How gene regulatory elements work

Question 1

Give an overview of how gene regulatory elements work?

Answer 1

We have a series of models to how we think they work

However, this isn’t 100% - but we will collate to the most likely

Question 2

What are the models of enhancer action?

Answer 2

Looping – the enhancer physically touches the promoter by looping out the intervening DNA

Tracking – enhancer-bound activators recruit RNA pol II and this complex scans the DNA until it reaches a promoter

Facilitated tracking – combination of looping and tracking – enhancer loops and then tracks along the chromosome until it finds a promoter

Linking – proteins bind between the promoter and enhancer form a protein structure that spreads along the chromosome until it finds an active promoter
It forms mini-loops in the chromosome, almost concertina the chromosome

Question 3

Enhancer action - describe the looping model?

Answer 3

Chromosome conformation capture (3C experiment) – detection of long range interactions

DNA binding proteins are treated with formaldehyde and cross links are formed to link interacting regions
6 bp cutter restriction enzymes are used to break up the DNA
The sample is diluted (promote intra-molecular interactions) and ligated
Cross links are reversed and proteins are removed - by heat treatment and proteolysis
PCR is used to detect if the spatially distant regions interact in the nucleus
The interactions fall off with distance

Question 4

Enhancer action - what do LCRs loop together to form?

Answer 4

An active chromatin hub
This hub is thought to interact with globin genes from the locus interact
Interacting with different genes at different stages of development
During foetal development, the gamma globin genes interact with the LCR
After birth, the delta and beta globin genes interact with the LCR

Showing the loop moves and interacts with different genes

Question 5

Enhancer action - what things has the 3C experiments revealed?

Answer 5

Mouse b-globin LCR also forms an active chromatin hub and that there is an interaction with the very 3’ end of the Locus

Other LCRs also form active chromatin hubs and looping to promoters

Interaction between other chromosomes with a low frequency

Question 6

Enhancer action - describe the interaction with the 3’ locus and ACH in mouse b-globin?

Answer 6

3’HS1 is a hypersensitive site downstream of the b-globin gene and is thought to delineate the 3’ end of the locus
CTCF binds the 5’ and 3’ hypersensitive sites and is thought to mediate the long range looping – compare with insulator function later (cohesin functions here too)

Question 7

Enhancer action - describe another LCR that forms an acrive chromatin hub, looping to promotors?

Answer 7

TH2 locus
TH2 locus co-ordinately regulates IL-4, IL-5 and IL-13
They activate B-cells

The LCR is at the end of the Rad50 gene (not involved in this gene)
Rad50 and Kif3a are not regulated by the LCR
Looping brings the promoters of IL-4, IL-5 and IL-13 together in a pre-poised state, early in T cell development

This is followed by looping later in development to touch the LCR
Rad50 and Kif3a are excluded from LCR/loop interactions

Question 8

Enhancer action - describe interaction between other chromosomes with a low frequency?

Answer 8

You can get chromosomes that interact between other chromosomes

Naïve T cells can differentiate to TH1 (expressing interferon gamma) or TH2 (expressing IL-4, IL-5 and IL-13)
Prior to differentiation the interferon gamma promoter interacts with the TH2 LCR
This is lost following differentiation to TH1 and TH2 potential way to coordinate locus activation

Called - ‘kissing chromosomes’

Question 9

Describe the argument for looping being the answer for enhancer action?

Answer 9

3C provides strong evidence that enhancers physically touch the promoter

But
1. Modelling of the chromatin fibre suggests that interactions over several kb will be inefficient
Experimentally – site-specific recombination between elements separated by several kb is inefficient in mammalian cells, compared to elements separated by 1 kb

2. Insulators work in a position-dependent way
   So how can this be if the enhancer simply loops over to touch the promoter? – Theoretically, it could by-pass the insulator

Question 10

Enhancer action - describe tracking/facilitated tracking evidence?

Answer 10

An insulator was placed between an enhancer from the b-globin LCR (HS2) and a globin gene promoter (e globin)
This prevented enhancer-mediated activation and the spread of histone acetylation from the enhancer to the promoter

Question 11

From tracking evidence, how are chromosomes folded?

Answer 11

Chromosomes are arranged into a series of Topologically Associating Domains (TAD)
Up to 1 mega base long and bound at the base
CTCF binding sits usually lie at the boundaries of the TAD
11 zinc finger transcription factor
Interactions are promoted within the TAD and are significantly reduced with regions outside the TAD

Question 12

Describe cohesin?

Answer 12

CTCF interacts with cohesin
Cohesin is a 3 protein ring - it is a motor
Cohesin holds sister chromatids together following replication until mitosis
Provides a potential way to form chromosome loops

Question 13

Describe loop extrusions in TADs?

Answer 13

Loop extrusion within the TADs forms sub-domains within TADs
Cohesin is loaded onto the chromosome and mediates loop extrusion until it hits a barrier on the chromosome
This might be a CTCF binding site

Cohesin can also mediate enhancer/promoter interactions within the loop
Other factors can do this too such as YY1 and mediator

Question 14

Enhancer action - describe the theory of the linking model?

Answer 14

Linking model – proteins bind between the promoter and enhancer form a protein structure that spreads along the chromosome until it finds an active promoter

Chip protein- regulator of the Cut locus in Drosophila is essential for enhancer function
Proposed that - Chip organises chromatin between enhancer and promoter
Homologues of Chip in vertebrates – LIM domain homeodomain proteins – e.g. LMO2

Question 15

What is the problem with the theory of the linking model?

Answer 15

Problem: This does not explain how the enhancers for some genes lie in the introns of other genes – e.g. the human alpha globin locus

No evidence for the linking model in its original form
Instead, LMO-2 can stabilize (link) enhancer/promoter interactions once the loop is formed

Question 16

Give an overview of the difference between Enhancers and LCRs?

Answer 16

Enhancers
Estimated to be 10^5 - 10^6 per mammalian genome – i.e. 1 per 3000 to 30000 bp
Defined in transient transfection assays
Now defined by specific chromatin signatures of H3K4me1 and p300 binding

LCRs
Defined as giving position independent, copy number dependent expression in transgenic mice
Some LCR hypersensitive sites have enhancer function e.g. human b-globin HS2
Other globin LCR hypersensitive sites do not act as enhancers in transient assays - they only function when integrated into the genome, e.g. HS3 and HS4

Question 17

What are insulators?

Answer 17

They have two functions - enhancer blocking and to act as a barrier to heterochromatin

There are 3 models for how insulators act as enhancer blockers

Question 18

What are the models for how insulators act as enhancer blockers?

Answer 18

Model 1 - promoter decoy model
The enhancer interacts with the insulator and this prevents its interaction with the promoter
Problem: what stops the enhancer looping over the top of the insulator?

Model 2 - tracking model
If enhancers find promoters by tracking, the insulator can block the enhancer as it tracks along the chromosome

Model 3 - structural model
The insulator organises the chromosome into topologically constrained loops where interaction with a promoter in a different loop is prevented
Problem: How is it possible in 3D space to prevent interaction between an enhancer and a promoter in an adjacent loop?

Question 19

Describe CTCF binding?

Answer 19

Purification of the factors that bind to enhancer blocking insulators demonstrated CTCF binding
The CTCF/Cohesin complex can promote insulator function by forming loops

These loops can:
1. Form an obstacle to tracking to prevent interactions being established between the promoter and the enhancer
2. Prevent promoters outside the loop from interacting with the enhancer
However, if an enhancer and promoter lie within the same loop, their interactions are promoted

Question 20

What is the role of CTCF in tethering insulators to nuclear structures?

Answer 20

CTCF is needed for enhancer blocking
CTCF sites lie adjacent to nuclear laminar associated domains (LADs)
CTCF is not needed for barrier function
This suggests CTCF association with LADs - functions in enhancer blocking

Question 21

What are CTCT sites associated with?

Answer 21

Nuclear Lamina
By associating repressed regions with the nuclear lamina, this separates active domains better than simply forming looped structures
Restricting the movement of the loops (even within 3D space) - by tying them down

Two mechanisms enhancer blocking:
Physical barrier via CTCF/cohesin binding/looping
Tethering to nuclear structures

Question 22

What is the evidence for insulators acting as a barrier?

Answer 22

Insulators protect from gene silencing by preventing the spread of heterochromatin

The barrier function of cHS4 insulator is mediated by USF1 and USF2
USF1 and USF2 recruit histone acetyltransferases to provide a barrier to the spread of heterochromatin
Heterochromatin spreading is mediated by HP1 binding to H3K9me3
This recruits the H3K9 methyltransferase to modify adjacent nucleosomes
A peak of acetylation is found at cHS4 - this is lost upon mutation of USF sites
By acetylating H3K9, this prevents the spread of repressive heterochromatin

If the lysine is acetylated it can then also be methylated

Question 23

What are transcription factories?

Answer 23

Proposed pre-formed regions in the nucleus with a high concentration of RNA pol II
Genes themselves move to these factories for transcription
The polymerase remains immobile within the factory and the DNA is spooled through

Question 24

What is any evidence for transcription factories?

Answer 24

Early evidence:
Pulse cells with biotin-CTP – see nascent transcripts in distinct foci rather than throughout the nucleus

More recent evidence:
Antibodies against pol II (ser2 phosphorylated CTD) show pol II is present at distinct foci
FISH evidence that genes move out of their chromosome domains when active

Genes loop out from chromosomes to go to regions of the nucleus where they are expressed

Question 25

What doesn’t fit with the transcription factory model?

Answer 25

How can more than one polymerase transcribe a gene at the same time?
The promoter would need to loop back into a factory from which it has just left
It is known that 2-10 polymerases occupy highly transcribed genes at the same time

A- and b-globin proposed to occupy the same factory BUT the mean distances between the “associated” globin loci is 500-800 nm
The average diameter of a transcription factory is ~70 nm

GATA-1 is a transcription factor for globin genes but it does not co-localise to the factory where the b-globin genes is proposed to be transcribed

Question 26

What is involved in a relaxed version of the transcription factory model?

Answer 26

Regions with increased concentrations of RNA pol II and transcription machinery exist within the nucleus
These factories are not necessarily pre-formed but genes move to these regions for transcription
Recent evidence suggests these factories are dynamic
Other genes also associate with these regions, leading to a transcription zone
These do not exist as rigid structures where pol II is immobilised but instead pol II can move along the gene - just more concentrated there

Question 27

What are advantages/potential dangers with transcription within factories?

Answer 27

Advantage:
Factories allow concentration of splicing and transcription machinery to increase efficiency of the numerous proteins involved in transcription coming together for transcription initiation

Potential dangers:
1. Splicing factors also associated with these factories and this poses the risk of trans-splicing
2. Chromosome translocations - IgH and c-myc associate with the same transcription factory
These genes are involved in a frequent translocation, leading to Burkitt’s lymphoma