MNase digestion — nucleosome phasing
26 different histone modifications mapped genome wide. in 20kb of yeast chrIII
chromosome conformation capture
mapping 300 nm
the 30 nm fiber mode
- solenoid model, regular 30 nm model
- zigzag, 3 nucleosomes / turn, less regular
- non-existent in vivo, only in dilute in vitro preps
- using MNase to map nucleosome-nucleosome contact maps.
- different fiber folding predict different peaks
- challenge, need to repair ends of MNase digested DNA while crosslinked to allow ligation
- claim: Nuclesome resolution HiC yeast map. semi-blocky structure on scale of 100 nucleosomes.
- clear relation between local gene structure
- in yeast squares on scale 2kb
- is this really contact or is it co-accessabililty?
- difference between triangle and mountain
- triangle structure are highly expressed.
- tandem orientation genes more likely in same block.
- divergent genes, clean break at promoter.
- (I suspect this is all transcription driven not topology driven)
- no evidence for regular 30 nm fiber
- ‘Gene loops?’
- +1 touches N-2 nucleosome etc maybe just as well or better than the N.
- ‘gene crumples’ rather than ‘gene loops’
- access to data about DNA as a chromatin polymer
Pt II: inheritance of acquired characteristics
- compare high protein vs. low protein fed males
- hundreds of (liver genes) can distinguish the difference between dads
- upregulated genes enriched in cholesterol and lipid biosynthesis
- downregulated are nothing in particular
- see phenotype differences as well.
- other work
- starve males in utero, up-regulate glucose and colesterol
- in utero during dutch hunger winter — increased diabetes etc. bodies hoarde calories
- connection between metabolic phenotype and later generations
- hypothesis ‘sperm epigenome?’
- also data showing molecules in the sperm fluids
- artificial insemination doesn’t work in mice. Need to do IVF.
- phenotype less penetrant than natural mating but still passable. Can sequence the rest of the sperm sample or the blastocyst. Look for cytosine methylation patterns.
- these methylation patterns and histone modifiation patterns don’t seem to carry the info.
- small RNAs?
- small RNAs (under 40bp).
- types: 70% are tRNA fragments. Also 19bp RNAs and microRNAs.
- most abundant form is 25-30% of all small RNA in sperm
- see differences in these guys in males with different parental effects
- tRNAs directly linked to metabolism, so this is a reasonable messanger. The particular ones here are logical choices.
- tRNAs are used in retro-element replication. (e.g. HIV packaging)
- tRNAs prime reverse transcriptase for example. Also true for endogenous retro-elements
- can out-compete other
- are these refuse from earlier sperm metabolism or functional?
- charged testes tRNAs don’t correlate with deitary effects of sperm on tRFs.
- testes small RNAs vs sperm — testes don’t make lots of tRNA fragments. more abundant in sperm (50 fold).
- tRNA fragments accumulate during sperm maturation in epididymis.
- deep sequence somatic cells from epididymis, find lots of these. (transfered from soma to germline?)
- Sperm maturation
- change lipid composition
- gain 200 proteins (despite being translationally inactive) shipped in vesicles from epididymis.
- isolate and sequence epididymesomes, similar tRNA profile as mature sperm.
- mostly come from distal epididymis, not proximal.
- do immature caput sperm have tRNA fragments?
- yes but less than mature ones.
- Reflections
- increasingly find other examples of soma-germline communication
- mostly mediated by small RNAs
- evidence against this hypothesis? Find high levels of intact tRNA in caput sperm.
- follow up
- genetically express modified label tRNAs in somatic cells, look for these in germline.
- make library of tRNAs express in virus, look for trasnfection into germline cells.
Biogenesis
- Epididymisis as a sensory organ for gamete RNA engineering
- try to squirt in epididymesomes into oocytes.
consequences
- knockdown GlyC tRNA, see strong overexpresseion of ~30 genes.
- genes that are de-repressed in knockdown are repressed in low protein sperm.
- All 25 genes are regulated by MERVL, an endogenous retro-element
- MERVL uses tRNA leucine (not Gly) for its replication. odd.
- turned on in 2 cell totipotent stage. Turns off in 4 cell stage and back on in 8 cell stage.
- in an ES cell colony, 1:100 cells is oct4 negative and is MERVL positive, cells cycle in and out of this state.
- FACS these cells and implent. MERVL negative cells are pluripotent (can’t make extra-embryonic tissues)
- MERVL cells are totipotent, also make placenta.
- phenotypes seen are similar to uterine implantation phenotypes. Possible model:
- tRNAs affect implantation and placenta vs embryo growth?