This document describes a study investigating the mechanisms underlying X chromosome counting in female embryonic stem cells (ESCs). The researchers conducted an RNA interference screen targeting proteins involved in chromosome structure to identify those necessary for the unique organization of X-linked genes observed in XX ESCs, which is a signature of counting. Knockdown of the loading factor Nipbl for the cohesin complex decreased doublet signals and increased singlet signals without changing cell morphology. Knockdown of Smc2, a subunit of the condensin complex, had no effect. In total, 15 targets were identified that may affect counting.

1. An
Assay
for
Coun,ng
I
HAVE
2
X’S,
BUT
WHO’S
COUNTING?
LENA
BENGTSSON,
KAREN
LEUNG
PHD,
SAILAJA
PEDDADA
PHD,
LEEANNE
GOODRICH,
JESSELYNN
LABELLE,
BARBARA
PANNING
PHD
PANNING
LAB,
DEPARTMENT
OF
BIOPHYSICS,
UNIVERSITY
OF
CALIFORNIA,
SAN
FRANCISCO
Conclusion
The
knockdown
of
Nipbl,
a
loading
factor
for
the
cohesin
complex,
which
regulates
chromosome
structure,
results
in
a
decrease
in
the
singlet/doublet
signals
and
an
increase
in
singlet/singlet
signals,
without
an
obvious
change
in
cell
morphology.
Follow
up
experiments
need
to
be
performed
in
order
to
understand
how
Nipbl
controls
this
unusual
chromosome
organiza@on
that
may
regulate
coun@ng.
Knockdown
of
Smc2,
a
subunit
of
the
condensin
complex,
has
no
effect
on
the
singlet/
doublet
frequency
and
may
not
play
a
role
in
X
chromosome
coun@ng
in
female
ESCs.
All
results need to be repeated, but 15 targets were identified that may have an
effect on counting.
Funding
for
this
project
was
provided
by
CIRM
grant
TB1-­‐01188.
A
special
Thank
You
to
all
the
amazing
scien@fic
minds
in
the
Panning
Lab
–
Barbara
Panning,
Karen
Leung,
Sailaja
Peddada,
Leeanne
Goodrich,
Betsy
Mar@n,
Joel
Hrit,
Dale
Talbot
and
Assen
Rougev.
Their
guidance,
pa@ence,
and
support
made
this
project
possible.
Abstract
Female
placental
mammals
(ex.
mice
and
humans)
undergo
X
Chromosome
Inac@va@on
(XCI)
to
equalize
X-­‐linked
gene
dosage
between
XX
females
and
XY
males.
Because
it
is
necessary
for
female
survival,
XCI
is
an
important
developmentally
regulated
epigene@c
process.
Early
in
post-­‐implanta@on
development,
female
embryos
inac@vate
one
of
their
two
X
chromosomes
(Xs).
XCI
also
occurs
ex
vivo
when
female
embryonic
stem
cells
(ESCs),
which
have
two
ac@ve
Xs,
are
differen@ated.
The
silent
X
is
highly
condensed,
forming
a
structure
called
the
Barr
Body.
XCI
does
not
occur
in
cells
with
one
X,
such
as
XY
male
cells
or
female
cells
that
have
lost
one
X
(XO).
Cells
with
mul@ple
X’s
(XX,
XXX,
XXY)
silence
all
but
one
X,
sugges@ng
that
cells
are
able
to
“count”
their
Xs.
The
mechanisms
behind
how
Xs
are
counted
are
currently
unknown.
The
Panning
lab
has
iden@fied
a
signature
of
coun@ng,
an
unusual
organiza@on
of
X-­‐linked
genes
in
female
ESCs.
I
carried
out
a
screen
to
iden@fy
proteins
necessary
for
this
cytological
signature
of
coun@ng.
The
screen
focused
on
proteins
involved
in
regula@on
of
chromosome
structure,
since
we
hypothesize
that
chromosomal
structural
proteins
may
be
important
in
determining
the
unusual
organiza@on
of
X-­‐linked
genes
in
XX
ESCs.
Using
this
approach
I
hope
to
gain
a
mechanis@c
understanding
of
how
Xs
are
counted.
Being
aware
of
the
mechanisms
behind
XCI
will
help
us
to
understand
how
cells
developmentally
regulated
gene
expression
and
improve
our
understanding
of
stem
cell
biology.
Smc2
esiRNA
Oct4
esiRNA
Untransfected
Nipbl
esiRNA
Assay
for
Gene
Knockdowns
in
Female
ESCs
• 12
well
plate
RNAi:
• 9
targets
• Oct4
(RNAi
Control)
• GFP
(Nonspecific
Control)
72
hours
Xist
Fig.
1
-­‐
esiRNAs
silence
effec@vely
in
ESCs.
Brighfield
and
GFP
fluorescence
images
of
ESCs
bearing
an
EGFP
transgene
transfected
with
indicated
esiRNAs.3
Fig.
3
-­‐
Representa@ve
phase
and
RNA
FISH
images
of
esiRNA
KDs
in
female
ESCs.
A.
Untransfected
control.
B.
Oct4
esiRNA
KD
showing
differen@ated
morphology
(phase)
and
Xist
clouds
(RNA
FISH).
XaXa
ESC
Soma,c
cell
X
Chromosome
Inac,va,on
Noncoding
RNA
(Xist)
is
upregulated;
coats
future
Xi.
Differen,a,
on
Silenced
X
(Xi/
Barr
Body)
Xist
coated
Phase
images
RNA
FISH
Stages
of
XCI:
Coun,ng
à
Choice
à
Silencing
XaXi
Female
ESC
Undergo
Gene
Dosage
Compensa,on
During
Differen,a,on
RNA
fluorescent
in
situ
hybridiza@on
(FISH)
probes
for
X-­‐
linked
genes
produce
a
unique
singlet/doublet
(S/D)
pinpoint
paiern
in
female
mouse
ESCs
indicates
X
chromosome
coun@ng.2
♀
mouse
ESC
cell
with
RNA
Xist
FISH
probe
Singlet
Doublet
Xist
DAPI
U,lizing
RNAi
to
Deplete
(Knockdown)
Protein4
• RNA
FISH
for
Xist
RNA
FISH
for
Xist
• Phase
images
• Live
cells
cytospun
&
fixed
Sister
chroma@ds
Xist
cloud
Undifferen,ated
ESCs
Differen,ated
XY
or
XO
ESCs
XX
ESCs
X-­‐linked
loci
Autosomal
loci
RNA
FISH
Signals
Ques,on:
What
proteins
are
necessary
for
S/D
signals
and
coun,ng?
Answer:
Deplete
proteins
(RNAi)
à
Cell
Morphology
(Phase)
à
FISH
• One
of
two
classes
of
Structural
Maintenance
of
Chromosomes
(SMC)
proteins
might
have
an
effect
on
the
singlet/doublet
cytological
signature.
• NIPBL,
a
cohesin
loading
factor,
forms
a
dimer
that
is
essen@al
for
loading
the
cohesin
complex
onto
sister
chroma@ds
and
also
plays
important
roles
in
stabilizing
cells6
gene@c
informa@on,
repairing
damaged
DNA,
and
controlling
the
ac@vity
of
certain
genes
that
are
essen@al
for
normal
development.
• Smc2
&Smc4
-­‐
Components
of
the
condensin
complex,
which
contains
the
SMC2
and
SMC4
heterodimer,
and
three
non
SMC
subunits
that
probably
regulate
the
complex.7
Xist
Brighfield
GFP
fluorescence
A B
References
1.
Kathrin
Plath,
Susanna
Mlynarczyk-­‐Evans,
Dmitri
A.
Nusinow,
Barbara
Panning
Xist
RNA
and
the
mechanism
of
X
chromosome
inac@va@on.
Annu
Rev
Genet.
2002;
36:
233–278.
Published
online
2002
June
11.
doi:10.1146/
annurev.genet.36.042902.092433
2.
Mlynarczyk-­‐Evans,
S.,
Royce-­‐Tolland,
M.,
Alexander,
M.
K.,
Andersen,
A.
A.,
Kalantry,
S.,
Gribnau,
J.,
&
Panning,
B.
(2006).
X
Chromosomes
Alternate
between
Two
States
prior
to
Random
X-­‐Inac@va@on.
PLoS
Biology,
4(6),
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doi:10.1371/journal.pbio.0040159
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Fazzio,
T.
G.,
J.
T.
Huff,
et
al.
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RNAi
Screen
of
Chroma@n
Proteins
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Tip60-­‐p400
as
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A,
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D,
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GL,
et
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in
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cells.
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doi:10.1038/nmeth.2398.
5.
Michelson
Medical
Research
Founda@on;
hip://michelsonmedical.org/wp-­‐content/uploads/2014/05/
interference_f1_large.jpg,
20
May
2016
6.
Horsfield,
J.,
C.
G.
Print,
et
al.
(2012).
"Diverse
developmental
disorders
from
The
One
Ring:
dis@nct
molecular
pathways
underlie
the
cohesinopathies."
Fron@ers
in
Gene@cs
3.
7.
Watrin,
E.,
A.
Schleiffer,
et
al.
"Human
Scc4
Is
Required
for
Cohesin
Binding
to
Chroma@n,
Sister-­‐Chroma@d
Cohesion,
and
Mito@c
Progression."
Current
Biology
16(9):
863-­‐874
8.
Chromosome
condensa@on,
amazing
evidence
of
design;
hip://reasonandscience.heavenforum.org/t2086-­‐
chromosome-­‐condensa@on-­‐amazing-­‐evidence-­‐of-­‐design,
20
May
2016
20x
Phase
20x
Phase
20x
Phase
20x
Phase
Xist
Xist
Xist
Xist
e
Fig. 2- Gene silencing by RNA interference(RNAi).5
Fig. 4- Examples Cohesin and Condensin Complexes.8
Fig.
5
–
NIPBL
plays
a
role
in
cohesin
loading
on
the
sister
chroma@ds.6
Results
Results continued
esiKnockdown counts that resulted in a RNA FISH singlet/doublet (S/D) signal pattern percentage
outside of 45-54% range as compared to the negative controls (NC, Mock GFP) are potential targets of
interest.
Singlet/Singlet (S/S), Doublet/Doublet (D/D), and Total X Cloud (TC) signal pattern ranges also considered, but not
discussed.