13th Feb - TFs = key determinants of cell fate

Question 1

What are master transcription factors?

Answer 1

Switches on appropriate cell type specific genes and switches off non-appropriate genes through the induction of TFs

Question 2

Give an example of a master TF

Answer 2

Oct 4 –> stem cell pluripotency

GATA-1 –> erythrocyte development

Question 3

What is the key limitation in inducing certain types of adult cells from ESCs?

Answer 3

Our knowledge of TFs

Question 4

How is pluripotency maintained?

Answer 4

Through Oct4, Sox2 and Nanog

Question 5

How were Oct4, Sox2 and Nanog identified as the main pluripotency factors by Boyer (2005)?

Answer 5

ChIP-chip for Oct4, Sox3, Nanog

• Found they co-occupy many target genes
• -E.g. HOXb1, GOLGA6, IER5L
• -Half the promoter regions bound both Both Oct4 and Sox2 were also bound by Nanog

Question 6

What is GATA-1?

Answer 6

The master TF for erthryocytes which binds to the GATA sequence

Question 7

How was GATA-1 identified as binding to the GATA sequence?

Answer 7

through a gel-shift assay

Question 8

Outline the pathway of GATA-2

Answer 8

GATA-2 creates the oligopotent precursor (CMP) –> GATA-1 creates the megakaryocyte erythyroid precursor (MEP) –> GATA1 and KLF1 induce a red blood cell, GATA1 and Fli-1 induce platelets

Question 9

What are the binding sites shown through Chip-seq for GATA1?

Answer 9

5749 binding site
Only 13% of which bind at the promoter
Targets itself GATA-2, KLF-1 etc. potentiating red blood cell differentiation

Question 10

What did KLF1 Chip-seq by Tallack show?

Answer 10

That it is erythroid specific
Most binding sites are distal i.e. enhancers
There is a big overlap between GATA1 and KLF1 binding sites

Question 11

How is de-differentiation prevented in erythrocytes?

Answer 11

GATa1 and GATA2 are expressed mutually exclusively

Question 12

How can the transcriptome be reprogrammed?

Answer 12

By replacing the master TF

Question 13

Who first demonstrated cell reprogramming in 1987?

Answer 13

Davis, Weintraub and Lassar

Question 14

Outline the experiment of Davis, Weintraub and Lasser (1987)

Answer 14

Treated mouse C3H/DT1/2 embryonic fibroblasts with 5-azacytidine converting them to myoblasts
Found 3 genes which can conver embryonic fibroblasts to myoblasts

Question 15

What is MyoD1?

Answer 15

A myoblasts specific cDNA which can alone convert a fibroblast to a myoblast, it shares a coding region with myc

Question 16

Give an example of transdifferentiation

Answer 16

Fibroblasts + (myoD)–> Muscle cells
Monocytic precursors + (GATA1) –> erythroid megakaryocytic cells
Fibroblasts + (Oct4, sox2, Klf4, Myc) –> iPSCs

Question 17

Who won the nobel prize in 2012?

Answer 17

Yamanake for the discovery that mature cells can be reprogrammed to become pluripotent

Question 18

How did Yamanaka (2006) create an iPSC?

Answer 18

He identified 24 candidate genes
Detected pluripotent state by resistance to G418
Introduced each gene into mouse embryonic fibroblasts
Then to identify which were critical they examined the effect of withdrawal of individual factors

Identified Oct4, Sox3, Klf4 and Myc were essential

(Nanog wasn’t required as this is induced by Sox2 and Oct4)

Question 19

What is the frequency of successful iPSC derivation?

Answer 19

About 1 in 10000 cells

Question 20

What is the main obstacle to de-differentiation?

Answer 20

Epigenetics

Question 21

What is Waddington’s epigenetic landscape?

Answer 21

Concept that each valley represents a cell fate decision, once it reaches the bottom it has lost its potential as it accumulates DNA methylation