1. Examining
the
role
of
WDR-­‐23
by
using
C.
elegans
mutants
Hanna
Kiani1,
Jacqueline
Y.
Lo1,2,
Sean
P.
Curran1,2
University
of
Southern
California
1.
Leonard
Davis
School
of
Gerontology;
2.
Dornsife College
of
Letters,
Arts,
and
Sciences
Department
of
Molecular
and
Computational
Biology
Overview
SKN-­‐1
is
a
transcription
factor
that
plays
a
vital
role
in
C.
elegans
stress
response
and
longevity.
As
a
transcription
factor,
SKN-­‐1
binds
to
specific
DNA
sequences
and
initiates
transcription
of
stress
response
genes.
SKN-­‐1
is
regulated
by
diverse
signals
that
control
metabolism,
development,
and
stress
responses.1 WDR-­‐23
is
a
negative
regulator
of
SKN-­‐1;
it
functions
with
Cul4/DDB1
ubiquitin
ligase
(a
complex
which
regulates
DNA
damage
response,
DNA
replication,
and
chromatin
remodeling)
to
regulate
nuclear
abundance
and
activity
of
SKN-­‐1
in
C.
elegans.
When
SKN-­‐1
enters
the
nucleus,
it
is
prevented
from
accumulating
by
WDR-­‐23;
WDR-­‐23
interacts
with
the
Cul4/DDB1
complex
and
targets
the
transcription
factor
for
proteasomal
degradation.
WDR-­‐23
represses
SKN-­‐1
protein
levels,
nuclear
accumulation,
and
activity.2 In
WDR-­‐23
mutants,
however,
WDR-­‐23
is
not
able
to
suppress
SKN-­‐1
levels
and
SKN-­‐1
is
always
active.
Because
of
uninterrupted
SKN-­‐1
activation,
WDR-­‐23
mutants
are
able
to
withstand
more
oxidative
stress
than
N2
wild-­‐type.
We
have
studied
such
WDR-­‐23
mutants
in
environments
of
oxidative
stress
and
measured
their
survival
in
comparison
to
N2
wild-­‐
type
C.
elegans to
characterize
the
role
of
WDR-­‐23
and
its
effects
on
SKN-­‐1.
References:
1.
PLOS
Genetics:
The
Conserved
SKN-­‐1/Nrf2
Stress
Response
Pathway
Regulates
Synaptic
Function
in
Caenorhabditis Elegans.
N.p.,
n.d.Web.
10
Jan.
2016..
2.
Choe,
Keith
P.,
Aaron
J.
Przybysz,
and
Kevin
Strange.
Molecular
and
Cellular
Biology.
American
Society
for
Microbiology
(ASM),
n.d.Web.
10
Jan.
2016.
Acknowledgements:
Curran
Lab
USC
Davis
School
of
Gerontology
Figure
1:
wdr-­‐23 mutants
on
As
treated
SKN-­‐1/
L4440
RNAi.
RNA
interference,
or
RNAi,
is
a
biological
process
where
RNA
molecules
inhibit
gene
expression
by
causing
the
destruction
of
specific
mRNA
molecules.
wdr-­‐23 mutants
have
greater
SKN-­‐1
activation.
By
exposing
these
mutants
to
SKN-­‐1
RNAi,
we
are
able
to
knock
down
SKN-­‐1
levels
and
compare
survival
with
mutants
that
have
been
treated
with
a
control
RNAi.
Seven
different
wdr-­‐23 mutants
were
tested
on
5
mM
arsenite
treated
RNAi
plates.
The
mutants
we
used
were
lax
124,123,211,134,129,101,
and
126. L4440
was
used
as
a
control
RNAi
strain
and
skn-­‐1 RNAi
was
used
to
knock
down
activated
SKN-­‐1
in
the
mutants.
The
number
of
dead
and
alive
worms
per
plate
were
counted
after
4
hours.
As
shown
here,
mutants
on
skn-­‐1 RNAi
had
higher
levels
of
death
than
mutants
on
L4440/control
RNAi.
The
higher
death
rates
in
skn-­‐1 knock
down
worms
supports
the
claim
that
SKN-­‐1
plays
an
integral
role
in
survival
and
longevity
of the
wdr-­‐23 mutants.
Figure
3:
qPCR
of
lax134 and
N2
shows
higher
levels
of
SKN-­‐1
activation
in
wdr-­‐23 mutants.
qPCR
is
a
method
used
for
transcriptional
quantification.
qPCR
quantifies
mRNA
transcripts
to
indicate
how
many
mRNA
copies
of
a
gene
is
being
made.
RNA
is
used
as
a
template
to
be
reverse
transcribed
into
DNA
(cDNA),
which
is
then
used
for
qPCR.
We
used
genes
that
are
known
to
be
turned
on
by
SKN-­‐1
as
targets
we
are
interested
in
quantifying
the
expression
of.
We
extracted
RNA
from
synchronized
L4
N2
wild-­‐type
and
lax134 (a
WDR-­‐23
mutant)
grown
on
OP50
to
reverse-­‐transcribed
into
cDNA.
We
used
snb-­‐1,
a
gene
which
normalizes
for
the
amount
of
cDNA,
as
our
control
gene.
We
then
used
gst-­‐4,
gcs-­‐1,
and
ugt-­‐11 to
help
determine
levels
of
SKN-­‐1
activation.
These
three
detoxification
enzymes
get
activated
by
SKN-­‐1;
therefore,
there
are
more
copies
of
these
three
genes
when
SKN-­‐1
is
active.
(a)
The
Ct
number
is
the
number
of
cycles
it
takes
for
the
qPCR
to
reach
the
threshold;
the
higher
the
Ct
number,
the
less
cDNA
is
in
the
sample.
The
dCt
number
is
the
normalized
Ct
value
calculated
by
subtracting
the
snb-­‐1 Ct
number
from
the
sample
Ct
number.
The
ddCt
number
is
the
compared
Ct
value
between
lax134and
N2.
We
are
then
able
to
calculate
the
relative
amounts
of
each
gene
in
each
cDNA
sample.
(b)
Looking
at
the
below
qPCR
data
and
calculations,
we
are
able
to
see
that
lax134 has
much
greater
levels
of
gst-­‐4,
gcs-­‐1,
and
ugt-­‐11.
Higher
levels
of
detoxification
enzymes
that
are
activated
by
SKN-­‐1
indicate
greater
SKN-­‐1
activation.
We
can
conclude
that
the
wdr-­‐23
mutant
lax
134 has
greater
SKN-­‐1
activity
than
N2.
Figure
2:
Cross
between
lax
213
hermaphrodite
and
gen-­‐1::gfp male.
gen-­‐1 is
a
resolvase enzyme
that
snips
Holliday
junction
in
cases
of
recombination.
In
addition
to
SKN-­‐1,
WDR-­‐23
interacts
with
GEN-­‐1.
Since
gen-­‐1 is
not
a
transcription
factor,
we
cannot
measure
its
activity
through
analysis
of
gene
transcription
rates
like
we
did
for
SKN-­‐1.
We
have
crossed
lax213,
a
WDR-­‐23
mutant,
with
gen-­‐1::gfp, to
identify
changes
in
gen-­‐1 function
in
the
presence
of
a
lax213 mutation.
GFP
acts
as
a
tag
to
reveal
gen-­‐1 activity
in
the
animal.
Unlike
other
WDR-­‐23
mutants,
gen-­‐1 no
longer
interacts
with
WDR-­‐23
in
lax213.
Because
a
lax213 allele
mutation
disrupts
interaction,
that
region
in
the
protein
is
likely
to
be
important
or
necessary
for
interaction.
We
first
crossed
a
WDR-­‐23
mutant
hermaphrodite
(lax213 )
with
a
gen-­‐
1::gfp male.
The
F1
generation
results
in
a
213,+
gfp,+
genotype.
We
then
allowed
the
F1
generation
to
self,
resulting
in
the
F2
generation
which
consists
of
16
possible
genotypes.
To
isolate
the
desired
213,
213
gen-­‐1::gfp,
gen-­‐1::gfp we
single-­‐lysed
64
different
worms.
To
check
for
GFP,
we
ran
a
PCR
with
a
set
of
primers
that
will
amplify
a
single
band
when
GFP
is
present.
To
identify
the
lax213 mutation,
we
ran
a
PCR
and
restriction
enzyme
digestion
comparing
our
samples
with
N2.
After
RE
digestion,
the
N2
sample
would
cut
and
the
lax213 mutation
would
remain
uncut.
(a)
The
cross
between
the
F1
progeny
and
itself
(self)
reveals
sixteen
possible
genotypes
for
the
F2
population.
Out
of
the
sixteen,
we
need
to
select
for
the
213,
213
gen-­‐1::gfp,
gen-­‐1::gfp genotype.
(b)
When
the
two
parent
worms
mate,
the
F1
progeny
are
hermaphrodites
which
we
let
mate
with
themselves.
We
need
a
worm
who
is
not
wild-­‐type
for
either
mutation.
(a)
(b)
Ct snb-1 Ct dCt ddCt 2^-ddCt
gst-4
N2
lax 134
20.92
16.37
20.71
21.41
.21
-5.04
0
-5.258
1
38.0546277
gcs-1
N2
lax 134
21.63
19.91
20.71
21.41
0.92
-1.5
0
-2.42
1
5.35171022
ugt-11
N2
lax 134
27.34
25.06
20.71
21.41
6.63
3.65
0
-2.98
1
7.88986164
(a)
(b)
Future
In
the
future,
we
will
use
qPCR
to
test
for
SKN-­‐1
dependency
of
N2
and
lax134
C.
elegans on
L4440
and
SKN-­‐1
RNAi.
This
will
allow
us
to
identify
levels
of
expression
of
gst-­‐4,
gcs-­‐1,
and
ugt-­‐11
in
worms
that
have
SKN-­‐1
knock
down
to
worms
that
were
on
L4440
control.
We
will
also
be
able
to
compare
levels
of
expression
between
N2
wild-­‐type
and
lax134
after
exposure
to
both
L4440
and
SKN-­‐1
RNAi.
We
will
also
proceed
to
identify
the
relationship
between
gen-­‐1 and
lax213.
We
are
aware
that
WDR-­‐23
also
interacts
with
gen-­‐1,
but
do
not
know
why
or
what
changes
when
the
interaction
is
not
there.
We
will
use
gfp as
a
tag to
distinguish
changes
in
the
absence
of
the
interaction.