Lecture 5: Covalent modifications

Question 1

How can the histone code be read?

Answer 1

• Methylated lysines can be read by chromodomains e.g. HP1 and PHD.
• Acetylated lysines can be read by bromodomains e.g. Gcn5.
• DNA methylation is read by methyl C binding proteins e.g. meCBP.

Question 2

How can methylation of lysine residues be read?

Answer 2

• A methyl-lysine binding domain can distinguish different forms of methylation (monomethylation, demethylation etc).
• They can also distinguish different lysine residues and methylation sites. Sometimes domains will bind to the same site.
• Methyl-lysine binding can distinguish the same modification in different contexts.

Question 3

What are some of the functions of domains which bind H3K4me?

Answer 3

• Moving nucleosomes which are in the vicinity of a promoter.
• Writing or erasing chromatin modifications at other sites.
• Spreading the K4me modification.
• Inhibit DNA methyltransferases.
• Allow nucleosome movement only when other modifications are present.
• Recruit TFIID.

Question 4

How can histone modifications be spread through the genome?

Answer 4

• Histone modifications can be spread through domains.
• This is stopped by boundary elements.
• Many writers contain domains which allow them to bind to the modification which they deposit.
• SUV39 lysine methyltransferase spreads H3K9me with the chromodomain. This can be used to spread silencing.
• Gcn5 lysine acetyltransferase spreads H3K9ac with the bromodomain.
• SUV39 KMT binds directly to H3K9me3 in order to spread the modification. It can also bind to HP1.

Question 5

How does DNA methylation play a role in chromatin? What happens when this goes wrong (Rett syndrome)?

Answer 5

• DNA methylation influences how proteins interact with DNA.
• Some TFs bind methylated and unmethylated DNA equally well.
• Rett syndrome results from defects in MeCP2.
• MeCP2 binds methylated DNA.
• MeCP2 recruits enzymes called HDACs (histone deacetylases). Deacetylated histones are associated with genes that are switched off.
• Rett syndrome can be artificially induced by mutating or eliminating MeCP2 in mice. This can be reversed in adult mice by replacing the MeCP2.

Question 6

How does histone acetylation work?

Answer 6

Acetylating neutralises the positive charge on a lysine residue.
• Acetyltransferases add acetyl while HDACs take them away.
• Histone acetylation makes DNA more accessible and it tends to be found in regions where the genes have the potential to be expressed.
• For example, they are found at the promoters of active genes and origins of replication.

Question 7

How do chromatin remodelling ATPases function? How can we prove this? What are the different types?

Answer 7

ATPases can have both activating and repressing activities.
• ATPases can be recruited by TFs or pre-existing histone modifications.
• SWI/SNF has an activation function. It has a bromodomain.
• ISWI can have both activating and repressing activities.
• Mi-2 has a repressing activity. It has a chromodomain.
• Chromatin remodelling ATPases can remove or move specific nucleosomes to allow events such as transcription.
• They also space nucleosomes to ensure that all nucleosomes occupy the same position in a cell population.
• In the presence of CR ATPases, the nucleosomal DNA resembles naked DNA. It is more accessible to TFs.
• Naked DNA will be cut one every 10 bp by DNases. Nucleosomal DNA is cut less frequently. Adding ATP and CR ATPases will cause the DNA to have the same 10 bp pattern.