Nucleosomes and Chromatin Flashcards
Why does DNA need to be compact to be stored?
Human genome - 21,000 genes
46 chromosomes
A lot of information to store in a small space
What are the two principals used to package DNA?
- Neutralise negative charge of DNA with positively charged proteins (to neutralise the phosphate groups)
- Supercoil DNA to occupy smaller volume
How is DNA packaged in prokaryotes?
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
What are the positively charged proteins used in packaging of prokaryotic DNA?
Positively charged proteins that package the prokaryotic DNA are not yet identified but candidates include:
HU - wrapping
IHF and FIS - bending
H-NS - compaction
What are the steps of the process of DNA into a chromosome?
B-DNA (+ Histones) = Nucleosomes (+ H1) = 10 nm chromatin fibre 30 nm chromatin fibre Loops of 30 nm chromatin fibre (+ protein scaffold) = Chromatid
Describe how nucleosomes are formed?
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
What histones are used to form nucleosome?
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
How do histones interact with the DNA?
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
How is the 10 nm chromatin fibre produced?
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
How is 30 nm fibre chromatin formed?
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
What are the two models of 30 nm chromatin fibre?
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
How is a chromtid formed?
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
What happens to chromosomes during interphase?
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
What are the different types of genes? location?
Active genes - located centrally in the nucleous
Inactive genes - associate with the nuclear periphery or with peri-centromeric
What are the different types of chromatin?
Euchromatin - A site where genes can be expressed, dispersed during interphase
Heterochromatin - Transcriptional inactive, stays condensed during interphase
What are the types of heterochromatin?
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
How do chromosomes affect transcription?
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
How is the chromatin remodelled?
Chromatin contains ATP-driven complexes that remodel nucleosomes
They disrupt the interactions between histones and DNA, to make the DNA more accessable
What are the two methods of remodelling chromatin?
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
How does the chromatin remodelling complex work?
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
What other feature of chromatin is involved in gene expression?
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
How can histones help in gene expression?
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
How does PTM of histones help in gene expression?
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
What carries out acetylation of histone tails?
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
What happens to the acetylated histone tails?
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
What is gene silencing?
Promoting transcriptional repression
Histone acetylation is reversible via histone deacetylases (HDACs)
Histone methylation promotes gene silencing via histone methyltransferases (HMTs)
Acetyl allows, Methyl masks
How do methyltransferases methylate histone tails?
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
What is a feature of DNA methylation?
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
What is genomic imprinting?
Maternal and paternal inheritance can differ
It differs from defferential DNA methylation, during gametogenesis
What are some diseases on genomic imprinting?
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
When RNA polymerase starts working in transcription what happens to the chromatin?
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
Describe the chromosomes?
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
What are the centromere positions?
Metacentric: centromere in the middle
Acrocentric: close to one end
Submetacentric: intermediate position
The centromere position affects chromosome shape during anaphase of mitosis
What are the namings of the chromosome bands?
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
How do we prepare the probes for chromosome staining?
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
How is chromosome staining carried out?
- The cells need to be ‘arrested’ in metaphase using colchicine - to prevent association with microtubules
- Spread onto microscope slides
- Stain with fluorescent dye or Giemsa
○ Fluorescent - gives interbands, taken from earlier in S phase
○ Giemsa - gives a banding pattern to identify individual chromosomes
