Stem cells epi

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  • Created by: Sarah
  • Created on: 29-05-19 09:28
what are polycomb and trithorax proteins important for?
initiating the
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what are stem cells?
undifferentiated poliferation-competent cells capable of both self-renewing and differentiating into multiple specialised cell types
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what is developmental potential?
the capacity of fetal, embryonic or adult cells to self-renew and/or commit to distinct programmes of cell differentiation
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what is the 3 levels of developmental capacity?
totipotent, pluripotent and multipotent
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what is totipotent?
fertilised egg- it can give rise to all embryonic and extra-embryonic cell types
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what are pluripotent?
embryonic stem cells
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why are embryonic stem cells pluripotent?
can self-renew and give rise to all definitive embryonic cell types
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what is multipotent?
haemopoietic stem cells
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why are haemopoietic SCs multipotent?
they can self-renew and give rise to all haemopoietic cell types
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where do pluripotent ESCs arise?
in the inner cell mass of the preimplantation mouse embryo = the blastocyst
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what precedes the establishment of the pluripotent ground state in the mouse preimplantation blastocyst?
a burst of genome wide demethylation
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why do you have this burst of genome wide demethylation after establishing pluripotency?
to erase epigenetic marks present in gamete genomes (sperm and eggs)
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what follows genome wide demethylation?
a process of de novo DNA methylation
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when does de novo DNA methylation occur?
during implantation and formation of the epiblast
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what is the epiblast?
the future embryo- the outermost layer of the embryo before it differentiates into mesoderm and ectoderm
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what exhibits progressively reduced levels of methylation in the pre-implantation blastocyst?
gamete-specific Differentially Methylated Regions (DMRs)
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what does the re methylation in the zygote include?
re-establishment of sperm-contributed DMRs
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what was this evidence for? so like why do we have the whole global DNA demethylation in the preimplantation blastocyst?
global DNA demethylation in the preimplantation blastocyst removes epigenetic barriers to the acquisition of pluripotency
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when is de novo methylation?
during implantation and formation of the epiblast
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what introduces the de novo DNA methylation patterns?
DNA methyltransferases
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what are the specific DNA methyltransferases intorducing the de novo methylation?
DNMT3A and DNMT3B
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what is maintenance of the DNA methylation patterns in the dividing cell populations to persist long term done by?
DNA methyltransferase DNMT1
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what does DNMT1 do?
in DNA replication the strands get separated out to make a new strand from it- this new strand ins unmethylated so DNMT1 methylates the new strands
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what happens in the absence of maintenance methylation?
in proliferating cells DNA methylation patterns are lost from the epigenome
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what happens to DNA methylation patterns if maintenance methyltransferases are inhibited or lost?
DNA methylations can be diluted or lost passively as after repeated rounds of DNA replication the partner strand won't get methylated so it will be lost
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what enzymes can actively and specifically remove DNA methylations from the genome?
TET enzymes
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where does the pluripotent stem cell phenotype arise?
pluripotent stem cell arise in the preimplantantion embryo (ICM)
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when are embryonic SCs usually established like what days is the preimplantation embryo?
E3-4.5
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as the embryo implants (and undergoes de novo DNA meth) the postimplantation embryo the definitive embryonic structures form what? what day?
epiblast forms the egg cylinder postimplantation around E5.5
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when is the pluripotent ground state established?
when embryonic SCs arise int he ICM and persist in the epiblast of the post implantation embryo
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what happens as the preimplantantion embryo is created?
genome wide demethylation
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when does re-methylation occur?
after the embryo implants (postimplantation) and begins to develop into an egg cylinder with an epiblast
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what occurs as the preimplantation blastocyst forms to the postimplantation blastocyst?
wave of genome demethylation followed by a wave of re-methylation
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why do you get this?
erase the epigenomic imprints of the egg and sperm
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preimplantation what happens?
de methylation
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postimplantation what happens?
re-methylation
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what is the difference between the egg and sperm?
the oocyte they start off highly methylated whereas in the sperm there's no methyation
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what happens to these DMR regions in the egg genome?
about 50% of them become demethylated, you get a progressive loss of methylation at the egg derived DMR as development proceeds from 2 to 4 cell stage to ICM
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what happens to sperm DMRs?
sites unmethylated and then fully methylated and then as development proceeds you see demethylation up until implntation
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what happens to sperm DMRs at implantation?
you see an increase in the methylation of these sperm contributed methylation sites
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what is the embryo re-acquiring?
the embryo reacquires a set of methylation marks that were present in the sperm but it applies them to the sperm derived genome AND to the oocyte derived genome so both halves of the zygotic genome become re-methylated to acquire this pattern
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what happens in early embryogenesis to the methylome?
its dynamically reprogrammed so it's erased and then re-established
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when DMNT3A+3B do de novo methylation what are they doing?
take an unmethylated CpG and de novo methylation or apply methyl mark to both cytosines in tha cytosine dinculeotide
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what does DNMT1 recognise why?
hemi-methylated DNA because it maintains the methylation marks in cell replication so it recognises the daughter strand and puts marks on that
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what happens if maintenance methytransferases are lost?
then you lost the methylation of the genome it's diluted out and actively lost during DNA replication
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is the wave of demethylation across the embryo active or passive?
active- done by TET enzymes
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how do TET enzymes remove DNA methylation marks?
TET enzymes create hydroxyl-methyl cytosine -> additional steps -> full removal of the nucleotide to create a gap in the DNA -> gap repaired by base exicision repair process -> repairs it back to cytosine
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how do you know when there is active demethylation going on in cells when collect a piece of DNA?
if there is hydroxyl-methyl-cytosine- its being actively demthylated by TET enzymes
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what does the mosue blastocyst give rise to?
1) epiblast (pluripotent embryonic stem cells) 2) trophoectoderm (extraembryonic tissue)
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what is there a progressive loss of as embryonic stems give rise to all the lineages?
developmental potential
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what has a greater developmental potential the epiblast or the egg cylinder?
the epiblast
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what is Waddington's epigenetic landscape?
the idea that cells take a journey through an epigenetic landscape where they go from being highly totipotent to lineage restricted and terminally differentiated
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cell populations derived from the mammalian preimplantation blastocyst or post-implantation epiblast can be maintained as what?
in vitro can be maintained indefinitely as self-renewing pluripotent EX=SCS
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in the pre-implantation blastocyst what can the ICM give rise to?
embryonic stem cells
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what is the 2 different culture forms embryonic stem cells can be maintained as
2i culture serum-culture
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what does maintaining pluripotent ESCs in these 2 different cultures allow?
allows them to contribute to all the lineages of the developing embryo- both definitive embryonci lineages and the extraembryonic tissues
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what are epiblast derived SC like cells?
PLURIPOTENT embryonic SCs from egg cylinder PS stage embryo and put them into culutre then reintroduce them back into an embryo developmental potential is reduced they can only germ layers (mesoderm, endoderm, ectoderm) NOT extraembryonic
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what is pluripotency?
capacity to give rise to all differentiated cell types (ectoderm, mesoderm, endoderm, germline) when transplanted into a host embryo- cells from postimplantation epiblast are pluripotent
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what are the 2 distinct types of ESCs?
1) naive ESCs 2) Primed ESCs
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what do naive ESCs define?
the pluripotent ground state
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what can naive ESCs be culutred in?
serum cultured ESCs or 2i-cultured ESCs
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what are 2i-cultured ESCs?
derived from E3.5-4.5 preimplantation blastocyts cultured in medium containing FGF/ERK inhibitor PD03 and GSK3 inhibitor chiron
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why is is called 2i?
2 inhibitors
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what inhibitors are in 2i-culture?
GSK3 inhibitor (CHIRON) and FGF/Erk inhibitor PD03
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what are the serum-cultured ESCs?
derived from E3.5-4.5 preimplantation blastocyts cultured in serum-containing medium
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what are the 2 types of naive ESCs?
2i cultured or serum-cultured
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what are the 2 types of primed ESCs?
epiblast stem cells or epiblast-like cells/ epiblast SC like cells
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what can both naive and primed ESCs do in vitro?
self-renew and differentiate
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what do naive ESCs function as when transplanted into host blastocyts? difference with primed ESCs
naive ESC function as pluripotent SCs (all cell types) whereas primed ESCs are much less capable of doing this
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why are naive cells cultured in 2i medium containing inhibition of FGF and Wnt/GSK3 signalling?
2i medium promotes global progressive DNA demethylation and a transient increase in hydroyxymethylation
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what does the increase in transient hydroxymethylation suggest?
that it's being actively demethylated by TET enzymes
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what culture gives DNA demethylation?
2i culture
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what does the serum culture of naive ESCs do?
facilitates expression of pluripotency genes such as Nanog
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what are the ESCs derived from the preimplantation embryo known as? what do they define?
naive ESCs- define the pluripotent ground state
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where are ESCs derived from the post-implantation embryo known as?
primed ESCs- have a reduced level of pluripotency compared to naive
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what can both primed and naive ESCs do? what can only the naive do?
they can self-renew and differentiate in culture but only naive ESCs when they're reintroduced into host blastocysts function as pluripotent
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inhibitors in 2i culutre?
FGF signalling and GSK inhibitor (inhibits wnt signalling)
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what is the optimum way of maintaining naive ESCs?
in 2i-culture
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if you maintain naive ESCs in serum or 2i culture what do you see happen to the DNA methylation patterns?
the DNA demethylation is maintained over days- have a reduced level of methylation so they're actively demethylating their genome as hydroxylmethyly cytosine is transiently increased
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what is hydroxyl-methyl cytosine rise telling us?
its active loss of methylation by TET enzymes
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what gene did they find here that is a pluripotency gene involved in the establishment and maintenance of the pluripotent state?
nanog
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what is the difference between the nanog gene methylation in 2i and medium cultured SCs?
culture medium- lots of methylation across the genome, in 2i culture profound demethylation
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what does demethylation in serum culture of the nanog gene promote?
expression of pluripotency genes like nanog
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what is demethylation of specific CpGs in 2i-cultured naive ESCS dependent on and accompanied by?
TET2 demethylase and accompanied with downregulation of DNMTs
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what is a DNMT?
DNA methyltransferase- so you downregulate the thing making the methyl makrs and you upreg TET2 which takes them off = overall demethylation
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what do you see at D1, D3 and D4 individual CpGs in the genome in cells that are maintained in 2i culture for progressively long periods of time?
levels of 5-methyl cytosine drops right down (demethylated) and the levels of hydroxyl-methy-cytosine increase as they are being actively demethylated by TET2 and then this drops because hydroxyl-methyl-cytosine is an intermediate of demethylation
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simplified version of D1, D3 and D4?
decrease in methylated cytosines, transient increase in hydroxyl-methyl-cytosine that then falls
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what happens to these CpG sites in the TET mutant?
the sites are not as well demethylated - the loss of methylation is lower so most methylation is preserved
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how is the fact that methylation stays shown?
there's less hydroxy-methyl-cytosine
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do all locuses require TET2 demethylation (particularly D3 doesn't)?
no some CpGs could be demethylated without TET2 function- as there's other TET enzymes
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how do D1 + D4 behave?
it's been demethylate din WT 2i-culutre but in TET2 KO that demethylation is less profound
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what does this tell us?
demethylation is not a passive process it required the TET2 enzyme
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what is the TET2 accompanied by?
downregulation of de novo methylation methytransferases- DNMT3A+DNMT3B
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what did a WB show of DNMT3B in 2i medium for long period of times?
reduction of DNMT3B (at the same time you get active demethylation by TET enzymes)
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why would you get downregulation of DNMTT3B+3A when TET enzymes are demethylating?
so that the methylases don't counteract the work of the TET enzymes = ensures the genome is fully demethylated and rendered permissive for re-methylation
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what do naive ESC resemble?
ICM/epiblast cells from pre-implantation embryos
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what do primed epiblast like SCs resemble?
post implantation ESCs
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what did a transcriptomic analysis of changes in gene expression occuring in the transformation from the pre to post implantation embryo show?
group of genes that are expressed more abundantly than almost any other genes in both naive and primed ESCs
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what were these genes?
Oct4, Sox2 and Nanog
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What gene had greater expression in epiblast like cells than in ESCs?
Otx2
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what are Oct4, Nanog and Sox2 known to be involved with?
the creation of pluripotency
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what is the group of Oct4, and Sox2 known as?
OSKM group (Nanog isn't part of this group)
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what did forced expression of pioneer factors Oct4, Sox2 and Klf3 and myc in fibroblasts do?
reverses cell differentation in vitro yielding induced pluriptent SCs
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what did Yamanaka show?
If you infect fibroblasts with viruses containing these pioneer TFs- Sox2, Oct4, KLF4 and C-myc it converted them into pluripotent SCs if they were introduced into host blastocysts gould give rise to all somatic cell types+ some germline
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what are OSKM pioneer TFs powerful regulators of?
phenotypic plasticity (they can reverse differentation of fibroblasts)
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what is required to maintain pluripotency in embryos and ESCs (epiblast like and ESC)?
Oct4, Sox2 and Nanog
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how did they know Oct4, Sox2 and Nanog was required to maintain pluripotency?
they did Nanog and Oct4 KO ESCs- did qPCR of gene expression of cells
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what genes did they look at?
genes that are markers of differentation
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what happened with the Nanog and Oct4 KOs?
mutant ESCs begin to spontaneously differentiate into various types of endoderm
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what happens to the blastocysts in the Nanog KO?
you can form blastocysts but they can't implant
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what did they see when they KO Sox2 in ESCs?
progressive increase in expression of differentiated trophoectoderm markers so extraembyronic lineages
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what drops off in the KOs?
expression of pluripotency markers
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what do Oct4, Sox2 and Nanog recruit?
histone acetyltransferases - P300/CBP to their target genes/enhancers
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what does Oct4, Sox2 and Nanog recruiting histone acetyltrasnferases to their genes do?
transcriptionally activate pluripotency genes (Jarid2)
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what do Oct4, Sox2 and Nanog encode?
protieins that bind to cis regulatory sequences in pluripotency maintenance genes like Jarid 2 and they recruit an enzyme called P300 which is a HAT
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what does P300 HAT target?
lyisne 27 of histone H3 (H3K27)
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what is closely related to p300?
creb binding protein- another HAT- they have near-identical domain structures. P300 and CBP function interchangeably
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what do both P300 and CBP have?
a histone acetyltransferase domain that carries out the histone acetylation and a bromodomain which recognises acetylated histones
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what does having the HAT enzyme and a bromodomain allow P300 and CBP to do?
bind to pre-existing acetylation marks and make more in the vinicity then bind to them and make more so creates focus of histone acetylation
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what is recruited to pluripotency trarget genes in the naive ground state (Serum or 2i)?
Oct4, Sox2 and Nanog (pioneer TFs) along with Histone Acetyltransferases P300
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what do these HATs attach to the pre-existing acetylation marks with?
their bromodomain
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what else do these pluripotency genes contain?
histone lysine 4 methyl domains as well as PHD fingers
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why is it a critical function of the OSKM TFs to recruit HAT?
to sustain transcription of pluripotency genes by promoting their histone acetylation
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what do you see if you do a chIP seq analysis of a whole range of pluripotency maintenance genes and look at their promoters?
see peak of Oct4, Sox2 and Nanog and P300 binding
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what does the Oct4, Sox2 and Nanog and P300 complex do?
sits on pluripotency determing genes and promotes their transcription so each one colocalises peaks of these TFs and P300 binding
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what do a class of developmental regulatory gene promoter exhibit?
bivalent H3 histone modifications in ESCs
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what are these genes doing?
they are poised for transcription but are inactive
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what is the fact that they are poised for transcription but inactive a characterstic feature of
characteristic feature of the pluripotent state
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what are the bivalent modifications at the poised regulatory gene promoters locus?
H3K4me3 and H3K27me3- lysine 4 and 27 trimethylation on histone H3
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What does H3K4me3 do?
induction of neural differentation leads to transcription of neural fate genes
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neural fate genes?
Nkx2.2 and Sox21
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what does H3K27 methylation do?
induction of neural differentation leads to repression of non-neural genes
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non-neural genes incluse?
Pax5, Evx1
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these developental regulatory genes that are poised activation must be prevented in order for what?
pluripotency to be maintained- they are differentation and commitment genes so once activated ESCs leave the pluripotent state and start differenitiating
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what does the developental regulatory genes that are poised H3 contain?
methylated lysine 4 and lysine 27 on histone H3
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what is methylation of H3K27 carried out by? what's it associated with?
polycomb protein = enhancer of zest- associated with transcriptional repression
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what is H3K27 methylation associated with?
transcriptional repression
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what is H3K27 methylation associated with?
transcriptional activation
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where do you only find this bivalent modification of histone H3 (methylation of H3K27 and H3K4)?
In ESCs
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a bunch of neural differentiating genes showed what when they looke dat the methylation patterns of their promoters?
they are rich in H3K4 and H3K27 methylation- bivalent histone modification
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what happened when they differentiated these ESCs into neural cells and re-assess the levels of methylation?
some of them retain the H3K4 methylation mark and some retain the H3K27 methylation mark- but all removed one of the 2 marks
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what is lost when they differentiate into neural?
bivalency they become monovalently methylated
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what are genes that retain the H3K4 methylation marks transcribed in?
neuronal precurors- genes inactive in the neural precursors carry the H3K27 methylation mark
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why does the gene expression of these genes shown to be silent in ESCs?
they are covalently methylated with H3K4 and H3K27 methylation in ESCs.
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when differentiation is triggered to neural genes they express neural products of differentiation, what do the neural cell fate determinant genes retain?
the H3K4 methylation mark
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what genes retain the H3K27 methylation mark?
az
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where are the bivalent modifications detectable?
around the transcription start site (TSS)
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in what genes around the TSS are bivalent modifications in?
are in the subset of genes that are inactive but poised for the initiation at the TSS, the genes are inboth serum and 2i treated ESCs
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what are they accompanied by?
RNA polymerase II to start transcription when the time comes for differentiation
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the genes that have the bivalent mod of H3K27 and H3K4 methylation are silent and enriched in these but what are they associated with?
RNA pol 2 so these genes are all poised waiting to be induced to differentiate
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Bivalent Histone Modifications are detectable around the transcription start site (TSS) of genes that are transcriptionally what?
transcriptionally inactive but required for lineage differentation
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are the bivalent mods in serum cultured or 2i cultured SCs?
both
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The mammalian ortholog of Drosophila Trithorax, MIL2 is required for what?
H3K4 methylation
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what does trithorax do?
methylate H3K4
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where can trithorax MIL2 (mammalian trithorax) methylate H3K4?
at bivalent promoters in pluripotent ESCs
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H3K4 methylation required the activity of what?
trithorax protein MIL2
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what did they see in WT and KO MIL2?
MGL1 and DLX1 in WT have a strong peak of H3K4 methylation, MIL2 KO- methylation completely abolished
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what happened with the MIL1 KO?
the H3K4 methylation was unaffected- is specific to MIL2
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what does this tell us?
MIL2 is required for H3K4 methylation at bivalent promoters in pluripotent ESCs
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what happened to H3K27 methylation in MIL1 or MIL2 KOs?
was unaffected still get H3K27 methylation
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what does PCR2 (polycomb repressive complex 2) catalyse?
H3K27 methylation in ESCs
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how did they show PCR2 catalysed H3K27 methylation in ESCs>
western blot when they KO components of PCR2 like EED or SUZ then there was no trimethylation and less dimethylation of H3K27
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what did this tell us?
histone lysine 27 methylation requires the function of the polycomb repressive complex
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what does the H3K27 mark require?
Polycomb proteins- PCR2
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what does the H3K4 mark require?
trithorax function
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what is PCR2 required for in ESCs?
to maintain expression of pluripotency TFs in ESCs, to create the bivalency on genes when activated engage in commitment but are poised in inactivity
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how did they know PCR2 is required to maintain expression of pluripotency TFs in ESCs?
PCR2 KO lost expression of pioneer TFs
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what does PRC2 do?
PRC2 methylates H3K27
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what does PRC1 do?
PRC1 binds to H3K27me3
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what component of PRC2 creates H3K27me3?
Ez/EXH2
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what does uts partner Esc/EED do?
binds to H327me3 modifcations
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what does EXH2 and Eed working together enable?
create and bind H3K27me3 marks enabling local propagation of H3K27 in chromatin
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what does loss of PRC2 function cause to happen to ESC differentation?
causes spontaneous differentiation of ESCs into meso-endodermal tissue
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what is Polycomb proteins functionally antagonistic with?
trithorax proteins
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how does polycomb and trithroax proteins being functionally antagonistic work in the differentaiton of ESCs (MIL2 pathway)?
MIL2 methylates H3K4 -> CBP binds to H3Kme4 via its PHD finger -> CBP acetylates H3K27 allowing bromodomain binding but PRC2 methylates H3K27 prevents H3K27ac recog by bromodomain
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what does Bivalent H3K27 and H3K4 methylation mark?
a set of genes in pluripotent cells that are subject to lineage-specific transcriptional activation during cell division
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in ESCs what happens to bivalent genes?
they remain transcriptionally silent
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what does induction of differentation do to bivalent genes?
breaks the sulence by activating them
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what does loss of PRC2 function do?
promotes differentiation by enabling activation of these differentiation genes
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what activates the Oct4 sox2 Nanog (OSN genes)?
FGF/GSK3 signalling inhibition and TET demthylation of them
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what turns on pluripotency genes?
P300/CBP histone acetylation and PRC2
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what are pluripotency genes inhibiting?
bivalently modified lineage specification and cell differentiation genes from being expressed
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what helps the bivalent genes become turned on to activate differentation?
there silence is broken by MIL2 and CBP acetylating them and H3K27 methylation marks being removed
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