Topic08 - Epigenetics in Regenerative Medicine

Question 1

Define pluripotent embryonic stem cells

Answer 1

Pluripotent ESCs are undifferentiated cells that are capable of replicating themselves (self-renewal) and can also differentiate into highly specialised cell types (pluripotent)

Question 2

State the three lineages derived from a blastocyst

Answer 2

1. Early epiblast (ICM)
2. Primitive endoderm
3. Tropectoderm

Question 3

State the lineages of a blastocyst that are embryonic

Answer 3

1. ICM (inner cell mass)

2. Primitive endoderm

Question 4

Where are pluripotent ESCs derived from?

Answer 4

The inner cell mass (ICM) of a blastocyst

Question 5

State the conditions required for culturing naive ESCs

Answer 5

2i-LIF: serum free conditions supplemented with MEK and GSK3 inhibitors and Leukemia Inhibitory Factor

Question 6

State the conditions required for culturing conventional/metastable ESCs

Answer 6

Serum-LIF: serum-containing medium, Leukemia Inhibitory Factor + mouse fibroblast feeders

Question 7

In both naive and conventional ESCs, MEK-ERK signaling pathway is reduced. TRUE or FALSE?

Answer 7

TRUE. naive ESCs have active suppression of MEK-ERK while conventional ESCs have low activation of MEK-ERK. Both are thus pro-differentiation.

Question 8

What are EpiSCs?

Answer 8

EpiSC = epiblast stem cells, a type of primed pluripotent cells derived from post-implantation epiblasts.

Question 9

Differentiate ESCs and EpiSCs

Answer 9

1. ESCs are derived from the ICM of pre-implantation blastocyst, while EpiSCs are derived from post-implantation epiblasts.
2. ESCs do not depend on MEK-ERK signaling, while EpiSCs depend on MEK-ERK signaling.
3. ESCs exhibit global DNA hypomethylation, while EpiSCs do not.
4. ESCs do not exhibit X inactivation, while EpiSCs exhibit X inactivation.
5. ESCs exhibit low H3K27me3 on developmental regulators, while EpiSCs exhibit high H3K27me3 on developmental regulators.

Question 10

Why does X inactivation occur?

Answer 10

For dosage compensation between sexes – only a single X chromosome should be transcriptionally active.

Question 11

Briefly explain the cycle of X chromosome inactivation during development

Answer 11

1. imprinted X-p inactivation occurs at the 4 cell stage
2. X-p is reactivated in the ICM (ESCs), but X-p inactivation is maintained in the trophectoderm
3. Random X inactivation occurs in the epiblast (EpiSCs)

Question 12

State the ncRNA involved in X chromosome inactivation

Answer 12

Xist

Question 13

What is RNA-FISH?

Answer 13

RNA-FISH is a method for detecting and localising particular RNA molecules in fixed cells.

Question 14

State the proteins involved in maintaining the silencing of the X chromosome

Answer 14

PRC1, PRC2, DNMT1, macroH2A1/2

Question 15

Discuss the role of PRC2 in X-inactivation

Answer 15

Xist ncRNA promotes PRC2 binding to the X inactivation complex, resulting in the spread of H3K27me3

Question 16

Briefly discuss the various factors involved in the process of X inactivation and their roles.

Answer 16

1. Xist RNA is expressed and surrounds the X chromosome to be inactivated.
2. Xist RNA then recruits PRC1 and PRC2, conferring H3K27me3 and H2Aub1, resulting in gene silencing
3. Chromatin is further modified by hypoacetylation of histone H4
4. Histone variant macroH2A is incorporated.
5. DNMT1 further methylates promoters on Xi and represses gene expression.

Question 17

State the effect of pluripotency-inducing factors on Xist expression

Answer 17

Pluripotency inducing factors (such as NANOG) represses Xist expression

Question 18

State the pluripotency factors involved in transcriptional repression of Xist

Answer 18

Oct4, Sox2, Nanog, and Prdm14

Question 19

State the phase of the cell cycle in which canonical histones such as H2A, H3.1, and H3.2 are primarily expressed.

Answer 19

S phase. These canonical histones are deposited in a strict DNA replication dependent manner.

Question 20

State the histone variants that are expressed and deposited throughout the cell cycle

Answer 20

H2A.Z and H3.3 (these are deposited independent of DNA synthesis)

Question 21

List the three types of chromatin states.

Answer 21

1. Euchromatin
2. Bivalent domains (facultative heterochromatin)
3. Constitutive heterochromatin

Question 22

Describe the chromatin of undifferentiated ES cells

Answer 22

Homogenously spread and largely devoid of compact heterochromatin blocks.

Question 23

Describe the chromatin of differentiated cells

Answer 23

Heterogenous expression with distinct blocks of compaction (i.e. clusters of heterochromatin marked by H3K9me2/3 and HP1)

Question 24

What is HP1?

Answer 24

Heterochromatin protein 1

Question 25

Explain hyperdynamic binding, using HP1α and H1 as examples.

Answer 25

HP1α and H1 exhibit hyperdynamic binding in ESCs, binding loosely to chromatin in ESCs. Upon differentiation, the hyperdynamic proteins become immobilised on chromatin.

Question 26

ESCs do not exhibit silencing mechanisms, as ESCs primarily allow for active transcription of all genes. TRUE or FALSE?

Answer 26

FALSE. While there are abundant epigenetic marks indicative of active transcription, there are counteracting mechanisms that silence developmental regulatory genes, preventing premature differentiation. (Polycomb)

Question 27

Briefly describe the Mediator complex

Answer 27

Mediator is a multi-subunit complex that forms part oft the RNAPII transcription pre-initiation complex

Question 28

State the four Mediator components that form the CDK8 module

Answer 28

MED12, MED13, CDK8 (Cyclin-dependent kinase), and Cyclin C

Question 29

State the role of the Mediator (only) complex in relation to RNAPII

Answer 29

Mediator helps to recruit RNAPII and other PIC factors to the promoter.

Question 30

State the role of CDK8-Mediator in relation to RNAPII

Answer 30

CDK8 negatively regulates Pol II-Mediator interactions. CDK8-Mediator recruits and activates the super elongation complex (SEC) to promote Pol II release and elongation. SEC contains CDK9 which phosphorylates Ser2 on RNAPII CTD.

Question 31

Briefly describe the fate of CDK8-Mediator during re-initiation of transcription.

Answer 31

CDK8-Mediator cannot interact with Pol II, thus the re-initiation of transcription requires the release of CDK8.

Question 32

State the role of Mediator in relation to transcriptional activation by nucleosome displacement

Answer 32

Mediator interacts with TrxG proteins SWI/SNF and CHD1 chromatin remodeling complexes and contributes to nucleosome displacement

Question 33

State the role of Mediator in relation to DNA looping

Answer 33

Mediator interacts with TFs, ncRNAs, and Cohesin, bringing linearly separated DNA sequences together in close proximity, stabilising enhancer-promoter loops.

Question 34

State the roles of Cohesin

Answer 34

1. Cohesin connects sister chromatids during metaphase 2. Cohesin assists recombinational repair of DNA DSB by tethering the break site to the intact sister strand. 3. Cohesin also fosters interaction between distal enhancers and gene promoter.

Question 35

Chromatin architectural organisation (i.e. DNA looping) is associated with increased expression. TRUE or FALSE?

Answer 35

FALSE. Chromatin looping can cause coordinated expression or insulation of specific regions.

Question 36

State the canonical Yamanaka reprogramming factors

Answer 36

OCT4, SOX2, KLF4, c-MYC | OSKM

Question 37

State the issue with induced pluripotency.

Answer 37

It is a fairly slow and inefficient process.

Question 38

Describe the role of Mediator and Cohesin in reprogramming

Answer 38

ESC-specific long range interactions are re-established during reprogramming (and these long range interactions depend on Mediator and Cohesin)

Question 39

Define superenhancers

Answer 39

Superenhancers are dense clusters of enhancers enriched for multiple TFs that regulate temporal gene expression and imparts cell-specific transcriptional outputs.

Question 40

Superenhancers are the same in all somatic cells. TRUE or FALSE?

Answer 40

FALSE. superenhancers tend to be cell type specific.

Question 41

What are superenhancers enriched with?

Answer 41

Multiple TFs, H3K27ac, H3K4me1, DNAse hypersensitive sites (DHS), and MED1 (mediator)

Question 42

Briefly state the role of enhancers

Answer 42

Enhancers integrate extracellular signals with intracellular cell fate to generate cell-type specific transcriptional responses.