Molecular regulation of the niche Flashcards
What are the stem cell signatures?
- stem cell signatures:
- transcriptome
- epigenome
- proteome
Why is the Waddington’s epigenetic landscape presented as valleys?
As the cell differentiates it needs to make a series of decisions about which lineage to follow. The reason it presented as valleys is because usually it is a one way system and there would have to be a lot of energy put in to going back up (dedifferentiation)
What are epigenetic changes?
DNA and chromatin modifications that do not alter the primary nucleotide sequence
Give an example of an epigenetic change and how it occurs
- DNA can be methylated on cytosine residues by methyl transferases (Dnmt1,2,3)
- Dnmt3a and b de novo methylation while Dnmt1 works for maintnance of methyltransferase during DNA replication
- reversible methylation of the 5 position of cytosine by methyltransferases
- In mammals mainly occurs at CpG sites - CpG islands
How does methylation regulate gene expression?
- Methylation important as it can repress gene expression in 2 ways:
- orevents the binding of transcritpion factors
- modifies chromatin structure to repress transcription
What can abnormal methylation do?
Abnormal methylation silences tumour suppressor genes
What does methylation do to the DNA structure?
Methylated DNA forms compacted chromatin and so is less accessible for recombination and translocation
What is the effect of Dnmt knockouts in mice?
mouse ES cells express high levels of Dnmt3a and Dnmt3b, double knockout ES cells of both de novo methyltransferases continue self-renewal. However, these ES cells lose DNA methylation during long-term culture and then show a higher incidence of spontaneous differentiation toward trophectoderm than wild-type ES cells. Triple knockout ES cells lacking all three DNA methyltransferases are still viable, indicating that DNA methylation is not required for ES cell self-renewal.
Describe MeDIP-Seq
- Identifying DNA methylation MeDIP-Seq:
- Methylated DNA immunoprecipitation
- DNA of interest (disease and normal) is fragmented by sonication and denatured
- To separate the methylated from non-methylated DNA an antibody used that binds to the methylated - 5-methylcytosine (5mC)
- Separated by immunoprecipitation
- Isolated fraction (the methylated regions) are sequenced using a next generation sequencing platform
- Sequences mapped back onto genome to identify methylated regions
What happens to methylation during cell division?
DNA methylation patterns are inherited through cell division
Where can you find CpG islands?
CpG islands surround many genes - they are unmethylated in housekeeping genes; they are methylated in silenced genes
What histone modifications can you have?
- Histone modifications
- covalent modifications made by multiple ensymes and associated with heterochromatine and euchromatin
- acetylation, methylation phosphorylation and ubiquitination are the most weel understood histone modifications
How does bisulphite sequencing work?
- Samples treated with bisulphite
- Converts cytosine to uracil but 5-methylcytosine unaffected
- Treated samples sequenced and compared to determine methylation e.g. cancer and normal cells
- Targeted regions or whole genome approach (WGBS)
- Higher cost but greater resolution
Which genes in which methylation state are poised to be expressed?
- unmethylated genes with euchromatic histone patterns are poised to be expressed
- unmethylated genes with heterochromatic histone patterns are poised to be silenced
- methylated genes have heterochromatic histone patterns and are silenced
What is chromatin remodellling?
Chromatin remodeling is involved in the regulation of proper transcriptional activation at the promoter region by altering the histone-DNA contact.
What genes are found in stem cells?
- cell cycle inhibitors (p16,p18, p21, p17(
- chromatin modelling genes (to maintain chromatin in an open state)
- telomerase - very importatn to maintain the integrity of the genome
- DNA repair genes
Why do progenitor cells not respond as strongly to stimuli compared to HSCs?
in progenitor cells some genes can be more methylated so although the same amount of stimuli comes to them,and the number of receptors is the same they might not respond as strongly
What is the role of HoxB4 i HSCs?
- HSC have a lot of Hoxb4 and high expression of it is important for self renewal
- Hox transcription factors have emerged as important regulators of primitive hematopoietic cell proliferation and differentiation
- In particular, HOXB4 appears to be a strong positive regulator of hematopoietic stem cell (HSC) self-renewal
What is single cell transcriptomics useful for?
single cell transcriptomics - for comparison of cells and also allows us subdivision of cells we previously thought belonged to one cluster
How do you plot cells in pseudotime?
you harvest cells from the tissue in one go and then when you sort them in a particular way you can predict how they would develop in pseudo time (it is not real time but you can see what is possible to happen based on your data); if we know how the tissue is organised we could talk about pseudospace
What is the significance of proteome in SC?
transcription factors regulate gene expression and each other and can determine cell fate
What can deletion or overexpression of certain transcription facotrs lead to?
Deletion/overexpression of certain transcription factors leads to:
- deletion/expression of specific cell lineages
- reduction/increase of stem cell self renewal
How do post translational modifications affect cells?
post translational modification and protein levels affect a cell’s response to stimulus.
Why is studying proteomics of SC difficult?
It used to be very difficult to study proteomics of stem cells as you need quite a bit of protein to do it and stem cells are rare but with the improvements in mass spec we can study more and more of stem cells
What are some cell exrtrinsic ways to regulate stem cells?
- signalling pathways
- transmit signals inside stem and differentiating cells
- signals known to be important to stem cells:
- cytokines
- growth factors
- hormones
- cell-cell junction
- cell-matrix junction
