Saturday, April 27, 2013

Two more paper sumups: Germline Stem Cells

Eun S H, Gan Q, and X Chen.  "Epigenetic Regulation of Germ Cell Differentiation."  COCeBi 22, 2010: 1-7.

Epigenetics modify chromatin w/o affecting DNA sequence.  There are proteins who "write" on the modifications, "read" and act on the modifications, or "erase" the modifications.

Germ cells are "immortal" b/c they produce the next generation.  Epigenetics in germ cells might be involved in
1) meiosis and terminal differentiation
2) keeping the genome information good
3) erasing bad information

GSCs (germline stem cells) initiate gametogenesis.  Their chromatin structure maintains self-renewing and blocks differentiation.  Evidence: ISWI "Imitation Switch" is a chromatin remodeling factor.  it is essential for preventing differentiation (in mutants for iswi, the bag gene is expressed and induces differentiation).  One example is the NURF complex that contains the ISWI factor in male drosophila.

histone modifications also functions in maintaining stem-cellness.  Evidence: scny gene removes ubiquitin from histone H2B.  scny mutant leads to super acetylated H3 and super ubiquitinated U2B => open or loose chromatin -> active transcription of differentiation genes.

also, this chromatin landscape isn't permanently blocked from differentiation but described as "poised" to start differentiation upon stimuli.  in mammalian: have both activating H3K4methyl3 and repressive H3K27methyl3 going on and a paused RNA Pol II.  it prevents DNA methylation, which is a more stable block and would be difficult to remove for differentiation to happen at a snap.  in drosophila: not bivalent (i.e. not an active and a repressive histone modification at the same time) and not associated with paused RNA Pol II, but probably b/c drosophila doesn't have methylase anyway.

only germ cells do meiosis.  with meiosis, chromatin regulators and opposite-fxn histone modifying enzymes are downregulated.  at same time, celltype specific factors are unregulated (i.e. the tTAFs testis-specific TBP-associated facotrs in drosophila spermatocytes)

same/similar-fxn histone modifying enzymes work together though, maybe separated by time (i.e. in drosophila ovary, one isoform is in early germ cells and GSCs while the similar isoform is needed in later stage germ cells).  but in mice, meiosis was found to have both functional enzymes that are opposite fxn.

also during mammalian gametogenesis is when imprinting (DNA methylation) happens.  This might happen via a sort of weird transcription process that goes over modified genes in order to "read" and match it to the new DNA strands.  This happens while oocytes are arrested in meiosis but we're not sure about spermatocytes.  More research needs to be done.

in spermiogenesis in drosophila and mammals, histones are kicked out and replace with Tnps and Prms to super condense the DNA in super nuclei.  This process is also regulated by epigenetics.  Some examples include:
1) H4 S1A substitution/mutation messes up phosphorylation of H4S1 and results in failed sporulation in yeast
2) demethylation of H3K9me2/1 is necessary to increase expression of the Tnps and Prms in mice.
3) (see more in the paragraph)

in human sperm, about 4% of the genome still has regular histone nucleosomes, no Tnps or Prms or anything.  Specially, the Hox genes and imprinted genes that are necessary for early embryonic development.  but things like Nanog that are necessary for ESC pluripotency are off.

after fertilization, Prms get kicked out and given maternal histones.  some chromatin regulating proteins are involved in helping out.  the cells that will be the germ cells for this zygote's babies are the PGCs (primordial germ cells).  They get a genome-wide erasure of DNA methylation so as to get rid of any epimutations that happened between their ancestor's PGCs and this fertilization.

Proper histone modification control takes care of differentiation genes and fertility, but they also have been observed to ensure proper lifespan.

During the journey of germ cells, they need to maintain a correct epigenome, which is ensured by dynamic control, erasure, and re-establishment of the epigenome.  More needs to be done in the field, except it is hard to get a good amount of these GCs and PGCs (ethics!) so if we can has new technology that don't need as many cells, we will be good to go.  Also this will be useful for diseases due to germ cell differentiation fail (infertility, germ cell tumors, etc) but also regenerative medicine.

Tran V, Lim C, Xie J, and X Chen.  "Asymmetric Division of Drosophila Male Germline Stem Cell Shows Asymmetric Histone Distribution."  Science 338: 2012: 679-682.

question: stem cell activity is regulated by epigenetic, but do stem cells retain their epigenetic info?

male drosophila GSCs have been well characterizes and are easily identified.  they divide asymmetrically and can be examined at the single cell level.

histones are major carriers of the epigenome.  we created this switchable dual-color method to label and identify what is a new histone versus and old histone.  can be controlled spatially or temporally (by heat shock!).  labeled histones can get switched from green to red.

how? heat shock causes the recombinant gene to irreversibly shut down GFP-labeled old histone expression and initiate expression of new mKO-labeled histones.  if old histones are distributed btw daughter cells equally, over time any green color will be replaced by red color.  if old histones are kept in the GSC and new histones are kept in the daughter fated cell, then green will be specifically retained.

before heat shock: testes with transgenes showed green nucleus but almost no red.  after heat shock, red started appearing.  of all GSCs, about 70% of them will be in G2 phase, 21% in s phase, and <2% in mitosis.  entire cycle length is 16 hrs.  plus the two daughters will be connected after incomplete cytokinesis and are called "spectrosomes."  examine testes 16-20 hrs after heat shock, specifically the spectrosomes.

results: green preferentially held in daughter GSC, while red was preferentially delivered to fated daughter cell.  in contrast, for symmetrically dividing GSCs, both daughters had equal red and green.  also in contrast, this was observed with canonical H3 transgene, but not with variant H3.3 transgene.  also looking at during mitosis, anaphase pulled green chromatids toward future daughter GSC while the spindle pulled red chromatids towards future fated daughter cell.

change it up: ectopic activation of JAK-STAT signaling pathway.  previously shown that this causes overpopulation of GSCs.  they did it and found that the asymmetric green-red distribution didn't happen.  so equal distribution of old and new histones is also associated with this forced symmetrical divisions.

proposed mechanism: old and new histones are incorporated into separate chromatids during S, and are preferentially segregated at the spindle.

this may go towards understanding how epigenetic is maintained in SCs and reset in sibling cells that will differentiate.

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