M2 Fitz- Epigenetic regulation of chromatin

what is the structure of chromatin?

• the nucleosome is made up of ~147bp of DNA wrapped around an octamer of two H2A+H2B dimers and two H3+H4 dimers (histone subunits)

• the histone proteins are positively charged because they are rich in lysine, which interacts with the negative backbone of DNA

• nucleosomes are evenly spaced along the DNA into a 10nm fibre with ~60bp of linker DNA in between

• histone H1 can increase compaction of DNA by binding to linker DNA near the nucleosome

• we used to believe there was a higher order of packing into regular helical (solenoid/zigzag) 30nm fibres, but we now believe this higher order is much more disorganised with consecutive chromatin loops

what is epigenetics?

• the DNA sequence does not change, but the expression of its genes does change, due to molecules and mechanisms that produce a change which can be perpetuated at least through mitosis

what are histone variants?

• these can be involved in epigenetic regulation

• histone variants are histone gene copies that have significantly diverged in their structure from ‘canonical histones’, often to fill a different function

• eg. CENP-A is a histone variant that defines the location of the centromere (it is not defined by the DNA sequence), because CENP-A recruits kinetochore proteins to link to microtubules

what are post-translational modifications of histones and how can they change expression?

• histone PTMs are involved in epigenetic regulation

• histone tails extend out of the nucleosome and so they are very accessible to PTMs- these histone modifications act as signals for different expression profiles

• eg. to mark the repression or activation of regions

these can act directly by changing the nucleosome properties

• eg. acetylation of lysine neutralises its positive charge to weaken the interaction with negative DNA and make DNA more accessible

• eg. large modificaitons like ubiquitination significantly change the size of a histone to change the structure of a nucleosome

or indirectly, by affecting the recruitment of reader/effector proteins

• eg. heterochromatin protein 1 (HP1) binds to H3K9me3 modifications and recruit many different effectors

how are histone modifications deposited at particular sites?

• writers and erasers deposit and remove PTMs from histones

this can occur in multiple ways:

• by association with DNA-binding proteins- to specific DNA sequences or generic base-rich regions

• by association with transcriptional machinery-

• by association with nascent RNA- transcripts stil llinked to RNA polymerase can have recognition signals for binding proteins that recruit writers/erasers

• directed by other nearby epigenetic signals- polycomb repressive complexes (PRC) 1 and 2 bind to each other’s histone modifications and locally reinforce them

how can DNA methylations change expression?

• DNA can be methylated at multiple site

• in eukaryotes this is usually at carbon 5 of cytosine

• in vertebrates this is usually in a CG context

• these are usually repressive, because active transcription factors don’t recognise methylated cytosine and don’t bind, and because methyl-CpG-binding proteins (MBP) recognise it and recruit other factors

• writers de-novo methylate a cytosine, and maintenance methylation causes the cytosine on the oppostie strand (in a CG context) to be methylated as well

• erasers

DNA is often methylated at:

• inactive promoters

• constitutive heterochromatin

• gene bodies- to silence cryptic transcription (starting midway through a gene)

why have many phyla lost DNA methylation?

• the ancestral role of DNA methylation was not in regulating genes, it was just in preventing cryptic transcription

• it’s very useful, but DNA methylation is costly in causing DNA damage and transcription stalling, particualrly at gene bodies (where it is most important to not have DNA damage)

• it has co-evolved with repair pathways that prevent DNA damage, but this is energetically costly so many haven’t prioritised it

how are epigenetic marks preserved in chromatin replication?

• epigenetic marks must be perpetuated at least through mitosis

• it can also go through meiosis, particularly in plants

• DNA methylation stays on one strand

• histones are distributed evenly across each daughter strand

this leads to 50% dilution, so:

• DNA methylation is maintained by DNMT1 recognising hemi-methylated DNa to methylate the other strand

• a lot of PTMs reinforce themselves through reader-writer coupling

• histone variants are maintained by histone chaperones that replace canonical histones with histone variants when they are diluted

• however this means that chromatin domains can continue to spread across the chromatin fibre (and in 3D, to other chromatin fibres)

• this can be stopped by barriers like nucleosome depleted genes, areas of high nucleosome turnover, or by other opposing PTMs