Lecture 4: Chromatin

Question 1

What is chromatin? How is it structured and how does it interact with DNA?

Answer 1

• Euchromatin is more active (or has the potential to be). rRNA is the most extreme example. It still has nucleosomes though.
• Heterochromatin can be constitutive and facultative.
• Histones can be activating. They can bring transcription factors close together.
• Promoter area is known as the nucleosome depleted region (NDR).
• Nucleosomes are only really an impediment to initiation. They don’t really do anything to elongation.
• First histone as +1.
• NDR will change from active to inactive genes. The rest will largely remain the same.

Question 2

What are nucleosomes? How are they assembled/disassembled?

Answer 2

• There are 4 basic histone proteins: H2A, H2B, H3 and H4.
• Nucleosomes are two copies of each of the core histones and 147bp of DNA.
• Two nucleosomes are separated by linker DNA.
• Histone tails extend from the surface.
• Histone chaperones (e.g. FACT, CAF, HIRA) are required to assemble or disassemble nucleosomes.

Question 3

How does DNA methylation work? How are methyls added and removed?

Answer 3

• A cytosine is methylated on its 5 carbon. It is then followed by a guanine.
• TET is an enzyme which removes methyl groups.
• DMNT is an enzyme which adds methyl groups.
• Both enzymes are affected by metabolic processes in the cell.
• Paternal and maternal chromosomes are differentially methylated.

Question 4

What are imprinting control regions? Draw a diagram.

Answer 4

ICRs are areas of the genome where differential methylation occurs in the parental and maternal genomes.
• Methylation prevents the binding of CTCF.
• CTCF can be used to define boundaries and prevent enhancer interactions.
• During spermatogenesis, the ICR near Igf2 is methylated.
• It is not methylated during oogenesis.
• This means that in embryos/adults only one copy is expressed.
• If the ICR is methylated in eggs, overgrowth syndromes like BWS can occur.

Question 5

How do post-translational modifications to histones work?

Answer 5

Histones can be modified in a number of ways. PTMs are reversible and highly conserved. PTMs are influenced by the metabolic state of the cell.
• Histones can be modified by methylation, acetylation, phosphorylation, ubiquitylation, sumoylation, ADP-ribosylation, and other modifications.
• Combinations of PTMs define the function of local chromatin regions (histone code hypothesis). It’s still not entirely understood.
• Some PTMs lead to changes in structure.
• Some PTMs lead to binding sites for other proteins.

Question 6

How does ChIP work?

Answer 6

ChIP (chromatin immunoprecipitation) is a method which can be used to study DNA-protein interactions. ChIP-seq is a genome wide study.

1) Add a cross-linking reagent such as formaldehyde.
2) Lyse the cell.
3) Use sonication or enzymes to digest DNA into small fragments.
4) Add an antibody specific to the protein of interest. This precipitates out the DNA-protein complex.
5) Reverse the cross-linking.
6) Analyse the DNA. We can sequence it, use PCR etc.

Question 7

How are histones and their modifications distributed across chromatin?

Answer 7

Distribution of chromatin is very important for determining how genes are expressed.
• Many promoters have nucleosome depleted regions.
• TSSs are generally devoid of nucleosomes.
• Chromatin modifications define the architecture of a gene: enhancers, promoters and transcribed regions.
• H3K4me3 is enriched around TSSs.
• H3K4me1 is enriched around enhancers and more downstream.

Question 8

How do methylation and acetylation work?

Answer 8

• Lysines in histone tails are the most common modification target. Acetylation neutralises the positive charge. Methylation retains the charge (mono, di or tri).
• Arg can also be methylated (mono or di).
• Modification of one site can stop modification of another.
• Some sites are subject to only a single type but others have multiple states (e.g. lys 9 can be methylated or acetylated).
• Acetylation at H3 is a common change for active genes. Mostly at +1. Destabilize by reducing the interactions. E.g. H3K9.
• H3K4me3 is a modification at +1. However, removing it doesn’t really have much of an effect. Could be redundant modifications? It can recruit remodellers, histone modifying enzymes and transcription factors.
• H3K9me3 and H3K27me3 are repressive modifications. K27 recruits PRC1.