Plasticity of differentiation

Question 1

How do gene regulatory proteins self regulate expression

Answer 1

the gene transcribed maintains its own expression by binding to the DNA and causing transcription of the DNA that forms itself

Question 2

What are examples of epigenetic modifications

Answer 2

histone methylation, acetylation and phosphorylation

Question 3

What does SCNT prove regarding the genome?

Answer 3

it proves that certain factors exist to reprogramme the nucleus and reverse a cell from differentiated to an undifferentiated state

Question 4

What are the 2 main types of nuclear reprogramming and what are they?

Answer 4

de-differentiation = a differentiated cell reverting to a state of totipotent/pluripotency
transidfferentiation = a cell switching from one differentiated cell type to another

Question 5

What is needed for successful cell nuclear reprogramming?

Answer 5

changing the gene expression profile and what is/isnt expressed
resetting epigenetic mechanisms that stabilise gene expression such as DNA methylation and histone modifications

Question 6

State briefly what SCNT is?

Answer 6

nuclear reprogramming by nuclear transfer into eggs by meiotic metaphase II arrested oocytes
mimics natural fertilisation as egg factors reprogramme the somatic nucleus
pluripotency genes are switched on, DNA replication and cell division are initiated, a new organism/clone is generated

Question 7

How does SCNT induce a new pattern of gene expression in the transplanted cell?

Answer 7

the transcription factors in the implanted nucleus and outcompeting by reprogramming proteins in the oocyte which act on a nucleus to determine cell state

Question 8

What factor decreases the success rate of SCNT and why?

Answer 8

if adult do not nuclei are used. because they suffer major DNA damage and chromosome loss when being forced to undergo rapid DNA replication and cell division. epigenetic factors are nt completely reset and there is still persistent memory of previous gene expression. certain genes may fail to be switched off + pluripotency genes may be resistant to reactivation

Question 9

How can histone H3.3 account for epigenetic memory?

Answer 9

when incorporated into the chromatin that is transcriptionally active, it can help ‘lock in’ the gene expression state

Question 10

How does direct transcriptional reprogramming by nuclear transfer into meiotic prophase I oocytes work?

Answer 10

prophase I stage oocytes can directly reprogramme somatic nuclei to express stem cell marker genes. mechanism of SCNT can be analysed without damaging effects of enforced rapid DNA replication and cell division. hundreds of nuclei can be injected into the same oocyte and reprogrammed.
causes nuclear volume increase, chromatin decondensation, removal of histone modifications and chromatin protein exchange

Question 11

How to linker histones cause nucleosome remodelling and chromatin decondensation

Answer 11

the oocyte specific linker histone (B4 in amphibians and H1foo in mammals) is incorporated into the transplanted nuclei and is needed to express pluripotent genes. remodels the nucleosome to allow transcriptional regulators and RNA polymerase II to activate the DNA. the linker histones are swapped over time (somatic histones removed and replaced by oocyte histones) to affect chromatin condensation and turn genes on/off

Question 12

What happens to pluripotency genes during SCNT

Answer 12

reactivated by histone H3.3

Question 13

What events must occur to decondense chromatin

Answer 13

chromatin remodelling, demethylation of DNA, histone acetylation, activate differentiation gene in normal develpment and activation of repressed pluripotency genes during reprogramming

Question 14

What events must occur to condense chromatin

Answer 14

DNA methylation, histone deacetylation, repression of a differentiation gene during reprogramming, repression of a pluripotency genes during normal development

Question 15

How can cell fusion reprogramme a cell?

Answer 15

nucleus from each cell becomes exposed to each others transcription factors. the larger and more actively dividing cell typically dominates and caused direct transactivation of the gene sin the responding cell = nuclear swelling and chromatin decondensation

Question 16

How can you induce pluripotency by overexppressing TF’s?

Answer 16

trasnfection of oc4, klf4, sox2 and myc silences somatic gene expression and activates pluripotency genes

Question 17

What is a heterokaryon?

Answer 17

when both nuclei in the fused cells are present and are exposed to the TFs from each other

Question 18

What can a myoblast cDNA library be used for

Answer 18

screening for the regulatory factors that commit cells to becoming muscle cells
such as MyoD = responsible for converting fibroblasts to muscle cells

Question 19

What is a master switch gene

Answer 19

a single gene that acts as a master switch and initiates a complex programme of differentiation

Question 20

What are MRF’s and 4 examples

Answer 20

myogenic regulatory factors. can impose a muscle-specific programme of gene expression. expression of a single MRF = converts fibroblasts into committed myoblasts
MyoD, Myf5, myogenin, MRF4

Question 21

What is the structure of MRF’s

Answer 21

contain a transactivation domain
contain a basic region where the DNA binds as it recognises the E box (CANNTG)
Has a helix-loop-helix motif which mediates heterodimerisation with the E class of bHLH proteins such as E12 and E47

Question 22

What happens when either myoD or myf5 are knocked out from a cell

Answer 22

skeletal muscle is still present as they compensate for the loss of each other

Question 23

What happens when myoD, myf5 and mrf4 are all knockout of a sample

Answer 23

no myoblasts and no skeletal muscle

Question 24

What happens when only myogenin is knocked out and why is this?

Answer 24

myoblasts still present but functioning skeletal muscle not formed as myogenin is required for terminal differentiation

Question 25

How are MyoD, myf5 and mrf4 activated

Answer 25

by external signals such as Wnt and shh. once activated they are then self sustaining and activate myogenin for terminal differentiation

Question 26

What happens to myoblasts in a culture when growth factor is withdrawn

Answer 26

triggers cell cycle arrest and myogenic regulatory factors mediate the beginning of differentiation

Question 27

What events mean that proliferating myoblasts express myoD and myf5 but do not differentiate

Answer 27

cdk4/cyclin-D phosphorylate MyoD and myf5 meaning they are degraded so in low levels. they also phosphorylate RB promoting cell division.
inhibitory HLH proteins such as Id are expressed at high level. I’d forms a heterodimer with MRF’s or R proteins and lacks a DNA binding basic domain so prevents binding to DNA

Question 28

What happens when growth factors are withdrawn and myod and myf5 cause cell cycle arrest/beginning of differentiation?

Answer 28

myoD/myf5 expression is upregulated. they activate transcription of the p51 family which inhibits cyclin-cdk. means that myoD, myf5 and RB are no longer phosphorylated
causes G1 arrest
Id expression declines and MRFs can bind to DNA

Question 29

How does MyoD activate transcription of muscle specific genes

Answer 29

myoD binds an enhancer upstream of itself by positive feedback
also activates mef2 expression which cooperated with myoD and other myogenic bHLH/E protein heterodimer

Question 30

What does myoD do regarding TFIID?

Answer 30

myoD activates TAF3 expression which interacts with TRF3 bound to the TATA box. forms a complex which replaces TFIID and activates the expression of muscle genes