Molecular Regulation of Stem Cells

Question 1

Explain what is meant by cell state

Answer 1

Functionally, the cell state is a description of what the cell can do (neurons transmit stimuli), or their potential (ES cells are pluripotent). Molecularly, the cell state is the result of protein expression and activity. RNAs also play key roles.

Question 2

What is a transcription factor?

Answer 2

Protein with a DNA binding domain that binds to specific DNA sequences and control gene expression - a key part in determining the cell’s identity.

Question 3

What is somatic cell reprogramming?

Answer 3

Taking a terminally differentiated cell, forcing the expression of a few TFs, and thereby making the cell go back to an ES cell-like state

Question 4

Describe what is meant by a TF network

Answer 4

The idea that TFs regulate eachother in a network of positive and negative interactions. Also, the network contains main and supportive transcription factors.

Question 5

What is meant by a Class 1 TF? (In ES cell network)

Answer 5

An essential transcription factor - deletion of them results in differentiation/death. Vital in maintaining the self-renewal of ES cells. “Loss-of-function” effect.

Question 6

What is meant by a Class 2 TF? (In ES cell network)

Answer 6

These factors can be individually deleted while still maintaining ES cell self renewal. In some cases, forced increased expression of these factors can cause the maintainence of self renewal even when factors wouldnt normally allow. This is why it’s called “gain-of-function”. Deleting more than one at a time results in differentiation or death.

Question 7

Do TF levels matter?

Answer 7

Many TFs only need to be present above a certain threshold. Some TFs need to be expressed in certain quantities, and deviation from the optimal amount can result in a change in fate of the cell. Oct4 expression is very tightly regulated and an increase/deletion -> differentiation. Forced expression of defined amount of Oct4 -> stops differentiation

Question 8

How can the same TF have different roles in different contexts?

Answer 8

Because being expressed in different cell types leads to a different function. This is because it may be part of different networks which changes its activity.

Question 9

How does Sox2 function differently in different contexts?

Answer 9

Sox2 is essential for ES cells and its deletion causes differentiation. Also essential for the maintainence of TE stem cells. It binds in different locations in each type of stem cell. It’s part of 2 different TF networks so regulates different genes in different cells.

Question 10

How does Klf2/4 function differently in different contexts?

Answer 10

Important class 2 TFs in ES cells. Klf2/4/5 substitute for eachother. Forcing expression of Klf2/4 keeps cells undifferentiated in the absence of LIF. Klf4 is essential in the skin and expressed when epidermal cells begin differentiating. Klf4 deletion -> no barrier properties of the skin. Early developmental stages require Klf2 for blood vessel maintainence, absence -> early embryos die of generalised haemorrhaging. Kl2/4 in later life help create tight barriers - no evidence to support this function in ES cells

Question 11

How is the activity of TFs regulated by other TFs?

Answer 11

TFs can physcially interact with other TFs, influencing the binding site that the TF binds to

Question 12

How is the role of Oct4 regulated in mouse ES cells and in differentiating endoderm?

Answer 12

In ES cells, Sox2 and Oct4 form a DNA binding complex that results in the expression of ES cell specific genes. When the cells differentiate into endoderm, they maintain Oct4 expression but shut down Sox2, replacing it with Sox17 - the new binding partner. This creates a new complex which allows the regulation of a different set of genes. Forcing Sox17 expression in ES cells leads to binding of Oct4 to places in the DNA that are associated with endoderm.

Question 13

How can we identify TF networks?

Answer 13

To get a global view of all the genes expressed we analyse RNA. Tells us which TFs are expressed and the output of the network (which genes are being regulated).

Question 14

How can chromatin immunoprecipitation be used to identify TF networks?

Answer 14

Use chromatin immunoprecipitation followed by DNA sequencing, we can figure out where in the genome TFs bind. TFs bind to promoter/enhancers. Promoters surround TSS so easy to tell when they’re bound. Enhancers can be far away, downstream, upstream, within, or within another gene so harder to identify.

Question 15

How are histones positioned to activated genes?

Answer 15

For a gene to be active, the nucleosome has to be removed from the TSS. Nucleosome remodelling complexes have this job, and they are recruited by cell type specific TFs to target genes.

Question 16

What is the histone code?

Answer 16

The covalent modifications that belong to the amino acids on the N-terminal tails of the histone proteins which protrude from the nucleosome. The modified amino acids can act as recognition sites for protein complexes that activate/inhibit transcription.

Question 17

How is constitutive heterochromatin usually marked?

Answer 17

Often marked by the addition of 3 methyl groups to lysine 9 of histone 3, denoted by H3K9me3 (trimethylation). Heterochromatin protein 1 binds the modification and with other factors, condenses the chromatin. Also constitutive heterochromatin is marked by high levels of DNA methylation

Question 18

How does an acetyl group attached to a lysine lead to activation of transcription?

Answer 18

Acetylation of lysine neutralises its positive charge, reducing the electrostatic attraction between lysine, DNA, and histone

Question 19

How are enhancer elements often marked?

Answer 19

Often with H3K4me1 and H3K27ac is added when they’re activated.

Question 20

List some modifications associated with activation

Answer 20

Promoters marked with H3K4me3. H3K27ac.

Question 21

Which other types of post-transcriptional modifications of histone tails are there?

Answer 21

Mono, di, tri, methylation, acetylation, phosphorylation, SUMOylation, ADP-ribosylation

Question 22

What is the meaning of a bivalent modification?

Answer 22

The promoters of the genes are marked by both H3K27me3 and H3K4me3 (repression and activation), so the gene is poised, ready to be turned of/off depending on signals. This occurs in ES cells for developmentally regulated genes, ie. genes that will be upregulated in differentiation.

Question 23

What is a modification associated with repression?

Answer 23

H3K27me3 of a nucleosome near the promoter.

Question 24

What is DNA methylation?

Answer 24

When a CpG is methylated by DNA methyltransferases. C is methylated at 5’ C. Most of the Cs in CpG dinucleotides are methylated, because DNAmts recognise this sequence.

Question 25

Why is the CpG sequence underrepresented in the genome?

Answer 25

5mC is susceptible to spontaneous deamination, converting it to a T. This results in a TG mismach, which is repaired. Mismatch repair mechanisms preferentially repair Ts to Cs, they can repairs Gs to As so some of the Cs are lost.

Question 26

What is one of the roles of CpG methylation?

Answer 26

Silencing the expression of repetitive DNA elements such as retrotransposons

Question 27

What are CpG islands?

Answer 27

Long runs of multiple CpGs, commonly found in gene promoters. 50% of human promoters contain a CpG island

Question 28

What is the state of DNA methylation in CpG islands?

Answer 28

In the vast majority of genes, CpG islands are unmethylated, and will never become methylated - it seems they are resistant

Question 29

What are the rules of DNA methylation?

Answer 29

1) Most Cs in CpGs are methylated
2) DNA methylation usually associated with repression
3) CpG islands found in promoters and are DNA methylation resistant
4) If CpG island methylation occurs, it’s usually associated with repression
5) Methylation of CpGs in gene bodies is associated with activation
6) 5mC can be oxidised to form 5-OHmC and others

Question 30

Explain the role of DNA methylation in the epiblast of the preimplantation embryo

Answer 30

Has low levels of DNA methylation, which can be captured in ES cells. As these pluripotent cells differentiate, the DNA methylation levels increase because different parts of the genome are shut down in order to differentiate

Question 31

What is a topologically associated domain?

Answer 31

TADs are units of the genome which has boundaries defined by insulator domains. Regulatory elements within one TAD cannot cross the boundaries of that TAD. They’re thought to be established in early development. Deletion of TADs can affect the genes expressed and the levels they’re expressed at

Question 32

What is the importance of epigenetic regulation in stem cells?

Answer 32

ES cells can tolerate the deletion of much epigenetic machinery (complete removal of DNA methylation and loss of histone modifying enzymes). They can self renew, but their differnetiation is impaired. So, epigenetic regulation is important in allowing cell state transitions. Also applies in the embryo, where proper development requires intact epigenetic machinery. TFs and epigenetics work together, TF can bring an epigenetic regulator to the DNA, which can make the DNA more accessible for further TFs.

Question 33

How can metabolism result in the regulation of gene expression in stem cells?

Answer 33

Mouse ES cells rely heavily on oxidative phosphorylation. Epiblast stem cells rely on anaerobic glycolysis. Mouse ES cells tolerate glycolysis inhibition but EpiSC don’t. Many intermediates from energy production also play a role in signalling and regulation of transcription. Acetyl-coA is the universal substrate of histone acetyltransferases. Some histone deacetylases use NAD+ as a cofactor. Evidence in yeast and mammalian systems that modulating metabolism impacts epigenetic states

Question 34

How can the cell cycle be involved in modulating stem cell activity?

Answer 34

HSCs in Go can be in a transient and reversible quiescent state. Intrinsic and extrinsic factors push HSCs to reenter the cycle cycle, leading to renewal or differentiation. TFs that regulate differentiation also regulate cell cycle. MyoD and MEF2 are TFs for skeletal muscle that have been shown to regulate cell cycle exit. MyoD is a bHLH factor - phosphorylated in progenitors and inactive. Cdks that maintain prolif in undifferentiated progenitors also phosphorylate and inactive MyoD. CBFA1 and Runx2 regulate differentiation and cell cycle exit in osteoblasts

Question 35

How do physical forces regulate stem cell activity?

Answer 35

Cells can sense various properties such as stiffness and shape as well as stretch or sheer from fluid flow. The mechanical signals are transduced and change the properties of the cell.

Question 36

What is a clonal assay?

Answer 36

Plating cells at very low density, allowing each cell to replicate, forming a colony where all the cells are clones

Question 37

How did Martin Evans use clonal assays to derive ES cells?

Answer 37

Used clonal assays with different media in order to determine which media grew EC cells the best and then used this to derive ES cells.

Question 38

What is wrong with growing the ES cells with medium (containing FCS) on feeder cells?

Answer 38

We don’t know what’s in the FCS and media. We don’t know if the effect is due to the feeder cells or the medium and don’t know what exactly it is from the feeder cells causing the maintainence of ES cells

Question 39

When feeder cells are removed, ES cells die or differentiate. Form a hypothesis on the role of the feeder cells in maintaining ES cells

Answer 39

The feeder cells secrete a soluble protein into the medium that allows the maintainence of the ES cell self renewal

Question 40

What is the factor that the feeder cells release?

Answer 40

The fibroblasts release LIF, which is required for self renewal of ES cells

Question 41

Explain the signalling pathway of LIF

Answer 41

LIF binds to TKR called LIF-R, which dimerises with co-receptor gp130. This activates JAK which phosphorylates the receptor on key tyrosine residues, which STAT3 then binds to. JAK phosphorylates STAT3 at key tyrosine residues, which causes its dissociation from the receptor, dimerisation, and entering of the nucleus. STAT3 then binds to DNA where it upregulates expression of a number of target genes

Question 42

Describe the role of the MEK-ERK pathway in ES cell self renewal maintainence

Answer 42

LIF activates MEK-ERK pathway, and it is not required for ES cell self-renewal. Blocking the pathway enhances self-renewal. MEK-ERK is activated by many ligands, one being FGF, which is thought to drive differentiation.

Question 43

Which kind of properties would a key pluripotency TF have?

Answer 43

In vitro: Expressed in ES cells, has a loss of function phenotype, and a gain of function phenotype
In vivo: Expressed in Epiblast, has loss of function phenotype

Question 44

Why might KO of a gene not always lead to the expected outcome ie. loss of function?

Answer 44

Redundancy - Another substitute gene takes on the role

Question 45

Where are Oct4 and Nanog expressed in the embryo?

Answer 45

Oct4 is expressed throughout the whole ICM ie. PE and Epi. Nanog expressed in the Epiblast

Question 46

Where are Oct4 and Nanog expressed in ES cells?

Answer 46

They are both expressed in every ES cell in a colony

Question 47

What happens when Oct4 is knocked out of mouse embryo? What does this tell us about the role of Oct4?

Answer 47

The embryo doesn’t make it past the ICM stage, all you get is TE cells. Same for in culture. This shows us that Oct4 is required to make the ICM

Question 48

Does Oct4 have a gain of function or loss of function phenotype?

Answer 48

Loss of function in vivo and in vitro. Gain of function in vitro. It is gain of function in vitro because when overexpressed, the cells differentiate.

Question 49

What is the role of Nanog in ES cells?

Answer 49

Promotes ES cell renewal, has a gain of function phenotype. Overexpression of Nanog leads to increased self renewal, even independent of LIF.

Question 50

What happens if Nanog is knocked out in ES cells and in the embryo?

Answer 50

In the embryo, if Nanog is KO, you don’t get an epiblast. If Nanog is knocked out in ES cells, they can survive, and are pluripotent, but they are prone to differentiation.

Question 51

In the Epiblast, why do Nanog and Oct4 drive the expression of FGF4?

Answer 51

Because the epiblasts job is to differentiate - pluripotency is transient. Therefore it makes sense for the pluripotency TFs to drive expression of TFs needed for differentiation.