Epigentics v Genetics Flashcards
What is significant about gene expression and DNA
Pluripotent stem cells differentiates into all the different cells of the body
The DNA within the nucleus doesn’t change as the cells differentiated - even though they have very different phenotypes and gene expression
proved by John Gurdon in cloning experiments and further proved by the cloning of Dolly the sheep
Describe differential gene expression?
There are transcription factors expressed in different cell types
There are also epigenetic factors
Together they can determine which genes are turned on/off
What is epigenetic regulation?
Epi - means on top off, so these alterations are on top of the normal DNA sequence
Epigenetic - a change in the state of expression of a gene that does not involve a mutation, but that is nevertheless inherited (after cell division) in the absence of the signal (or event) that initiated that change
DNA methylation
Some histone modifications e.g. By polycomb/trithorax protein complexes
The epigenetic marks maintain the memory of previous states of activity/inactivity
They do NOT establish them
Tissue specific gene expression relies on the combination of the activity of specific transcription factors plus chromatin modifications
What does control of specific gene expression rely on?
Upstream transcription factors - very specific ones and not just general
They bind to short regulatory DNA sequences = control sequences or control elements
They are transactivating factors
All promotors have these binding sites for various transcription factors
General transcription factors are expressed in ALL cell types
Transcription activators have a DNA binding domain and an activation domain
The DNA-binding domain is responsible for determining which promotor is activated
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 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
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 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
What are DNase I hypersensitive sites?
Hypersensitive sites are found at the promoters and enhancers of expressed genes
They are generated by the binding of transcription factors and the displacement of histone octamers
They reflect changes in chromatin structure as the nucleosome was blocking access to the DNA
DNase I digests hypersensitive sites about 100 fold more rapidly than sites in the rest of the genome
How can we map DNase I HS?
Indirect end labelling
- Add titrated DNase I in nuclei = cleave the DNA at the HS
- Prepare DNA - we have many broken DNA ends
- Cut with restriction enzyme
- Electrophoresis/southern blot and a probe with a region next to the restriction site
- Calculate the distance of the HS from restriction site in order to map the place where the HS occurs
ATAC Seq
This uses transposase - bits of bacterial DNA that are mobilised around the bacterial genome
Tn5 - is a cut and paste transposase (enzyme) that has been isolated
Transposase is pre-loaded with sequencing primes, which preferentially attacks open chromatin - it is inserting sequencing primers (also cut the DNA)
We the PCR amplify the cut DNA, sequence and map the sequencing will read back onto the genome
Advantage - uses 50,000 cells rather than 1-2 million
