Lecture 4 - Chromatin dynamics and the histone code

Question 1

Which PTM corresponds to which domain and corresponds to which chromatin state

Answer 1

Methylation is code for chromodomain and code for heterochromatin. Acetylation is code for bromodomain and code for euchromatin.

Question 2

What drives heterochromatin formation

Answer 2

Trimethylation of lysine 9 on histone H3 (H3K9 -> H3K9Me3)

Question 3

Steps to heterochromatin formation

Answer 3

1) H3K9 methyl transferase 2) HP1 chromodomain binds to H3K9Me3 3) HP1 self-oligomerizes thus cross-linking nucleosomes into more-tightly packed chromatin

Question 4

Model to heterochromatin formation is called a _________________ and these can _______ and _________ histone codes

Answer 4

positive feedback loop, reinforce and spread

Question 5

what HP1 does in addition to self-oligomerization. effect ?

Answer 5

Binds and recruits H3K9 methyl transferase. effect = heterochromatin tends to spread along chromosome once initiated

Question 6

Euchromatin tends to ________ along the chromosome because of a __________

Answer 6

spread. positive feedback loop.

Question 7

what H3K9 HMT stands for

Answer 7

H3K9 histone methyl transferase

Question 8

__________ can isolate adjacent chromosomal domains and _________ them. They can separate _______ chromatin from spreading _______ and _____-coated ____________

Answer 8

Boundary elements. distance spread. active chromatin. silenced and HP1 coated heterochromatin

Question 9

what is the evidence of the link between gene expression and chromatin structure

Answer 9

DNase 1 digestion

Question 10

DNase 1 is an ___________. It digests _____ DNA by cleaving it only between the __________. The result is ________ of DNA that are _______ bp long. _________ chromatin is protected from DNase 1 cleavage.

Answer 10

endonuclease. naked. nucleosomes. fragments. 150. Condensed

Question 11

Prediction in a DNase I digestion experiment

Answer 11

Genes should be more susceptible to DNase I digestion in cells where they are expressed

Question 12

DNase I cleavage experiment was done on ___________ gene, expressed in _____________ but not in _________ cells. What is expected ?

Answer 12

chicken globin. 14-day erythroblasts. MSB. Globin gene expected to be cleaved into fragments when DNase I added to 14-day erythroblasts but not in MSB cells.

Question 13

What does DNase I cleavage on DNA from different cell types of the same organism show ?

Answer 13

All genes of an organism are contained in all its cells but gene expression determines cell type (or cell type determines gene expression)

Question 14

Steps of southern blotting hybridization (in general) (5 STEPS)

Answer 14

1) DNA cleaved w/ restriction enzymes 2) Gel electrophoresis 3) On filter paper in an alkaline solution, fragments transfered from gel to nitrocellulose using alkaline solution (capillary action) 4) Nitrocellulose map incubated with labeled DNA probe 5) Location of fragment hybridized to probe is revealed by autoradiography or fluorescent imaging

Question 15

Why use southern blotting (and probes) if gel electrophoresis seperates fragments

Answer 15

Because when a whole genome is fragmented, there are too many fragments so you can’t resolve (identify) any particular DNA fragment as a discrete band on the gel

Question 16

Why do we use a blot (replica of the gel on the nitrocellulose)

Answer 16

Probes do not readily diffuse into the original gel

Question 17

How do you obtain a probe

Answer 17

cloned restriction fragment that is radiolabelled (ex: radioactive isotope of the phosphorus in phosphodiester bond)

Question 18

Southern blotting with the globin gene and DNase I digestion STEPS + length of globin gene

Answer 18

1) Isolate chromatin 2) DNase I digestion 3) Remove proteins 4) Cut pure DNA with restriction enzyme (BamH1) (will cut fragment with the gene if the gene hasn’t been digested 5) Restriction fragments seperated by gel electrophoresis 6) Detection of globin gene (4.6 kb) by Southern blot hybridization w/ DNA probe for globin gene

Question 19

You can shut down a gene by expressing a ________or a ____________ (enzymes)

Answer 19

methyl transferase. deacetylase

Question 20

Why acetylation opens chromatin

Answer 20

Positive charge on lysine tail changes to neutral and it reduces its ability to interact with DNA negative phosphate backbone AND ABILITY TO INTERACT WITH ADJACENT NUCLEOSOMES.

Question 21

what technique can help identify if PTMs are related to global gene activity (acetylation related to transcription and methylation related to transcription repression) and what does it use

Answer 21

Chromatin immunoprecipitation (ChIP) uses antibody specific to acetylated histone tail.

Question 22

ChIP briefly explained by comparison

Answer 22

Antibody affinity chromatography (proteins bound with antibody that is specific to them hang up) but with chromatin

Question 23

How can ChIP be used to determine which genes are expressed in a particular cell type

Answer 23

Antibody specific to acetylated N terminal histone tails binds to these. In column, you obtain fractions with these histones and their DNA. By amplifying these specific DNA sequences, you obtain multiple copies of the expressed gene.

Question 24

Steps to ChIP (in ChIP where you want to amplify a gene that is expressed)

Answer 24

1) Isolate and shear chromatin mechanically 2) Add antibody specific for acetylated N terminal histone tail 3) Immunoprecipitate 4) Release immunoprecipitated DNA and assay by PCR

Question 25

How chromatin modification works for specific gene (regulation of expression)

Answer 25

Transcription factors (activators or repressors) bind to enhancers or silencers (sequences) and recruit enzymes (coactivators/corepressors) that modify histone tails

Question 26

What modification(s) promote(s) chromatin decondensation

Answer 26

Acetylation (acetyl transferase)

Question 27

What modification(s) promote(s) chromatin condensation

Answer 27

Deacetylation (deacetylase) or H3K9 or H3K27 Methylation (methyl transferase)

Question 28

All enhancers are ____ which means that they have to be physically in ______ with the gene they are affecting

Answer 28

cis. line

Question 29

2 main steps in transcriptional control

Answer 29

1) (pioneer) TF binds to regulatory sequences in condensed chromatin and recruits chromatin remodelling enzymes 2) TF (other one) that will bind to mediator complex can now bind their specific sequences (that were closed before) and bind the mediator complex (goal is stable elongation ?)

Question 30

REMINDER : Point of mediator is to link RNAP II to complexes that open chromatin ?

Answer 30

NO. HETEROCHROMATIN/EUCHROMATIN SPREADS ALONG DNA