A project concerning a method called EDGE to achieve subregion/cell-type specific expression in the Entorhinal Cortex

1. Enhancer-Driven Gene
Expression in the EC
Αριάδνη Αλιμπέρτη
Χρήστος Μάντης
Γιώργος Κόντης

2. 1
Introduction

3. Promoter vs Enhancer based methods
Promoter
based methods
are widely used
among
scientists for
targeted
expression
It is known that
millions of
enhancers exist
while only
thousands of
promoters and
genes
This suggests that
the same gene is
expressed in
different cell types
via the activation 0f
distinct enhancers
Therefore,
the focus of
the paper is
targeted
gene
expression
induced by an
enhancer

4. EDGE basics
EDGE (Enhancer-DrivenGene Expression) is
a method based on the specificity of the
enhancers for targeted expression
A necessary step for the use of EDGE was to
identify specific enhancers in the targeted
brain region
Subregion specific expression
Cell type specific expression

5. Entorhinal Cortex as a target
01
02
03
04
Entorhinal
Cortex (EC)
MEC
(medial)
LEC
(lateral)
RSC
(retrosplenial) ACC
(anterior
cingulate)
Using enhancers specific for those
subregions (MEC/LEC), subregion
and cell type specific expression
was achieved

6. 2
Methods

7. Identifying cis-regulatory elements
10%
31%
Microdissection of specific brain
regions in the EC
Promoters and
putative
enhancers were
identified using
ChIP-seq based
on H3K27ac
signal
10% of all promoters
were active only in
one subregion
(2.045)
31% of all the active enhancers
in one subregion, were
identified as unique to that
specific region (18.185)

8. Targeting unique enhancers
1 2
34
5 6
Previous data was combined with
that from other studies, making a
unified data set of enhancers
Enhancers were filtered
based on the H3K27ac signal
The set was clustered two times:
1. Hierarchically (neuronal/non
neuronal cells)
2. K-means (tissue type)
Subregion specific enhancers
were filtered to ensure that they
are not active in other tissues
Enhancers were assigned
to targeted genes based
on the GREAT algorithm
Selection of enhancers was
prioritized based on:
1. Specificity of H3K27ac signal
2. Conservation across 30 species

9. Injection construct
Putative
enhancer1 tTA
gene3
Minimal
promoter2 WPRE (post-
transcription
regulatory
element)
4
mRNA
stabilizing
sequences
5

10. The making of transgenic lines
The construct was
inserted in mouse
embryos via
pronuclear injection
in order to make
transgenic lines
8 MEC and 2 LEC
enhancers were selected
for the creation of
injection constructs
At least 3 embryos
were made containing
the same construct,
that had similar
expression patterns
The created
lines were
crossed with
tTA-reporter
lines
From 105 genotypically
positive founders only
41 expressed in the EC

11. 3
Results

12. Subregion specific expression
One MEC enhancer was associated
with the Kitl gene
The Kitl gene expresses broadly
throughout the brain
Results showed an enriched
expression of the gene in the layer-II
of MEC
01
02
03

13. Subregion specific expression
Similar results
were observed
for 4/8 MEC and
2/2 LEC
enhancers
Specific
enhancers can
drive subregion
specific
expression of non
specific genes
Even less specific
enhancer can give
rise to subregion
specific
expression
≈

14. Using EDGE one can
dissect out the individual
genetic components that
underlie the expression of a
non specific gene in multiple
cell types

15. Cell type specific expression
Two MEC enhancers were selected
for targeted cell type expression
Both MEC enhancers expressed
in excitatory neurons
Excitatory neurons in the EC are typically
subdivided in two groups:
Calbindin cellsReelin+ cells
Reelin+ cells
Calbindin cells

16. Cell type specific expression
The one MEC enhancer
expressed exclusively in
reelin+ cells
The other MEC enhancer
expressed in both cell
types
While the one
showed specificity
for stellate cells
the results of the
other may be
interpreted as
specific for
biological
subpopulations

17. 4
Critiques

18. Cell type specific expression
Only 2/8 MEC
enhancers and 0/2
LEC were tested for
cell type specificity
Although the
results were
promising,
they could be
misleading
Results could
confirm/reject the
effect of:
Co-regulated
network
Mosaicism of
insertion site

19. Subregion specific expression
Although 6/10
selected enhancers
showed specific
expression in the EC,
expression in other
brain areas occurred
To ensure this
happened due to
positional effects
further experiments
should be done

20. Authorship
The syntax and
grammar was
often
incomprehensible
In many cases
there was no
coherence
between the
paragraphs
Sometimes
having simple and
short sentences is
the best choice

21. 5
Bibliography

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26. Thank you for your
attention!