BIO 200 lecture 21 - McGill PDF

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BIO 200Chromatin, epigenetics, and the histone code But how can the transcription complex assemble on DNA when it is all wound up? DNA is almost never accessible in the cells (it’s not naked)  it’s all wound up o Its surrounded by the proteinaceous coat that is made up of histones called Chromatin ...

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BIO 200 Chromatin, epigenetics, and the histone code But how can the transcription complex assemble on DNA when it is all wound up? -

DNA is almost never accessible in the cells (it’s not naked)  it’s all wound up o Its surrounded by the proteinaceous coat that is made up of histones called Chromatin

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Chromatin  allows us to wind up that DNA strand so we can compact it into a tiny nucleus

 DNA is not naked in cells  It is associated with histone proteins (H2, H3, and H4) that bind it strongly and wind it up into high-order structures called nucleosomes Heterochromatin vs Euchromatin -

When we think of chromatin we can look at it in two ways: 1. Euchromatin = delicate and thread-like a. The delicate and thread-like chromatin is being actively transcribed 2. Heterochromatin state = densely packed and considered with transcriptionally repressed regions a. Heterochromatinized regions are not actively transcribed  they are silenced

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How does the cell make choices about where chromatin should be delicate and transcribed vs. regions that are condensed and heterochromatinized?

 Euchromatin is delicate and thread-like 

It is abundant in actively transcribing cells

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It may represent DNA that Is unwound to provide a transcriptional template

 Heterochromatin is a condensed form of chromatin that localises at the nuclear envelop often neat the nuclear pore 

Is considered transcriptionally inactive

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Transcriptionally inactive regions of the genome are maintained in a “heterochromatinized” state

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Their transcription could be detrimental to the cell/organism

How was this all worked out? The power of yeast genetics and biochemistry -

How do we regulate these higher order configurations that affect transcriptional regulation? = by looking at lower order organisms (particularly yeast)

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Bakers yeast has helped us to understand about how you can silence complete regions of chromosomes (it is easy to manipulate, and their genetics are clear)

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One of the means of understanding chromatin silencing = by using a simple yeast (saccharomyces cerevisiae) function o The yeast will reproduce and divide in a haploid form 

when things are really good this yeast is always in its haploid form

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when thing go bad this yeast will revert to a diploid form through mating

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how does this yeast mate? o Baker’s yeast is also referred to as the budding yeast  the budding occurs when the mother yeast grows to a certain point and a daughter starts to bud off

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The moment that the mother cell starts budding she switches her mating type  there are two mating types = can be either alpha or a o Every time the mother cell (in her haploid form) gives rise to her daughter  she switches her mating type so that in the very worst conditions she can actually mate with the cells that are next to her (have to be different mating types in order to mate)

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The information from the mating type is all wound up in two different places on the extremes of chromosome three in yeast

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The a-type mating information  which includes the gene for the transcription factor, the promoter, etc. is all on the right-hand side in the region called HMRa

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The -type mating information  which includes the gene for the transcription factor, the promoter, etc. is all on the left-hand side in the region called HML

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If both these regions are expressed  the yeast will be confused o Instead they are silenced while they are out there in order for the yeast to take on a mating type o In order for the yeast to take a mating type = this region has to be nonreciprocally recombined into the center region where it will be expressed  as long as it in the extremities it won’t be expressed (silenced)

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This says something about those locations  they have to be somewhat responsible for shutting down the transcription of those two important factors

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What is also important = it’s not just RNA Pol 2 specific  can take an RNA Pol 3 gene and introduce it to those regions and the Pol 3 gene will never be expressed

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Also, can take an enzyme from bacteria, introduce it into a yeast molecule  the enzyme modifies specific nucleotides in the DNA sequence (it will methylate A’s) but it cannot access HMR and HML  so the A’s are not methylated in those situations o Meaning that there is something that physically constrains those areas and doesn’t allow access to the DNA

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After many experiments, it became clear that: o Somehow the histones were involved  some regions of histones are critical to maintain the silencing o There are proteins that are important for interacting with these regions and setting up some higher order configuration that changed that part to silence the transcription of those genes in that area

 Three genetic loci on chromosome III genetically control the mating type of Saccharomyces cerevisiae  The HML and HMRa loci must be silenced otherwise the cells will be diploid a/  and cannot mate  Transcriptional repression depends on silencer sequences 

This silencer works outside the context of mating and can even block expression of tRNA genes (RNA Pol 3)

 Genetic experiments indicate that histones affect repression, whole regions around telomeres behave similarly Genetic screens identified factors required for repression of the silent mating type loci -

The proteins that were identified (that are involved with the silencing of transcription) are: 1. RAP 1  interacts with the telomeres (telomeres have to be silenced) a. You do not want gene expression in the telomeres 2. SIR 1 (silent information regulator)  interacts with RAP1 at these extremities of the chromosome to change the high-order structure and block transcription 3. SIR 2,3, 4 (host of other SIR proteins)  together when they start to build up (in regions of the chromatin) they will give rise to a repression of a transcription processes around where they are accumulating a. SIR 2 was found to have some enzymatic activity  removes acetyl-groups off the histone tails b. Those hypoacetylated histone tails are then recognized by more SIR 2,3,4  positive reinforcement (the more you get the more you hypoacetylated, the

more come in)  end up spreading of this repressive influence over a region of the chromatin (heterochromatin spreading)  RAP 1: 

Binds to DNA in the region of the silencer

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Also binds to repetitive sequences in telomeres

 SIR 1: 

Cooperates with RAP 1 and is important for binding the silencer region in the silent mating type loci

 SIR 2, 3, 4 

Binds to hypoacetylated histone tails (H3, H4) and recruits SIR2

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Forms large complexes with telomeric DNA

Transcriptional repressors may act through histone deacetylation complexes (HDACs) -

What does a deacetylase activity? o It is important in certain situations where the histones are interacting with the DNA o Under normal circumstances  the histones are positively charged and interact strongly with the negative phosphate backbone of the DNA o When they become acetylated  that electrostatic interaction become neutralized o Regions of Euchromatin (or regions where there is ongoing transcription) are associated with acetylation of specific histone tails  the acetylation relaxes the chromatin 

This relaxation allows access to transcription factors or others (DNA binding transcription factors)

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This whole idea of changing the histone tails has been coopted by a number of DNA binding factors (some that are repressive and some activating) o (using yeast as an example) a DNA binding transcription factor (Ume6 =has a classic DNA binding domain) interacts with a sequence within the DNA

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But in doing so it also tethers to another repressive domainthat interacts with a multi subunit protein complex

o This protein complex  has a protein (Sin3) and another protein (Rpd3) 

Rpd3 = has histone deacetylase activity  some histone deacetylase enzyme is going to be recruited to a gene (based on this DNA protein interaction

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This activity will deacetylate all the histone tails in the vicinity of that gene

o When you deacetylate the histones  that neutralization that you got through the addition of acetyl groups is eliminated and immediately go back to interacting very strongly with the DNA (they close up and condense) o In doing so you can shut down the transcription of specific regions by recruiting the Sin3 and Rpd3 complex  which will deacetylate all the histone tails near it -

Co-repressor

Those things are called Corepressors o A number of transcription repressors use these enzymes containing multi subunit protein complexes in order to actually change the chromatin around it  to shut it down

 Positive charge of N-terminal histone tail interacts electrostatically with the DNA phosphate groups  Acetylation neutralizes the electrostatic interaction and permits complex formation  Rpd3p is a histone deacetylase and demon states deacetylase activity  Specific targeting requires Ume6p (which binds URS) and finally Sin3p ...While activators often recruit Histone Acetyl Transferases (HATs) -

The alternative is also true for Coactivators

o Where DNA binding transcriptional activators will not only interact with the DNA sites that they recognize but also recruit in protein complexes that will modify the histone tails by adding acetyl groups o By adding the acetyl groups  you neutralize the charge between these histone tails and the negative phosphate backbone of the DNA o Doing so, you open up the chromatin  it becomes delicate, light and transcribeable -

(in the diagram – an example of yeast) a classical transcription factor Gcn4

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Gcn4 = DNA binding domain  it has an activation domain (since transcription factors are modular)

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The activation domain interacts with the multi protein complex that includes Gcn5  that is capable of acetylating histone tails

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These histone acetyltransferases (HATs) combined with DNA binding transcription factors = coactivators o These things affect the higher-order conformation of the chromatin in order to change transcriptional efficiency

 Some transcriptional activator can overcome the repressed chromatin state by inducing acetylation of histone tails through associated proteins (ie… Gcn4p and Gcn5p, GBP, p300) The Histone Code leaves its mark… -

Acetylation and deacetylation are not the only marks the occur on the histone tails o The histone tails are really important  (in addition to all the structural things they do) they are a scaffold for a number of signals

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The signals are put down on particular residues within those histone tails and each one of those modifications might be read by different transducer

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The marks that are put down on those histone tails = Chromatin writers

o they are enzymes that will put mark down on histone tails (in specific regions around specific genes) -

those marks are then read/ recognized by chromatin readers that will see these marks and will carry-out the appropriate processes downstream

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some of the marks include o methylations o phosphorylation o ubiquitylations 

these will be read by proteins that will affect some sort of downstream event

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mostly when you acetylate these histone tails  usually associated with transcriptional activation (the other marks are hard to generalize)

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For example: Methylation of particular lysine’s in H3  can give rise to radically different outcomes o The methylation of lysine 4 on histone 3 (H3 K4)  usually associated with transcriptional activation marks o If we put methylations on lysine 9 on histone 3 (H3 K9)  usually associated with condensation/ compaction of chromatin to give rise to heterochromatin o The methylation marks that are read by these effectors are very different and outcomes are very different

 Specific modifications ont ails of H3 and H4 induce changes in chromatin structure typical of EUCHROMATIN vs HETEROCHROMATIN … but be careful not to generalise  Methylation on H3K4  active  Methylation on H3K9 inactive

Antibodies against modified histone tails can be used for Chromatin Immunoprecipitation (ChIP) -

We can understand how various genes are being affected by these marks as long as we have antibodies to those particular antigens

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Can get antibodies that have been raised to all these histone modifications

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If you want to know if your gene is associated with some high-order change at the chromatin level,  you can carry-out ChIP

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For example: you can take an antibody to H3 K4 trimethylation and incubate that antibody with a sample to DNA that you collected/sheared from cells o That antibody will recognize that mark though the entire genome and then precipitate it using beads that have been conjugated to protein A o When you have the precipitate and wash off all the non-specific stuff

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Want to know if the gene of interest in the precipitate  carry out PCR o Using primers that are specific to the genes of interest o If you amplify something it is probably there, and it was somehow associated with the mark you were interested in (either if it was transcriptionally activated or repressed)

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Alternatively, if you want to look beyond a single gene  use next generation sequencing and sequence the whole genome o Get all the genes that had this particular mark associated with them  get a spectrum of the entire genes that are affected by them

 By using reversible crosslinking agents proteins bound to chromatin can be isolated using antibodies and the sequence of the bound DNA can be determined

1. Using known primers if you want to know whether a specific gene is affected 2. By using NGS the entire genome can be analyzed to determine what regions of the genome are being affected  Using HGS the entire genome can be surveyed to provide information about the genes (loci) affected by the marks Too much of a good thing: Dosage Compensation -

Females are mosaic  made up of patches that express not all the same genes (men are expressing all the same genes)

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Why are women different? o We know that most women have two X chromosomes and most men have one X chromosome you can’t normally have more than one dose of a chromosome (or if you have too little you should die) o Theoretically with a double dose of the X chromosome  fatal

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Turns out that very early of embryogenesis  all the cells in the embryo have to decide o There’s a counting mechanism that determines if you are a male or female o If the mechanism says you are female (by counting your sex chromosomes) then you have to inactivate one of your X chromosomes = X inactivation o Each cell will inactivate one X  then every cell division after will respect that inactivation

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With the inactivation of one X chromosome  it also shuts off the transcription of that X chromosome o Can see that through spreads of chromosomes that have been stained for activating marks (acetylation) o The inactivated X chromosome doesn’t have the regions of the histone light up

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Female mammalian cells will always inactivate one X chromosome and give rise to Barr Bodies (electron dense  it is the inactivated X chromosome)

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If the female has more than two X chromosomes  it will inactivate all except for one (make more Barr bodies)

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We see evidence of this in nature = the fur colors on the calico cat

 Females must inactivate one X chromosome during embryonic development 

Inactive X chromosome are referred to as Barr Bodies

 Once inactivated the targeted X-chromosome will remain inactive in all the cells of the organism  The patched nature of the fur of calico cats/ tortoise shell colored cats indicate regions where the inactivation of the X chromosome was altered 

Therefore, providing coat color information from two different X chromosomes

XIST

is a

seXIST gene product… -

When the inactivation take place  the mechanism that is associated with shutting down the X chromosome = mediated by a long non-coding RNA (there is no proteins involved in the initial step)

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The transcription of a gene called XIST  encodes a long non-coding RNA and will NEVER translate to give rise to a protein o It’s an RNA that coats the X chromosome o (in doing so) it sends a message that this is the chromosome that we choose to inactivate and respect that in every cell division there-after

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Once that initial chromosome is coated with XIST RNA  it is not required any longer (the chromosome is heterochromatinized and transcription is reduced)

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Only the initial step/initial mechanism requires the XIST RNA  everything else is propagated through these histones modifying readers and writers

 The XIST locus encodes long non-coding RNA  It binds to discrete regions of the X chromosome and spreads along the X to eventually X-tinguish gene expression  Although many mechanisms have been proposed it is still not clear how this RNA functions and what is involved in its activation or specificity Epigenetic marks have to be propagated -

Epigenetic traits = transmitted independently of the DNA sequence itself  you can have identical DNA sequences, but the outcomes are very different (based on the way the DNA is being read/transcribed/expressed)

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Examples of epigenetic traits: o Inactive X o Developmental restrictions 

Epigenetic modifications ensure that we make legs where they are supposed to be by repressing those genes outside of the body plan where they legs should be expressed

o Imprints = affect the genes in a parental manner 

the genes that are inactivated/activated in a male’s gametes are very different from the genes inactivated/activated in a female’s gametes (parental specificity)

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these often these include methylation marks on the DNA

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the DNA methylation marks can be recognized by proteins that were recruited in that big Sin3 complex o these marks very often change the vicinities of those regions where the DNA has been methylated  to radically modify the histone tails and the overall highorder chromatin configuration

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many of the histone marks are propagated through the activities of enzymes called histone methyltransferases (or methylases)  things that add on methyl groups/ that remove methyl groups o important for propagating a signal that is present on histone tails after every single cell division

 DNA marks (methylation) are read by specific proteins then used to modify histones in proximity through mSin3 recruitment  Histone marks (ie…H3K9 methylation) can nucleate and histone methyltransferases to repress gene activity across an entire genetic region 

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