Chromatin structure and histone code

Question 1

Define Chromatin

Answer 1

The nuclear complex of DNA and associated proteins that forms chromosomes within the nucleus of eukaryotic cells

Question 2

What is the function of chromatin?

Answer 2

Packing long DNA molecules into a more compact and denser shape to fit into the nucleus and to protect the structure of DNA and sequence

Question 3

What do the chromosomes consist of?

Answer 3

1. DNA
2. Histone Proteins
3. Non-histone proteins
4. Non-coding RNA
5. Almost all the DNA packages into the 1st level (nucleosomes). Much of the DNA packages into higher-order structures.
6. Chromosomes arms and bands are distinct and mutually exclusive

Question 4

Describe the 2 points about mitosis

Answer 4

1. Chromatin condenses in prophase and the chromosomes are visible.
2. During interphase, the chromatin is organised into heterochromatin and euchromatin

Question 5

Compare and contrast heterochromatin and Euchromatin

Answer 5

On table

Question 6

Define Consecutive heterochromatin and Facultative heterochromatin

Answer 6

Constitutive heterochromatin: invariably heterochromatin containing highly repetitive sequences of DNA which are genetically inactive and serves as structural element of the chromosome, for example the telomeres and centromeres.

Facultative heterochromatin: regions on chromosomes which become heterochromatin in certain cells and tissues. Potential for expression at a particular point of the development. For example, the X chromosome in female somatic cells is inactive.

Question 7

What does ‘Chromatin shows plasticity’ mean?

Answer 7

• Chromatin shows plasticity: enabled by choice of histone variants, modifications of DNA bases, and reversible post-translational modifications (PTM) of histone tails.

Question 8

Describe the Nucleosome

Answer 8

• First level of chromatin packing is the nucleosome.
• Nucleosomes were first in in electron microscopy as “beads on a string”.
• The DNA double helix is wound around 8 histone subunits (two molecules each of 2A, 2B, 3 and 4 known as histone octate). Note N-terminal tails outside the octamer core. Histones have tails which are positively charged amino acids, this interacts with the negative phosphate groups in DNA, this electrostatic attraction allows the DNA to store around the outside/wrap around the histone octate. This keeps the DNA in place compacting the DNA by a factor of 6 (otherwise DNA would be 2m long) still not enough. Histone 1 binds to the outside and to linker DNA. The combined loop of DNA around the histones is the nucleosome.
• Multiple nucleosomes are coiled together, which then stack on top of each other. This results in a fibre of nucleosomes called chromatin, which is then condensed to a 30nm chromatin fibre. This is then looped and coiled more, using other proteins to form the chromosomes, which only form when cells are dividing.
• When chromatin is extracted at physiological salt concentration, much of it appears as a 30nm thick fibre. This fibre is made up of nucleosomes tightly packed together. The 30nm fibre can be further compacted to form 80-100nm fibres
• Compaction of nucleosomes can form higher order structures, e.g.
o Linker histones (e.g. H1)
o Interaction of histone tails with adjacent nucleosomes
o Binding of packing proteins to histone tails
• 146bp of DNA wrapped around the histones core (1.7 turns of DNA).
• H1 protein wraps another 20 base pairs of “linker DNA” resulting in two full turns around the octamer. It helps to stabilize the zig-zagged 30 nm chromatin fiber.
• Nucleosomes are joined by linker DNA (20bp) that runs between them. Long chain of nucleosomes gives the appearance of” beads on a string” (10nm).
• Nucleosomes tightly pack together into a fiber of 30nm (2nd level).

Question 9

Define nucleosome dynamics

Answer 9

During transcription, or DNA replication, they must be removed from the DNA in front of the polymerase and replaced behind the polymerase.

Question 10

Define Chromatin loops, Cohesion and the mechanism of action

Answer 10

Chromatin loops (looping): stretches of genomic sequences that lie on the same chromosome are in close physical proximity to each other.

Cohesion: Proteins rings that bind to the DNA and facilitate looping

Chromatin loops provide favorable environment to processes such as DNA replication, transcription, and repair.

Question 11

TADS is the next level of chromatin structure

Answer 11

Organisation in Topological Associated Domains (TADs): Highly conserved chromatin domains that shape functional chromosomal organisation. Function not fully understood, their disruption lead to diseases. They are conserved.

Question 12

Describe chromosome territories

Answer 12

Chromosome territories: non-overlapping domains/regions of the nucleus occupied by uncondensed chromosomes. They are composed of TADs. This is done by staining, showing the different regions corresponding to the different chromosomes

Question 13

Describe Epigenetics

Answer 13

1. Heritable and reversible changes in gene expression which do not involve a change in the DNA sequence
2. As a result of external or environmental factors, as part of development program

Question 14

What do histone modifications do and what are the 4 different types

Answer 14

Histone modifications modify the structure of the histone which alters gene expression which also alters the chromatin structure.

1. Histone Phosphorylation
2. Histone Ubquitlation
3. Histone acetylation
4. Histone methylation

Question 15

Describe histone methylation

Answer 15

On image

Question 16

Describe histone acylation

Answer 16

Histone tails are acetylated by histone acetyl transferases (HATs) and deacetylated by histone deacetylases (HDACs).
Most common: acetylation of lysines.
Acetylated histones are generally associated with relaxed chromatin and gene expression and deacetylated histones with closed chromatin, silencing of genes.

Aberrant acetylation is associated with several solid tumours and haematological malignancies.

HDACi as emerging drugs in cancer treatment (e.g. vorinostat (SAHA) in T-cell lymphoma).

Question 17

Describe histone phosphorylation

Answer 17

Histone tails are phosphorylated by protein kinases and dephosphorylated by proteases.
Can occur on serines, threonines and tyrosines.

Phosphorylation of H3S10 and H3S28 is involved in chromatin condensation during mitosis and meiosis, as well as in chromatin relaxation linked to transcription activation.

Question 18

Describe Histone Ubiquitylation

Answer 18

Ubiquitin ligases and deubiquitinating enzymes.
Primarily on lysines of histones H2A and H2B.

H2Aub is more frequently correlated with gene silencing, while H2Bub is mostly associated with transcription activation

Question 19

Define Histone code and Protein readers

Answer 19

Histone Code - combinations of post-translational modifications on the same histone’s tail/s.

Code readers- protein complexes that read combinations of marks: chromodomains specifically recognize methylated residues, while bromodomains bind acetylated residues. This read leads to chromatin remodeling (open or closed).