic modifications that allow only
the genes that are needed to be expressed
in heart cells to be expressed.
Epigenetic modifications accumulate
and change in response to the environment,
so that would be in response to stress, diet,
toxin exposure, and most of these are set
during early development.
So maternal nutrition, stress, toxin exposure
can impact gene expression for the lifetime
of her child or of her offspring.
And I want to give you an example of that in mice here.
This is a pretty striking example.
These two mice are genetically identical.
Genetically identical.
So think of them as like identical twins
or clones of one another.
Their DNA is exactly the same,
but why do they look so different?
This one's orange, this one's brown,
this one is obese, and this one is a normal weight.
This one also was very susceptible over its entire lifetime
to cancer development.
While this mouse on the right is not,
it has a normal chance of developing cancer.
So what's the difference?
The mother of this mouse on the left,
while this mouse was in utero,
the mother ate a diet that was a very poor diet,
that contained very few
methyl groups in the food that she ate.
And methyl groups are found
and it's sort of basic nutrition.
It's anything that you think is kind of healthy food,
fresh, more plant-based foods have more methyl groups
and it's just like it's basic food
that's generally healthy for you are usually
are relatively high in methyl groups.
So this mouse's mother, while this mouse was in utero,
it had a very poor, methyl poor diet.
While this mouse on the right,
its mother while it was in utero,
ate a normal sort of healthy diet
that contained plenty of methyl groups.
Why do you need methyl groups
and why is this associated with epigenetic modifications?
Well if you think about all the DNA methylation that occurs
and the histone modifications that include methylation
of histones, well those need methyl groups
in order to form properly.
So this mouse on the left,
while it has the same exact kind of DNA
as this mouse on the right,
it has a very different pattern of epigenetic modifications,
the result because of the diet
its mother consumed while this mouse was in utero
compared to this mouse.
So as I'm sure you're aware
and I'm sure you express to your patients as well,
the diet and stress, the toxin exposure that a mother
exposes her child in utero to
can affect not only the way that child develops initially,
but also how its genes are expressed
for its entire lifetime, for its entire lifetime.
So huge implications on the overall health of that child.
All right, let's go back to what we were talking about,
where does gene regulation occur?
So we talked about transcriptional regulation
and transcription factors being present or not
and epigenetic modifications,
let's talk about some post-transcriptional regulation,
specifically splicing variants.
And where are we talking about that?
That is after mRNA has been copied off of the DNA
for a particular gene, you have your mRNA,
but it's not quite ready for showtime.
So you know, it's not quite there,
not quite ready to be translated by the protein.
There's another stage that we have yet to talk about
and that would be splicing.
What do I mean by splicing?
Well I apologize that this
particular image is a relatively low resolution,
but it actually is a nice way to show
what I wanted to sort of piece together for you.
So if we're thinking about a gene,
so let's say this is a gene,
so it's your DNA along your DNA strand,
it's in your nucleus, right?
Here's your promoter,
which is upstream of the start of your gene.
Well, so far we've been talking about a gene
as coding for a protein and it does,
but there are some parts of it that don't actually code,
that are non-coding,
even within the stretch of sequence that is called a gene
there are regions called introns
or intervening sequences.
Introns do not code for amino acids,
do not code for protein,
and they have to be cut out of the RNA
that has been transcribed before that RNA is ready
to be translated into protein.
This process is called splicing.
Splicing, so that just means cutting out the introns
and sticking together the exons in the order,
remaining in their linear order.
So if this is your primary transcript
that was copied directly off of your DNA,
basically what's going to happen is that
you can take like a little pair of scissors
and cut right here,
cut right here on either side of this intron,
and glue stick these two exons together
and you do the same thing here,
you cut that out and you cut that out
and you glue stick these two exons together
and you do that again
for every one of your introns that's found in a transcript.
All introns are always removed and that leaves, what?
It basically leaves an mRNA transcript,
which is shorter than what was originally transcribed,
copied from that particular gene,
but only contains sequences which are coding,
only contains sequences which are going to
provide ribosomes, the information for
knowing which amino acids to assemble together
to properly make a protein.
Okay, I know that can be a little confusing,
I know that can be a little confusing, so
think about that for a minute
and let me know if you have questions on that
and we're gonna build off of that
as another mechanism for gene regulation here
in the next slide.
Okay, so let's build off of that.
Let's say you have your exons, right?
Depicted here in these different colors.
This is all one gene.
One gene has many different exons, okay?
So each exon codes for a part of a protein,
so each exon codes for a part of a protein.
And when they're all stuck together in the proper way
the single transcript, the single mRNA
can be read and will be read
and translated into a single protein.
Okay, but where can we get some variability?
So we talked about there being 25,000 genes.
Well there are over a hundred thousand different proteins.
How is that possible if we only have 25,000 different genes?
Well, the cells pretty smart
and it's come up with a kind of a cool way to do it
and it does it in sort of a modular fashion.
And what do I mean by that?
Well it kind of takes each of these exons as a model
and can stick them together remaining in its linear fashion,
but can choose, for example,
in one version of the transcript,
it can choose to only include, say exons one, two,
oh we'll skip exon three,
we'll include exon four, five, and six.
So that's one type of mRNA
that can form from this single transcript.
You could also then say, oh, and instead I'll do exon one.
In this case the cell doesn't actually need the protein
to have this particular region.
So it's going to cut that out
and instead do one, three, doesn't need four,
let's say it needs five and six,
so it'll form this particular version.
Or it can form any other version of this,
just remember it always stays linear, so
exon one always has to come before,
so you can't say for example,
do exon one, then four, then two, then six, then three.
It has to be linear so
you can basically remove certain exons
or not include them in the final mRNA that gets translated,
but you can't rearrange the order of exons.
Okay, I know that's confusing,
but just think about it for a second
and really look at this picture
'cause I think this particular picture demonstrates it well.
The end result of which would be
three different versions of a protein
or three different proteins I guess you could say
that are similar to one another,
but are not identical because they contain different,
this would be a single protein.
I know it's shown as like different blobs,
but those are all strung together
as one long chain of amino acids that then forms
these different structures.
And I want to mention that this is not a rare event
where you have alternative splicing occurring.
This occurs in 90% of genes.
So most genes you can have different
what are called splice variants.
Splice variants, which is just,
say this is one splice variant,
this is another splice variant, this is another
which are each translated into different versions of
protein, which can have very different function.
Just want to mention here, introns are cut out of the RNA.
We never ever, ever, ever change our DNA.
Think of DNA, our actual genes,
think of those as like read only files.
So you can open it, you can look at it, you can copy it,
but you cannot change it.
You cannot make any changes to the DNA, so as a result,
all of the changes are occurring really at the RNA level.
Let's look at an example of gene regulation
and this would be more transcriptional control where
this might be very applicable in some of your practices,
specifically response to estrogen
to prepare for possible pregnancy.
So estrogen, as you probably know, is a hormone.
It is what we call a small molecule, so it's not a protein.
Estrogen is not a protein, it is a small molecule.
It's produced in females, it's produced by the ovaries,
but I'm sure you're all aware of estrogen therapy, which
basically is you can take a pill.
Birth control pills contain estradiol or estrogen
as well as hormone replacement therapy
can also contain estrogen.
So these are small molecules
and they're small molecules that have a big impact.
And here's why, they actually affect gene expression.
So cells respond to estrogen in part
through changes in gene expression.
Estrogen receptor acts, so an estrogen receptor is a protein
that we produce in certain cells.
Actually in a lot of our different cells
contain estrogen receptor, it's quite interesting.
I mean primarily we focus on cells in breast tissue
and say in the uterus,
but certainly there are other cells
that also express estrogen receptor,
including some cells in the heart, cells in the brain,
and in other locations.
And you can start to imagine the impact of that
as we talk through the dramatic impact
that estrogen can have on gene expression.
So estrogen receptors, again, proteins that bind to estrogen
act as transcription factor
when estrogen is present in the cell.
So they enter the nucleus
and bind to specific sequences in the DNA
called estrogen responsive elements or EREs
and this is just really like a specific sequence, so
I don't actually recall off the top of my head
what the sequence is, but it's like a specific
set of six bases in a row
say that the estrogen receptor
when it's bound to estrogen will bind to
wherever it finds it in DNA.
And they can activate transcription of genes
with these estrogen response elements in their promoters.
When estrogen is absent,
estrogen receptors cannot bind to DNA.
So some genes that code for cell proliferation proteins
have these estrogen response elements
and thus their transcription
is activated in the presence of estrogen.
Not all cells express estrogen receptor,
thus not all cells are responsive to estrogen,
but as I mentioned, there actually are quite a few that do.
Cells in breast tissue and uterine lining
along with certain other tissues proliferate
in preparation for possible pregnancy.
So let's take a look at what that actually means.
If we have estrogen, let's say being shown with this
purple molecule here, it enters the cell
so it actually can just diffuse into the cell.
So this is again, a small molecule, not a protein,
but a small molecule diffuses into the cell
can bind to the protein that we
have present in certain cells, right?
So this estrogen receptor is present.
The estrogen molecule binds to this protein.
The estrogen receptor can now act as a transcription factor.
So remember what is a transcription factor?
It's a protein that binds specifically
to certain sequences on DNA
and specifically in a promoter region.
So this particular gene here
has within its promoter region an estrogen response element,
which just simply means the sequence of DNA that
the transcription factor binds to specifically.
So the estrogen receptor
with its bound estrogen will bind to the promoter
for say this particular gene.
And let's say this gene is involved in
inducing cell proliferation.
So the estrogen receptor
with its estrogen binds to the promoter.
In this particular diagram,
they're calling these coactivators,
what I was calling like the scaffolding proteins,
and you know, transcriptional machinery all can now bind
and you have transcription of this particular gene.
Transcription means that the mRNA is produced, remember,
and now what happens to mRNA once it's produced?
It has to be spliced in order to remove those introns
and just allow the exons to be present.
So let's say now you have your fully formed mature
messenger RNA which exits the nucleus,
and in this diagram, they're not showing translation.
But remember ribosomes will bind to this mRNA
and will produce the specific proteins
that this gene encodes.
These specific proteins,
let's say in this case for this gene,
let's say these proteins play a role in cell proliferation,
so increasing the amount the cell will grow and divide.
And so that's what occurs as a result.
So the cell will, let's see,
let's just do a diagram of what would happen here.
You have high levels of estrogen,
it gets into the cell, estrogen receptor can go in
and bind to the promoter for these
cell proliferation genes, activates them,
turns on transcription,
the mRNA for those genes gets translated,
so you have these functional proteins
that are involved in proliferation,
and boom, you have these cells dividing like crazy,
specifically breast or uterine cells.
And the breast tissue is proliferating
in preparation for lactation
and the uterine cells are proliferating
in preparation for possible pregnancy.
As we all know, in the normal menstrual cycle,
there are various levels of estrogen that occur
and in the high peak levels of estrogen,
this is when, say in the example of breast cells,
you have the most proliferation.
And this is when you know breast tissue will actually
increase during the cycle as a result of estrogen
because estrogen is, what?
It's activating estrogen receptor, which is a
transcription factor which will bind to
say cell proliferation genes in their promoter
and induce their transcription,
which then results in cell proliferation.
And as this goes through,
so you have a breast cell proliferation
and increase in the tissue density at that time.
And then when pregnancy does not occur,
the estrogen levels will decline
and as a result you lose that proliferation
and the cell starts to die off and
the tissue returns to its normal density and normal state.
The reason why I wanted to bring up this particular
example is because we will discuss in more detail
breast cancer and its genetic implications.
But I wanted to start you out here
with just a basic understanding
of how estrogen receptor works
because it is a transcription factor.
All right, let's summarize what we have learned.
Cells need to regulate the extent
to which certain genes are expressed to control
how much of a specific protein is around
to perform functions as needed.
Remember, not all proteins should be present all the time,
only really in response to certain environmental cues
or for the specific function of a particular cell.
The genes are regulated both
transcriptionally and post-transcriptionally
and by that I mean
the both in how much mRNA is actually transcribed
or when mRNA is actually transcribed.
And then for post-transcriptionally,
once the mRNA has been formed in the splicing of that mRNA
and which exons will make it on
to the final mature mRNA product
and therefore will affect which protein
is actually translated.
Transcriptional regulation involves both
the presence and absence of transcription factors
and also access of transcription factors to promoter
controlled by epigenetic modifications.
So you have two levels here, you have both
whether or not transcription factor is present,
but also can the transcription factor even reach
its promoter or bind to its promoter
and that's the epigenetic modification side of things.
Post-transcriptional regulation affects
the final mRNA sequence through alternative splicing
as we discussed and if you have questions on that,
I'm more than, or that or anything,
I'm more than happy to discuss with you.
It seems like it's working out really well
to just go ahead and post those onto the discussion board
because if you have a question about it,
probably someone else does and
I'm sure everyone would be happy to
see both your question and the response to that
or clarification to that.
So please feel free to post away
any questions that you might have.
So what's next in this module?
We're going to switch gears a little bit
and start talking about two aspects
that really fit hand in hand with one one another
and those would be DNA replication
and then meiosis and mitosis, which would be cell division.
DNA replication being the process of actually
creating exact duplicates of all of your DNA
in preparation for one cell becoming two cells.
So and that process of one cell becoming two cells
is mitosis and the process of forming
egg or sperm cells is called meiosis.
And we'll get into all of the details of that next, woo.
So, thank you for hanging with me through this lecture
and, again, please let me know
if you have any questions or comments.
So remember, you will get this,
you are absolutely getting there,
think of how much farther along you are now
than you were at the beginning of module two.
I mean, even if you already knew
a lot of what we covered in module two,
it was a lot of refreshing, I hope,
and you know, you're making a lot of progress
and getting there and you are going to get there.
We are absolutely going to get there,
we're gonna get there together.
So if you have questions, let me know,
and otherwise I will talk with you in the next lecture.