Molecular regulation of stem cells

Question 1

What is somatic reprogramming

Answer 1

• Take a terminally differentiated cell force the expression of 3-4 TF for pluripotency and reprogram the cell back to ES cell state

Question 2

What are the important classes of TF in ES cells

Answer 2

essential TF that is required to maintain self-renewal (loss-of function) and factors that we can delete immediately which forces cells to express high levels (gain of function)

Question 3

Role of Oct4

Answer 3

• Involved in self-renewal of undifferentiated ES cells
  
  • Gene expression lead to stem cell differentiation
  • Plays role in determining fate of both ICM and ES cells and the ability to maintain pluripotency
  • Marker for undifferentiated cells

Question 4

why is the level of OCt4 closely regulated

Answer 4

too much or too little expression will lead to differentiation

Question 5

Function of LIF

Answer 5

induce terminal differentiation

Question 6

Function of Sox2

Answer 6

○ Essential for maintaining self-renewal or pluripotency of undifferentiated ES cells
○ When deleted causes ES to differentiate

Question 7

How to show that same TF can have different functions

Answer 7

Sox2 important in both ES and TS have ○ Binding sites of SOX2 in ES and TS are very different because each cell type express a different combination of TF
§ SOX2 can form part of two different TF networks

In the Klf family, Klf2,4 play role in reprogramming fibroblasts back to ES cells state but also have roles in barrier function and embryonic

Question 8

How to show that activation of genes depends on where they bind and what this shows

Answer 8

• In endoderm, OCt4 switches binding partner, when in complex with Sox17, it is able to regulate different set of genes
  
  • If we force Es ells to express Sox17, we can force OCt4 to bind to regions of DNA specific for endoderm, places it would not normally binding to ES cells

This also showed that given a cell state is not just the result of the individual TF that are expressed, but rather the exact combination of the TF that are expressed

Question 9

What is Nanog

Answer 9

• TF that helps ES cells maintain pluripotency

- Deletion lead to differentiation of ESCs

Question 10

how to identify TF network

Answer 10

via RNA-sequencing

1. It tell us which transcription factors are expressed and therefore which are the potential candidates for our transcription factor network
2. It Gives us the output of the network, the other genes that the transcription factors are regulate in the cell 

Then use chromatin immunoprecipiation followed by DNA sequencing CHip-seq to figure out where the TF bind within the genome which can be promoters or enhancers

Question 11

How do epigenetic affect gene expression

Answer 11

• Regulate what genes are active or inactive by fine tuning how accessible different regions of the DNA are to transcription factors or RNA polymerase

Question 12

What are the main epigenetics modification

Answer 12

histone positioning and packaging, histone modification, DNA methylation, Topologically associated domains

Question 13

What is histone positioning

Answer 13

• Nucleosome have to be repositioned or removed away from start site by nucleosome remodelling -> GENE TO BECOME ACTIVE
  
  • Genome divided into
    
    • Euchromatin (open DNA)
    • Heterochromatin (DNA is closed and less accessible)

Question 14

What is the histone code

Answer 14

set by histone modifying enzymes of defined specificity and read by non-histone protein that bind in a modification sensitive manner
§ Provides a rich source of epigenetic information

Question 15

what are the different kinds of histone modification

Answer 15

acetylation, deimination, methylation, ubiquitination and phosphorylation

Question 16

What are the 4 types of modification on histone 3

Answer 16

H3K9me3 - Constitutive heterochromatin marked by adding three methyl groups to lysine 9 of histone 3 Modification bound by heterochromatin protein 1 (HP1) -> condense chromatin
H3K27me3 Adding three methyl groups to lysine 27 of histone 3 gene repression
H3K4me3 Promoters marked by tri-methylated lysine 4 on histone 3 Decompaction of chromatin -> gene activation
H3K27ac Acetylated lysine 27 on histone 3 Neutralises the positive charge of lysine residues so the electrostatic attraction between the negatively charged DNA and the histone are reduced

Question 17

role of histone modification in ES cells

Answer 17

In ES cells many developmentally regulated genes- , genes which are not expressed in ES cells but that will become upregulated during differentiation

- Are marked bivalent histone modifications 
	- Promoters of these genes are marked by both h3k27me3 and h3k4me3
	- Genes are therefore to exist in a poised state, ready to be turned on or off depending on which type of cell the ES cell differentiate into

Question 18

What is the role of dna methylation

Answer 18

to silence expression of repetitive DNA elements (eg. Retrotransposons)

Question 19

What are the exceptions in DNa methylation

Answer 19

• CpG islands

	- Multiple CpG commonly found in promoters of genes 
	- Unmethylated and never become DNA methylated at any stage of development 
	- Resistant to methylation

Question 20

Rules in DNA methylation

Answer 20

1. Most cytosines in CpGs in the genome are methylated.
   
   2. DNA methylation is generally thought to be associated with repression – such as in silencing of repetitive elements, in heterochromatin and in the inactive X-chromosome in females.
   3. CpG islands are found in promoters and are generally resistant to DNA methylation.
   4. When DNA methylation of a CpG island does occur, it is associated with gene repression. However, it is not required for silencing – most promoter CpG islands never become DNA methylated, and the associated gene can be silenced despite this.
   5. Methylation of CpGs in gene bodies is actually associated with gene expression – why this is the case is currently unclear.

Question 21

function of topologically associated domain

Answer 21

to keep enhancers from being used by the wrong gene

Question 22

What are the four types of chemical signaling

Answer 22

autocrin, paracrine, juxtacrine and endocrine

Question 23

Importance of epigenetic regulation in stem cells

Answer 23

• ES cells tolerate deletion of much of the epigenetic machinery
  
  • Epigenetic regulation is important to allow transitions in cell states
    
    • When a different set of genes has to be turned on, while others have to be turned off
    • When they tolerate deletion, they are often impaired in differentiation

Question 24

How do signalling pathways influence stem and progenitor cell behaviour

Answer 24

1. drive stem and progenitor cells toward timely differentiation
2. Keep cells undifferentiated
3. Guide cell movement/ recruitment
4. Direct cells towards a particular lineage

Question 25

How do signalling pathway drive stem cell and progenitor cell to differentiation

Answer 25

• Fibroblast Growth Factor 4 (FGF4) recognised by receptors in the receptor tyrosine kinases (RTKs) family
  
  • Pluripotency-associated transcription factor (Oct4 and Sox2) binds upstream of FGF4
    
    • TF necessary to maintain the pluripotent ES cell state also produce the factor that drives their demise

Question 26

How to keep cells undifferentiated

Answer 26

When leukaemia inhibitory factor binds to receptor, cause phosphorylation and activation of STAT3 and STAT3 binds the promoter and enhancer of pluripotency-associated TF important for maintaining TF network in ES cells, counteract the effect of FGF4 by sustaining pluripotency

Question 27

How to guide cell movement/recruitment

Answer 27

1. Cells in the genital ridges produce SDF-1
   
   2. Signalling molecule is sensed by CXCR4, receptors in the surface of PGCs
   3. PGCs migrate towards increasing concentrations of SDF-1
   4. IF expression of SDF-1/ CXCR4 reduced -> fewer PGCs reach the genital ridges

Question 28

Importance of SDF-1/CXCR4 signalling

Answer 28

• Important for mobilising and recruiting progenitor cells from the bone marrow to an injury site
  
  • Important for recruiting circulating progenitors to the site of injury -> homing
  • Platelets and other cells at the injury site release SDF-1 which is sensed by CXCR4-expressing HSCs
  • Increased SDF-1 results more cells leaving the bone marrow niche

Question 29

How to direct cells towards a particular lineage

Answer 29

• When gastrulation begins, localised signalling pathways start to act on epiblast and instruct a fate to the cells
  depends on their position within the embryo, as the position dictates how far they are from key signalling centres

Question 30

How do signalling pathway influence cell behaviour/fate

Answer 30

drive expression of canonical genes (activated in all cell types) and express different cell type-specific TF to respond, post-translational modification , modulating other pathways ,

Question 31

TGF-B pathway

Answer 31

• Activation of TGFb (inhibit cell cycle progression)pathway result in phosphorylation, dimerization and nuclear translocation of TF SMAD2/3
- SMAD2/3 binds to promoter and enhancer of many genes associated with pluripotency
- Binding of SMAD2/3 is dependent on Oct4, if OCt4 is knocked down, Smad2/3 cannot be recruited to the same site
• TGFb is important in early muscle cell (myotubes) and pro-B cell
- Smad2/3 binds to different site in the genome and assocaite with different specific TFs
• Myod1 in myotubes
• Pu.1 in Pro-B cells

Question 32

Give an example in post-translational modifications

Answer 32

• Phosphorylation of Basic helix-loop-helix (bHLH) TF
  
  • Control the differentiation of various cell types eg. Neural and myocyte differentiation
    Eg. Ngn2 (TF expressed throughout neurogenesis)
  • Ngn-2 is phosphorylated in early stages
  • P-Ngn2 decreased stability and binds DNA poorly
  • Ngn-2 increased stability and enhanced chromatin binding
  Therefore, removing the phospho-group or mutating the a.a so they cannot be phosphorylated in Ngn2 pushes cell towards neural differentiation
  -

Question 33

How to modulate other pathways ?

Answer 33

1. receptors can activate multiple intracellular pathways
2. intracellular mediators talk to each other
3. intracellular mediators compete for a common co-regulatory subunit

Question 34

Important regulators of stem cell behaviour

Answer 34

1. metabolism, ES rely on oxidative phosphorylation, epiblast Sc rely on anaerobic glycolysis
2. Cell cycle - regulating entry into the cell cycle and the duration of various cell cycle affect Sc properties
3. Physical forces - mechanical cues can change properties of cells

Question 35

What might you find in a gene promoter?

Answer 35

1. Core promoter– where the basal transcriptional machinery bind.
   
   2. Transcription factor binding sites– which add a regulation to gene expression. If the gene is expressed then transcription factors that help to activate gene expression will be bound here. If it’s off, you may find transcription factors that repress expression instead.
   3. A CpG island– more than 50% of human gene promoters contain a CpG island.
   4. Histone modifications– these will vary based on whether or not the gene is expressed. For instance, in an expressed gene there may be H3K4me3 marks. At a gene that is not expressed H3K27me3 might be found.

Question 36

Which techniques might be useful to investigate a transcriptional network in a new type of stem cell?

Answer 36

1. RNA-seq– to find out which genes are transcribed
   
   2. ChIP-seq– to show where transcription factors are bound. You could also do ChIP-seq to show where different histone modifcations are found in the genome
   3. Knockout or knockdown studies– this would involves deleting or reducing the expression of key transcription factors to see the effect on the stem. If the stem cell dies or differentiates (or its function is impaired in any way) then you might be able to draw conclusions about the function of the transcription factor. If the transcription factor can be deleted with no consequence, then it is unlikely to be a critical factor in regulating that stem cell
   4. Reprogramming– once you have a list of candidate genes, you could test them in an assay similar to that used by Yamanaka. For instance you could try overexpressing them in a fibroblast to see if the cell reprograms towards your stem cell of interest. Of course, this relies on you being able to grow the stem cell in culture in the first place…

Question 37

What are the key components that might contribute to maintaining the stem cell state?

Answer 37

1. Transcription factors
2. Epigenetics – DNA methylation, histone modifications, non-coding RNAs
3. Signalling pathways
4. Metabolism
5. Cell cycle
6. Physical forces