Relationships between chromatin structure and control of eukaryotic gene transcription Flashcards
What happens if there is an alteration in the cis regulating sequence in germ cells?
It is passed onto the next generation
What happens if there is an alteration in the cis regulating sequence in somatic cells?
It is passed onto the progeny of that cell
What are epigenetic changes?
- Changes to the chromatin structure which modulate gene expression but DO NOT alter the DNA sequence:
- Reversible covalent modifications which sit ON TOP of the DNA sequence
What happens to any epigenetic changes which occur in early embryogenesis?
Why?
They can be erased in the definitive germ line
In order to make the germ line totipotent (all genes in the germ line are potentially activatable again)
What does ‘totipotent’ mean?
Can become ANY cell in the embryo
What did Conrad Waddington do in 1940?
Invented the term epigenetics, using an epigenetic landscape:
- Epigenetics expressed CHANGE as the journey of a cell progresses
- Cells and lineages are created - go thorough ‘epigenetic landscape’ which has lots of decisions in it
- Series of decisions are spatially and temporally (time) organised - like a map
- Genes encode components of the epigenetic mechanisms
HE IDENTIFIED mutants in drosophila where disruptions in the epigenetic landscape was apparent
What are ‘the building blocks of chromatin’?
Nucleosomes (DNA associated with histones)
What are the subunits of a histone?
2 x H2A
2 x H2B
2 x H3
2 x H4
What is the charge of an octomer?
positive
What do the N-terminal tails of the octomer subunits act as?
‘Tagging sites’ for covalent modifications which affect gene transcription
What is the structure of transcriptionally active chromatin?
De-condensed
Sometimes completely stripped of nucleosomes
What is the structure of transcriptionally INactive (silence) chromatin?
Why?
10nm condensed into a 30nm fibre which is coiled further
Makes the structure transcriptionally IMPERMEABLE
What are the covalent modifications which occur on the N-terminal tails?
1) ACETYLATION - addition of acyl groups to LYSINES in the amino tails
2) METHYLATION - can be mono, di or tri-methylated
On which residues does methylation occur?
Arginine or Lysine
On which residues does acetylation occur?
Lysine
What modifications occur on lysine?
What does this mean?
BOTH methylation and acetylation
These modifications are competing
What do Histone Acetyltransferases (HATs) do?
How do they do this?
Add acetyl groups to lysines in the N-terminal tails of histones
WITHOUT any specificity of the enzyme (can acetylate any site, but with direction)
What enzyme removes acetyl groups from the N-terminal tails?
Histone DEacetylases
What are the Histone Acetyltransferase enzymes and how are they regulated?
CREB-binding protein
PCAF
GCN5
They are all subject to DIFFERENT regulatory influences which govern their activity to specific loci in the genome
What do histone methyltransferases (HMTs) do?
How do they do this?
Add methyl groups - writes the epigenetic CODE
- Done by many methylases which are SITE SPECIFIC
- Respond to specific signals which direct them to the specific site
(some sites are regulated by MANY methylases and some are regulated by only ONE)
What is the ‘histone code’?
Epigenetic modifications on DNA, laid down by site specific histone methyltransferases and non site specific histone acetyltransferases
Each mark has a different biological meaning
What enzyme removes methylation?
Histone DEmethylases
Can methylation and acetylation happen on the same lysine AT THE SAME TIME?
No - one modification must be removed before the other can be added
Do HAT/Histone deacetylases and HMT/Histone demethylases have binding capacity?
No
They are recruited to specific loci by complexes which contain DNA binding domains
Where are lysine acetylation marks found?
ALWAYS on ACTIVE genes
Where are lysine/arginine methylation marks found?
On active OR inactive genes - DEPENDANT on where they are found
Which methylation marks mark ACTIVE genes?
On genes:
H3-K4 (Histone H3 - lysine 4)
H3-R17 (Histone H3 - arginine 17)
Which methylation marks mark INACTIVE genes?
On genes:
H3-K9 (Histone H3 - lysine 9)
H3- K27 (Histone H3 - lysine 27)
Are methylation and acetylation marks reversible?
What does this mean?
Yes
Even if the marks are relatively stable, gene expression can be changed
What does acetylation of lyisines create?
Created binding sites for TRANSCRIPTIONAL ACTIVATORS that contain a BROMODOMAIN
(recruits part of the components of the transcriptional machinery
What are bromodomain transcription activators?
Epigenetic code readers
What does methylation of H3K27 and H3K9 create?
Biding sites for transcriptional REPRESSORS which contain a CHROMODOMAIN
What the acetylation status of a gene DIRECTLY proportional to?
The transcription levels of that gene
What does methylation of H3K4 create?
What does this include?
Binding site for transcriptional ACTIVATORS with contain a PH domain
Includes epigenetic code readers
What are the code readers?
Translate the epigenetic code into functional gene REGULATION
Transcription factors which are recruited to the sites of acetylation and methylation
What do transcriptional activator proteins do?
Bind SPECIFICALLY to DNA sequences and recruit transcriptional initiation machinery
Through COMBINATIONS of different chromatin remodelling/histone modification
How can chromatin be remodelled? (3 ways)
What do these allow?
1) Selective nucleosome remodelling - relaxation to make more accessible for the transcription complex
2) Selective histone removal - to reveal particular parts of the DNA for particular transcriptional components (eg. TATA binding proteins)
3) Selective histone replacement - with unusual properties
Why is chromatin remodelled?
After the binding of transcriptional activator proteins, which have been recruited by methylation and acetylation
In order to for the transcriptional machinery to be recruited and be able to bind to the DNA (RNA pol II, general TFs, mediators)
In what way can histones be modified?
Recruitment of code writers (Acetyl, methyl)
Recruitment of code readers (Bromodomains, Chromodomains, Zinc fingers)
What do transcriptional repressors do? (2 things)
1) Interfere with the transcriptional activator
2) Converts chromatin to a closed structure to prevent transcription
How do transcriptional repressors interfere with transcriptional activators? (3 ways)
1) Competitive DNA binding
2) Masking the activation surface
3) Direct interaction with the GTFs - so activators cannot activate it
How do transcriptional repressors convert chromatin to a closed structure? (3 things)
1) Recruitment of chromatin remodelling complex
2) Recruitment of histone deacetylases
3) Recruitment of histone methyl transferase (to H3-K9 and H3-K27)
What does the Polycomb Group of proteins include?
What do these proteins do? (2)
PRC1 and PRC2
Includes proteins which can generate or recognise repressive chromatin modifications (histone code writing AND reading)
These proteins restrain the expression domain of a specific Hox gene to a specific AP domain
(Present in a lot of developmental processes)
Where was PRC discovered?
What happens in mutants?
In drosophila
In mutants - ALL segments of the drosophila express ALL of the hox genes
What is the ‘enhancer of zeste’ in mammals and what does it do?
Ezh2 - Enhancer of Zeste homologue 2
A histone mehyltransferase code writer which forms the enzymatic core of a polycomb repressive complex 2 (PRC2)
Mediates the methylation of H3-K27 (lysine 27)
What is Pc?
What is its function?
Polycomb protein (chromodomain)
A code reader which is contained within PRC1 (polycomb repressive complex 1)
Recruited to and binds to histone marks (H3K27 - methylation of lysine 27) laid down by EzH2 (a histone methyltransferase in PRC2)
What are DNMTs and what do they do?
What does the result of this act as?
DNA methyltransferase
Adds methyl groups to CYTOSINE residues creating 5methyl-cytosine
Acts as recruitment sites for transcriptional repressors (MeCP2)
How do DNMTs and EZH2 work together?
Physically interact to reinforce each others effects
What are CpG nucleotides?
Cytosine nucleotide followed by a Guanine nucleotide in the LINEAR sequence
What is MeCP2 and what does it do?
Methyl-CpG-Binding protein
- Binds to methyated CpG dinucleotides
- Interacts with histone deacetylses and histone methyltransferases to help transform acetylated nucleosomes into methylated nucelosomes
What are calico cats and what gender are they?
Why?
Have BOTH black and orange fur - Female
They are heterozygous for 2 alleles of an X-linked coat pigment gene:
- In the black patch - orange allele inactivated
- In the orange patch - black allele inactivated
What happens early in embryogenesis in a female mammalian embryo?
One of the X sex chromosomes (maternal or paternal) in each somatic cell are chosen RANDOMLY to undergo permanent, stable inactivation
The progeny of this cell will all have this X in an inactive state
What is the mechanism of X-inactivation?
1) Synthesis of a non-coding RNA (Xist) from the X-inactivation centre (XIC) on the chromosome destined for inactivation
2) Xist spreads across the chromosome
3) Recruitment of a polycomb complex which recruits DNA methylases
4) DNA methylases decorate the entire chromosome where-ever there is Xist RNA (H3-K9 and H3-K27)
5) Promotes chromatin condensation and trasncriptional repression
This is communicated to the other X chromosome (which doesn’t inactivate)
What is a Barr Body?
A highly condensed inactive X chromosome which sits at the periphery of female somatic cells
Why do calico cats also have white fur?
No pigment there
Describe Agouti viable yellow
Agouti viable yellow is a dominant mutation of the agouti gene
Causes obesity and yellow fur due to high levels of the agouti gene
Caused by the insertion of an IAP (retroviral like transposon) into the Agouti locus
IAP is sensitive to DNA methylation - causing OVEREXPRESSIOn
If folic acid is ingested in the maternal diet, what happens to the Agouti viable yellow phenotype?
It is repressed and the offspring will look like wild-type mice
What is the polycomb gene highly involved in?
Cancer (tumor progression)
