4 - Histone Modification

Question 1

Define epigenetics and epigenomics

Answer 1

Epigenetics refers to ‘the study of heritable changes in gene expression or cellular phenotype caused by mechanisms other than changes in the underlying DNA sequence’.

Epigenomics refers to ‘the sum of genome-wide epigenetic patterns, distinguishes and defines one tissue from another, stem cells from somatic cells, and aged from young cells’.

Question 2

List the pathway for environmental gene programming

Answer 2

1. Epigenator (differential environment/signal)
2. Epigenetic initiation (in nucleus, DNA binding factors, ncRNA etc.)
3. Epigenetic maintainer (histone/DNA modifiers)

Question 3

Describe histone modifications

Answer 3

• Covalent attachments to amino acids of histones
• Affect affinity to both DNA and other proteins
• Can affect nucleosome and higher order DNA structures
• Predominately (but not exclusively) added to extended histone tail (of H3)

THEY ARE POST-TRANSLATIONAL MODIFICATIONS

Question 4

List the four major types of histone modifications and the amino acid modified

Answer 4

• Acetylation (Lysine)
• Methylation (lysine and arginine)
• Phosphorylation (Serine and threonine)
• Ubiquitination (lysine)

Question 5

What is evidence for a histone code?

Answer 5

• Specific modifications correlate with specific processes
• Specific modification patterns and sequences
• Interactions of specific modified residues with classes of protein domains

Question 6

What is ChIP?

Answer 6

Chromatin immunoprecipitation

It investigate the interaction between proteins and DNA in the cell. It aims to determine whether specific proteins are associated with specific genomic regions, such as transcription factors on promoters or other DNA binding sites, and possibly defining cistromes.

ChIP also aims to determine the specific location in the genome that various histone modifications are associated with, indicating the target of the histone modifiers

Question 7

Give the basic steps of a ChIP assay (6)

Answer 7

1. Lyse cells and shear DNA
2. Add pre blocked protein G agarose beads
3. Add primary antibody of interest
4. Immunoprecipitate to enrich for fragments bound by protein of interest
5. Reverse cross links and treat with proteinase K
6. Detect and quantify precipitated DNA through PCR and hybridization methods

Can also use ChIP on chip (array based, with an antibody specific to one type of histone modification or etc.)

Question 8

Histone modifications correlate with specific genomic processes. List some localized (gene or region specific) and some broad (regional or genomic) genomic processes which can be detected through identification of histone modifications.

Answer 8

Localized

• Transcription
• Silencing
• DNA repair

Broad

• Chromatin condensation
• Apoptosis
• DNA repair

Question 9

List two enzymes which catalyze histone tail modifications.

Answer 9

Histone deacetylases (HDACs)

• Remove acetyl groups from lysine
• Part of multi protein complexes
• Silences genes

Histone acetyltransferases (HATs)

• Acetylate conserved lysine amino acids
• Neutralizes positive histone, and therefore renders DNA more accessible to transcription factors
• Eg. CREB-binding protein

Question 10

List 3 types of factors that can affect histone modification

Answer 10

• Dietary factors
• Drugs
• Metabolism