6th Feb - TFs 1

Question 1

What are transcription factors?

Answer 1

Adaptor molecules that detect regulatory sequences in the DNA and target the assembly of protein complexes that control gene expression

Question 2

How were enhancers originally identified?

Answer 2

As SV40 DNA elements that could upregulate host beta-globin gene expression

Question 3

What are enhancers?

Answer 3

DNA elements that activate gene expression from a distance, irrespective of their orientation

Question 4

Roughly how many active enhancers are present per cell?

Answer 4

10000-15000

Question 5

What is 3C?

Answer 5

Chromosome conformation capture

1. Cells are crosslinked with formaldehyde to link chromatin segments that are in close spatial proximity
2. Chromatin is fragmented by restriction digestion or sonication
3. Crosslinked fragments are ligated to form unique hybrid DNA molecules
4. DNA is purified and analysed
   - –This is where the variation of methods occurs
   - –Traditional uses individual PCR to detect single ligation products one at a time
   - –4C uses inverse PCR to generate genome wide interaction profiles for single loci

Question 6

How were enhancers and their local interactions identified across the genome?

Answer 6

Using 3C

Question 7

What histone modifications are common at inactive enhancers?

Answer 7

H3K27me3

H3K4me1

Question 8

What histone modiciations are common at active enhancers?

Answer 8

H3K4me1

H3K27ac

Question 9

What are pioneer TFs?

Answer 9

(master TFs)

Bind independently of nucleosomes, preceeding other factors binding

Question 10

What are super enhancers?

Answer 10

Multiple enhancers in close proximity combined, they determine cell fate

Question 11

What are hotspots?

Answer 11

Large nucleosome free regions to which multiple factors bind

Question 12

How were enhancer RNAs discovered?

Answer 12

Through GRO-seq

Question 13

What are the three models of eRNA function?

Answer 13

• Noise from unspecific recruitment of PolII i.e. PolII binds on any open DNA soon realises its in the wrong place and terminates transcription
• Mechanism of enhancer function - released from the transcribed enhancer and somehow communicates with the promoter
• eRNAs play an important functional role

Question 14

What is specificity?

Answer 14

The difference in Kd between one site and another

Question 15

What are the different ways TFs have evolved to overcome transient DNA binding and lack of specificity?

Answer 15

Variable spacing - half sites seperated by variable length spacers

Multiple DBDs - e.g. Oct1 can bind to different DNA sites using different arrangments of its 2 DBD motifs

Multi-meric binding - Elk1 can bind as either a monomer or dimer

Alternative structural conformations - SREBP can bind to different DNA sites by adapting alternate structural conformations

Question 16

What percentage of genes encode TFs?

Answer 16

About 6-7%

Question 17

How is co-operative binding utilized by TFs?

Answer 17

Gives enhanced complex stability due to co-operativity which allows binding to lower affinity aites
OR
Inter protein interactions alter protein DNA contacts, altering DNA binding specificity

Question 18

What is the stucture of the Mad-max DNA binding motif?

Answer 18

Helix-loop-helix

Question 19

Why do zinc fingers contain zinc?

Answer 19

It holds the structure together, as disulphide bonds can’t be maintained in the reducing atmosphere of the nucleus