Lecture 22 - Chromatin, Epigenetics, and the Histone Code

Question 1

What has been used as a model organism to study transcriptional mechanism?

Answer 1

Yeast

Question 2

What are silenced regions in chromosome 3 associated with?

Answer 2

Yeast Mating Types

Question 3

What mark neutralizes the charge between histones and DNA?

Answer 3

Acetylation

Question 4

On which residues do methylations typically take place?

Answer 4

Lysine

Question 5

What does H3K4 methylation result in?

Answer 5

Activation

Question 6

What does H3K9 methylation result in?

Answer 6

Inactivation

Question 7

What process condenses the chromatin to ensure that transcription doesn’t take place?

Answer 7

Heterochromatinization

Question 8

What is dosage compensation? How is it done?

Answer 8

It is when female mammals have to shutdown one X chromosome to equalize the dose of gene expression of the X chromosome.

It is done by heterochromatinizing an X chromosome to form an inactive Barr body (highly condensed chromosome).

Question 9

What does XIST do?

Answer 9

It coats the entire X chromosome and thus shuts down most of the transcription of that chromosome.

Question 10

How is the initial choice made for which X chromosome should be turned into a Barr body?

Answer 10

There is a competition between the expression of XIST and an antisense RNA to it (TSIX).

Question 11

What histone mark ensures that the appropriate X chromosome is heterochromatinized after each cell division?

Answer 11

H3K9 Trimethylated Histones

Question 12

What is open active chromatin called?

Answer 12

Euchromatin

Question 13

Suppose you want to activate a gene downstream of a gene that is recognized by a DNA-binding domain. What must you pair with the DNA-binding domain to make this work? Why can this work?

Answer 13

You must pair it with a transcriptional activation domain from any well-characterized transcription factor.

You can do this because transcription factors are modular.

Question 14

What is a heterologous element?

Answer 14

It is a element that does not belong in the cell being tested and comes from different cells or organisms.

Question 15

Suppose you introduce a lac operator (cis-regulatory sequence) and lac repressor (lacI) joined with a strong transcriptional activation domain like VP16 into a eukaryotic cell. What will happen?

Answer 15

LacI will interact with the lacO and then VP16 will change the chromatin region in the area and activate transcription. This will co-opt the cellular apparatus to transcribe genes at very high efficiency (particularly, viral genes).

Question 16

What are SWI/SNF? What do they do?

Answer 16

SWI/SNF are chromatin remodelers that come from yeast genes.

One of the subunits of of the remodelers has homology to a helicase, but uses ATP hydrolysis to shift around the entire environment of the nucleosome which exposes regions of the DNA that would normally be hidden in the nucleosome or histone-bound high order complexes.

Question 17

Which molecular process is very important for development? Why?

Answer 17

Transcriptional regulation is very important for development because it is needed to change the transcription of each precursor cell type and eventually the fully differentiated cell types.

Question 18

What protein is often the first step in a multitude of steps that give rise to the differentiation of a lineage of cells?

Answer 18

Pioneer Transcription Factors

Question 19

What do pioneer transcription factors do?

Answer 19

They bind to a region of exposed DNA and recruit major effectors that change the chromatin configuration around the binding site.

Question 20

What is important for the termination or differentiation of an entire lineage of cells?

Answer 20

The initial chromatin change caused by pioneer transcription factors is important for this.

Question 21

Why is chromatin “looped out” to increase efficiency?

Answer 21

Chromatin gets looped out so that regions far downstream and far upstream of the gene that have to be transcribed come together, potentially linked by enhancers or other things. The outcome is that you make it advantageous for these general transcription factors to access their promoters and then transcribe their downstream genes.

Question 22

What is responsible for looping out chromatin?

Answer 22

The Mediator

Question 23

How does the mediator cause looping out?

Answer 23

The mediator mediates the interactions between upstream or downstream cis-regulatory elements, like enhancers (or UAS in yeasts). These elements bridge together and the chromatin between them forms a loop.

Question 24

Why is looping out advantageous?

Answer 24

Looping out enhances the interaction of key general transcription factors and RNA pol II with the promoters they should be interacting with and the genes they should be transcribing.

Question 25

What indirect evidence exists for looping out?

Answer 25

If you take complexes that have looped out, cut it using enzymes, and re-ligate the bits back together, you get regions of re-ligated chromatin that are thousands of bases away from eachother.

Question 26

How many major domains does the mediator have?

Answer 26

3

Question 27

Why is it often that if you eliminate key subunits of the mediator, the transcription of a class of genes will be eliminated?

Answer 27

It appears that the individual subunits interact with individual transcriptional activation domains. As such, this elimination occurs because the individual subunits confer a specificity to the mediator to interact with a class of transcription factors.

Question 28

What regions of the mediator are critical to interact with RNA pol II?

Answer 28

The Middle and Head

Question 29

How do enhancers connect to the mediator?

Answer 29

The enhancers interact with DNA-binding proteints that interact at the same time with the head of the mediator.

Question 30

Where do transcriptional activators interact with the mediator?

Answer 30

The Tail

Question 31

Why is the TATA box not actually as important as we initially thought it was?

Answer 31

It isn’t as important because transcription does not occur linearlly.

Question 32

What makes it advantageous for the general transcription factors and RNA pol II to come together so that RNA pol II can proceed from initiation to elongation?

Answer 32

The proximal binding sites and the proteins that interact with them (to activate the transcription of downstream genes) and far upstream and downstream cis-regulatory elements (enhancers) are brought together by the mediator so that the region is more advantageous to bind the general transcription factors and RNA pol II.

Question 33

What are alleles?

Answer 33

They are variants of genes on different chromosomes.

Question 34

How can two alleles respond to an enhancer at the same time?

Answer 34

Both alleles can be brought together by RNA pol II/mediator complexs in the same liquid-liquid condensate so that they can be activated at the same time.

Question 35

What are P-granules?

Answer 35

P-granules are liquids made up of RNA and proteins.

Question 36

What are P-granules important for?

Answer 36

P-granules (through phase separation, phase transition, or formation of liquid-liquid) bring together very important proteins so that they can actually carry out their function.

Question 37

How do P-granules behave (metaphorically)?

Answer 37

They behave like drop of baslsamic vinegar in oil.

Question 38

What is enhancer RNA?

Answer 38

It is RNA being made at regions in enhancers.

Question 39

What can you see if you mark the mediator with JF646 and RNA pol II with Dendra2 and then later block transcription? What does this indicate?

Answer 39

You can see that the mediator and RNA pol II are together within the cell at sites of initiation. When you block transcription, these sites go away. This indicates that the sites are dependent on transcription.

Question 40

Why may intrinsically disordered domains be critical for the liquid-liquid condensates containing the mediator and RNA pol II?

Answer 40

The elements required for transcription are found in this condensate, and since they are intrinsically disordered, a chromosome can leap into them and be activated by all of the necessary components for transcription.