Lecture 3 - Chromatin Structure

Question 1

what is DNA packaged into?

Answer 1

chromatin

Question 2

what is the function of chromatin?

Answer 2

to compact DNA

Question 3

composition of chromatin

Answer 3

composed of small basic proteins called histones

Question 4

what are the 2 types of histones?

Answer 4

core histones

linker histones

Question 5

core histone structure

Answer 5

highly conserved

all have a globular domain (atleast 3 alpha helices) and an N-terminal domain which is highly basic

globular domains all fold in the same way - classic histone fold

2 histones interact in a histone handshake

Question 6

how do core histones bind DNA?

Answer 6

form repeating units called nucleosomes

Question 7

what is a nucleosome?

Answer 7

147bp of DNA wrapped twice around an octamer of histone proteins

Question 8

what is an octamer?

Answer 8

central H3-H4 tetramer + 2 flanking H2A-H2B dimers

octamer is not stable unless there is DNA wrapped around it

Question 9

nucleosome assembly and structure

Answer 9

tails are flexible and protrude out of the structure

by wrapping DNA around nucelosome, it restricts access to DNA
• chromatin has an inhibitory effect on transcription

Question 10

how are nucleosomes organised?

Answer 10

1. DNA passes directly from 1 nucleosome to the next - 10nm fibre
2. linker histones such as histone H1 binds to the DNA between nucleosomes - bind at the points where DNA enters and leaves the nucleosome
3. the 30nm fibre is formed

Question 11

what evidence do we have to show that chromatin inhibits transcription?

Answer 11

in vitro reconstruction experiments

in vivo nucleosome positioning experiments

genetic studies in S.cerevisiae

Question 12

how does the nucleosome have conflicting roles in the nucleus?

Answer 12

nucleosome is required to compact DNA but the compaction of DNA inhibits transcription

Question 13

3 mechanisms for modulating the structure of chromatin

Answer 13

histone variants

post translational modification of histones

ATP dependent chromatin remodelling

all mechanism are interlinked

Question 14

histone variants

Answer 14

encoded by genes that differ from the highly conserved major types

expressed at very low levels

affect chromatin dynamics

Question 15

post-translational modification of histones

Answer 15

mainly N-terminal tails are modified

• acetylation
• methylation
• ubiquitation
• phosphorylation

Question 16

how do histone modifications affect its transcriptional state?

Answer 16

could directly alter chromatin folding/structure

could control the recruitment of non-histone proteins to chromatin
• influences recruitment of transcriptional machinery

Question 17

histone lysine acetylation

Answer 17

acetylation is mediated by HATs and is readily reversible by HDACs

acetylation state is highly dynamic

Question 18

what are HATs?

Answer 18

Histone Acetyl Transferases

Question 19

what are HDACs?

Answer 19

Histone Deacetylases

Question 20

how does acetylation work?

Answer 20

lysine residue on histone tail is acetylated by acetyl-CoA along with HAT

acetyl group released by HDACs and water

Question 21

how are HATs recruited?

Answer 21

activators recruit HATs to specific promoters

act as a bridge between UAS

they don’t have their own DNA binding domains, they interact with other proteins that do contact DNA directly

some HATs are part of the general transcription machinery

Question 22

how does the acetylation of histones mediate transcriptional activation?

Answer 22

direct influence on chromatin structure

directs the recruitment of bromodomain proteins

Question 23

how does acetylation directly influence chromatin structure?

Answer 23

changes the charge on lysine

positive histone interacts with negative DNA to cause compaction - acetylation reduces ability of tails to interact with DNA

less compaction = more translation

Question 24

how does acetylation direct the recruitment of bromodomain proteins?

Answer 24

specific acetylated lysine residues are recognised by proteins with bromodomains

bromodomain proteins often promote transcription

Question 25

how do bromodomain proteins promote transcription?

Answer 25

helpers TFIID bind to the promoter found in proteins that promote transcription bromodomain slots acetyl-lysine into the groove

Question 26

examples of bromodomain proteins

Answer 26

Bdf1 - | TAFII250 -

Question 27

Histone methylation

Answer 27

Can occur on lysine (& also arginine) Proteins mediating methylation are HKMTs (Histone Lysine Methyl Transferases) Lysine’s may be mono, di or tri methylated by HKMTs – which often contain a SET domain Methylation is not readily reversible, but demethylases do exist Methylation does not affect charge so probably only has minor is any influence on chromatin structure

Question 28

Lysine methylation & control of transcription

Answer 28

A variety of different domains have been shown to function as methyl-lysine binding modules Specific methylated lysine’s are recognised by specific proteins Depending upon the context methyl-lysine residues can function as either activating or repressing marks – depends on the residue being methylated

Question 29

What lysine residues are activating & which ones are repressing?

Answer 29

H3 Lys9 – repression H3 Lys27 – repression H3 Lys4 – activation H3 Lys36 – activation