L6: Histones and Packaging Flashcards
How long is the length of DNA in a diploid cell?
Approx. 2m long
How long is one complete turn of the double helix in DNA?
Approx. 10 bases - 3.4nm long
What is DNA packaged into?
Chromatin
What is the DNA fundamental unit of chromatin?
Nucleosome
What does the nucleosome consist of?
Histones
What can histones give a greater understanding of? How?
Can isolate and characterise biochemical properties to give a clearer understanding of the crystal structure of the nucleosomes
What 3 features make up chromatin? What are their roles?
- DNA = trying to package this
- Proteins = form part of the nucleosome
- non-coding RNA = small amount to keep transcriptional regulation of genes
What are histones?
- The most common nuclear proteins which are highly abundant
- Form half of all protein in the nuclei
What is the ratio of histones in the nucleus to the mass of DNA?
Ratio of 1:1
What is the function of non-histone components?
- Vary by species
- Allow higher levels of DNA packaging
What are the 4 core histones?
H2A
H2B
H3
H4
What is the linker histone?
H1
What properties do histones have?
- Small
- Highly positively charged
- Highly conserved - important role in the cell
Which histones are the most highly conserved?
- H4 and H3 the most followed by H2A and H2B
- The most highly conserved proteins in a eukaryotic cell
How conserved is the H1 histone? How is this histone different to the others?
- 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
When will DNA exist in the double helix shape?
- 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
Summarise the packaging levels of DNA
- From 10nm fibre to 30nm fibre, up to 300-700nm
- Then highly compacted chromatin in metaphase
Summarise metaphase
- Splitting of chromosomes to transfer genetic information from one cell generation to the next
- Protects the genome in the mitotic phase
What is level 1 of packaging?
Nucleosome
What makes up the nucleosome?
- 4 core histones - H2A, H2B, H3, H4
- There are two molecules of each of these core histones
What term is used to describe the nucleosome core of histones?
Octameric core
What is around the octameric core?
- 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
How many H1 molecules are in the octameric core?
1
What mass of protein is made up of the 4 core histones?
108Kda (da=daltons)
What connects one nucleosome to another? What does this do to the mass?
- 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
What ratio is mass of DNA to histones in a nucelosome?
1:1 ratio
130Kda of DNA
108Kda of core histones
24Kda of H1
- Highly conserved in every living organism
What is the first process towards the formation of the octameric core?
Heterodimerisation of H3 and H4
What is formed in the heterodimerisation of H3 and H4?
- 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
What shape do the H4 and H3 dimers form?
- 2 H4 and H3 dimers (histone handshakes) formed
- These join together to form the centre of the octameric core in a horseshoe shape
What happens to the H2A and H2B histones in the formation of the octameric core?
- 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
What term is used to describe the shape of the nucleosome?
Canonical nucleosome structure
What is a dyad axis?
-where DNA that wraps around the nucleosome, comes to an end and crosses over
What is located in the middle of the dyad axis, at the cross over point, and what is the function?
- H1 is located at the cross over point on the dyad axis
- Prevents DNA splippage from around the octameric core
What term is used to describe the point where H1 binds in a dyad axis?
Dyad access
What structure is found on every core histone?
N-terminal tail
What is the function of an N-terminus tail?
- 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
What effect can a change in the canonical histones have on packaging?
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
What are the 4 histone variants for H2A?
H2A.Z , macro H2A, H2A.X, H2A.Bbd
Describe macro H2A
- 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
Describe H2A.X
- 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
Describe H2A.Bbd
- Vertebrate specific
- Depleted on the inactive X chromosome
What % of amino aicds are different and similar in variant H2A.Z compared to H2A?
60% different
40% the same
How does a change in variant change the packaging of DNA? Use H2A as an example
- 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
How many different variants does H3 have?
- 5
- H3.1, H3.2, H3.3, Cenp.A, H3.lt
What’s interesting about the H3 varaints?
- 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
Where is Cenp.A found?
Enriched in the centromeres and telomeres
What is the role of H3.3?
- 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
What is the overall outcome from the introduction of variants in the nucleosome?
- 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
Which two histone variant signify transcriptionally active regions?
H2A.Z and H3.3
Which histones signify transcriptionally silent regions, with no genes present as these are the centromeres and telomeres?
- Cenp A and H3
- Don’t want genes present here as the centromere will bind to the mitotic spindle and be pulled apart
Why is access to some nucleosomes restricted?
- DNA does not follow a smooth path around the nucleosome
- Some located on the inside by the octameric core will be inaccessible
What is the second level of packaging?
- 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’
How is the second level of packaging described?
A successive row of individually, equally spaced nucleosomes along your DNA packaging it up
What is the packing ratio in a 10 nm fibre?
6-7
What is the third level of packaging?
The 10 nm fibres coiled to form the
30 nm solenoid
How does the 30 nm solenoid form?
- 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
What is the packing ratio at level 3?
Around 40
What is the fourth level of packaging?
- 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
What is the fourth level packing ratio?
680
What is the fifth level of packaging?
- 700 nm fibre
- Described as the coiled coil and requires loops of chromatin to coil again to form the condensed fibre
- Highly compacted
What is the fifth level packing ratio?
What is the sixth level of packaging?
- 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
When is transcription able to happen in a eukaryotic cell?
Have to go back to the 10 nm structure (the simplest structure)
Could transcription happen at any level of packaging higher than the
10 nm?
- 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
Why is packaging important?
- 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
What is chromatin?
Compaction of DNA by the association with the nucleosome
What are the different types of chromatin?
- Euchromatin - found in the P and Q arms of the metaphase chromosome - contains the genes
- Heterochromatin - in the telomeres and centromere - no genes
What is the function of the centromere?
- Primary constriction
- mediates chromosome cohesion
- spindle attachment
- chromosome segregation
How do the different types of chromatin differ as stains on the interface in microscopy?
- Euchromatin is lightly stained - genes in the nucleus
- Heterochromatin is dark - highly compacted/condensed even at the interface
What are the two types of heterochromatin?
- Constitutive
- Facultative
- Both really highly condensed even in interphase
- Generally do not contain genes
- Repetitive DNA sequences
- Replicated late in S phase
What % of the genome is inactive and what % is in the highly condensed form?
- 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
What is constitutive heterochromatin?
- All cells of a given species will package the same regions of DNA into constitutive heterochromatin
- This is in the centromeres and telomeres
What happens to a gene that is expressed in the constitutive heterochromatin?
Very poorly expressed because the highly packaged region will have to unravelled down to the 10 nm fibre for transcription - poor efficiency
What is one chromosome that is mainly constitutive?
- 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
Where is the majority of constitutive heterochromatin found on a chromosome?
Normally on the Q arm
What is facultative heterochromatin?
- DNA packaged in facultative heterochromatin is not consistent within the cell types of a species
- Regulated and is often associated with morphogenesis or differentiation
What is an example of a facultative chromosome?
- 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
What is the main difference between facultative heterochromatin and euchromatin?
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)
When are heterochromatic regions replicated?
- Very late in S phase
- Have to unravel the structure and decondense to allow replication or transcriptional machinery to have access
What are the characteristics of euchromatin?
- 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
