Molecular Biology Gene Regulation in Eukaryotes

Hello, and welcome back to www.educator.com.0000

Today's lesson will be on gene regulation in eukaryotes.0003

First, we will talk about transcriptional regulation and we will also talk about the regulation of translation.0008

Finally, we will talk just a little bit on epigenetics.0013

Let us first talk about our eukaryotic transcriptional regulators.0020

Mainly, we are going to talk about our transcription factors.0025

There are many different types of transcription factors, usually characterized by the specific domain by which they bind DNA.0028

We have homeodomain proteins, we have zinc finger proteins, we have leucine zippers, loop helix proteins, as well as HMG proteins.0037

HMG just stands for high mobility group.0052

We also have insulator proteins, in which these are proteins that bind between the enhancer and the promoter in a gene.0055

They suppress transcription.0065

If you remember, enhancers are usually far upstream of where the core gene element itself starts.0066

Up here, our transcription factors, they can be either enhancing or repressive.0076

Compared with our prokaryotes, transcriptional regulation and regulation in general, for eukaryotes is much more complex.0083

Usually, there are more regulators and longer length of regulatory sequence in the DNA.0094

Let us first start off with an example of transcriptional regulation.0106

We have a gal-4 and gal-1.0110

Gal-4 is a protein and gal-1 is a DNA sequence.0113

We are looking at this in the yeast saccharomyces cerevisiae.0118

The gal-4 protein will bind to a site upstream of the gal-1 gene.0125

This is going to increase transcription by gal-1 gene, by a thousand fold.0138

What this would look like, gal-4 binds as a dimer.0145

What we see here, if we draw this out.0150

Right here, this right here, this arrow, that shorthand form of saying that is where promoter is and gal-1 that is our gene.0170

This right here is a UAS, when upstream activating sequence.0179

There are 4 sites here at which the gal-4 protein will bind.0186

It will bind as a dimer.0198

Between the UAS and the gal-1 gene, is about 235 base pairs.0200

The binding upstream over here will affect how this gene is transcribed.0213

We can increase, this is gal-4, there are g4.0221

This can cause an increase of transcription about a thousand fold.0226

For example you are supposed to only make one mRNA0231

or in a certain period of time you make a thousand times more than that.0235

Let us talk about our locus control regions.0242

First off, we are going to draw this out.0245

I guess we will use red.0251

Here is our locus control region.0256

Then, we have our globin genes.0267

That should be gamma not Δ.0294

This is Δ, this one is β.0307

Here is our cluster of globin genes.0320

We have locus control regions right.0340

Locus control regions are just long range regulatory elements that enhance expression of our linked genes.0342

This helps function in a copy number dependent manner.0353

It is actually tissue specific.0360

For this one, for example, this allows this locus control region.0362

This allows ordered transcription of downstream genes.0369

What that is really saying is that we have selective expressions of our β globin genes in our erithrocyte cells.0375

This is an example talking about our selective.0406

Our erithrocyte cells are also known as red blood cells.0413

Our locus control regions allow for order transcription in downstream genes.0416

Here is our locus control region.0421

Here are downstream genes.0425

We have our 5 globin genes.0427

We have ε, we have gamma G, gamma A, Δ and β.0429

During human development, all 5 genes are transcribed sequentially, meaning, first ε, all way through to β.0433

What happens here is that we have euchromatin position.0442

If we remember correctly, euchromatin is open, more transcriptionally available.0447

Heterochromatin is closed, less transcriptionally available.0452

Euchromatin position will change and heterochromatin formation, reformation will follow behind.0456

This is something that I want you to just keep in mind because we will be talking about epigenetics later.0463

How euchromatin and heterochromatin positioning affects the transcription of genes,0472

as well as the replication of DNA sequence.0480

Here are locus control regions, this is one example.0488

Let us move on to signal transduction.0494

Signal transduction occurs when we have an extracellular signaling molecule,0498

activating a specific receptor located on the membrane of the cell.0506

If I draw it out, here is a cell and here might be a receptor.0514

Here is our extra cellular signaling molecule.0524

When this molecule binds the receptor, it is going to trigger a chain of events, inside the cell it creates some sort of response.0529

That response is going to be different, based on which pathway is being activated.0544

Some of our examples that we will talk about today are NF kappa B,0550

the JAK/ STAT pathway which is very important for immune system regulation.0555

We have sigma molecule growth factors, seven-spanner receptors which are important for sensing light, smells, serotonin.0559

And then, we have nuclear receptor proteins that are important for the signaling of our steroid and non-steroid molecules.0572

This is an important one that you will learn more in biochemistry.0590

If you have not taken that yet or if you decide to take a nutrition class, something like that.0594

First off, NF kappa B.0603

NF kappa B is a dimer protein meaning it has two parts.0607

We have to have two pieces of that protein.0612

This protein helps control transcription, as well as cytochime production in cell survival.0617

Cytochimes are just small protein molecules that are involved in immune system regulation.0622

Ligands which means it is just something that is binding, can cause our NF kappa B0628

to turn on more than 150 genes in the inflammatory system and the immune system,0637

as well as during development in the cytoplasm.0642

I mentioned that the NF kappa B is a dimer.0646

That dimer is made up of the P50 and the P60 protein, therefore, it is a heterodimer.0650

This might be 50, this would be 65, then we have our heterodimer.0657

Let us talk a little bit more about NF kappa B.0662

NF kappa B, as I said, plays a large part in the immune system and so forth.0667

NF kappa B, for one example, can be inhibited when the P50 subunit binds to a cancer suppressor protein.0672

If it is inhibited, therefore, it cannot help with transcription or increase transcription, for the rest of its purposes.0686

A second example of how NF kappa B maybe regulated, it can be inhibited by its binding to another protein called I kappa B.0696

NF kappa B is bound or it can be bound and inhibited by the inhibitory protein I kappa B.0710

What you can do to release this inhibition is phosphorylate I kappa B, that will cause I kappa B to release NF kappa B.0720

At this point, NF kappa B can travel to the nucleus, it was originally in the cytoplasm.0734

At that point, once NF kappa B is in the nucleus, it can go ahead and regulate any transcription, as it normally would.0743

At the same time, this phosphorylated I kappa B is no longer bound to NF kappa B, can be ubiquitinated.0752

Ubiquity, if we remember back to the protein unit, is just a small protein and that can have many different outcomes.0761

In certain outcomes, when a protein is ubiquitinated, it is set to the prodiuzome and degraded,0771

broken down, thrown into the trash.0778

These are two separate examples how NF kappa B can be regulated, in which case,0781

if it is inhibited, it cannot affect transcription.0785

If it is not inhibited and able to act in the nucleus, then it can affect and be a regulatory protein.0791

Next, we have the JAK/ STAT pathway.0801

This pathway is turned on by a cytochime.0803

For an example, interferon α, that will attach to the cell surface receptors.0807

First of all, let us write out what is JAK.0812

JAK is a kinase, it is called Janus kinase.0823

Remember, kinases are responsible for phosphorylating things.0832

We have STAT, STAT stands for signal transducer and activator of transcription.0837

The JAK/ STAT pathway, if we are talking about the previous slide.0878

If we have our cell, here is our nucleus.0887

We have a receptor and we have something binding to that receptor.0893

In this case, the cytokine is binding.0902

JAKs, the kinases come together.0906

Those JAKs will activate the kinase which phosporylates pairs of our STAT.0911

Those phosphorylated STAT monomers will dimerize.0919

The stats are also monomers.0923

They will be phosphorylated.0933

We will just say that it is a yellow piece, that causes them to dimerize.0934

They do that via a specific domain on the STAT molecule called an SH2 domain.0954

We will call the nucleus in purple over here.0970

That STAT dimer will travel into the nucleus and bind a specific DNA sequence, related to transcriptional enhancement.0975

This, once again, shows you that a molecule from outside the cell can affect what happens inside the cell,0987

through a series of events that happens to, in this case, deal with the binding of a molecule to receptor,0995

a phosphorylation of other proteins.1006

Those proteins being sent into the DNA or being sent into the nucleus, to bind to DNA.1009

At that point, it will affect transcription.1016

Another example of sigma transduction would be our signal molecule growth factor.1022

A couple examples being epidermal growth factor or EGF, as well as insulin.1028

Remember, insulin is the protein that is sent out by the body, by the pancreas, in response to glucose in the blood, blood sugar.1033

When you eat something, you have carbs, those get broken down, absorbed from the stomach through the intestine into the blood.1046

Insulin is released, insulin takes that glucose into the cells.1055

How this works is that, we have a binding to the receptor, activating a dimeric, a two protein,1061

RTK which is a receptor thyrosine kinase enzyme.1076

The dimerization requires the adapter proteins grab to an SOS.1082

That is just some extra information for your knowledge.1088

We have this signal causing the RTK dimerization.1092

That dimer binds and activates a protein called RAS, which will bind the GTP molecule.1102

That RAS will then bind to and activate another protein called RAF.1110

RAF is what is called a map KKK which is monogen activated protein, we usually call this MAP.1115

It is a MAP kinase kinase kinase meaning it phosphorylates a MAP kinase kinase.1127

It is quite heavy nomenclature but we will understand it, as we go to the next step.1138

This RAF which is our enzyme, a specific MAP kinase kinase kinase.1143

RAF will then phosphorylate, since it is a kinase, it will phosphorylate MEK.1149

MEK is a MAP kinase kinase.1155

MEK, once it is phosphorylated, will phosphorylate a MAP kinase, a MAP K.1162

This MAP K is a different type of kinase, than the one that we talked about up here,1175

it is a kinase that is called a serine threonine kinase.1182

What are the differences between these kinases?1187

The RTKs, they phosphorylate a thyrosine amino acid.1189

This one down here will phosphorylate proteins at certain amino acids of either a serine or a threonine residue.1199

Now that MAP kinase is active, it is still a kinase.1213

MAP kinase will phosphorylate a bunch of other transcription factors.1218

These transcription factors, now many of them by the phosphorylate, will be activated.1224

And now, they can go and bind to the DNA in the nucleus and enhance transcription.1231

It is a very complex process, many steps, but the whole outcome is to affect the transcription happening in the nucleus.1239

We have other ways of regulating transcription.1252

Utilizing certain proteins called seven-spanner receptors.1257

What is a seven-spanner receptor?1262

If this is the cell membrane, we have a protein that crosses the cell membrane 7 times, 1, 2, 3, 4, 5, 6, 7.1266

Maybe that is its N terminal tail and this is its C terminal tail.1284

What we have here is it is crossed 7 times, 1, 2, 3, 4, 5, 6, 7.1288

That is what is a seven-spanner receptor is.1296

There are several different seven-spanner receptors.1299

How do these work?1303

Very similar, in the fact that there is ligand binding that is activating a process.1306

Specifically, we have ligand binding, activating trimeric proteins called G proteins.1313

Trimeric meaning 3, 3 different polypeptide chains.1320

Each one of these subunits, we have an α subunit, a β subunit, and a gamma subunit.1328

In order, how this happens, we have the ligand binding activating that protein.1339

When GTP binds, our α subunit that will activate adenylyl cyclase, which is an enzyme.1347

This requires GTPAs activating protein also known as GAP.1363

Adenylyl cyclase normally converts ATP to cyclic AMP.1372

We talked about this in the previous lesson, this adenylyl cyclase enzyme.1376

The CAMP, we have also talked about this, will bind to protein kinase A to activate it.1383

Protein kinase A, otherwise known as PKA, will then be able to phosphorylate many different proteins.1391

Some of which can be transcription factor.1401

PKA, once activated can phosphorylate transcription factors which will bind to certain pieces of DNA in the nucleus and affect transcription.1403

Protein kinase A can also another protein called CREB which will bind to a specific DNA sequence called CRE.1416

As well as, be bound by CBP and P300 protein which is CBP stands for creb binding protein, CREB BP.1429

CBP300, this protein is normally considered a transcriptional activator.1444

It turns on the transcription of genes.1450

There are two different ways a protein kinase can affect gene expression.1453

Let us talk about another example.1465

We actually have two examples in this one.1467

Let us talk about how we have two different diseases, cholera and whooping cough, otherwise known as pertussis.1470

Cholera, cholera is caused by the action of the bacteria vibrio cholerae.1479

This vibrio cholerae produces a toxin called the cholera toxin.1495

How does this work, what is the mechanism of action?1500

The cholera toxin catalyzes an ADP ribosylation.1504

That is basically adding a ribose group to ADP together.1509

This happens via the use of our old friend NAD, nicotinamide adenine dinucleotide.1514

We have talked about this one before and it is useful in energy.1523

This ATP ribose will bind to the α subunit of the G protein, as we talked about in previous slide,1529

which does not allow the GDPAs activating protein, GAP, to activate GTPAs.1538

Therefore, you cannot convert GTP to GDP.1545

What is that even mean?1551

What that means that we have constitutive activation of our adenylyl cyclase enzyme.1553

Constitutive means it is always on.1562

If our adenylyl cyclase enzyme is always on, that means that we are continually turning ATP into CAMP.1566

If we have a bunch of CAMP, that is going to keep PKA, protein kinase A, constitutively active.1577

If PKA is always active, it is always going to be phosphorylating things.1587

In these certain cases, it will phosphorylate certain transcription factors to turn on gene expression and1594

can lead to types of diseases or the types of symptoms from cholera or whooping cough that are common.1601

Whooping cough, specifically, is caused by the bacteria bordetella pertussis and1614

that bacteria also produces a toxin called the pertussis toxin.1621

This pertussis toxin acts in a little different way than the cholera toxin does.1625

Let us talk about this, we have pertussis toxin binding to a protein called G sub I.1632

G sub I is a G protein inhibitor.1640

Normally, G sub I will bind to and inhibit adenylyl cyclase, opposite of cholera.1647

Cholera adenylyl cyclase is always on.1660

G sub I normally turn adenylyl cyclase off.1663

However, we have pertussis toxin binding to that inhibitor preventing that normal inhibition.1667

Therefore, we end up with the same outcome.1680

We have constitutive activation of adenylyl cyclase, leading to continual conversion of ATP to cyclic AMP.1683

Meaning, continual activation or constitutive activation of protein kinase A,1694

leading to transcriptional regulation at increased rates.1701

These are just a couple of ways how we can utilize the previous material in this unit,1708

to understand how a couple different bacteria create their symptoms that we see for medical purposes.1715

Going on to a different type of transcriptional regulation.1728

We have what are called nuclear receptor proteins.1732

These are proteins found within cells that are responsible1736

for sensing both steroid and thyroid hormones, as well as certain other molecules.1739

First off, we have our steroid hormones.1749

For example, our androgens and estrogens, our sex hormones, our glucocorticoids which are involved in water retention.1752

They are going to bind our cytoplasmic nuclear receptor protein homodimer.1761

Let us say dimer of the same two things, two different polypeptides.1768

The steroid hormones, these will bind our NRP homodimer.1774

This is what is called a type 1 complex.1782

The type 1 complex will head the nucleus, bind to an inverted repeat DNA sequence called a hormone response element or an HRE.1787

At that point, once the HRE is bound then transcription can be regulated, either in a positive or negative manner.1802

A second way that nuclear receptor proteins act is sensing hormones, such as vitamin A or a thyroid hormone.1811

They will bind to NRP heterodimers.1822

In this case, it is two different proteins.1826

This is what is called a type 2 complex.1836

This type 2 complex is already in the nucleus.1840

It will bind direct repeats in the nuclear DNA and then affect transcription regulation.1845

A third way to look at our NRPS and how they might affect transcription1855

would be in an unbound heterodimic NRP, not bound by hormone.1862

They can cause deacetylation of histone tails, the lysine residues on histone tails.1868

These heterodimeric NRPs that are bound by a hormone, such as this right here, can cause the acetylation of those tails.1876

Remember, when a histone, when its tails are acetylated, depending on the histone code, it is very complex.1887

It often is related to the loosening of chromatin meaning transcriptional activation.1899

Whereas, the deacetylation of histone tails usually means the compaction of chromatin,1908

meaning decreased transcription and replication.1915

Let us move on to something that is also regulating transcription but in a completely different way.1923

Actually, this is a really cool mechanism.1932

This is what is called RNA interference.1937

This is a method that is actually been adopted by many research labs.1939

I will show you an example in a couple slides to actually choose what genes get silenced or expressed.1945

This is coordinated by a complex called the RISC complex.1958

The RNA induced silencing complex.1964

It is a ribonuclear protein complex meaning it has both RNA and protein.1970

This complex has been most well studied for having a function in the degradation of a specific target mRNA.1977

If you target mRNA to be degraded, that will decrease the level of the transcripts available to be translated.1988

Therefore, in S sense it is functioning to decrease gene expression of a specific gene.1996

We have very important players.2006

We have RNase enzymes, RNase is an enzyme that breaks down RNA molecules.2009

These RNase enzymes called dicer and drosha, trim double stranded RNA2016

to form either small interfering RNA, sRNA, or microRNA, miRNA.2024

There RNAs then get incorporated into the RISC complex leading to specific mRNA targeting, and then degradation.2035

It targets to a specific mRNA and does not allow translation to take place.2046

It is actually really cool.2052

This process we, call RNA interference or RNAi.2055

We find this in many eukaryotes.2061

It was thought to be developed as a very important mechanism of gene silencing and viral infection defense.2063

Let us talk about the RISC pathway, in a little bit of detail.2076

First of all, what is an activated RISC complex?2080

That composes the RISC ribonuclear protein, as well as a micro RNA or small interfering RNA and an ATP molecule.2083

How does this all go down?2096

First off, dicer and drosha cleave a double stranded RNA into short 21 to 23 base pair fragments,2100

with a two nucleotide 3 prime overhang.2111

Simply, it looks like this, where this is two nucleotides, that is two nucleotides long.2115

This is our 3 prime, 5 prime, 3 prime, 5 prime.2137

This whole thing is 21 to 23 base pairs.2145

This is double stranded RNA.2156

That is the first thing that happens.2165

Then, we bring in a different RNase called argonaut which is also termed slicer.2167

The RNase argonaut then associates the single stranded RNA with the RISC complex,2178

to act as a complementary strand to whatever the target mRNA is.2190

This double stranded piece right here, that has been processed by dicer and drosha2198

can be separated into single strands and that is what argonaut is working with.2206

The complex will bind to our target mRNA, whatever piece of gene material2210

that you do not want to end up turning into a protein.2219

The complex binds to the target mRNA and silences it.2223

Silencing meaning it is not been turned into a protein, no translation.2226

There are two different ways this occurs.2232

If we are talking about an miRNA being used, micro RNA.2234

The micro RNA in the RISC complex binds to the 3 prime untranslated region,2242

that is a part of an RNA, and is untranslated.2249

Therefore, it would not have turned into protein anyway2252

but is a great spot for regulatory proteins to bind and affect whether something is translated or not.2256

MiRNA in that RISC complex, binds to the 3 prime UTR with a mismatch.2262

It will block transcription.2270

The other way that we can affect translation, if we have a small interfering RNA in the RISC complex,2274

binding to the mRNA without a mismatch.2285

This will cause RISC to cleave the mRNA.2291

Cleaving it vs. just blocking transcription.2296

mRNA degradation happens at a certain spot in the cell called a P body.2304

mRNA degradations localized in P bodies which are found in cytoplasmic space.2314

And often stain darkly, that you can look under a microscope.2321

Importantly, we have activated RISC, the RNA inducing complex,2329

being implicated in formation of nuclear heterochromatin.2339

Meaning, we can have RISC actually not just cleaving these mRNAs but can actually somehow be a part of epigenetic changes.2342

Actually not even getting transcription available, not even allowing transcription to happen.2358

That is kind of cool.2364

Here is an example of the RISC pathway.2369

Actually, how scientists have been able to utilize it.2371

What we would have is viral, if there is an infection, they make double stranded RNA.2378

You can break that into srRNA via dicer and drosha.2386

Those double stranded RNAs can enter into the RISC complex, argonaut can take a single stranded RNA2391

and utilize that either as an miRNA or srRNA, to either degrade the mRNA2398

or to affect possibly the epigenetic state of the heterochromatin.2406

What scientists can do, they can target individual genes and silence them.2414

Here we see a wild type, a normal petunia plant.2420

These two are lab created petunias, utilizing RNA interference, RNAi, using the RISC pathway.2426

What they have done is through selective targeting of the mRNA specific for the color,2442

they have silenced them in the white portions.2448

This is pretty cool.2453

RNAi can be used other than just for cool phenotypes.2455

It can actually possibly be used for more functional things such as something that we will talk about a little later,2464

maybe gene therapies, something similar to that.2471

That is your transcriptional regulation.2479

We will just have a few slides, a few instances of translational regulation.2482

Just like prokaryotes, eukaryotes mostly regulate transcriptionally.2486

However, they can regulate translationally.2494

You can have global regulation of translation meaning you regulate all translation,2498

not specific genes, by phosphorylating protein EIF2 that we talked about before.2505

This is eukaryotic initiation factor 2.2515

If you phosphorylate EIF2 that protein cannot bind GTP.2519

If you cannot bind GTP, you are not able to bring the starting codon, the charged tRNA with the initiating methionine.2525

You cannot bring that to the ribosome because this EIF2 GTP complex is required to bring that to the 40S ribosome.2537

If you phosphorylate EIF2, you cannot translate anything.2547

Another way of global regulation, we have phosphorylate of EIF2,2554

another form of global regulation is competitive binding of the 5 prime cap of mRNA.2559

Normally, that mRNA, remember, we have a cap.2566

That cap is normally bound by EIF4-G.2572

Phosphorylated regulatory proteins called 4EBP, binding protein, the cap is normally bound by EIF4-E which is then bound by EIF4-G.2583

If these EIF4-E is bound by these 4E binding proteins right, then the EIF4-E will not allow,2614

the binding of 4EBP will not allow EIF4-G to bind.2669

Therefore, you will not allow translation to occur.2673

This can also be used to regulate specific mRNAs but in general, if you affect the binding of initiation factors for translation,2679

binding the actual mRNA you can affect all mRNAs that are undergoing translation.2690

We can talk about a couple different ways of translational regulation.2698

This is what is called translation dependent regulation.2703

This is when translation is already started and it is in the elongation phase.2706

We have two different types, we have nonsense mediated mRNA decay and we have nonstop mediated mRNA decay.2712

Nonsense mediated mRNA decay is when you come across an mRNA that has a premature stop codon.2723

What you want to do is degrade those mRNAs so that you do not even have to utilize the energy and2734

the amino acids of the whole process to make this short protein which will end up being degraded by the prodiuzome anyway.2741

You can degrade mRNAs with the premature stop codon, by removing either the 5 prime cap2751

or the 3 prime poly-A tail of the mRNA.2760

In which case, you have now freed up either one of those ends to nucleolytic digestion.2764

Exonucleases can now come in and just chew it up and degrade it, the mRNA.2771

We have another thing called nonstop mediated mRNA decay.2777

This occurs when a ribosome is working on an mRNA that wacks a stop codon.2781

This can be pretty detrimental to the cell because if there is no stop codon,2789

basically the ribosome is stuck with that mRNA attached to it, as well as the protein coming out.2794

It just stalled, nothing can happen, you cannot release the polypeptide.2812

You cannot separate the ribosome, which means you cannot then translate a new mRNA.2817

Without that stop codon, the ribosome is stalled, it cannot do anything.2825

We want to be able to fix that.2833

This is rescuing, ribosome is translating mRNAs without a stop.2836

If you do not have a stop codon, that poly-A sequence in the tail gets translated,2842

and that Poly-A, the AAA codon gives you lysine.2849

What this does, that causes the ribosome to stall.2854

It acts as a signal that there is something wrong.2858

What happens is we have eukaryotic releasing factors 1 and 3,2862

binding to the ribosome and associating the ribosome from the mRNA.2869

At that point, the endonuclease which actually is an unknown endonuclease at this point,2892

researchers are still dwelling into this information.2899

This endonuclease will degrade the mRNA from 3 prime to 5 prime.2902

The protein, if it is enable to fold properly which is very likely due to all of the extra lysines,2908

will likely just be signaled to be degraded and be sent to the prodiuzome.2917

These are two different ways that you can have translational regulation, actually once translation has occurred.2922

Transcriptional regulation and translational regulation, and now I’m on to epigenetics.2936

We touched over this a little bit, when we talked about chromatin organization.2942

I think it was unit 5 but we are going to talk a little bit about this again, give you a little more detail.2946

Epigenetics, what is it?2956

That is inherited patterns of gene expression resulting from chromatin alteration.2958

It is not in alteration of the DNA sequence that is heritable.2964

It is not a change in the base sequence, A, C, T, G.2969

But it is a heritable, passing from generation to generation,2974

it is a heritable trait of extra non DNA related changes, in terms of the basic cell.2980

But it could be due to just small changes, such as a methylation, such as acetylation.2996

It can be on the DNA sequence, as well as on the nucleusome itself.3004

This can happen, we will talk about three different ways, DNA methylation,3017

the nucleosomes or the histone proteins, as well as something called polycomb repressors.3023

A very important point of epigenetics is that DNA sequence does not act alone in passing genetic information to future generations.3028

Meaning, the methylation state, let us say of DNA or of histone proteins.3039

The methylation state usually has to do with what genes are silenced or active, that is able to be sent from generation to generation.3046

DNA methylation usually occurs at CPG sites or CG sites.3059

The p that just says it is a C right next to a G, only separated by a phosphate which is what is in the backbone of DNA.3064

DNA methylation occurs in our CPG sites via the DNA methyltransferase enzyme.3073

What you do is you methylate cytosine to get 5-methylcytosine.3081

That can keep that methylation even through the next generation.3086

However, one drawback to this is that actually spontaneous deamination can turn this 5-methylcytosine into thymine overtime.3092

Remember, this cytosine, even as a 5-methylcytosine, will base pair with a guanine.3103

If it gets deaminated, it now becomes a thymine.3113

On the other strand that was not touched, it is a guanine.3120

What you need to do now is, we see that this is in a proper DNA pair, there is a DNA repair that needs to come on.3124

If this T is repaired back to a C, no harm no foul.3133

If this T is not repaired but the G is repaired to an A, now we have a difference in the original base sequence.3145

An epigenetic change has actually turned into a genetic change that gets passed on to next generation.3157

This can cause DNA mutations, if not properly repaired.3167

CPG islands, I just want to talk about because you will hear that a lot, probably,3173

if you read some research papers or if your professors are talking about genetics and especially DNA methylation.3177

CPG islands are regions of DNA with the high frequency of CG sites.3184

There are usually about 200 base pairs in length and they are associated with promoter regions of a lot of our mammalian genes.3191

Usually, when you see CPG island, that is something that should bring to your mind, we are in the promoter of a gene.3199

Not all the time, but very frequently.3209

Methylation of many of these CPG sites will silence gene transcription,3213

leading to heterochromatinization, the formation of heterochromatin.3219

The methylation pattern, as I talked about before, it can be passed on to daughter cells.3228

This methylation will prevent the binding of a transcription factor called SP1 to the CPG islands.3233

The binding of SP1 is part of transcriptional enhancement.3242

We also have this other protein involved with DNA methylation called MECP2.3250

This is a binding protein of those methylated CPG islands.3257

It is a repressor protein that binds to those islands in DNA.3263

It will act as a transcriptional repressor.3268

It decreases transcription by recruiting histone deacetylase.3272

Remember, if you deacetylate histones that usually compacts our chromatin,3275

meaning there is no room for transcription of machinery to get in and go through making your mRNA.3281

A phosphorylated MECP2 protein has a decreased infinity for methylated CPG sites, and not only to an increase in transcription.3291

MECP2 repressor protein, phosphorylate it, it would not bind, therefore, it leads to transcriptional activation.3309

SP1 transcription factor, that is an activator protein.3317

Let us look at an example using MECP2.3324

We have a protein called BDNF, brain derived neurotrophic factor.3329

It is a protein implicated in learning a memory class disease.3335

Basically, being able to retain your memories and reorganize them.3338

This BDNF is release when our dendrites which is a neuro cell, get depolarized, activated.3344

BDNF release can lead to a phosphorylation of in MECP2, meaning, it will now not be able to bind the DNA very well,3351

meaning you have an increase in transcription.3362

On a related but different specifically from this BDNF, if you have a mutation in the gene for the MECP2 protein3368

which is also the MECP2 gene, this will cause a disease called rett syndrome.3378

This is a syndrome that has clinical manifestations very similar to autism.3385

It will affect the neural function.3390

In addition to the autism like symptoms, you have stereotypical hand movements3393

like handwringing or repeatedly putting your hands in your mouth.3399

It is almost unconscious.3403

These are some slides that we have already seen in the chromatin organization lecture.3410

But I wanted to just have them again so that we can reference them.3416

Remember, we a histone core, we have 8 histone proteins, we have 2 dimers of H2 and H2B,3420

one tetramer of H3 and H4.3429

147 base pairs of DNA wound around.3433

It is about 1.65 times around the core.3437

We also have linker DNA, 20 to 60 base pairs.3440

Just that nucleosome will compact DNA about 6 fold.3446

When you add in the linker histone H1, you can compact even more into your 30 nm fiber from your 10nm,3452

that will compact DNA about 40 fold.3462

You can even compact even more than that, utilizing scaffolding so that you can make a bunch of loops.3466

We have talked about the histone code before, there are post translational modifications of our N-terminal histone tails.3477

That is part of the epigenetic code.3485

If we remember, phosphorylation usually adding a negative charge, acetylation positive charge.3487

If you are adding a negative charge that is going to decrease its interaction with DNA.3494

It will loosen it, making it more transcriptionally active.3502

Acetylation adding positive charge.3506

That is neutralizing the negatively charged lysine which is actually going to open you up.3513

Methylation will increase the interaction of DNA meaning transcriptional repression.3519

You also have ubiquitination which can be binding site for either transciptional activators or repressors.3524

We have talked about the histone code being very complicated.3534

You can silence, you can have gene expression, gene silencing.3537

You can have chromosome condensation, you can have DNA damage repair,3541

based on several slight changes in the epigenetic code of the histone.3547

We talked about nucleusome assembly.3556

When you replicate your DNA, you have to replicate your nucleosomes.3558

Some of those nucleosomes get reused in the next generation.3563

What those nucleosomes, whatever they had for epigenetic changes, methylations, ubiquitinations, phosphorylations,3569

acetylation, whatever, that will be transported with the nucleosome to the daughter cell.3578

That can propagate the epigenetic status, from one generation to the next.3587

What is very important are those parental H3 H4 tetromers.3600

We have talked about the inheritance of the chromatin state.3606

Histone acetlytransferases combined acetylated tails using its bromo domain.3611

Therefore, spread the acetylation which would decrease the condensation of the heterochromatin,3617

to make it more euchromatic, therefore, transcriptionally active.3631

Or you can have the same thing happening with histone methyltransferases3636

binding methylated regions via its chromo domain, causing a condensation.3641

Therefore, more heterochromatization IE transcriptional repression.3649

We will just write this real quick.3657

Histone acetyltransferase binds acetyl groups via its bromo domain leading to a looser state3660

which is more likely going to be a transcriptional activation.3679

We have histone methyltransferase via its chromo domain, binding methylated histone tails3685

which will tighten, condense, leading to a more likely repressed state.3698

Chromatin remodeling, it is all part of epigenetics.3711

As I said on the previous slide, when we have histone acetyltransferase,3715

as well as chromatin remodelers like the swi/snf complex,3718

that allows you to loosen up the interaction of DNA with the histones and allow your transcription of machinery to come in.3722

Therefore, being in an activated state.3732

If you have your histone deacetylases and your histone methyltransferases, that is going to cause a tighter association with DNA.3735

Therefore, less room for your big RNA polymerase and all your initiation factors to come in and allow transcription.3743

This is a transcriptionally repressed state.3751

Our last example, I believe, we have methylation of DNA and histones seeming to be correlated.3757

Usually, when both DNA and histone tails are methylated, this will be our transcriptionally repressed state.3765

Acetylation of histones leads to loosening of DNA around the histone, offering a high likelihood of replication or transcription.3773

Acetylation, high likelihood of replication and transcription.3784

Methylation, transcriptionally repressed state.3792

Finally, just as a mentioned, we have our polycomb repressors.3801

These polychrome repressors just are a family of proteins that can remodel chromatin,3806

such that epigenetic silencing will take place.3813

PRC, that is just polycomb repressor complex or repressive complex.3818

PRC2 protein complex will bind DNA and try methylate your histone 3 at lysine 27.3825

This will cause a repression.3834

The PRC1 protein complex will also repress transcription.3841

We have another protein complex called the MLL protein complex which will reverse this repression by demethylating H3K27.3846

A couple things I want to point out is that, the PRC1 protein complex, a mutation in this,3859

if you are a mutant, like they can make in labs, mutants die perinatally.3870

Meaning, either at the time of birth or shortly after no more than two weeks, in under a couple of weeks.3879

The PRC2 mutants are what are called embryonic lethal, meaning they never get born in the first place.3896

These are really important complexes.3913

One more thing to show you how important they are is that, over expression, if PRC1 and PRC2 are too active,3915

either too many around, they are repressing too much.3925

If they are over expressed that is actually correlated with a more severe and more invasive types of cancer.3927

Your cancer is likely to be, you are worse off if you have over expression of your polycomb repressor complexes.3942

That is the end of the lesson today, I hope you enjoyed our lesson.3952

I hope I see you back for our next one.3957

Thank you for joining us at www.educator.com, I hope to see you again.3963