Epigenetic regulation of stem cell developmental potential Flashcards
What are stem cells?
Undifferentiated proliferation-competent cells capable of both self-renewing and differentiating into multiple specialised cell-types
What is developmental potential?
The capacity for embryonic, foetal or adult stem cells to self-renew and/or commit to distinct programmes of cell differentiation
What are the 3 types of stem cell developmental potential?
- Totipotency
- Pluripotency
- Multipotency
What is the potency of a fertilised egg? (2)
- Totipotent
- Gives rise to all embryonic and extra-embryonic cell types
What is the potency of embryonic stem cells? (2)
- Pluripotent
- Can self renew and give rise to all definitive embryonic cell types
What is the potency of adult stem cells e.g. haematopoietic stem cells? (2)
- Multipotent
- Can self-renew and give rise to all haematopoietic cell types
Where are pluripotent embryonic stem cells found? (2)
- Inner Cell Mass of preimplantation stage embryo (blastocyst)
- Gives rise to germ layers (endoderm, ectoderm, mesoderm)
What happens to CpG methylation upon zygote formation? (3)
- Genome wide active CpG demethylation to erase epigenetic marks from gamete genomes
- Persists into early preimplantation stages of development
- Followed by de novo DNA methylation
What are DMRs? (2)
- Differentially methylated regions
- Genomic regions with different methylation statuses among multiple samples (tissues, cells, individuals etc.)
What are the gamete-specific differences seen in de novo methylation? (3)
- Oocyte contributed DMRs decrease and remain low/decline further
- Sperm contributed DMRs decrease and then a subset are re-established during re-methylation
- This is evidence that global DNA demethylation in the preimplantation blastocyst removes epigenetic barriers to acquisition of pluripotency
What are the 2 types of enzymes involved in re-methylation?
- De novo methylation done by DNA methyltransferases DNMT3A and DNMT3B
- Long term persistence of methylation in dividing cell populations requires maintenance DNA methyltransferase DNMT1
What happens to DNA methylation in dividing cells? (3)
- Fully methylated DNA becomes hemi-methylated after DNA replication and cell division
- DNMT1 converts hemi into fully methylated DNA (maintenance)
- Methylation modifications would be diluted/lost passively in the absence of maintenance methyltransferases in proliferating cells
How does active demethylation occur? (3)
- TET demethylases do a series of oxidation reactions on 5-methylcytosine
- Forms an intermediate recognised by thymine deglycosylase which removes the base from the DNA
- Base excision repair (BER) process restores the double stranded DNA by replacing with a normal cytosine
What does the blastocyst give rise to? (2)
- Epiblast (pluripotent embryonic cells)
- Trophectoderm (differentiating extraembryonic tissue)
What is the difference between day 6 and day 7 mouse blastocyst? (2)
- Day 6 epiblast cells are pluripotent, best time to derive pluripotent stem cells
- Day 7 egg cylinder cells are multipotent ectoderm, mesoderm and endoderm layers
How do you get embryonic stem cells? (2)
- Take cells from epiblast of preimplantation blastocyst and can maintain in 2i culture indefinitely
- Get self-renewing pluripotent embryonic stem cells (ESCs)
What is the definition of pluripotency?
Cells have the capacity to give rise to all differentiated cell types (ectoderm, mesoderm, endoderm, germline) when experimentally transplanted into a host embryo
How are embryonic stem cells maintained in a state of pluripotency in vitro? (3)
- Inhibition of FGF signalling via MEK inhibition (downstream of FGF)
- Inhibition of GSK3 which is a negative regulator of wnt signalling
- Results in inhibition of FGF signalling and potentiation of wnt signalling
What are the OSN transcription factors? (2)
- Oct4, Sox2 and Nanog
- Required in ESCs to maintain pluripotency
What is the difference in ESCs in 2i culture vs serum? (3)
- Demethylation of Nanog in 2i culture compared to in serum
- Better for maintaining pluripotency because demethylation allows expression of pluripotency genes such as Nanog
- Presence of hydroxymethylcytosine in 2i culture ESCs is proof of active demethylation to maintain pluripotency
What is the effect of forced expression of the OSKM group in fibroblasts?
Reverses differentiation and makes them induced pluripotent stem cells (iPSCs)
What is the OSKM group? (2)
- Pioneer transcription factors Oct4, Sox2, Klf4, Myc which are powerful regulators of phenotypic plasticity
- Includes OSN factors Oct4 and Sox2
How do the OSN factors maintain pluripotency in ESCs? (3)
- Oct4, Sox2, Nanog form a complex at genes that are required to maintain pluripotency/undifferentiated cell state e.g. Jarid2 in ESCs
- Recruit histone acetyltransferase p300/CBP which performs H3K27ac modifications to promote gene expression
- H3K27ac prevents H3K27me by PRC2 at pluripotency genes
Which residues do histone acetyltransferases target? (2)
- Non-specific so will add acetyl groups to multiple lysines in the N-terminal tails of ALL core histones at transcriptionally active genes
- H3K27 (methylation target of E(z)) acetylation by p300/CBP prevents H3K27 methylation
