Histone modifications and transcription factors

Question 1

What is gene expression?

How is it viewed today?

Answer 1

The process of bringing the genome to life, through mechanisms which manufacture and regulate the protein complement of a cell.

Prior it was thought that each step worked as a singular component
… however, the process is now discovered to work as a ‘genome conveyor belt’, with each step being linked to the next to ensure quality control.

Question 2

5 facts about Swi/SNF:

Answer 2

1) are present in all eukaryotes
2) required for many transcription events
3) do not bind to specific DNA sequences

4) Complex of 10 proteins:
- ATPase, uses ATP dependent energy production
- Helicase like domain
- Bromodomain, interacts with modified histones

5) modifies DNA by causing histone sliding

Question 3

Why is Swi/SNF required and what are the steps?

Answer 3

DNA and histone core have around 100 close contact between them.
Furthermore, they have a high thermal stability ( dissociation occurs at <65C).
So Swi/SNF is needed to minimise this energetic penalty through the use of ‘loop transmission’

1) (translocation domain of…) Swi/SNF translocates DNA from histone surface, through the use of ATP-dependent energy. Loop forms

3) Translocation domain releases DNA - which rebinds to nucleosome
But loop propagates around histone core - altering relative position of nucleosome compared to DNA

4) Transcription factors are now able to access DNA through this looping

Question 4

How does bacterial transcription work? Why is it different

Answer 4

Bacteria’s don’t have histones. So transcription is much easier and only consists of 3 steps

1) repressor bound the DNA
2) activator binds, translocation of repressor and recruitment of polymerase
3) transcription starts

Question 5

3 types of polymerase…

Answer 5

Polymarase 1: ribosomal RNA
Polymerase 2: mRNA
Polymerase 3: tRNA

Question 6

What are basal transcription factors and what are the steps?

Answer 6

Basal transcription factors are used for transcription of all polymerase 2 promoters

the transcription factor 2 D (TFIID) complex constitutes as this. It acts with several proteins including TBP

1) TBP binds to TATA box in promoter region, facilitating the binding of TFIID
2) TFIIB recruited which stabilised structures
3) Structure conformation recruits TFIIF which acts with RNA polymerase 2 - both bind

4) TFIIE and TFIIH recruited, pre-initiation complex is formed. TFIIH is a complex of 10 proteins consisting of:
- ATPase
- Kinase: phosphorylates pol 2 serine 5 residue in C terminal to start transcription
- Helicase

5) Once transcription starts, all tf’s leave bar TFIID, which is kept to facilitate continual transcription
6) Once transcription stops, polymerase is recycled, dephosphorylated and re-associated with TFIIF

Question 7

What are specific transcriptional factors?

Answer 7

Transcription factors that aid in increasing transcription of specific genes
Through the binding of enhancer sequences:
Cis-acting elements that operate from long/short distance, up/downstream, or different chromosomes

E.g Glucocorticoid receptor. Made up of:

• Transactivating domain
• DNA binding domain (DBD), P box
• Ligand binding domain (LBD)
• 2 zinc fingers
• D box for dimerisation

Glucocorticoid receptors have been found to use enhancers to facilitate transcription factories.
On exposure to glucocorticoid - 3 target genes on different chromosomes make inner chromatin contact

Question 8

Use the regulation of INFbeta promoter to explain mechanisms at work in transcriptions:

Answer 8

Upon viral infection…

PRD- positive regulatory domain

1) NFkB locates to PRDII sequence on promoter region - this recruits HMGA which binds to promoter changing its intrinsic curvature and so facilitating binding of NFkB.

2) Binding recruits transcription factors IRF 3 and 7 (function as dimers) and C-jun and ATF2 (function as dimers).
Transcription factors are able to bind to their respective sequences (PRDIII and PRDIV) after HMGA1 protein binds to promoter sequence and further straightens curvature.

3) Binding of transcription factors creates enhanceosome which recruits PCAF (histone acetyl transferase), that acetylates:
- H3 lys9
- H4 lys8
- HMGA1 lys71 strengthens NFkB binding

4) after a kinases phosphorylates H3 ser10, PCAF is able to acetylate H3 lys 14
5) CBP (creb binding protein) which works in complex with RNA polymerase 2 and Swi/SNF, translocates PCAF.
6) Swi/SNF conducts histone modification which makes TATA box present
7) TBP form the TFIID complex binds to TATA box, creating a bend in DNA which results in nucleosome sliding (36bp downstream)
8) Transcription can start

Question 9

What is combinatorial transcriptional regulation?

Answer 9

Combinations of transcription factors causes expansion in regulatory complexity

1 in 4 genes humans are TF’s

However can’t fully explain complexity:
- mRNA and protein diversity… result in a complex transcriptome and proteome

Question 10

Activation of transcription:

Give example

Answer 10

Multiple ways to activate activation factors:
… ligand binding, phosphorylation, addition of a second subunit, unmasking.

Example: steroid receptor

Bind of steroid hormone to LBD domain, results in chaperone proteins hsp90 (heat shock proteins) translocating.
This reveals nuclear localisation signal (NLS) domain, and this conformational change of receptors increases affinity to steroid response element.