Lecture 2 - DNA methylation, histone modification, nc RNAs Flashcards
Outline DNA methylation from lecture 1
- DNA methylation can be recognised by methyl-cytosine binding protein
- S-adenosyl methionince methyl donor to the DNA methyltransferases
- methylation on cystenines - 5-methyl-cytosine
- can rpomote a closed chromatin structure
- can be maintained through DNA replication
- used by lots of eukaryotes but not all
- assocaited with control of gene expression and genome defence
What are the diferent patterns of DNA methylation
Two main types: Mosiac/Global
- Mosaic DNA methylation
- fungi e.g. Neospora craasa
- methylates transposons not genes to supress transpson movement
- Mosaic DNA methylation
- plants, e.g. arabidopsis
- all transposons methylated and around 50% genes methylated
- methylation supressed the activity of spurious promoter elements in genes
- methylated genes are typically housekeeping genes
- Mosiac DNA methylation
- animals e.g. Ciona intestinalis
- methylates some genes, doesn’t methylate transposons to supress (supress by histone modiifications)
- Global DNA methylation
- animals e.g. homo sapiens
- whole genome predominantly methylated in CG configuration
- some small segments unmethylated, typically gene promoters
- Global DNA methylation
- plants e.g. zea mays
- similar pattern to arabidopsis, some genes methylated/not
- high # of transposable elements which are always methylated
What are the features of DNA methylation in mammals?
- absolutely required for correct developemtn
- tissue-specific differences in DNA methylation profile
- occcurs mainly on CG (Cp(phosphodiester linkage)G) dinucleotides (heritable) but increasing evidence that CHH (CpHpH) (non-symmetrical) methylation in important in some tissues/cell types (ED cells, brain, placenta) (H any base that isn’t G)
- Most CGs are methylated except for CpG islands
- Unmethylated regions corresponse to promoters of genes
Why are there fewer CpG pairs than would be expected in the mammalian genome?
DNA is constantly subjected to chemical reactions leading to spontaenous deanimation
Methylcytosine converts to thymine
Cytosine converts to uracil (can be ercognised and repaired - not mutagenic)
If thymidine is not repaired, this can persist.
Methylcytosine is mutagenic because it heavily in a heavy methylated genome, slowly get a loss of the cytosine base.
What are CpG islands and how to they form?
- CpG islands are regions (300-3000bp) in promoters that are typically not methylated and so have a higher than average CpG content as C base is not lost (5mc is mutagenic -> thymidine)
- around 60% of genes have CpG islands
- remain unmethylated throughout development
What CpG islands typically remain unmethylated throughout development? Others may change methylation status at key stages in development, how can abberant CpG methylation lead to disease?
- those that are methylated are those that need to be turned off and stay off e.g. master regulators of stem cells
- In cancer, tumor supressor genes have CpG islands aberrently turned off in cancer
What techniquies are there for assessing DNA methylation profiles?
- methylation-sensitive restriction enzymes (cobined with PCR or southern blotting)
- won’t cut if the DNA is methylated
- With PCR - have primers that span the region. If the DNA is methylated get a product, if not methylated get no cut
- Limitations: only tells info from a snigle base not about methylation occuring anywhere else
- Anti-methylcytosine antibodies to immunoprecipitate methylated DNA (5mhc or 5mc) followed by next generation sequncing
- take DNA
- shear to smaller fragments
- use antibodies to precipitate out regions of DNA
- look at whole genome sequences
- which regions precipitate out with antibodies
- look at a single gene
- was the gene interested in precipitated out
- Limitations: sheared DNA, may haave bits that are several kb long, can only say that a methylation occurs somewhere in the region
- Bisulphide sequencing
- gives single base resolution
- treatment by sodium bisulphite followed by desulfonation
- cytosine convert to uracil
- 5-methylcytosine is unaffected
- sequencing revealt C to T transitions for unmethylated cytosines
- but doesn’t distinguish between 5-methylcytosine and 5-hydroxymethyl-cytosine
- TET-assocatied bisulfite sequencing
- TET involved in the extrasteps
- glycosylation of 5hmc (stays as cytosine whole way through) mc won’t be glycosylated
- Use TET oxidation: mC oxidised to caboxycytosine
- During the bisulphite treatment: carboxycytosine -> carboxyuracil -> read as cytosine
- compare to traditional bisulfite sequencing to determine between 5hmc/mc
Describe the basic unit of packaging of the chromatin
Basic unit of packaging in the nucleosome
- octamer of 4 core histone proteins: H2A, H2B, H3 and H4
- linker histon: H1
- core histones can be replaced by variants
- nucleosomes can be remodelled
- histones can be covelently modified
- dynamic - associated of DNA with histones can be tightened/loosened
Outline histones and histone modification
Four core histone particles
- H2A
- H2B
- H3
- H4
- small (102-135 amino acids)
- basic (rich in lysine and arginine) - good for packaging DNA
- N terminal tails where the modification takes place
What are the main different types of core histone moficiations?
- acetylation (e/g/ lysine, K-ac)
- methylation (e.g. lysine, K-me)
- phosphorylation (e.g. serine, S-ph; Threonine, T-ph)
Modifications are dynamic
- Get different modifications on the tails relating to different information.
- Cell can distinguish between modifications of different amino acids.
- methylation can be active (Kme4) or repressive (Kme9) depending on where it occurs on the tail
Why is most known about the modifications of histone H3?
- may be easier to raise antibodies
- particular technology
How do histone modifications affect chromatin structure?
- Directly: disrupt contacts between nucleosomes in order to unravel chromatin
- e.g. acetlyation of lysines neutralises the basic charge
- Indirectly: recruitent of non-histone proteins (histone code hypothesis) that affect the chromatin structure
Describe how modifications of histones directly affect chomatin structure
- lysine is normally positively charged (histones are highly basic)
- If it is acetlyated this neutralises the charge, DNA less tightly bound to the nucleosome
- Histones are highly basic - changing a few lysine makes a differenece
- site directed mutagenesis to change the charge of histones, changing just one has an effect
- This modification is dynamic and reduced the charge-dependent interaction between histones and DNA
Give examples of where histone acetylation/deacetylation has a biological effect
The making of a queen bee
- honeybee queen and workers are genetically identical
- queen larvar get fed royal jelly thoughout development and into adulthood
- contains phenyl butyrate (histone deacetylase inhibitor)
- inhibiting DNA methylation in larvae mimmicks the effects of royal jelly
- large-scale DNA methylation differences
What results from the inhibition of histone deacetlyation by phenyl butyrate?
Pushing to the acetlyated state of lysine
- open chromatin configuration
- loss of DNA methylation
- activation of gene expression
- more accessible to enzymes that demethylate
