Chromatin Dynamics and the Histone Code

Question 1

What is the model for heterochromatin formation driven by histone H3 trimethylation on lysine 9 (H3K9)?

Answer 1

1) H3K9 methyl transferase tri-methylates lysine histone tails
2) Binding of HP1 chromodomain
3) HP1 self-oligomerizes (positive feedback loop) / cross-linking of nucleosomes into more-tightly-packed chromatin

Question 2

What do acetylated histone tails bind?

Answer 2

BROMODOMAINS, which open up chromatin.

Question 3

How does the positive feedback loop of methylated chromodomains work?

Answer 3

HP1 (and other chromodomain proteins) bind and recruit H3K9 methyl transferase, which propagates the reaction, thus “forming” more heterochromatin.

Question 4

What stops/controls the positive feedback loops used by Euchromatin and Heterochromatin?

Answer 4

BOUNDARY ELEMENTS, which bind non-histone proteins.

Question 5

What is one of the evidences of the link between gene expression and chromatin structure?

Answer 5

DNase I ENDONUCLEASE DIGESTION EXPERIMENT

• This showed that Heterochromatin / non-transcriptionally active chromatin was not digested, because it was protected for DNase I digestion by the nucleosome’s structure.

Question 6

In the DNase I experiment, what animal, what cells and what gene did they use?

Answer 6

• CHICKEN
• MBS (chicken leukemic cell line )and 14-DAYS ERYTHROBLASTS
• CHICKEN GLOBIN GENE

Question 7

What does acetylation of a histone tail do to a nucleosome?

Answer 7

• The LYSINE tail becomes NEUTRAL
• REDUCED BINDING to DNA

This opens up chromatin and separates nucleosomes

Question 8

How is the identification of chromatin regions containing modified histone tails done and how are those modifications determined?

Answer 8

All is done by CHROMATIN IMMUNOPRECIPITATION (ChIP)

This experiment id one using specific antibodies that bind to different groups. The mix is then centrifuged, which separates the histones to which the antibody binded to (the ones we are looking for) and the ones that the antibody did not bind to.

Question 9

What are the steps of a chromatin immunoprecipitation (ChIP) experiment?

Answer 9

1) Mechanical SHEARING of DNA
2) Add ANTIBODIES
3) IMMUNOPRECIPITATE / CENTRIFUGE
4) Release immunoprecipitated DNA and ASSAY BY PCR
5) AMPLIFY DNA sequence

Question 10

Which enzymes can act as chromatin co-repressors?

Answer 10

DEACETYLASE and H3K9 METHYL TRANSFERASE

Question 11

What are the steps of a DNase I Endonuclease Experiment?

Answer 11

1) ISOLATE Chromatin
2) DNase I Endonuclease DIGESTION
3) REMOVE PROTEIN
4) CUT pure DNA with restriction enzyme
5) Restriction fragments separated by GEL ELECTROPHORESIS
6) Detect DNA fragment by SOUTHERN BLOT HYBRIDIZATION with DNA probe

Question 12

What is the main co-activator enzyme responsible for modification of histone tails?

Answer 12

ACETYL TRANSFERASE