Regulation of ES Cells in Culture Flashcards
What is Oct-4?
- A pou domain TF – pou factors promote cell proliferation
- Unique to and universal in pluripotent stem cells
- Not expressed in invertebrates
- First detected in the oocyte and zygote, but expression low until you reach the morula stage
- At the blastocyst stage, it is only present in the ICM
What is meant by Oct-4 dose dependance?
• Oct-4 activity is dose dependent (Hiwa et al, 2000)
− Tetracycline used to turn off Oct-4
− Basal level of Oct-4 maintains pluripotency
− Decrease expression by 50% leads to TE
− Increase expression by 50%, leads to differentiation into the 3 germ layers
- So don’t need 10-fold changes, just subtle changes
- This is probably because this TF works in complexes with Nanog and Sox-2 to maintain pluripotency and also complexes with other factors involved in differentiation.
- Increasing levels of Oct-4 changes the equilibrium between the different molecules present around the chromatin, so you get activation of genes involved in differentiation
How does Oct-4 act in combination with other transcription factors?
• The correct combination of TF expression determines the maintenance of pluripotency or early cell commitment.
• Oct-4 acts as either a repress or an activator of gene transcription depending on its association with co-factors:
− Sox2 and Oct-4 act together on enhancer elements to enhance transcription
− FoxD3 and Oct-4 act at promoters of endoderm gnes to suppress transcription and therefore suppress differentiation.
Why is Oct-4 needed?
Oct-4 is an essential transcription factor for the maintenance of the ICM
• Oct-4 deletion results in formation of only trophectoderm
• Expressed exclusively in blastomeres, pluripotent early embryo cells and germ cell lineages
• Knockout of Oct-4 leaves cells in a transient indeterminate state (Nichols et al, 1998) (supported by the observation that Troma-1 positivity is only expressed by 50% of mutant embryos) but this is rapidly superceded by commitment to trophoectoderm differentiation.
–> Primarily responsible for repression of trophoblast genes.
What is Sox-2?
- HMG-box transcription factor family
- Bends DNA to make available regulatory elements for TF interaction
- Forms a complex with Oct-4 to regulate ES cell self-renewal
Why is Sox-2 needed?
- Knock down of Sox-2 in mouse embryo leads to developmental arrest at the morula
- Knock out of Sox-2 is peri-implantation lethal, with loss of the epiblast
Therefore, Sox-2 is needed for both the maintenance of the ES cell population and trophoblast formation.
What did Nichols et al, 1998 discover about FGF4?
- FGF4 is considered a key Oct-4 target
- Oct-4 mutants have reduced FGF4.
- Sox-2 is normally expressed in a similar pattern to Oct-4 in the ICM
- H19 is normally expressed in trophoblast cells
- In FGF4 induced mutant cells in culture, they do not express Sox-2 however they do express H19 → suggesting they are trophoblastic
- Demonstrates that FGF4 cannot rescue pluripotency in Oct-4 deficient embryos – which is noteworthy considering it is considered a key Oct-4 target.
What is Nanog?
- Divergent homeodomain protein
- Named after Ti Nan Og – the celtic land of the ever young
- Expressed in pluripotent cells
- Expressed first slightly at the morula stage, but greatly increased at the blastocyst.
Why is Nanog needed?
- Nanog knockout → Cells form PrE and trophectoderm (induced by upregulation of Cdx2, Gata4 and Gata6), NOT Epiblast
- Nanog function requires Oct-4 → there is an Oct-4 binding site within Nanogs promoter
- Overexpression of Nanog promotes self-renewal independent of LIF – suggesting Nanog by be downstream of LIF
So seems to be essential for formation of stable ICM.
Nanog null murine ES cells cannot form gametes in chimeras - so Nanog must be essential for gamete formation.
• Nanog seems to delay, rather than block, differentiation – that is, the threshold of differentiation is increased rather than abolished.
–> Primarily responsible for repression of extraembryonic endoderm.
What is the relationship between Oct-4 and Nanog?
- Both Oct-4 and Nanog mRNA are decreased after either Oct-4 RNA or Nanog RNA inhibition
- Gives us the idea that they likely regulate each other
- Removal of Nanog gives 47 fold induction of TE marker hCG, and a 12 fold induction o the endoderm marker hAlbumin.
- Removal of Oct-3 gives 3-4 fold induction of TE markers.
Epigenetic Regulators - what characteristic does ES cell chromatin display?
That of transcriptionally permissive euchromatin.
Epigenetic Regulators - what is lineage specification typified by?
A decrease in acetylation and concominant increase in heterochromatin formation, indicating that restriction of developmental potential is associated in a marked decrease in genome plasticity.
Name some examples of epigenetic changes
- Replacement of histone H1 with a version that binds more tighly to chromatin inhibits ES cell differentiation
- Genetic manipulation of H3 histone can cause accelerated differentiation
- Activating H3K4me3 is present in pluripotent cells
- Inactivating H3K27me3 is present in differentiated cells
Consistent with the idea that the chromatin of pluripotent cells is open, studies have shown that tissue specific genes which are expected to be inactive may be in a semi-permissive transcriptional state in ES cells, eg) active epigenetic markers noted in ES cells at discrete sites within the B cell locus.
Example of miRNA that enhances self-renewal.
miR290 → mediates the stability of pluripotency through depression of Oct-4 inhibitor. Role in germ cell development
Examples of miRNA that enhance differentiation
- miR145 → can cause degredation of the triad
- Let7 → stem cells have low let7, differentiating cells have high. Regulated by lin28 – the triad TFs activate Lin28 to maintain pluripiotency.
