Nucleosomes and Chromatin

Question 1

Why does DNA need to be compact to be stored?

Answer 1

Human genome - 21,000 genes
46 chromosomes

A lot of information to store in a small space

Question 2

What are the two principals used to package DNA?

Answer 2

1. Neutralise negative charge of DNA with positively charged proteins (to neutralise the phosphate groups)
2. Supercoil DNA to occupy smaller volume

Question 3

How is DNA packaged in prokaryotes?

Answer 3

Single DNA molecule that becomes a double-stranded DNA circle = ‘bacterial chromosome’
Packaged with proteins in a nucleoid, which is 80% DNA by mass
The nucleoid has 100 independent negatively supercoiled domains

Question 4

What are the positively charged proteins used in packaging of prokaryotic DNA?

Answer 4

Positively charged proteins that package the prokaryotic DNA are not yet identified but candidates include:
HU - wrapping
IHF and FIS - bending
H-NS - compaction

Question 5

What are the steps of the process of DNA into a chromosome?

Answer 5

B-DNA (+ Histones) =
Nucleosomes (+ H1) =
10 nm chromatin fibre
30 nm chromatin fibre 
Loops of 30 nm chromatin fibre (+ protein scaffold) =
Chromatid

Question 6

Describe how nucleosomes are formed?

Answer 6

B-DNA + 8 histones (octamer)
146 bp of B-DNA wrap around a core of positvely charged histone proteins
The histones interact with the sugar-phosphate backbone of DNA
Forming 142 H bonds, along with disulphide and salt bridges
B-DNA is wrapped 1.65 turns around the histone octamer

Question 7

What histones are used to form nucleosome?

Answer 7

H2A, H2B, H3 and H4
Central H3/H4 tetramer and two H2A/H2B dimers = histone octamer
Histones contain a lot of lysine and arginine in the N-terminus (used for PTMs)

They are highly conserved = critical functions
But many have PTMs
Variations are associated with specific functions in initiation/termination and in the formation of telomeres/centromeres

Question 8

How do histones interact with the DNA?

Answer 8

Histones bind only to the inner face of B-DNA
The mainly bind via the sugar-phosphate backbone via hydrogen bonds, salt bridges, and helix dipoles
They interact with the phosphate oxygens and hydrophobic interactions with the deoxyribose ring

Question 9

How is the 10 nm chromatin fibre produced?

Answer 9

The 146 bp DNA nucleosome is added to histone H1 - a linker histone
H1 binds at the entry and exit points of the nucleosome

It plays a role in condensing chromatin fibres and regulating access of other proteins to the DNA

Question 10

How is 30 nm fibre chromatin formed?

Answer 10

Under physiological conditions (increase in sat concentration) the chromatin condenses further into ‘zig zag’ structures to form fibre with a diameter of 30 nm
= 42-fold compaction in total

This is a higher order structure

Question 11

What are the two models of 30 nm chromatin fibre?

Answer 11

Solenoid model - equal zig zags, quite wide and short

Zig-Zag model - longer and thinner but unequal zig zags (some wide and some thinner)

The structure indicates which DNA sequences are likely to be folded next to each other, so when these sequences are bound by proteins that regulate transcription, which regions could interact
BOTH the structures are correct

Question 12

How is a chromtid formed?

Answer 12

Loops of 30 nm fibre (15 to 30 μm) are attached to a protein scaffold
The loops enter and exit the scaffold at almost the same place
Each loop forms a close circle at the base of the scaffold = maintain negative supercoiling

This forms the metaphase chromosome

Question 13

What happens to chromosomes during interphase?

Answer 13

They each occupy discrete nuclear territories
Showing genes on different chromosomes can be frequently packaged next to each other in the nucleus

This was discovered by FISH - fluorescence in situ hybridisation

Question 14

What are the different types of genes? location?

Answer 14

Active genes - located centrally in the nucleous

Inactive genes - associate with the nuclear periphery or with peri-centromeric

Question 15

What are the different types of chromatin?

Answer 15

Euchromatin - A site where genes can be expressed, dispersed during interphase

Heterochromatin - Transcriptional inactive, stays condensed during interphase

Question 16

What are the types of heterochromatin?

Answer 16

Consitiutive - permenantly condensed, near centromeres, lacks genes and are full of repetitive DNA

Facultative - condensed only at certain stages of development, genes can be switched on/off

Question 17

How do chromosomes affect transcription?

Answer 17

Nucleosomes can ‘hide’ or occlude transcription factor binding sites
We need a ‘linker’ - gap of DNA between nucleosomes
Chromatin needs to be remodelled to allow transcription initiation to begin (the TATA box needs to be exposed) - from heterochromatin to euchromatin

The aim is to generate enough space for the pre-initiation complex to form

Question 18

How is the chromatin remodelled?

Answer 18

Chromatin contains ATP-driven complexes that remodel nucleosomes
They disrupt the interactions between histones and DNA, to make the DNA more accessable

Question 19

What are the two methods of remodelling chromatin?

Answer 19

A chromatin remodelling complex binds at DNA entry and exit point pushing a section of DNA on whilst pulling at the nucleosome and either:

Sliding - histone octamer slides along the DNA strand to a new location

Displacement - the histone octamer is relocated to a different DNA strand

Question 20

How does the chromatin remodelling complex work?

Answer 20

They ‘walk’ up DNA strands driven by ATP hydrolysis

The remodelling complex binds to DNA, putting torsional strain on the DNA and decreased its local twist, the twist then diffuses along the DNA (translocates)
This loosens the histone octamers grip on the segment on DNA

Question 21

What other feature of chromatin is involved in gene expression?

Answer 21

HMG proteins are the architectural proteins in the protein scaffold
They help regulate gene expression as it causes the DNA to bend by as much as 70° toward its major groove
= facilitate the binding of other regulatory proteins to the DNA

Question 22

How can histones help in gene expression?

Answer 22

The tails of histones can be PTM e.g.
Acetylation of Lys 
Methylation of Lys and Arg 
Phosphorylation of Ser and Thr 
Ubiquitination of specific Lys

Question 23

How does PTM of histones help in gene expression?

Answer 23

Acetylation, phosphorylation and ubiquitination all reduce the electronic charge = more negative
This weakens the histone-DNA interactions promoting the chromatin decondensation

Methylation increases basicity and hydrophobicity = stabilises the chromtin

Question 24

What carries out acetylation of histone tails?

Answer 24

Histone acetyltransferases (HATs) 
It uses acetly-CoA as the acetyl donor group
It is attached to the tail via a isopropionyl group

Portions of HAT subunits are structural homologs of histones H3, H4 and H2B

Question 25

What happens to the acetylated histone tails?

Answer 25

The acetylated Lys residues are recognised by transcriptional coactivators (like the SH2 domain) called bromodomains
They have a deep hydrophobic pocket with a specific binding site for the acetylated Lys residues

They are comprised of antiparallel four-helix bundle

Question 26

What is gene silencing?

Answer 26

Promoting transcriptional repression
Histone acetylation is reversible via histone deacetylases (HDACs)
Histone methylation promotes gene silencing via histone methyltransferases (HMTs)

Acetyl allows, Methyl masks

Question 27

How do methyltransferases methylate histone tails?

Answer 27

They use S-adenosylmethionine (SAM) as their methyl donor

Methyltransferases flip their target bases out of the DNA double helix
Eukaryotic DNA is methylated on cytosine residues forming 5-methylcytosine residues

Question 28

What is a feature of DNA methylation?

Answer 28

DNA methylation is eukaryotes is self-perpetuating (it has the power to continue indefinately)
Due to the palindromic nature of DNA, it allows methylation patterns on parental DNA to be generated in the same pattern on the daughter strand
Carried out via DNMT1 protein - methylates hemimethylated DNA

Maintenance methylation = stable inheritance, with all cells having th esame differentiated phenotype

Question 29

What is genomic imprinting?

Answer 29

Maternal and paternal inheritance can differ

It differs from defferential DNA methylation, during gametogenesis

Question 30

What are some diseases on genomic imprinting?

Answer 30

Prader-Willi syndrome (PWS) - 5000 kb deletion of the paternally inherited chromosome 15, causing small hands/feet, obesity and mental retardation

Angelman syndrome (AS) - deletion of the same region but the maternally inherited chromosome 15, causing mental retardation, uncoordinated and random inappropriate laughter

Question 31

When RNA polymerase starts working in transcription what happens to the chromatin?

Answer 31

The RNA polymerase II displaces the histone octamer
The H2A/H2B dimer needs to be evicted onto the chaperone FACT to reform the nucleosome behind the polymerase
Therefore it is not shifted in tact

Question 32

Describe the chromosomes?

Answer 32

1-22 autosomes
Largest = chromosome 1
Smallest = 21
They differ around 5 fold in size

Chromosomes are arranged in pairs
They are grouped according to size and centromere position

Question 33

What are the centromere positions?

Answer 33

Metacentric: centromere in the middle
Acrocentric: close to one end
Submetacentric: intermediate position

The centromere position affects chromosome shape during anaphase of mitosis

Question 34

What are the namings of the chromosome bands?

Answer 34

Small arm = p (petite)
Long arm = q (“not-p”)
They have regions numbered within each arm
Sub-regions are known as ter, p-ter and q-ter

They have major and minor divisions
+ after the name = extra copy of the terminus of the
- before = missing a copy

Question 35

How do we prepare the probes for chromosome staining?

Answer 35

Probes specific to each chromosome were prepared by PCR
Probes labelled with different fluorescent dyes for each chromosome (chromosome paint)
Probes used to hybridise to metaphase chromosome spread on microscope slides

Question 36

How is chromosome staining carried out?

Answer 36

1. The cells need to be ‘arrested’ in metaphase using colchicine - to prevent association with microtubules
2. Spread onto microscope slides
3. Stain with fluorescent dye or Giemsa
   ○ Fluorescent - gives interbands, taken from earlier in S phase
   ○ Giemsa - gives a banding pattern to identify individual chromosomes