Visualizing Notch-induced chromatin looping through the use of a split

nano-Luciferase complementation assay

Aswin Bikkani
Raphael Kopan, PhD.
Department of Developmental Biology
Cincinnati Childrens Hospital Medical Center
 What is chromatin looping?

There are 2 levels of

looping

1st - The genome is

organized into
Topologically associated
domains (TADs)
correlated with
transcription

2014 Nature
 What is chromatin looping?

Looping of enhancers to
promoters is also
correlated with
transcription
Variable Distance

Any given enhancer can be

very far from a promoter
and transcription site
looping is how it gets there

2014 Nature
Chromatin looping is important

There are many homologous genes that

have been found between man and mouse
these are mostly in the coding region.

It is much harder to compare the noncoding

regions.

What does this mean?

It has been shown that a mutation in CTCF,

a chromatin architecture regulator, can turn
on oncogenes and lead to gliomas by
changing the boundaries between chromatin
domains.
 How do you detect looping?

Snapshot, lot of material,

background from things that
are close enough to
crosslink by chance

These systems require

killing the cell and are all
population level approaches

Image Credit: Kadauke, Blobel (2009).

Questions that cannot be answered with Chromosome Conformation Capture

Is looping necessary for an entire How stable are loops?

transcription cycle, or can it fall apart after a
while?

What is the relationship between a

Do loops open and close? particular looping event and transcription of
the relevant gene?

How does looping change through a cell Can we create a system to answer these
cycle? questions?
 The Notch pathway is
a highly conserved DAPT
cell-to-cell signaling
system that regulates
cell fate and
differentiation in
metazoans (animals).

Kopan and Ilagan, 2009

???
 Complementation Assay: SpDamID not in vivo

(Hass et al. 2015)

 How SpDam has been used to detect looping
Notch
D
Complex dCas9 AM

Guides

deactivated
Cas9 from
Crispr-Cas9

Notch
D GATC
Complex
Integrating over
dCas9 AM time Live
Cells until
you have to kill
them.
 Late,
Evidence of looping using Split DamID
Notch+Runx1-dep.

(hours) (hours)

Wisp2
-20kb RBPAMErt2 -10kb -6kb -4kb dCas9D
FAIRE:
Control
Notch

Early, mK4 wt+N1E

Notch-dep. mK4 Runx1+N1E

(hours)
 How we are hoping to detect looping
Notch
SmBit
Complex dCas9 LgBit

Guides

deactivated
Cas9 from
Crispr-Cas9

Notch
SmBit
Complex Light Watch
everything
dCas9 LgBit
happen in real
time
The Hypothesis

Notch
dependent?

Hypothesis:
Notch signaling
regulates known targets
such as Wisp2 by
inducing looping of
enhancers to promoters
 What we expect to see in various scenarios

DAPT Notch Off

Nucleus
DNA

Cell
Nucleus

DNA
 Cell Treatment What the DNA looks like

Nucleus DAPT Notch Off

DNA

DAPT Notch Off

Nucleus

Guides

DNA
 Cell Treatment What the DNA looks like
(maybe)

Nucleus

DNA

Nucleus

Guides

DNA

Light Light
 Nucleus

Guides

DNA

DAPT Notch Off

Light Light
In the Presence of
Wisp2 Guides

No DAPT

DAPT

DAPT Release
DIC DIC with Luminescence overlay in green
Overlay shows evidence of light coming from nuclei
No DAPT, No Guides No DAPT, Guides DAPT, Guides
Acknowledgements
Dr. Raphael Kopan, PhD
Dr. Matt Hass, PhD
Dr. Eric Brunskill, PhD
Dr. Mustafa Turkoz, PhD
Julie Lander
Daniel Brissette