Transcription Factor Modification

Question 1

How can eukaryotic genes be regulated by transcription factor modification?

Answer 1

Genes can be regulated by the presence or absence of a transcription factor
OR
By converting a transcription factor between active/inactive states
Therefore a signal is needed to activate from inactive states of the TF

Question 2

What are the advantages of transcription factor activation regulation?

Answer 2

Allows rapid response
Allows regulation in response to external factors
Allows fine tuning of transcription level of specific genes

Question 3

What are the types of transcription factor activation?

Answer 3

They can be activated in a number of ways from its inactive state
1. Factor activated by binding to ligand - nuclear hormone receptors

2. Factor activated by protein modifications - phosphorylation, acetylation and ubiquitination (most common)
3. Factor activated by cleavage of a larger precursor - notch receptor or SREBP TF
4. Factor activated by dissociation of an inhibitor protein - NFkB; HSF-1

Question 4

Describe factor activated by binding to a ligand?

Answer 4

The nuclear hormone receptors are activated by binding of ligand (hormone)
Examples
1. Thyroid hormone binding directly activates the receptor
2. Glucocorticoid binding releases an inactive form of the receptor from HSP90
The active GR plus ligand then moves to the nucleus to activate gene expression

Question 5

What is the mechanism of activation - binding to a ligand?

Answer 5

In the inactive state - there can also be co-repressor proteins that are part of HDAC’s

The binding of a ligand induces a conformational change in nuclear hormone receptors
This triggers DNA binding and gene activation
Binding of ligand also permits interaction with co-activators such as CBP (HAT), SRC-1 (HAT) and SRC-3 (HAT)

Question 6

Describe factor activated by cleavage of a larger precursor? Examples?

Answer 6

Example - Notch signalling
Notch receptor consists of E and M chains - connected by disulphide bonds
E - extracellular domain and M - membrane domain
When the signalling protein (ligand) delta binds the E chain - the M chain dissociates
This allows cleavage of the M chain and release of the Notch tail that activates transcription

Example - SREBP TF
It normally binds cholesterol and sits in the ER
If cholesterol levels fall - the whole complex moves to the Golgi
Here the SREBP is cleaved and is moved to the nucleus

Question 7

Describe factor activated by dissociation of an inhibitor protein? Example?

Answer 7

Example - HSF-1
HSF-1 is normally bound by HSP90 and is inactive
Following heat shock, HSP90 dissociates from HS-1
HSP90 (chaperone) binds unfolded proteins, formed during heat stress
Release from HSP90 (inhibitor) allows HSF-1 monomer to trimerise (following interaction with eEF1A and HSR-1) and bind to DNA at the HSE (heat shock element)
Activation of HSF-1 requires further phosphorylation

Question 8

Describe factor activation by protein modification?

Answer 8

The activation of transcription factors can take place via a number of covalent modifications:
Phosphorylation
Acetylation
Ubiquitination

Question 9

Give an overview of phosphorylation?

Answer 9

Often in response to cell surface receptor signalling
Can increase protein activation and increase protein/protein interactions
Increase interaction with co-activators
Addition/removal of phosphorylation allows dynamic activation
Allows fine-tuning of transcription through protein degradation

Transcription factor phosphorylation can by direct via second messengers or via kinase cascades
Phosphorylation of transcription factors allows a transcriptional change based on growth/environmental signals

Question 10

Phosphorylation - give an example of cell surface receptors?

Answer 10

Some transcription factors are phosphorylated directly from cell surface receptors
Cytokines (cell growth factors) activate the JAK/STAT pathway
Cell surface receptors transmit the signal to Janus Activated Kinases (JAKs) that phosphorylate STAT transcription factors
Phosphorylation causes dimerisation of STATs and translocation into the nucleus where they bind specific target genes

Question 11

Phosphorylation - what is the receptor tyrosine kinases role?

Answer 11

The binding of ligands to TRK activates the Ras/Raf/MAPK kinase cascade
Signalling cascade allows amplification of signal - at each point
Growth factors bind receptor tyrosine kinases
This triggers auto-phosphorylation and activation of Ras
Ras binds GTP, converting it to an active form
This phosphorylated Raf, that in turn phosphorylates other kinases in the pathway

Question 12

Phosphorylation - describe the MAPK cascade?

Answer 12

This triggers activation of SRF and early response genes
Starvation of cells of growth factors, followed by addition of serum, results in the activation of “Early Response Genes”
Many of these were oncogenes and were first activated by growth factors
The MAPK pathway phosphorylates Ternary Complex Factor (TCF) and Serum Response Factor (SRF)
This triggers protein/protein interactions between them and the binding to Serum Response Elements (SRE) in the promoters of the early response genes – e.g. fos and jun

Therefore the phosphorylation of the TCF allow it to bind to DNA on the SRE in the promotor

Question 13

Give an overview of acetylation?

Answer 13

Often via HATs that are co-activators at promoters
Usually increases transcription factor activation
Increases transcription factor binding to DNA
Increases protein/protein interactions
Positive or negative effects on protein stability

Examples of acetylated transcription factors: p53, GATA-1, EKLF, nuclear hormone receptors, c-myc, c-myb, GATA-3, E1A, E2F, c-jun, NFkB

Question 14

Give an overview of the experimental evidence to support acetylation of transcription factors?

Answer 14

GATA-1 = a erythroid transcription factor
Incorporation of 14C acetyl groups shows GATA-1 TF is acetylated
This was strange as it was only known that histones were acetylated

Mass spectrometry of acetylated peptides of GATA-1 was used to map the acetylation sites
To find if GATA-1 is acetylated in vivo - IP with anti-GATA-1 antibody and probed western with anti-acetyl lysine antibody
It was found GATA-1 acetylation is required for erythroid differentiation
Acetylation of GATA-1 increases GATA-1/DNA binding and therefore transcriptional activity

Therefore the HATs were also acetylating transcription factors
The recruitment of coactivators with acetyltransferase activity can lead to the acetylation of transcription factors at promoters, boosting their activity

Question 15

Give a summary of activation of GATA-1 by acetylation?

Answer 15

GATA-1 is acetylated by histone acetyltransferases
Acetylation:
Stimulates GATA-1/DNA binding
Stimulates GATA-1-dependent transcription
Enhances GATA-1/protein interactions
Is required for proper GATA-1 function during haematopoietic differentiation
Modulates GATA-1 levels

Question 16

Give an overview of ubiquitination?

Answer 16

Ubiquitin - 76 amino acids, is added to lysine
Generally triggered by other protein modifications (phosphorylation)
Polyubiquitination leads to protein degradation
TF polyubiquitination and activation appear to be very tightly linked
This is a mechanism to regulate the level of gene transcription

Addition of four or more K48-linked ubiquitin molecules leads to protein degradation

Question 17

What does ubiquitination require?

Answer 17

Ubiquitination requires E1, E2 and E3 ubiquitin ligases
The first ubiquitin is linked to the target protein via a lysine in the target protein and glycine at the C-terminus of ubiquitin
Forming a thiol group
The ubiquitin chain is built up by linking glycine at the C-terminus of ubiquitin to lysine’s within ubiquitin
K48 is the typical lysine used in ubiquitin to form a poly-ubiquitin chain that signals protein degradation

Question 18

Describe the 26S proteasome?

Answer 18

This consists of a 20S catalytic subunit and 19S Caps
Different components of the proteasome have distinct functions
This de-ubiquitinates and then the peptide is pulled through - and degraded
The ubiquitin can be recycled

The degradation of highly active transcription factors - allows the level of transcription to be regulated
Meaning transcription initiation absolutely depends on the de novo activation of a transcription activator

Question 19

Describe the ubiquitin-mediated degradation - black widow model?

Answer 19

Example - GCN4 is lost from Promoters via ubiquitin-mediated degradation
The kinase Srb10 phosphorylates promoter-bound GCN4
This recruits the E3 ubiquitin protein ligase SCFcdc34
Ubiquitination of GCN4 triggers its loss from promoters (via ubiquitin degradation)

Srb10 is part of the RNA pol II enzyme
When the activator, GCN4 contacts the basal transcription machinery, this triggers its phosphorylation and degradation
Mechanism to automatically destroy the active form of the transcription factor - almost as soon as it is activated

Question 20

What are unstable transcription factors?

Answer 20

The transcription activation and degradation domains overlap in many unstable transcription factors
If artificially linked a degron to a transcription factor, this can lead to its degradation

Question 21

What is some other evidence that active transcription factors are degraded?

Answer 21

Subunits of 19S proteasome have been detected at promoters by ChIP
The c-myc ubiquitin protein ligase was found at promoters by ChIP
The oestrogen receptor was found to cyclically occupy the S2 promoter, indicative of its degradation following pol II release
– Metivier, Gannon, Reid

Question 22

What is the model linking transcription factor degradation and Pol II transcription?

Answer 22

1. The activator interacts with the basal transcription machinery
2. This recruits ubiquitin ligases that modify the transcription factor
3. This ubiquitination recruits the 26S proteasome
4. The proteasome simultaneously destroys the activator and promotes pol II elongation

This limits uncontrolled transcription by (1) destroying the activator each time the promoter fires and (2) ensuring that it is activators at the promoter that are destroyed by the proteasome

Question 23

How is transcription factor degradation actually signalled?

Answer 23

Mutation of the phosphorylation or acetylation sites decrease GATA-1 ubiquitination
Acetylation increases GATA-1 transcriptional activity and causes its ubiquitination

Phosphorylation preferentially triggers the degradation of acetylated GATA-1
Mutate phosphorylation sites – acetylated GATA-1 increases
Prevent phosphorylation with inhibitors - acetylated GATA-1 increases

Phosphorylation preferentially triggers the degradation of active GATA-1:
Mutate phosphorylation sites – transcription increases

Question 24

How is MAPK phosphorylation involved in transcription factor degradation signalling?

Answer 24

This signals degradation of acetylated GATA-1
MAPK signalling via cytokines binding to cell surface receptors can modulate the degradation of active GATA-1 and therefore the level of GATA-1 dependent transcription

Because both acetylation and phosphorylation are needed to signal GATA-1 ubiquitination, this provides a way for phosphorylation to signal degradation of the active transcription factor