Oliver Rando: I: Structural biology of the yeast genome II: Mechanical disassembly and reassembly of mammalian reproduction

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.


  • Epididymisis as a sensory organ for gamete RNA engineering
  • try to squirt in epididymesomes into oocytes.


  • 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?
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