6. the molecular regulation of stem cell fate

Question 1

what is epigenetics?

Answer 1

DNA and chromatin modifications that do not alter the primary nucleotide sequence

Question 2

what are histone modifications?

Answer 2

covalent modifications made to histone tails

Question 3

what does HDAC do?

Answer 3

HDAC removed the acetyl group from histones

this promotes gene silencing as it promotes the formation of heterochromatin

Question 4

what does HAT do?

Answer 4

HAT adds acetyl group to histones

this promote gene activation as it promotes the formation of euchromatin

Question 5

what type of genome modification is most easily changed?

Answer 5

histone modification

Question 6

what DNA residue is methylated?

Answer 6

cysteine

Question 7

what enzyme adds methyl groups to DNA?

Answer 7

DNA methyl-transferases

Dnmt1, Dnmt2, Dnmt3

Question 8

what happens to the methylation pattern when a cell replicates?

Answer 8

it is inherited

Question 9

where are CpG islands found?

Answer 9

surrounding genes

Question 10

what is DNA methylation associated with?

Answer 10

silencing genes

Question 11

what treatment can be use to determine what residues are methylated and how does it work?

Answer 11

Bisulfite treatment

• this converts cysteine to uracil
• methylated cysteines are protected
• sequence DNA

Question 12

describe the epigenetics of house keeping genes

Answer 12

• DNA is in-methylated

- euchromatin

Question 13

what is the chromatin of an embryonic stem cells like?

Answer 13

euchromatin

Question 14

when a cell differentiates, what occurs to its epigenetics?

Answer 14

• genes of certain lineages that the cell is never going to express are silenced
• they are methylated and stored as heterochromatin

Question 15

what are required in order to bring a cell back to pluripotency?

Answer 15

chromatin remodellers and DNA methyl-transferases

Question 16

what is expressed in high levels in LT-HSC (and not further down the lineage) and what happens when this is knocked out?

Answer 16

Dnmt3a

differentiation is obstructed

Question 17

competitive transplantation was carried out with KO LT-HSC, describe how this was done

Answer 17

• mice with Mxl-Cre crossed with floxed Dnmt3a, BM transplanted into lethally irradiated WT mouse
• WT and KO cells genetically marked so they can be followed and transplanted 1:1
• pIpC added to induce deletion

Question 18

what was the result of this competitive transplantation?

Answer 18

• more Dnmt3a KO HSC were seen in peripheral blood than WT HSC
• KO HSC show different distribution of progeny than normal (more B cells and less T cells)

Question 19

what was concluded from the competitive transplantation?

Answer 19

larger number of HSC seen in peripheral blood when Dnmt3a KO as differentiation is being blocked

Question 20

what was seen in the proteome of Dnmt3a KO HSCs?

Answer 20

• genes for multipotency and HSC genes were expressed at much higher levels than WT
• genes for differentiation expressed at lower levels than WT

Question 21

what was seen in the genome of these Dnmt3a KO HSC?

Answer 21

multipotency genes that are methylated in WT HSC are un-methylated in KO

Question 22

name four types of genes that are linked to stemness and so are expressed at high levels in stem cells

Answer 22

1. cell cycle inhibitors
2. chromatin remodellers
3. telomerase
4. DNA repair genes

Question 23

what genes are expressed at low levels in stem cells?

Answer 23

genes of differentiation are expressed in low levels so that stem cells are poised to differentiate

Question 24

why are cell cycle inhibitors expressed at high levels in stem cells?

Answer 24

to maintain quiescence

Question 25

why are chromatin remodellers expressed at high levels in stem cells?

Answer 25

to maintain chromatin in an open state

Question 26

why is telomerase expressed in high levels in stem cells?

Answer 26

to maintain telomere length when stem cells replicate

Question 27

name a disease that affects the bone marrow and what is it caused by?

Answer 27

Fanconi anaemia is caused by defects in DNA repair which leads to increased cell cycle arrest in stem cells and progenitors

Question 28

what did the transcriptomic analysis of lots of stem cells during an injury response show?

Answer 28

there is lots of variation between stem cells which becomes even more obvious during an injury response

Question 29

describe the diffusion map which includes multiple different types of cell populations

Answer 29

- each cells transcriptome has a 3D position on the diffusion map - cells with similar transcriptomes are close to each other

Question 30

describe pseudo time on the diffusion map

Answer 30

the diffusion map represents a journey through time as the cell differentiates

Question 31

describe pseudo space on the diffusion map

Answer 31

in a tissue as cell that is derived from another cell will be close in the tissue

Question 32

why do we use the word pseudo when referring to the diffusion map?

Answer 32

because we cannot actually see this process happening

Question 33

what are microRNA?

Answer 33

non-coding RNA that is involved in the post translational regulation of gene expression

Question 34

in addition to transcription factors, what else may regulate differentiation of a stem cell?

Answer 34

microRNA expression

Question 35

what can the deletion of TF in stem cells lead to?

Answer 35

- deletion of certain lineages | - reduction in stem cell self-renewal

Question 36

what can over expression of certain TF in stem cells lead to?

Answer 36

- expansion of certain lineages | - increase of stem cell self-renewal

Question 37

what has very little proteomic analysis been done on stem cells?

Answer 37

proteomic analysis requires lots of cells and stem cells are rare

Question 38

when a HSC proteomic study was carried out on mice why was this so hard?

Answer 38

stem cells needed to be pooled from 600 mice

Question 39

what are the two types of mechanisms that control stem cells?

Answer 39

cell intrinsic and cell extrinsic

Question 40

what do signalling pathways do?

Answer 40

couple the extracellular environment to transcription and cell fate of stem cells

Question 41

name 5 different extracellular signals that are known to regulate stem cells

Answer 41

1. cytokine 2. growth factors 3. hormones 4. cell-cell junctions 5. cell-matrix junctions

Question 42

give an example of three different signalling pathways that are found throughout development and in somatic tissues?

Answer 42

Wnt, Notch and Shh

Question 43

describe the signalling in mice epidermis and how this affects hair growth

Answer 43

- Wnt signals for stem cells to proliferate - Notch signals for differentiation - Shh for proliferation and this results in hair growth

Question 44

describe the signalling in the intestine and how this affects cell fate

Answer 44

- Wnt signals stem cells proliferations - Notch signals TA proliferations - Wnt and Notch signalling antagonise each other to determine where cells will become secretory or absorption

Question 45

name 4 different non-protein cell extrinsic factors that regulate stem cells and why are these becoming easier to study?

Answer 45

1. ROS 2. matrix stiffness 3. environment shape 4. pressure better technology is being developed

Question 46

give an example of how ROS regulate stem cells

Answer 46

elevated levels of ROS limit the lifespan of HSC by interfering with their quiescence

Question 47

give an example of how pressure regulates stem cells

Answer 47

specific genes expression is associated with the types pressure applied to MSC - dynamic tension regulates fibroblastic and oestogenic associated genes - compression up-regulates genes associated with cartilage