Molecular Regulation of Stem Cells Flashcards

You may prefer our related Brainscape-certified flashcards:
1
Q

Explain what is meant by cell state

A

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.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
2
Q

What is a transcription factor?

A

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.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
3
Q

What is somatic cell reprogramming?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
4
Q

Describe what is meant by a TF network

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
5
Q

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

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
6
Q

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

A

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.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
7
Q

Do TF levels matter?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
8
Q

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

A

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.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
9
Q

How does Sox2 function differently in different contexts?

A

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.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
10
Q

How does Klf2/4 function differently in different contexts?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
11
Q

How is the activity of TFs regulated by other TFs?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
12
Q

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

A

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.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
13
Q

How can we identify TF networks?

A

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).

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
14
Q

How can chromatin immunoprecipitation be used to identify TF networks?

A

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.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
15
Q

How are histones positioned to activated genes?

A

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.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
16
Q

What is the histone code?

A

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.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
17
Q

How is constitutive heterochromatin usually marked?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
18
Q

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

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
19
Q

How are enhancer elements often marked?

A

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

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
20
Q

List some modifications associated with activation

A

Promoters marked with H3K4me3. H3K27ac.

How well did you know this?
1
Not at all
2
3
4
5
Perfectly
21
Q

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

A

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

22
Q

What is the meaning of a bivalent modification?

A

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.

23
Q

What is a modification associated with repression?

A

H3K27me3 of a nucleosome near the promoter.

24
Q

What is DNA methylation?

A

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.

25
Q

Why is the CpG sequence underrepresented in the genome?

A

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.

26
Q

What is one of the roles of CpG methylation?

A

Silencing the expression of repetitive DNA elements such as retrotransposons

27
Q

What are CpG islands?

A

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

28
Q

What is the state of DNA methylation in CpG islands?

A

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

29
Q

What are the rules of DNA methylation?

A

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

30
Q

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

A

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

31
Q

What is a topologically associated domain?

A

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

32
Q

What is the importance of epigenetic regulation in stem cells?

A

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.

33
Q

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

A

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

34
Q

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

A

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

35
Q

How do physical forces regulate stem cell activity?

A

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.

36
Q

What is a clonal assay?

A

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

37
Q

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

A

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.

38
Q

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

A

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

39
Q

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

A

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

40
Q

What is the factor that the feeder cells release?

A

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

41
Q

Explain the signalling pathway of LIF

A

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

42
Q

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

A

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.

43
Q

Which kind of properties would a key pluripotency TF have?

A

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

44
Q

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

A

Redundancy - Another substitute gene takes on the role

45
Q

Where are Oct4 and Nanog expressed in the embryo?

A

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

46
Q

Where are Oct4 and Nanog expressed in ES cells?

A

They are both expressed in every ES cell in a colony

47
Q

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

A

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

48
Q

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

A

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.

49
Q

What is the role of Nanog in ES cells?

A

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

50
Q

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

A

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.

51
Q

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

A

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.