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Genome Organization: Chromatin & Nucleosomes

    Medium, 4 examples, 5 practice questions

  • The genome is a highly organized structure than includes both nucleic acid and proteins.
  • Chromosomes often vary in size, number, and density between different organisms.
  • Genome size is roughly directly correlated with the complexity of an organism.
  • Genome density is roughly inversely correlated with the complexity of an organism.
  • Nucleosome and chromatin modifications can be responsible for transmitting information to future generations without changing the DNA sequence, also known as epigenetic inheritance.

Genome Organization: Chromatin & Nucleosomes

Generally, the more complex an organism, the ________ the genome.
  • More dense
  • Less dense
  • Shorter
  • Less complex
Which of the following is not one of the three requirements for proper chromosome duplication and segregation?
  • Centromere
  • Origin of replication
  • Telomeres
  • Translation
What is the first phase of Mitosis?
  • Anaphase
  • Metaphase
  • Prophase
  • Telophase
Phosphorylation of histone tails adds ______.
  • Methyl groups
  • Positive charge
  • Negative charge
  • Small peptides
Methylation of DNA and histones is often correlated, resulting in what chromatin state?
  • Repressed
  • Activated
  • Unchanged
  • Damaged

*These practice questions are only helpful when you work on them offline on a piece of paper and then use the solution steps function to check your answer.


Genome Organization: Chromatin & Nucleosomes

Lecture Slides are screen-captured images of important points in the lecture. Students can download and print out these lecture slide images to do practice problems as well as take notes while watching the lecture.

  • Intro 0:00
  • Lesson Overview 0:09
  • Quick Glossary 0:24
    • DNA
    • Gene
    • Nucleosome
    • Chromatin
    • Chromosome
    • Genome
  • Genome Organization 1:38
  • Physically Cellular Differences 3:09
    • Eukaryotes
    • Prokaryotes, Viruses, Proteins, Small Molecules, Atoms
  • Genome Variance 4:27
    • Humans
    • Junk DNA
    • Genes Compose Less Than 40% of DNA
    • Chart
  • Example 1 8:32
  • Chromosome Variance - Size, Number, and Density 10:27
    • Chromosome
    • Graph of Human Chromosomes
  • Eukaryotic Cell Cycle 12:07
  • Requirements for Proper Chromosome Duplication and Segregation 13:07
    • Centromeres and Telomeres
    • Origins of Replication
    • Illustration: Chromosome
  • Chromosome Condensation 15:52
    • Naked DNA to Start
    • Beads on a String
  • Mitosis 16:52
    • Start with Two Different Chromosomes
    • Split Into Two Diploid Cells
    • Prophase
    • Prometaphase
    • Metaphase
    • Anaphase
    • Telophase
    • Cytokinesis
  • Cohesin and Condensis 21:06
    • Illustration: Cohesin and Condensis
    • Cohesin
    • Condensin
    • Illustration of What Happens
  • Cohesins 27:23
    • Loaded During Replication and Cleaved During Mitosis
    • Separase
  • Nucleosomes 27:59
    • Histone Core
    • Eight Histone Proteins
    • Octamer of Core Histones Picture
  • Chromosome Condensation via H1 30:59
    • Allows Transition to Compact DNA
    • When Not in Mitosis
  • Histones Decrease Available Binding Sites 32:38
  • Histone Tails 33:21
  • Histone Code 35:32
    • Epigenetic Code
    • Phosphorylation
    • Acetylation
    • Methylation
    • Ubiquitnation
  • Example 2 38:48
  • Nucleosome Assembly 41:22
    • Duplication of DNA Requires Duplication of Histones
    • Old Histones Are Recycled
    • Parental H3-H4 Tetramers Facilitate the Inheritance of Chromatin States
  • Example 3 46:00
  • Chromatin Remodeling 48:12
  • Example 4 53:28

Transcription: Genome Organization: Chromatin & Nucleosomes

Hello, and welcome back to, today's unit is on genome organization.0000

We will focus on chromatin and nucleosomes.0006

As an overview, we will first go over a real quick glossary of terms, just so that we are on the same page.0010

Then, we will mainly focus on our genome variance and our nucleosomes.0016

Our quick glossary, just in case we do not know any of these terms.0024

DNA, that is our genetic material that we find inside each cell.0028

A gene is going to be our molecular unit of heredity.0034

This is a piece of DNA that will encode any type of functional product, whether that is a protein or RNA.0040

We have a nucleosome and that is going to be the fundamental subunit of chromatin, 0048

that is just a basic unit of DNA packaging in eukaryotes.0053

These consist of DNA wound around a histone octamer -- we will talk further about what that is.0059

We have chromatin. This is a complex of DNA protein, DNA and protein that forms chromatins and we only find this in eukaryotes.0068

Then, we have a chromosome and that is a tightly packaged and organized structure of DNA and proteins,0080

including our histone, as well as non-histone proteins.0086

Finally, a genome, that is just a complete copy of the entire sequence of DNA of an organism.0090

Let us talk about how a lot of these fit into play.0101

If we go from the most zoomed in, we have genes which consists of introns and exons.0106

We have intergenic regions, non-genes.0117

All of that is a piece of DNA, these are the smallest units.0119

If we zoom out a little bit, genes to DNA.0125

DNA, we can compact that DNA, compact it over and over and over again by winding unit around nucleosomes.0133

Nucleosomes can compact more and more, forming types of chromatin.0144

Chromatin is just DNA and protein interactions.0150

This would be an example of a type of chromatin down here.0157

As we compact even more, we have all of our chromatin compacting into chromosome.0162

If we remember, as humans, we have 23 sets of chromosomes, we are diploid so we have 46 chromosomes.0171

Remember, we have chromosomes 1 to 22 and then we have the X and Y chromosomes.0181

Let us talk about the physical cellular differences between eukaryote and prokaryote quickly.0191

Eukaryote, these are cells with nuclei, if we look down here that is a eukaryote and prokaryote.0197

The biggest one is that we have a nucleus in eukaryote.0204

In a prokaryote, they do not, they have what is called a nucleoid 0210

which is very similar as to where their DNA and protein are held together but it is not enclosed in a membrane.0212

Eukaryote may also have some extra things such as mitochondria, that prokaryote do not normally have.0221

Eukaryote are the biggest, we are anywhere from 10 microns to 15 microns, which is micrometers.0229

We can be even bigger than that.0242

Smaller than eukaryote are prokaryote, a bacteria types of things.0245

Smaller than that is virus, the thing smaller than viruses are what make up all of life.0249

We have our atoms, our small molecules, our proteins, these are going to be found in all of our viruses, prokaryote, and eukaryote.0257

It is important to know that there is a variance of genomes.0268

Since, we are going to talk a lot about human stuff in this entire course.0274

We will also talk about prokaryote, bacteria, specially our E coli.0281

We want to know the relationship between all of them.0286

First, let us talk about humans, most of our genome does not code for anything.0291

It is not composed mainly of genes, it is composed mainly of intergenic regions.0298

More than 60% of the human genome does not code for a gene.0305

46% of the total genome, 46% of the total DNA is repetitive sequences and that is often referred to as junk DNA.0312

But that is a misnomer and that is not a term that we should be using because,0324

just because we do not know that it has some sort of function, it does not mean that it actually does not have a function.0329

There are studies coming out every year that is finding what normally was thought of as junk DNA or even non-coding RNA, 0336

coming out as being coding for something or as being involved in some sort of gene regulation.0344

You can regulate the way that genes are expressed higher or lower, even in the non-gene sequences, the intergenic regions.0353

Genes compose less than 40% of the total DNA, and of that, less than 5% is actually the exon.0365

The exons are what is actually going to be added into the protein.0373

It has the instructions to how to make a protein.0379

The introns always cleaved out of the mRNA.0382

If we look down at this chart that I have made down here, I have four different organisms on here.0385

I have a very simple virus which is the bacteriophage virus.0392

It infects bacteria, it is called φ X174.0399

Then, we have our old friend E coli, and then drosophila melanogaster.0403

E coli is a bacterium, drosophila melanogaster is the common food fly, and then, we have us, the humans.0409

In general, genome size is roughly positively correlated with the complexity of an organism.0417

The bigger the genome size for the more complex organism.0429

The smaller the genome size for the lease complex individual.0437

This is size of DNA, in terms of mega bases, thousands of bases.0444

Also, another general thing you can talk about is that genome density, the amount of genes per base pair sequence,0454

basically, is roughly negatively correlated with the complexity of the organism.0464

A very simple organism like the bacteriophage has a much higher density of genes found in its genome than humans, 0471

which we have a very a low genome density, or gene density in our genome.0480

We have about 20,000 genes but that is spread out over more than 3 billion bases,0485

whereas the bacteriophage has 11 genes but it has an extremely small genome.0494

We are talking about 5000 bases.0502

For our first example, let us talk about how big the human genome is.0514

I said, it is over three billion base pairs long and that is just the haploid genome.0520

We are a diploid organism so we have twice the amount of that.0525

L, the length of just that 3 billion base pairs, if we were to spread it all the way out, that is a meter, close to 3 feet.0529

The diploid length, the doublet, that is 2 meters, about 6 feet around of DNA.0541

The diameter of human cell is only about 10 to 15 micrometers.0550

What does that really mean?0558

Can we understand the size of that?0562

If our cell was actually the size of a basketball, if we blew it up the size of a basketball, and everything else accordingly.0565

Then, our genome would actually be able to stretch from the earth to the moon.0577

Just based on the size comparison.0586

This to me says that, we need a really good way of compacting that DNA.0591

Because we cannot have the 6 feet of DNA inside a cell, that you cannot even see with your naked eye.0598

We need to compact that really well and we need a way of uncompacting things, decondensing or condensing things,0608

based on whether we need to do things such as replication, transcription, repair, so on and so forth.0616

That is what this unit is all about.0624

First of all, let us talk about chromosome variant.0629

We are going to see the size and density variance, even in between a single cell.0634

We will have chromosome variance, in terms of number, in between different organisms.0642

Once again, a reminder, a chromosome, we have a tightly packaged and organized structure of DNA0648

and proteins including both histone and non-histone proteins.0654

Right here, we have a graph showing human chromosomes.0658

We have our total number of genes, in pink here.0663

We have the total base pairs, in green.0668

As we can see, between individual chromosomes in the same,0673

we have not only a difference or variance between chromosomes in genes, but we also have a variance in terms of base pairs.0679

If we look it to, they are fairly similar in size, such as 1 and 2.0693

We also have a difference of variance in gene density because we have so many more genes, for barely that much more base pairs.0700

This is something that we should always keep in mind, that chromosomes are going to vary by size, number, 0715

and density between organisms and even within the same organism.0722

First off, let us talk about the eukaryotic cell cycle and why do I bring this out of nowhere.0729

The reason being, let us go back to this because we have talked about in one of the previous unit.0736

We will talk about it in the next unit again.0742

Remember, we have our two growth phases, G1 and G2.0746

We have S phase which is where DNA replication occurs.0750

We have mitosis, anaphase, that is where we have the splitting of our cells into two new daughter cells.0758

The reason I talk about this is that, we have to know that when we replicate DNA,0774

we are going to have to replicate a lot of other things relating to DNA.0781

One of these things that we are going to have to do is, yes, we duplicate DNA and then we segregate our DNA, or separate it.0788

In prokaryote, that happens at the same time.0796

In eukaryote, that happens at two different phases.0799

We duplicate in S phase, we segregate in anaphase.0801

Duplication, specifically segregation, relies on centromeres and telomereses.0809

Duplication is going to rely on our origins of replication.0817

If I draw out a chromosome, just a real rough drawing, we will just draw that like this.0823

We have already duplicated it, let us say that.0832

Let us start with before it is duplicated.0837

If we were on one chromosome, along the chromosome in eukaryote, 0840

we are going to have multiple places where we have origins of replication.0846

That means that, that is where the DNA synthesis during replication can begin.0851

If we are talking about a bacteria, it has a single origin of replication.0857

That is because it is circular, if you replicate it at one place,0862

it will actually start replicating in both directions until it ends at the terminal point.0866

Since, eukaryote, we do not have a circular chromosomes, we have linear chromosomes.0872

We start at many different places, that way it ensures that we can finish replication before we move through S phase.0880

There is our origin of replication.0888

After we have replicated our chromosomes, we are going to have a centromere.0890

A centromere is going to be a bunch of repetitive sequent in the chromosome.0898

That is where our two chromosomes are going to bind together during our anaphase.0904

It can be in the center, it can be on one end or the other, so on and so forth, and that is fine.0910

Telomeres are always going to be at the ends of our chromosomes.0915

These are telomeres sequences and this is just repetitive sequences that capped the ends of our chromosomes and 0922

allow us to have some protection from nucleolytic cleavage, as well as having adhesions,0928

binding into another chromosome which would be a very bad thing.0938

We need centromeres and telomereses, as well as origins of replication0943

to proceed through proper duplication and segregation of chromosomes.0948

Remember, I said, we need to have a way to condense our chromosomes quite a lot.0955

We have our naked duplex DNA to start with, then, we can compact it with nucleosomes.0959

We are going to talking more about nucleosomes in this lecture.0967

This is just a nice little preview.0971

This is our beads on a string or 10 nm fiber.0973

That can even more compacted, when you add in linker histone H1 to make the 30 nm solenoid fiber.0979

That can be extended even more by a chromosome scaffolding and eventually condensed in mitosis to look like the stereotypical X.0989

It looks like that X, real dark X chromosome.1001

Not X in the sex chromosome, X is in the letter X.1005

We need all this to happen.1014

I showed on the last one, we have that mitotic chromosome being extremely condensed.1016

The reason these seem to happen in mitosis is that, we need to be able to separate our daughter strands.1021

We need to make sure that everything is condensed into their individual units, their individual chromosomes,1027

so that we can properly segregate everything, that nothing gets tangled up.1034

If we remember mitosis, we start with, in this example, two different chromosomes.1038

You duplicate those chromosomes then you split that one cell into two different daughter cells.1044

Each one of which has the exact copies of that first red chromosome and that second blue chromosome.1051

Real quickly, we will go over what mitosis is, it is split into multiple phases.1059

First off, we start with prophase.1065

Prophase is when we start condensing our chromatin into chromosomes.1067

This is where we are going to start to get more and more condensed.1072

In pro metaphase, we have the nuclear envelope starting to disintegrate.1076

That nuclear envelop needs to disintegrate so that you can start moving the duplicated chromosomes away from each other.1084

Remember, once we start mitosis, we have already gone through S phase.1092

We already have twice as many chromosomes.1097

We have two times in our chromosomes that we want in every cell.1100

We have the nuclear envelope disintegrating.1105

We have microtubules coming in to grab onto those chromosomes.1107

The microtubules attached to kinetochores.1114

kinetochores bind to the centromere of the chromosome.1117

We have kinetochores binding on here.1126

Kinitocour is binding on the centromere.1130

And then, that allows the microtubules to attach to the kinitocour.1133

Eventually, they pull in either direction separating the chromosome.1140

It breaks the centromere apart.1147

In metaphase, the next step, this is all occurring throughout entire mitosis not just the pro metaphase.1151

In metaphase, we have the chromosomes aligning at the metaphase plate.1158

It is basically the midline of the cell.1162

The next part of mitosis is called anaphase and that is when the chromosome split.1169

The centromere breaks, you can break the centromere apart.1177

Or you break the chromosomes apart at the centromere.1181

The chromosome split and the kinitocour microtubules.1186

The microtubules attached to each kinitocour.1189

A kinitocour is attached to the centromere.1194

Those are being pulled in opposite directions.1197

It has to be at each pole of the cell.1201

This pole and this pole, which will eventually be two separate daughter cells.1205

In telophase, we have these decondensing chromosomes.1211

They are starting to not be as compacted, they are starting to release a little bit.1215

The get reformed, or what forms around in newly is new nuclear envelopes.1220

We start to do what is called cytokinesis.1229

The cytokinesis is the actual separation into two different daughter cells.1233

We have the cells pinching in, this is what we call a cleavage furrow.1240

The cell starts pinching in and eventually pitches off to have two separate cells.1248

In this case, each one of these cells has the exact amount of chromosomes as the original parent cell, 1254

before it went through S phase.1262

We have to talk about a couple of really important protein complexes,1269

when we talk about mitosis and we talk about proper chromosome segregation.1275

We have what are called cohesin and condensins.1280

This is cohesin and this is condensin.1284

Cohesins are loaded during replication.1297

Condensins are being loaded during N phase.1303

What actually happens is, if we look at a chromosome, this is a duplicated chromosome,1308

we have cohesins using this little hinge, binding around the chromosomes.1319

This is binding our sister chromatid together.1336

This is in S phase.1344

Normally, when we go into mitosis, we have prophase, we still have our chromosomes.1350

We still have our cohesion, holding the sister chromatids together, this is prophase.1372

In prophase, we are starting to condense our chromosome.1393

How we can do that is, condensin will come in metaphase.1396

In metaphase, we will go over here.1409

We still have our condensins.1426

I will make this easier.1436

Those are our cohesions, condensins will be in red.1438

Our condensins, in metaphase, will start binding these together.1447

Our condensins, after replication, compact each sister chromatids individually, making it even more and more.1457

And then, if we move on toward anaphase.1468

In anaphase, we actually have our condensins still on there.1483

Let us write this out, anaphase.1495

But our cohesins have been cleaved, our condensins are still there.1501

Our cohesins get cleaved in anaphase.1508

What we have is, this kind of becomes a little squiggly because it is no longer bound to each other.1510

It is squiggly here too.1523

It is no longer bound there.1529

It is important that cohesins get cleaved because that allows a separation of these sisters.1530

This one can go to one cell, this one can go to another cell but we still have a little compaction.1539

Eventually, once we have separated them into different cells, the nuclear envelope starts to form,1545

these condensin can then start to be cleaved.1551

What we have, just to show us right here, SMC, all these are cohesins and condensins, 1555

they are related proteins, a part of the SMC complex.1562

SMC stands for structural maintenance of chromosomes.1566

It is important that we have this compaction.1588

Cohesin and condensins play a large role in the actual condensing or compaction of DNA.1591

Remember, we have our cohesin binding sister chromatids together.1599

Condensins bind sister chromatids individually.1619

In this slide, I already talked a little bit about but I just want to reiterate.1645

We have our cohesin being loaded during replication and being cleaved during mitosis.1649

They are cleaved during anaphase.1655

They are cleaved via the enzyme separase.1657

This occurs right as we are moving into anaphase.1665

It is actually cutting up the end of metaphase and 1670

separase is what then allows the sisters to be segregated into individual cells.1673

One thing that I just want to mention about the previous slide is that after replication,1686

the microtubules have attached to the kinetochores.1692

They pull in opposite directions until those cohesin proteins get cleaved.1696

Separase is in there, cleaving, as well as kinetochores are pulling apart.1702

Eventually, that allows you to proceed into anaphase.1706

Now that we talked about the mitotic chromosomes, we need to talk about what about we are not in mitosis?1713

We still need to have some sort of compaction of the chromosome, that is occurring via nucleosomes.1722

We have what is called the histone core.1730

The histone core is composed of 8 histone proteins.1736

These histone proteins are positively charged, that is great because negatively charged DNA will attract positively charged proteins.1741

What we have, if we look down here at the octamer of core histones, 1754

we have two dimers of H2A and H2B, meaning there is two of each of those.1763

We have a tetramer, the H3H4 meaning we have two of each of the H3 and H4.1770

Around that, we have 147 base pairs of DNA, and that DNA is wound around that nucleosome, wound around 1.65 times.1776

The DNA wound around that histone octet 1.65 times.1796

We have what is called linker DNA and that is what will link many different nucleosomes together.1805

Remember, we are not just going to compact one little spot.1811

The linker DNA between those nucleosome is anywhere from about 20 to 60 base pairs.1814

A single nucleosome will compact DNA about 6 fold.1821

If they are just in the nucleosome, and we have a bunch of nucleosome all the way throughout.1825

Just nucleosomes, remember, we are looking at the 10nmm fiber here, a 6 fold compaction.1831

We need to compact it even more, we have 3 billion base pairs.1849

Over 2 meters of cells, when we are in diploid, 1/1 meter of cells when we are in haploid form.1853

We need to condense even more.1860

What can help with that is histone H1, also known as the linker histone.1862

That allows the transition from this 10 nm fiber which we just talked about,1868

which is just the nucleosomes and linker DNA, into the 30nm solenoid.1877

This helps compact DNA about 40 fold, from 6 fold to 40 fold between the 10 nm and the 30 nm fiber.1886

When you are not in mitosis, further compaction of DNA can be mediated by the organization of these 30 nm filament into loops.1897

These are our 30 nm filaments in loops.1909

That can consist of 40 to 90 bases per loop and that can associate with scaffolding.1915

We can get more and more compaction because we need it.1922

That is the simple aspect behind, we need all this compaction and there is an intelligent design behind this.1928

If we want talk mitosis, you can get even more condensed and eventually get to that mitotic chromosome.1938

But these, you cannot really do much with, there is no replication,1944

no transcription, none of that stuff happening during mitosis, no repair.1949

Histones, as they bind DNA to compact it, they take up available binding spots.1961

Because proteins like to bind DNA but they do not like to bind DNA when there is already something there.1968

These histones will decrease any available binding spot on the DNA.1975

Meaning, it basically makes it unavailable for anything to bind.1982

Therefore, affect whether maybe is transcribing or replicating or being repaired1988

or having interactions in bringing two pieces of DNA together.1996

Our histone tails, we have talked about the histones that are in the middle.2002

In this case, we see H2A is in yellow, H2B is in red.2007

I will write this in black, just in case you cannot see the yellow.2028

H3 is in blue and H4 is in green.2032

We have the histone octet, in the middle we have DNA wound around it, remember 1.65 times.2039

What I want to point out is, we will use a purple color.2047

Look at these coming out of the DNA, these are what are called N terminal histone tails.2054

The N terminal so the beginning part, if we think about N to C terminal.2066

These N terminal tails can interact with DNA.2070

This interaction with DNA can affect how anything, any further process may occur.2075

These histone tails can be post-translational modified.2087

Meaning, they could not have some types of groups,2091

some functional groups added to them and that will affect or alter the strength of their interaction with DNA.2095

If let us say, it strengthens the interaction of DNA, it makes it real tight, meaning it is not very available for protein to come in.2103

If the type of post-translational modification makes its interaction with DNA decrease,2111

then the DNA loosens around the nucleosome.2120

Therefore, that allows more proteins to possibly come in and maybe we can go through transcription, for instance.2124

These post-translational modifications on these N terminal tails have some sort of pattern that has been seen throughout history.2135

Scientists have theorized what is called the histone code.2149

What we see is that post-translational modifications of these N terminal histone tails,2153

somehow are related to epigenetics.2161

It is part of this epigenetic code along with, maybe DNA modifications,2164

like we have talked about methylation of DNA in other types of modifications of DNA.2170

These post-translational modifications of histone tails, maybe associated with certain modifications on DNA.2181

Together, they can be passed on to generations even though the DNA sequence itself has not been changed.2188

It is epigenetics, it is not the actual sequence is changed.2197

We have some sort of hereditary possible change.2200

This is when we are talking about the histone code.2204

Such things, such post-translational modifications of these tails, 2207

such as phosphorylation which would add a negative charge.2211

Acetylation which would add a positive charge.2217

We have methylation which is the addition of methyl group.2221

Ubiquitnation which is an addition of a small protein group.2224

What we can see is that, there are very highly conserved residues in these N terminal tails.2228

We find them in many organisms throughout.2235

It shows that there is likely a common theme, a common function of this.2237

What we see is that, methylation which will increase the interaction with DNA, and recruit proteins called chromoproteins.2244

Methylation of residues on the N terminal tails such as lysines and argentines, will recruit these chromoproteins.2261

What will eventually happen is that, these chromoproteins will actually somehow figure out a way2271

to spread or propagate this methylation to neighboring nucleosomes.2278

The same thing can happen via acetylation, adding positive charge,2285

including bromodomain proteins which would neutralize the negative charges of lysines.2290

Acetylation of lysines recruit bromodomai containing proteins which can spread that,2296

can be acetylation type modification to neighboring nucleosomes.2305

As I said, down here, with ubiquitnation, that can divide a binding site for transcriptional activators or impressers.2312

It can increase the rate of transcription or decrease the rate of transcription.2319

Remember, transcription is DNA to RNA.2323

An example of histone coding, just a lot of few.2330

If we have an unmodified histone 3, that usually codes for the fact these genes are silent, wrap around this nucleosomes.2334

If we have a histone H3, the H3 histone, protein, if it has a lysine, 2346

the amino acid is a K, 14 that means it is the 14th amino acid in the protein.2356

It is the 14th amino acid of the H3 protein being a lysine.2362

If it is acetylated, that often leads to increased gene expression, that is saying that transcription is active.2367

If we have the 9th amino acid which is also happens to be a lysine,2378

if that gets methylated 3 times with 3 different methyl groups, that will the silent.2383

Meaning, transcription is not going to be active.2392

There are few other traces to look at, one thing I want to point out is even the exact same histone,2396

H4, the exact same residue lysine 20, if you methylate it, you add one methyl group,2407

it will start chromosome condensation.2420

If you add a second methyl group, that will signal that DNA damage repair proteins to come in and 2424

fix some sort of damage that is been found on this DNA.2434

If you methylate it one more time, you are back to chromosome condensation.2440

They wait for DNA damage repair to occur, the chromosome has to be decondensed,2445

it has to be loosened from the nucleosome so that the machinery, the damage repair machinery can come in and fix things.2452

We go from condensed to decondensed, back to condensed, 2462

all by having a different modification to the same residue on the same histone.2472

We have talked about how the nucleosomes attach to DNA, how they interact.2485

We have not talked about how nucleosome are assembled or how they are replicated.2491

Remember, every time DNA gets replicated through S phase, we are getting twice the amount of DNA.2495

Therefore, we need twice the amount of nucleosomes because those DNA,2500

once put into daughter cells, are going to have to end up compacting again.2505

The duplication of DNA requires the duplication of histones.2509

This is going to occur in S phase just as the synthesis of DNA is going to occur.2513

We have all the histones being recycled.2521

We have all the H3 and H4 tetromers remain associated with one of the two DNA daughter strands.2525

And then, when we synthesize brand new H3 and H4 proteins in the tetromeric form, to bind the other daughter DNA strand.2532

And then, we have old H2A and H2B diomers completely being removed,2545

either as an old set can get back into the DNA or a brand new H2A H2B will jump in.2553

It is just a competitive interaction, it is just which one gets to be grabbed in by the DNA and added.2561

What is important to point out is that, chaperone proteins are involved in nucleosome assembly.2569

The chaperone proteins and what those are, they are just proteins that help these reaction occur.2575

We have what are called chromatin assembly factors COF1 and NAP1, 2584

being necessary for the proper assembly of nucleosomes.2596

COF1 is going to be responsible for bringing in new H3H4 tetromers to nucleosomes.2599

This will say that, this response will for H3H4.2607

NAP1 is responsible for bringing in new H2A and H2B diomers into nucleosomes.2614

Without these chaperons, nucleosomes likely will fail to assemble.2626

Even if they do, it will take quite a lot of time.2632

This can affect how quickly a cell would be able to get into, let us say, anaphase.2635

One of the last things that I want to talk about nucleosome assembly is that parental H3 and H4 tetromers.2647

Remember, one of those is going to stay completely with the original strand, the parental strand.2654

You will get brand new on the daughter strand.2664

Parental H3H4 tetromers will facilitate the inheritance of chromatin state.2668

Now, we are talking back to epigenetics.2675

This will provide a template for the propagation of any preexisting histone modifications.2678

These preexisting histone modifications could signal the fact that this gene should be active2687

or this other gene should be repressed.2695

That will affect what proteins possibly are getting synthesized or at least what RNA are being made via transcription.2699

This can help restore the parental chromosome state to any of the daughter cells.2709

These modifications can actually then recruit other modification enzymes of the histones,2715

to propagate this modification along adjacent histones and throughout the region of chromatin.2722

This can allow the actual passing from replication to replication, it can pass what is called the chromatin state.2729

Maybe this is methylated or maybe this is phosphorylated.2740

You start brand new, when you add new nucleosomes.2744

You can propagate the spreading of the modifications due to the parental H3 and H4.2751

As an example, our inheritance of chromatin state, we have enzymes called histone acetyl transferase or HAT.2762

They can bind acetylated histone tails, using what is called the bromodomain.2791

That will then allow HAT to move, here is DNA, here let us say that these are wrapped around nucleosomes.2807

These are nucleosomes.2824

If HAT binds, we are just going to say HAT is this.2833

If HAT can bind, it can bind there.2841

It can actually in a billion, it will bind either this acetyl group.2850

It can move to the next nucleosome, all in there again, adding another acetyl group,2857

and propagating this inheritance of the chromatin state.2866

The same can happen with histone methyltransferases, HMT, using their chromodomain.2871

Histone acetyltransferases, remember, use their bromodomains to bind acetylated histone tails.2879

Histone metyltransferases, HMT, use their chromodomain to bind methylated histone tails.2887

They could end up propagating, as well.2924

Remember, histone acetyltransferases often loosen the interaction of the nucleus on the DNA, making it more accessible.2927

Histone methyltransferases, often compact, they increase the binding affinity of the nucleosome in DNA.2935

Therefore, compact it even more and do not allow things to come in.2945

An example of that we can see in one of the next slides, talking about chromatin remodeling.2950

Remember, we have our DNA, we have to get it into our 10 nm fiber or 30 nm fiber, we need to compact it.2958

We have our basic unit of chromatin organization being our nucleosome.2969

Each one of these is a nucleosome.2973

This might be our 10 nm fiber.2979

This is a region of 30 nm fiber.2983

The degree of our nucleosomal packaging can affect any DNA processes, 2988

whether it would be transcription, repair, replication, any of that.2996

The ones that are in the less condensed state are what are called euchromatin or euchromatic regions, 3002

they are more transcriptionally active.3011

They are less condensed, they are the more likely other proteins can come in and bind.3014

Our heterochromatic regions are much more condensed and much less likely to have any type of activity occurring.3021

Nucleosome positioning in chromatin compaction, where are these nucleosome are positioned, maybe there is a gene there.3032

Maybe there is a gene in there, that will affect whether a gene is active or not.3040

If it is in an open region, it can be activated.3048

If it is bound around that nucleosome, maybe it is very likely it is not going to be active.3051

The positioning and the compaction can be altered by remodeling complexes.3058

If we look at this next slide, you can see what I mean.3064

In the top one, in the presence of histone acetyl transferase and the absence of our histone methyl transferase, 3069

which if we remember, usually causes the compaction of our DNA.3078

This chromatin will be much more loosely packed and it will be transcriptionally available.3085

That is due to our chromatin remodel or complex.3091

This helps move things around, maybe it is decondensing the nucleosomes or decondensing the fibers.3097

Maybe it is sliding these nucleosomes from away from this region3108

which should normally have a gene be transcriptionally repressed.3115

At this point, we want to activate it.3121

Sliding complex can move these nucleosome, maybe they were here to begin with and got moved over,3123

freeing up this part to be transcriptionally activated.3130

If we look down at the bottom, we have histone deacetylase, HDAC.3134

This is taking off acetyl groups which mean we are taking away 3141

what would normally loosen our chromosomes or loosen our DNA around the nucleosome.3145

That is going to compact.3151

Histone methyl transferase, the methylation is going to compact.3153

This is going to compact this more and more and it would not allow certain machinery to come in and do whatever job it wants.3156

If you look up here, we have had around no methylation, no HMT.3164

We have these remodeller complexes, we have allowed our region to be opened up that we can actually do something with.3170

We have allowed, if we are going to do transcription, we would allow RNA polymerase 2 to come in3180

and be able to start transcribing our mRNA.3186

Whereas, we do not have any room for RNA polymerase to come in and bind in between these really tightly bound nucleosomes.3192

As our last example, we are just going to talk a little bit about what we have mentioned so far.3210

I want to give you an example that I have talked a lot about histone modifications.3219

Methylation of DNA and methylation of histones, actually seems to be correlated.3229

When both your DNA and histone tails are methylated, this usually causes that repressed states.3234

Then, we have histone metyl transferase around, maybe the HDAC, the deacetylase.3243

We are not allowed to get all this machinery into the open spaces of DNA, because it is not very open, it is very condensed.3248

I have mentioned these two words before, heterochromatin and euchromatin.3257

What we see is that heterochromatin is usually transcriptionally repressed.3264

It is in repressed state, you cannot do anything in it.3270

It stains darkly with the Giemsa, Giemsa is the way that you can visualize proteins.3273

That is spelled this way.3280

You can do Giemsa and look at it under a microscope.3284

Heterochromatins stains very darkly because your DNA are so compact.3288

The euchromatin which is transcriptionally active, remember they are going to be more loose.3293

Therefore, it is going to stain much lighter with that Giemsa.3297

Just the last thing I want to leave you with, before we end today’s lesson,3302

is that we have the acetylation of histones leading to a loosening of the DNA around the histone.3306

The reason being is our neutralizing the positive charges of our lysine residues.3316

This loosening offers a likelihood of replication or transcription.3321

When acetylation of these tails occurs, we are more likely to be in the euchromatic state.3327

When we have methylation of DNA, we are more likely to be in the heterochromatic state 3340

because methylation of DNA is correlated with the methylation of histones, often.3352

That is the end of today’s lecture, I thank you for joining us.3359

Thank you for being here with, please come back and see me again.3363