pp 15

Question 1

What is a chromatin?

Answer 1

complex of DNA and protein in which the eukaryotic genome exists

Question 2

What is heterochromatin?

Answer 2

Chromatin that is condensed and inactive (not transcribed)

Question 3

What is Euchromatin?

Answer 3

Relatively extended open chromatin that is accessible to RNA polymerase

Question 4

What is the structure of chromosomes?

Answer 4

chromatin = histones (protein) + DNA (equal mass of both)
first folding is the nucleosome, which has a core of histone around which the DNA is wound. Then the folding produces a fibre 30nm in diameter.

Question 5

What is the basic unit of DNA packing in eukaryotes?

Answer 5

Nucleosomes

Question 6

What is the nucleosome?

Answer 6

A DNA-protein complex that consists of four pairs of different histones forming an octamer where DNA is wound. Another histone binds with “linker” DNA not coiled around octamer. The winding of DNA around nucelosome condenses length by 6 to 7 fold.

Question 7

What is the role of Core Histone?

Answer 7

H2A, H2B, H3, H4 - two of each core histone along with 146 bp core DNA make core nucleosome

Question 8

What is the role of Linker Histone? (H1)

Answer 8

associated with linker DNA, can be removed by mild treatment (high salt solution or mild trypsin digestion). Core nucleosome without H1 connected by linker looks like beads on a string

Question 9

What would the crystal structure of core nucleosome look like?

Answer 9

146 bp of DNA wrapped around a histone octamer core. Histones have long N-terminal tails that are highly basic (lysine) this is important for histone modifications
DNA wrapped around outside 1.7 turns in a left-handed superhelix

Question 10

What do histone tails mediate?

Answer 10

internucleosome connections that regulate the accessibility of DNA

Question 11

How do they mediate this?

Answer 11

The N termini have the N-terminal histone tails that are flexible. The core interacts tightly with DNA but tails have important contact between adjacent mucelosomes. They then operate important role in transcriptional regulation, where they can interact with regulatory proteins as well as be chemically modified, influencing interactions

Question 12

What is the importance of linker histone H1?

Answer 12

Stablilizes higher-order chromatin structures

Question 13

What are the arrangements of the fiber and how are they formed?

Answer 13

String of nucelosomes coil further into higher order chromatin structures creating a fiber. It can be solenoid or zig zag, both can happen but predominantly solenoid form.

Question 14

What does higher-order chromatin folding entail?

Answer 14

30 m, fibers account for most chromatin in interphase nucleus. Mitotic chromosomes require further folding. Radial loops is favoured for such higher order folding.

Question 15

How does the chromatin strucutre affect gene activity?

Answer 15

Histones are regulators of genetic activity. The tight packing of nucelosomes in chromatin makes DNA less accessible to transcription factors, which leads to lower transcription.

Question 16

How do histones repress transcription?

Answer 16

They lower transcription and oppose the effects of activator proteins binding to DNA

Question 17

What experiment shows histones repressing activity?

Answer 17

In vitro transcription of a gene assayed with and without histones. mRNA activity not seen with histones are added.

Question 18

Why do transcriptional activators and histones compete for DNA binding?

Answer 18

Since histones result in repression aka blocking access to promoters, and transcriptional activators act as activators but also as antirepressors.

Question 19

How is the chromatin structure regulated?

Answer 19

The nucleosome controls the accessibility of DNA to transcriptional machinery, therefore an “open” chromatin states are associated with active transcription whereas “closed” chromatin with repressed transcription

Question 20

What are the enxymes that regulated the arrangement of nucleosome in chromatin?

Answer 20

1. Chromatin remodeling complexes: slides nucleosome to a different location, ejects histones, from DNA or replaces them
2. Histone modifying enzymes: covalently modify N-terminal histone tails, influencing interactions between nucleosomes or with proteins that influence transcription.

Question 21

What is the mechanism for chromatin remodeling complexes?

Answer 21

• activation of many eukaryotic genes require chromatin remodeling. The complexes are ATP-dependent enzymes.
• ATP used to disrupt contacts between DNA and histones in nucleosome allowing it to reposition. When nucleosome covers a promoter, complexes can expose promoter by pulling DNA out of the nucleosome into a loop.
• when loop is tightened, promoter can be pulled out from repositioned nucleosome.
• Repositioning of a nucleosome over a previously exposed promoter results in repression.

Question 22

How are variant histone subunits made?

Answer 22

Can replace histone subunits within a nucleosome with variants. They differ primarily in susceptibility of histone tails to modifications. Variants promote gene transcription by stabilizing a more open chromatin state.

Question 23

What are the mechanisms of three histone variants?

Answer 23

1. open chromatin: H3.3 in, H3 out - locked open (maintaining transcriptionally active open state)
2. Chromatin: CENPA in, H3 out - kinetochore attachment sites (maintains kinetochore attachements)
3. Double stranded break in chromatin- H2AX in, H2A out - phosphorylation attracts repair proteins (attracting DNA repair enzymes)

Question 24

What type of modifications are N-terminal tails target of?

Answer 24

acetylation, methylation, phosphorylation

Question 25

What does acetylation of histone tail lysine’s do?

Answer 25

Neutralize their positive charge reducing affinity of the tails to negatively charged DNA backbone and negatively charged core of neighbouring nucelosomes. It can be reversed by deacetylation of histone lysine’s

Question 26

Why is acetylation or deacetylation of lysines important for gene regulation?

Answer 26

1. histone acetylation weakened interactions between nucelosomal histones with each other and DNA (gene activation)
2. Histone deacetylation: strengthens the interaction between histones and DNA (gene repression)

Question 27

How is acetylation catalyzed?

Answer 27

transfer of acetyl group to histone tails from acetyl- CoA catalyzed by histone acetyltransferases (HATs)

Question 28

How is deacetylation catalyzed?

Answer 28

Histone deacetylases (HDACs)

Question 29

What do proteins with bromodomain motifs recognize?

Answer 29

Acetylated histone lysine’s- help recruit larger protein complexes

Question 30

What is the importance of histone methylation / demethylation?

Answer 30

Methylation associated with gene activation or repression depending on position. Proteins that have chromodomain motifs recognize methylated histone lysines, help recruit other protein complexes that can remodel chromatin.

Question 31

What is the histone code and why is it important?

Answer 31

combination of histone modifications on a nucleosome near a gene control region affects the efficiency of that gene’s transcription. pattern modification is a “code”.
The code is epigenetic - information encoded not on the DNA sequence itself.

Question 32

What is the mechanism of activation and repression by histone modification?

Answer 32

1. altering the way histone tails interact with DNA and with histones in neighbouring nucleosomes, altering nucelosome cross-linking
2. attracting proteins that can affect chromatin structure and activity.

Question 33

How does epigenetic states get passed down through cell division?

Answer 33

Inheritance requires preservation of histone modifications during DNA replication.
Octamer comes apart, H-3H-4 tetramers remain bound to DNA, where they are randomly distributed between two daughter DNA duplexes
Old H2A-H2B not lost, and the new H3-H4 and H2A-H2B are required for new nucleosomes

Question 34

What are the two states maintained?

Answer 34

1. Maintaining active (open) chromatin state - propogated through bromodomain containing complex that contains HAT
2. Maintaining repressed (closed) state - propagated through chromodomain contained HP1, that recruits methyltransferases.