Boyes Epigenetics Flashcards
What do epigenetic factors do?
Bring about an inheritable change in expression of a gene without introducing mutation
What is the structure of a nucleosome?
H3/4 tetramer, 2xH2A/B dimers, H1 at entry and exit points.
147bp wrapped around with dyad 80p in
50 nucleotide linker
How many times does DNA wrap around the histone?
1.65 turns
What is the structure of a histone protein?
High R/K proportion, 2alpha helices conected by loops
How does the nucleosome remodelling complex work?
DBD binds entry point to push, forming loop
translocation domain binds at the dyad to pull 10bp via hinge
uses ATP
What increase in DNase1 hypersensitivity is found at promoters?
100x
How can promoters be mapped?
DNase digestion at promoters then restriction digests at known points, to find promoters based on size of fragment. Known as indirect end labelling
What is indirect end labelling?
Method of finding promoter sites
What additional functions do nucleosome remodelling complexes have?
spacing following replication
exposure of DNA
exchange of H2A/B dimerss
How are nucleosome remodelling complexes classified?
By ATPase subunit
What are the classes of remodelling complex?
SNI/SWF, ISWI, CHD, INO80
What is the structure of nucleosome remodelling complexes?
2-15 subunits, up to 2MDa
ATPase and helicase domains
other targetting domains
How can nucleosome remodelling complexes be recruited to a promoter?
Via activators or direct to chromatin
Where is the ovalbumin gene activated?
Oviduct
Where is the vitellogenin gene stimulated?
By oestrogen in the liver
How are X chromosomes inactivated?
DNA methylation of CpG islands in promoters
Does epigenetic memory exist in Drosphilia flies?
Yes, even though they do not have DNA methylation
What timescale can epigenetics occur on?
Accumulate over a lifetime
What methylation state does newly replicated DNA exist in?
Hemi-methylated
Which Dnmt is responsible for methylation after replication?
Dnmt1
Which enzyme is responsible for DNA methylation de novo?
Dnmt3a/b
When does Dnmt3 act?
During embryonic development
What is the structure of Dnmt?
Highly conserved catalytic C terminal domain
N terminal binds to other proteins
When is Dnmt1 recruited?
PCNA clamp association at replication fork
When is Dnmt3 not recruited?
When 3L regulatory unit dissociates
How are CpG islands protected from methylation?
Transcription by Pol 2 Ser5 recruits Set1 and Cfp1 to promoter
H3K4me3 opens chromatin structure
no association of 3L to dnmt3
Which novel proteins can be isolated from a promoter?
Set1 and cfp1
How can Set1 and Cfp1 be isolated from promoters?
HinP1 methylation sensitive digestion of GCGC sites
What is the structure of CpG islands?
1kb streches of G rich
Where are CpG islands found?
All housekeeping genes and 40% of tissue specific genes
How many CpG islands are the hapliod human genome?
25,500
What frequency are CpG islands?
1/10
What type of CpG islands have differential expression?
Weak promoters 1/20
What CpG frequency has no effect on transcription?
1/100
How does methylation repress transcription?
Directly by blocking major groove binding sites or indirectly by binding MBPs.
MECP1/2 has HDAC/Sin3a activity
When does CpG island methylation occur?
X chromosome inactivation and cancerous mutations
How can CpG islands be detected?
Bisulphite sequencing deaminates C to U, so only C left for sequencing are mC
Digestion with Msp1 (non specific) and Hpa11 (unmeth)
What is the histone code hypothesis?
Predicts that distinct combinations of modifications act to control chromatin structure and gene expression
Where do histone modifications act?
Exposed Lysine tails
Where does acetylation occur?
H3K14 binds bromodomains
H3K16 prevents compaction to a 30nm fibre
What type of proteins have bromodomains?
Coactivator/HATs associated with Pol2 elongator form
Remodelling complexes SWI/SNF and RSC which displaces H2A/B dimers
How is acetylation removed?
HDACs recruited after the Pol2 has passed
How can acetylation be observed?
Removal of nucleosomes alters sedimentation in centrifugation
Increased DNas1 sensitivity
How does acetylation activate chromatin?
Neutralises DNAphos - K/R interactions
Is acetylation epigenetic?
NO
How is H3K4me3 produced?
Set1 methyltransferase associated with Pol2 CTD phospho-ser5 at promoters
What type of domain does H3K4me3 recruit?
plant homeodomain (PHD) in remodelling complexes and HATs
Which histone methylations are activating?
K4, K36
Where is H3K4me1 found?
Active enhancers
Where is H3K36me3?
In gene body, associated with Set2 (Ser2)
How does H3K36 act?
Recruits HDACs to reset chromatin after transcription
What type of methylation does HP1 oproteins bind?
Chromodomains bind H3K9
How is H3K9 maintained?
Chromodomains of HP1 bind and recruit Suv39h set domain to act on adjacent nucleosomes to spread heterochromatin
Where can H3K9 be observed?
Position Effect Varigation in Droshphilia eyes by silencing in some cells producing patches of colours
What type of protein is Set1?
A trithorax protein
Which methylation do Polycomb group proteins act on?
H3K27me3
How do polycomb group proteins act?
PRC1 chromodomain binds H3K27me3 producing compaction
Recruits PRC2 Ezh SET to act on adjacent nucleosomes
What does H3K27me3 do?
Repression by compaction into heterochromatin. Hox genes silencing and X chromosome silencing
How is histone methylation maintained epigenetically?
Asf1 chaperones move Old H3/4 tetramers during replication, modifying enzymes stay attached to act on new tetramers brought by Caf1
How much heterochromatin is maintained epigenetically?
75% has HP1 or PRC proteins
Is Set a conserved sequence?
No
