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.
