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Theodora Koromila
1
, Fan Gao
1
, Yasuno Iwasaki
2
, Peng He
1
, Lior Pachter
1
, J. Peter Gergen
2
,
and Angelike Stathopoulos
1
,
*
1
California
Institute
of
Technology,
Division
of
Biology
and
Biological
Engineering,
1200
East
California
Blvd., Pasadena, CA 91125
2
Stony
Brook
University,
Department
of
Biochemistry
and
Cell
Biology
and
Center
for
Developmental
Genetics, Stony Brook, NY 1179
*corresponding author:
angelike@caltech.edu
Keywords:
Drosophila
melanogaster
,
Odd-paired
(Opa),
Zelda,
maternal-to-zygotic
transition
(MZT),
mid-blastula transition (MBT),
short gastrulation
(
sog
), ChIP-seq, ATAC-seq, Histone mark
Running title:
Late-acting pioneer factor Opa
.
CC-BY-NC-ND 4.0 International license
It is made available under a
(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint
.
http://dx.doi.org/10.1101/853028
doi:
bioRxiv preprint first posted online Nov. 23, 2019;
ABSTRACT
Pioneer
factors
such
as
Zelda
help
initiate
zygotic
transcription
in
Drosophila
early
embryos,
but
whether
other
factors
support
this
dynamic
process
is
unclear.
Odd-paired
(Opa),
a
zinc-finger
transcription
factor
expressed
at
cellularization,
controls
transition
of
genes
from
pair-rule
to
segmental
patterns
along
the
anterior-posterior
axis.
Finding
that
Opa
also
regulates
late
expression
through
enhancer
sog_Distal,
along
the
dorso-ventral
axis,
we
hypothesized
that
Opa
acts
as
a
general
timing
factor.
Chromatin-immunoprecipitation
(ChIP-seq)
confirmed
Opa
in
vivo
binding
to
sog_Distal
but
also
identified
widespread
binding
throughout
the
genome,
comparable
to
Zelda.
Furthermore,
chromatin
assays
(ATAC-seq)
demonstrate
that
Opa,
like
Zelda,
influences
chromatin
accessibility
genome-wide,
suggesting
both
are
pioneer
factors
with
common
as
well
as
distinct
targets.
Lastly,
embryos
lacking
opa
exhibit
widespread,
late
patterning
defects
spanning
both
axes.
Collectively,
these
data
suggest
Opa,
a
general
timing
factor
and
likely
a
late-acting
pioneer
factor,
heralds
in
a
secondary
wave
of
zygotic
gene
expression.
INTRODUCTION
The
transition
from
dependence
on
maternal
transcripts
deposited
into
the
egg
to
newly
transcribed
zygotic
transcripts
is
carefully
regulated
to
ensure
proper
development
of
early
embryos.
During
the
maternal-to-zygotic
transition
(MZT),
maternal
products
are
cleared
and
zygotic
genome
activation
occurs
(rev.
in
Hamm
and
Harrison,
2018;
Vastenhouw
et
al.,
2019)
.
In
Drosophila
embryos,
the
first
13
mitotic
divisions
involve
rapid
nuclear
cycles
(nc),
that
include
only
a
short
DNA
replication
S
phase
and
no
G2
phase,
and
the
nuclei
are
not
enclosed
in
separate
membrane
compartments
but
instead
present
in
a
joint
cytoplasm
(Foe
and
Alberts,
1983)
.
This
streamlined
division
cycle
likely
relates
to
the
fast
development
of
Drosophila
embryos,
permitting
rapid
increase
in
cell
number
before
gastrulation
in
a
matter
of
a
few
hours.
Dynamic
gene
expression
is
initiated
during
the
early
syncytial
stage,
as
early
as
2
.
CC-BY-NC-ND 4.0 International license
It is made available under a
(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint
.
http://dx.doi.org/10.1101/853028
doi:
bioRxiv preprint first posted online Nov. 23, 2019;
nc7,
and
continues
to
the
cellularized
blastoderm
stage
(Ali-Murthy
and
Kornberg,
2016;
Lott
et
al.,
2011)
.
Gene
expression
patterns
may
be
transient
or
continuous,
lasting
through
gastrulation
or
beyond
(Kvon
et
al.,
2014)
.
This
process
is
controlled
by
a
specific
class
of
transcriptional
factors
(TF),
called
pioneer
factors,
that
bind
to
closed
chromatin
cis-regulatory
regions
to
create
accessible
binding
sites
for
additional
transcription
factors
during
development
(Iwafuchi-Doi
and
Zaret,
2014)
.
A
pivotal
factor
in
Drosophila
embryogenesis
is
the
pioneer
TF
Zelda
(Zld),
a
ubiquitous,
maternal
transcription
factor
that
binds
to
promoters
of
the
earliest
zygotic
genes
and
primes
them
for
activation
(Harrison
et
al.,
2011,
2010;
Liang
et
al.,
2008)
.
It
was
unknown
whether
a
similar
regulation
exists
in
other
animals,
until
Pou5f1
(homolog
of
the
mammalian
Oct4)
was
shown
to
act
in
an
analogous
manner
to
Zld
in
that
it
controls zygotic gene activation in vertebrates
(Leichsenring et al., 2013)
.
The
exact
molecular
mechanism
that
supports
widespread
activation
of
zygotic
gene
expression
is
not
known,
but
several
regulatory
mechanisms
have
been
proposed.
One
model
suggests
that
a
decrease
in
histone
levels
occurs
over
time
that
provides
an
opportunity
for
the
pioneer
factors
that
herald
zygotic
gene
expression
to
successfully
compete
for
DNA,
in
order
to
access
and
activate
transcription
(Hamm
and
Harrison,
2018;
Shindo
and
Amodeo,
2019)
.
For
example,
Zld
is
a
pivotal
activator
of
the
MZT
as
it
increases
accessibility
of
chromatin
at
enhancers,
to
facilitate
binding
of
other
transcriptional
activators
to
these
DNA
sequences
and
thereby
allow
initiation
of
zygotic
gene
expression
(Harrison
et
al.,
2011;
Liang
et
al.,
2008;
Nien
et
al.,
2011)
.
Zld
binds
nucleosomes
and
is
considered
a
pioneer
factor
(McDaniel
et
al.,
2019)
.
Loss
of
Zld
leads
to
a
large-scale,
global
decrease
in
zygotic
gene
expression
as
many
enhancer
regions
remain
inaccessible
and
thus
non-functional
(Schulz
et
al.,
2015)
.
By
facilitating
chromatin
accessibility,
Zld
has
been
shown
to
influence
the
ability
of
morphogen
transcription
factors,
Bicoid
and
Dorsal,
to
support
morphogen
gradient
patterning
(Foo
et
al.,
2014;
Xu
et
al.,
2014)
.
While
Zld
is
clearly
pivotal
for
supporting
MZT,
some
genes
continue
to
be
expressed
even
in
its
absence
(Nien
et
al.,
2011)
.
In
particular,
as
chromatin
accessibility
in
the
early
embryo
has
recently
been
shown
to
be
a
3
.
CC-BY-NC-ND 4.0 International license
It is made available under a
(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint
.
http://dx.doi.org/10.1101/853028
doi:
bioRxiv preprint first posted online Nov. 23, 2019;
dynamic
process
(Blythe
and
Wieschaus,
2016)
,
it
is
possible
that
Zld
contributes
in
a
stage-specific
manner.
It
is
likely,
therefore,
that
other
pioneer
factors
also
exist
that
contribute
to
zygotic
genome
activation.
Furthermore,
the
embryo
undergoes
a
widespread
state
change
after
the
14th
nuclear
division,
and
this
developmental
milestone
is
classified
as
the
midblastula
transition
(MBT)
(Foe
and
Alberts,
1983;
Shermoen
et
al.,
2010)
.
The
cycle
slows
dramatically
and
nuclei
become
cellularized
relating
to
when
embryonic
programs
of
morphogenesis
and
differentiation
initiate.
Cell
membranes
emerge
to
encapsulate
nuclei,
forming
a
single
layered
epithelium.
In
addition,
at
nc14,
developmental
changes
relating
to
DNA
replication
occur;
namely
a
lengthened
S-phase
and
the
introduction
of
G2
phase.
MBT
is
also
associated
with
clearance
of
a
subset
of
maternally
provided
mRNAs,
large-scale
transcriptional
activation
of
the
zygotic
genome,
and
an
increase
in
cell
cycle
length
(Tadros
and
Lipshitz,
2009;
Yuan
et
al.,
2016)
.
We
hypothesized that other late-acting pioneer factors manage the MBT in addition or in place of Zld.
An
attractive
research
target
in
the
field
of
developmental
biology
is
the
transcription
factor
Zinc
finger
in
the
cerebellum
(ZIC
human
ortholog);
its
role
in
early
developmental
processes
has
been
established
across
major
animal
models
and
also
linked
to
human
developmental
pathology
(rev.
in
Aruga
and
Millen,
2018;
Houtmeyers
et
al.,
2013)
.
However,
the
founding
member
of
the
Zic
family
is
the
Drosophila
gene
odd-paired
(opa)
(Aruga
et
al.,
1996;
Hursh
and
Stultz,
2018)
.
opa
is
a
broadly
expressed
gene
of
relatively
long
transcript
length
(~17
kB)
that
initiates
expression
at
mid-nuclear
cycle
14
(Benedyk
et
al.,
1994;
Cimbora
and
Sakonju,
1995)
and
recently
was
shown
to
function
as
a
timing
factor
in
early
embryos
controlling
segmentation
(Clark
and
Akam,
2016)
.
Opa
protein
has
a
DNA
binding
domain
containing
five
Cys2His2-type
zinc
fingers,
and
shares
homology
with
mammalian
Zic1,
2,
and
3
transcription
factors.
It
is
categorized
as
an
atypical
pair-rule
gene,
as
it
is
expressed
in
a
broad
domain
within
early
embryos
rather
than
the
7-stripe
pattern
typical
of
other
pair-rule
genes.
Its
pair-rule
phenotype
relates
to
its
ability
to
act
as
a
timing
factor
to
control
the
transition
of
genes
with
pair-rule
4
.
CC-BY-NC-ND 4.0 International license
It is made available under a
(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint
.
http://dx.doi.org/10.1101/853028
doi:
bioRxiv preprint first posted online Nov. 23, 2019;
patterns
to
segmental
expression
pattern
(i.e.
from
7-
to
14-stripes)
(Clark
and
Akam,
2016)
.
opa
is
a
widely
expressed
gene
and
has
additional
functions
throughout
development.
For
example,
opa
mutant
embryos
die
before
hatching,
exhibiting
aberrant
segmentation,
but
mutant
embryos
also
exhibit
defects
in
larval
midgut
formation
(Cimbora
and
Sakonju,
1995)
.
During
midgut
formation,
it
regulates
expression
of
a
pivotal
receptor
tyrosine
kinase
required
for
proper
morphogenesis
of
the
visceral
mesoderm
(Mendoza-García
et
al.,
2017)
.
In
addition,
at
later
stages,
Opa
supports
temporal
patterning
of
intermedial
neural
progenitors
of
the
Drosophila
larval
brain
(Abdusselamoglu
et
al.,
2019)
.
Therefore,
opa
supports
a
number
of
patterning
roles
throughout
development,
several
relating
to
temporal
patterning.
In
addition,
previous
studies
had
suggested
that
Opa
can
influence
the
activity
of
other
transcription
factors
to
promote
gene
expression.
A
well
characterized
target
of
Opa
in
the
early
embryo
is
sloppy-paired
1
(
slp1
),
a
gene
exhibiting
a
segment
polarity
expression
pattern,
for
which
two
distinct
enhancers
have
been
identified
that
are
capable
of
responding
to
regulation
by
Opa
and
other
pair-rule
transcription
factors
(Cadigan
et
al.,
1994;
Prazak
et
al.,
2010)
.
One
of
these,
the
slp1
DESE
enhancer,
mediates
both
Runt-dependent
activation
and
repression
(Hang
and
Gergen,
2017)
.
Opa
is
clearly
a
central
player
in
slp1
regulation
by
supporting
Run’s
role
as
activator
at
DESE,
but
its
uniform
expression
pattern
does
not
provide
positional
information
as
other
pair-rule
gene
inputs.
Furthermore,
our
recent
study
showed
that
Runt
regulates
the
spatiotemporal
response
of
another
enhancer,
sog_Distal
(also
known
as
sog_Shadow;
Hong
et
al.,
2008;
Ozdemir
et
al.,
2011)
to
support
its
expression
in
a
broad
stripe
across
the
dorsal-ventral
(DV)
axis
on
both
sides
of
the
embryo
(Koromila
and
Stathopoulos,
2019)
.
Using
a
combination
of
fixed
and
live
imaging
approaches,
our
analysis
suggested
that
Run’s
role
changes
from
repressor
to
activator,
over
time
in
the
context
of
sog_Distal
;
late
expression
requires
Run
input
as
activator.
These
analyses
of
slp1
DESE
and
sog_Distal
regulation
supported
the
view
that
Opa
might provide temporal input into both these enhancers.
5
.
CC-BY-NC-ND 4.0 International license
It is made available under a
(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint
.
http://dx.doi.org/10.1101/853028
doi:
bioRxiv preprint first posted online Nov. 23, 2019;
The
current
study
was
initiated
to
investigate
whether
Opa
supports
late
expression
through
the
sog_Distal
enhancer.
Previous
studies
had
not
linked
Opa
to
the
regulation
of
DV
patterning.
Nevertheless,
through
mutagenesis
experiments
coupled
with
live
in
vivo
imaging,
we
provide
evidence
that
Opa
does
regulate
expression
of
the
sog_Distal
enhancer
and
that
its
role
is
indeed
late-acting,
occurring
in
embryos
at
mid-nc14
onwards
whereas
the
enhancer
initiates
expression
at
nc10.
Furthermore,
using
both
chromatin
immunoprecipitation
coupled
with
high
throughput
sequencing
(ChIP-seq)
as
well
as
single-embryo
Assay
for
Transposase-Accessible
Chromatin
using
sequencing
(ATAC-seq),
our
whole-genome
data
support
the
view
that
Opa
supports
patterning
in
the
embryo
by
serving
as
a
general
timing
factor,
and
possibly
a
pioneer
factor,
that
broadly
influences
zygotic
transcription
at
around
the
time
that
embryo
begin
cellularization
to
support
the
late
phase
of
the
maternal-to-zygotic transition.
RESULTS
Opa regulates the sog_Distal enhancer demonstrating a role for this pair-rule gene in DV axis patterning
In
a
previous
study,
we
created
a
reporter
in
which
the
650
bp
sog_Distal
enhancer
sequence
was
placed
upstream
of
a
heterologous
promoter
from
the
even-skipped
gene
(
eve.p
),
driving
expression
of
a
compound
reporter
gene
containing
both
tandem
array
of
MS2
sites
and
the
gene
yellow
including
its
introns
(Koromila
and
Stathopoulos,
2017)
.
The
MS2
cassette
contains
24
repeats
of
a
DNA
sequence
encoding
an
RNA
stem-loop
when
transcribed.
The
stem-loop
is
recognized
by
an
MCP-GFP
fusion
protein,
as
MCP
is
a
phage
protein
able
to
bind
the
RNA-stem
loop
whereas
GFP
provides
a
strong
green
signal
that
can
be
monitored
within
the
nucleus
of
Drosophila
embryos
at
the
site
of
nascent
transcript
production
associated
with
the
transgene
(Bothma
et
al.,
2014)
.
This
compound
reporter
gene
allows
assay
of
spatiotemporal
gene
expression
live,
through
MS2-MCP
imaging,
or
in
fixed
samples
through
in
situ
hybridization
to
gene
yellow
.
We
used
this
sog_Distal
reporter
to
assay
gene
expression
in
the
early
6
.
CC-BY-NC-ND 4.0 International license
It is made available under a
(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
The copyright holder for this preprint
.
http://dx.doi.org/10.1101/853028
doi:
bioRxiv preprint first posted online Nov. 23, 2019;