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Y1 Fundamentals of Life Science > L6: Histones and Packaging > Flashcards

L6: Histones and Packaging Flashcards

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1
Q

How long is the length of DNA in a diploid cell?

A

Approx. 2m long

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2
Q

How long is one complete turn of the double helix in DNA?

A

Approx. 10 bases - 3.4nm long

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3
Q

What is DNA packaged into?

A

Chromatin

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4
Q

What is the DNA fundamental unit of chromatin?

A

Nucleosome

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5
Q

What does the nucleosome consist of?

A

Histones

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6
Q

What can histones give a greater understanding of? How?

A

Can isolate and characterise biochemical properties to give a clearer understanding of the crystal structure of the nucleosomes

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7
Q

What 3 features make up chromatin? What are their roles?

A
  • DNA = trying to package this
  • Proteins = form part of the nucleosome
  • non-coding RNA = small amount to keep transcriptional regulation of genes
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8
Q

What are histones?

A
  • The most common nuclear proteins which are highly abundant
  • Form half of all protein in the nuclei
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9
Q

What is the ratio of histones in the nucleus to the mass of DNA?

A

Ratio of 1:1

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10
Q

What is the function of non-histone components?

A
  • Vary by species
  • Allow higher levels of DNA packaging
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11
Q

What are the 4 core histones?

A

H2A
H2B
H3
H4

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12
Q

What is the linker histone?

A

H1

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13
Q

What properties do histones have?

A
  • Small
  • Highly positively charged
  • Highly conserved - important role in the cell
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14
Q

Which histones are the most highly conserved?

A
  • H4 and H3 the most followed by H2A and H2B
  • The most highly conserved proteins in a eukaryotic cell
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15
Q

How conserved is the H1 histone? How is this histone different to the others?

A
  • Shows more divergence than the core histones, but is still highly conserved
  • Has less similarity between species but still has the same structure and function
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16
Q

When will DNA exist in the double helix shape?

A
  • Does not usually exist in the double helix unless acting as a linker between adjacent nuclear sites, when holding together two nucleosomes
  • Always packaged however
  • 2 nm in size
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17
Q

Summarise the packaging levels of DNA

A
  • From 10nm fibre to 30nm fibre, up to 300-700nm
  • Then highly compacted chromatin in metaphase
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18
Q

Summarise metaphase

A
  • Splitting of chromosomes to transfer genetic information from one cell generation to the next
  • Protects the genome in the mitotic phase
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19
Q

What is level 1 of packaging?

A

Nucleosome

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20
Q

What makes up the nucleosome?

A
  • 4 core histones - H2A, H2B, H3, H4
  • There are two molecules of each of these core histones
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21
Q

What term is used to describe the nucleosome core of histones?

A

Octameric core

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22
Q

What is around the octameric core?

A
  • 146 base pairs of DNA wrapped around the core in a left-handed superhelix of 1.8 superhelical turns
  • DNA make two turns around the core
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23
Q

How many H1 molecules are in the octameric core?

A

1

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24
Q

What mass of protein is made up of the 4 core histones?

A

108Kda (da=daltons)

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25
Q

What connects one nucleosome to another? What does this do to the mass?

A
  • Linker DNA to link one nucleosome to another
  • Makes total number of DNA bases up to around 200, which has an approximate mass of 130Kda
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26
Q

What ratio is mass of DNA to histones in a nucelosome?

A

1:1 ratio
130Kda of DNA
108Kda of core histones
24Kda of H1
- Highly conserved in every living organism

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27
Q

What is the first process towards the formation of the octameric core?

A

Heterodimerisation of H3 and H4

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28
Q

What is formed in the heterodimerisation of H3 and H4?

A
  • Both H3 and H4 form their own histone fold
  • Each histone fold then join together to form a histone handshake - highly associated with each other
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29
Q

What shape do the H4 and H3 dimers form?

A
  • 2 H4 and H3 dimers (histone handshakes) formed
  • These join together to form the centre of the octameric core in a horseshoe shape
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30
Q

What happens to the H2A and H2B histones in the formation of the octameric core?

A
  • Both form histone folds
  • 2 H2A and H2B combine to form 2 histone handshakes
  • These bind above and below the tetramer (H4 and H3) to form the octameric core
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31
Q

What term is used to describe the shape of the nucleosome?

A

Canonical nucleosome structure

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32
Q

What is a dyad axis?

A

-where DNA that wraps around the nucleosome, comes to an end and crosses over

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33
Q

What is located in the middle of the dyad axis, at the cross over point, and what is the function?

A
  • H1 is located at the cross over point on the dyad axis
  • Prevents DNA splippage from around the octameric core
34
Q

What term is used to describe the point where H1 binds in a dyad axis?

A

Dyad access

35
Q

What structure is found on every core histone?

A

N-terminal tail

36
Q

What is the function of an N-terminus tail?

A
  • Can act as signalling molecules
    -Signal transcription factors to come in or remodellers to remodel an area of the genome, or replication machinery
  • Binding point/ origin for replication and signalling to other nucleosomes
37
Q

What effect can a change in the canonical histones have on packaging?

A

A change in one of the canonical histones changes the interaction between the other histones in the octameric core, which will alter the path of the DNA around the nucleus - changes the packagng of the DNA

38
Q

What are the 4 histone variants for H2A?

A

H2A.Z , macro H2A, H2A.X, H2A.Bbd

39
Q

Describe macro H2A

A
  • Vertebrate specific
  • Enriched on the inactive X chromosome
  • Females have one inactive X chromosome which happens randomly, but will remain inactiive once so - the nucelosomes have macro.H2A instead of normal H2A
40
Q

Describe H2A.X

A
  • Damage to our DNA
  • A break will be signified by the expulsion of the normal H-2a and its replaced with H-2a x
  • The variant signals that the nucleosome is damaged
  • Needs to be fixed before replication can continue
41
Q

Describe H2A.Bbd

A
  • Vertebrate specific
  • Depleted on the inactive X chromosome
42
Q

What % of amino aicds are different and similar in variant H2A.Z compared to H2A?

A

60% different
40% the same

43
Q

How does a change in variant change the packaging of DNA? Use H2A as an example

A
  • Alters the stability between H2A and H2B dimers
  • Alters the binding of the dimers to the tetramer of H3 and H4
  • Binding of DNA is looser and the canonical nucleosome’s octameric core is larger
  • A larger octameric core means more DNA can be wrapped around the nucelosome and is loosely packaged
  • Regions of the genome that have a lineage that is transcriptionally active are more exposed to transcriptional machinery when the variant H2A.Z is present
44
Q

How many different variants does H3 have?

A
  • 5
  • H3.1, H3.2, H3.3, Cenp.A, H3.lt
45
Q

What’s interesting about the H3 varaints?

A
  • Very little difference in amino acid sequences - H3 is highly conserved
  • But still sufficient enough to change the structure of the tetramer of the octameric core
46
Q

Where is Cenp.A found?

A

Enriched in the centromeres and telomeres

47
Q

What is the role of H3.3?

A
  • Used to change the transcriptional process outside of replication
  • Allows genes to be turned on outside of replication and transcriptional machinery to gain access to the genome
48
Q

What is the overall outcome from the introduction of variants in the nucleosome?

A
  • Variants of histones H3 and H2A differentiate chromatin at
    centromeres, active genes and heterochromatin
  • Replacement of H3 with H3.3 marks actively transcribed loci by replication independent nucleosome assembly
  • Epigenetically silenced chromatin is enriched or depleted in an abundance of diverse H2A variants
  • All of this can cause changes to the interactions of either the H3 and H4 tetramer or the H2A and H2B dimers, which can cause a tightening (silence heavy packaging) or loosening of DNA around the octameric core, allowing transcriptional activity
49
Q

Which two histone variant signify transcriptionally active regions?

A

H2A.Z and H3.3

50
Q

Which histones signify transcriptionally silent regions, with no genes present as these are the centromeres and telomeres?

A
  • Cenp A and H3
  • Don’t want genes present here as the centromere will bind to the mitotic spindle and be pulled apart
51
Q

Why is access to some nucleosomes restricted?

A
  • DNA does not follow a smooth path around the nucleosome
  • Some located on the inside by the octameric core will be inaccessible
52
Q

What is the second level of packaging?

A
  • 10 nm fibre
  • Lowest level of packaging seen in the nucleus
  • DNA is packaged and each nucleosome is connected to the next
  • Structure of DNA is present in the linker DNA but its connected to another nucleosome
  • ‘Bead on a string’
53
Q

How is the second level of packaging described?

A

A successive row of individually, equally spaced nucleosomes along your DNA packaging it up

54
Q

What is the packing ratio in a 10 nm fibre?

A

6-7

55
Q

What is the third level of packaging?

A

The 10 nm fibres coiled to form the
30 nm solenoid

56
Q

How does the 30 nm solenoid form?

A
  • Requires 6 nucleosomes to coil and in the centre is a H1 histone
  • H1 is essential for the higher order forms of packaging - changes to the variant of H1 will change the packing of the solenoid
57
Q

What is the packing ratio at level 3?

A

Around 40

58
Q

What is the fourth level of packaging?

A
  • The 300 nm solenoid
  • Each loop contains 60-100 kilo bases of DNA tethered by a non-chromatin protein component
  • Each loop is then tethered to a scaffold by non-histone scaffold proteins
  • Controlled packaging
59
Q

What is the fourth level packing ratio?

A

680

60
Q

What is the fifth level of packaging?

A
  • 700 nm fibre
  • Described as the coiled coil and requires loops of chromatin to coil again to form the condensed fibre
  • Highly compacted
61
Q

What is the fifth level packing ratio?

A
62
Q

What is the sixth level of packaging?

A
  • Metaphase chromosome
  • P arm (short arm) and a Q arm (long arm)
  • Centromere in the middle and the telomeres at the ends
  • The compacted DNA and nucleosomes
    Highest form of packaging
63
Q

When is transcription able to happen in a eukaryotic cell?

A

Have to go back to the 10 nm structure (the simplest structure)

64
Q

Could transcription happen at any level of packaging higher than the
10 nm?

A
  • Could be initiated at the 30 nm fibre stage
  • Providing the promoter is accessible to allow chromatin remodelers to come in and bring the 30 nm fibre back to the 10 nm fibre to allow transcription
65
Q

Why is packaging important?

A
  • A way of regulating the gene expression that’s going to be happening within each of your cell lineages
  • Different parts of the genome will be packaged in different ways
  • Important because within a lineage there will be a particular transcriptional cassette that needs to be turned on - required genes will be at a lower level of packaging
  • Some genes are never on and are silenced - more highly packaged as they are not required
66
Q

What is chromatin?

A

Compaction of DNA by the association with the nucleosome

67
Q

What are the different types of chromatin?

A
  • Euchromatin - found in the P and Q arms of the metaphase chromosome - contains the genes
  • Heterochromatin - in the telomeres and centromere - no genes
68
Q

What is the function of the centromere?

A
  • Primary constriction
  • mediates chromosome cohesion
  • spindle attachment
  • chromosome segregation
69
Q

How do the different types of chromatin differ as stains on the interface in microscopy?

A
  • Euchromatin is lightly stained - genes in the nucleus
  • Heterochromatin is dark - highly compacted/condensed even at the interface
70
Q

What are the two types of heterochromatin?

A
  • Constitutive
  • Facultative
  • Both really highly condensed even in interphase
  • Generally do not contain genes
  • Repetitive DNA sequences
  • Replicated late in S phase
71
Q

What % of the genome is inactive and what % is in the highly condensed form?

A
  • 90% is inactive - silenced
  • But only 10% is in the highly condensed form /as heterochromatin - mainly constitutive region
  • Will carry different epigenetic marks to keep them silent but packaged above the 10 nm level
72
Q

What is constitutive heterochromatin?

A
  • All cells of a given species will package the same regions of DNA into constitutive heterochromatin
  • This is in the centromeres and telomeres
73
Q

What happens to a gene that is expressed in the constitutive heterochromatin?

A

Very poorly expressed because the highly packaged region will have to unravelled down to the 10 nm fibre for transcription - poor efficiency

74
Q

What is one chromosome that is mainly constitutive?

A
  • Y chromosome
  • Very small and hardly any genes
  • Chromosome that determines male gender
  • Highly packaged
  • Only genes available are the ones to be expressed from the Y chromosome on the P arm
75
Q

Where is the majority of constitutive heterochromatin found on a chromosome?

A

Normally on the Q arm

76
Q

What is facultative heterochromatin?

A
  • DNA packaged in facultative heterochromatin is not consistent within the cell types of a species
  • Regulated and is often associated with morphogenesis or differentiation
77
Q

What is an example of a facultative chromosome?

A
  • The inactive X chromosome
  • Females have two active X chromosomes - one of these is randomly deactivated in early development and remains this way for every cell, even after replication
  • Each active X chromosome is condensed and takes on the facultative heterochromatin form
78
Q

What is the main difference between facultative heterochromatin and euchromatin?

A

A sequence in one cell that is packaged in facultative heterochromatin (genes poorly expressed) may be packaged in euchromatin in another cell (and the
genes expressed)

79
Q

When are heterochromatic regions replicated?

A
  • Very late in S phase
  • Have to unravel the structure and decondense to allow replication or transcriptional machinery to have access
80
Q

What are the characteristics of euchromatin?

A
  • Lightly stained regions of chromosomes
  • More ‘open’ chromatin configuration during interphase - found in 10 nm fibre in transcription
  • Replicated early in S phase
  • Contains both transcriptionally active and inactive genes (30 nm fibre when inactive)
  • Differential histone modifications to keep inactive genes silent

Y1 Fundamentals of Life Science (17 decks)

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