of 25
Pervasive SUMOylation of heterochromatin and piRNA pathway
proteins
Maria Ninova
*
1,2
,
Hannah Holmes
1
,
Brett Lomenick
3
,
Katalin Fejes
-
T
ó
th
2
, Alexei
A.
Aravin
*
2
1
Department of Biochemistry
,
University of California Riverside
3401
Watkins Drive, Boyce Hall
, R
iverside, CA 92521, USA
2
Division of Biology and Biological Engineering
&
3
Proteome Exploration Laboratory
of the Beckman Institute
,
California Institute of Technology
1200 E. California Blvd.
,
Pasadena, CA 91125, USA
*
Corr
espondence:
Maria Ninova
mninova@ucr.edu (lead contact)
Alexei Aravin aaa@caltech.edu
Abstract
Genome regulation involves complex and highly regulated protein interactions that are often
mediated through post
-
translational modifications (PTMs). SUMOylation
the covalent attachment of the
s
mall
u
biquitin
-
like
mo
difier (SUMO)
is a conserved PTM in eukaryotes that has been implicated in a
number of essential processes such as nuclear import, DNA damage repair, transcriptional
control,
and
chromatin organization.
In
Drosophila
,
SUMO is essential for viability and
its depletion
from the
female
germline causes infertility associated with
global
loss of heterochromatin, and
illicit
upregulation of
transposon
s
and lineage
-
inappropriate genes.
However, the specific targets of SUMO and its mechanistic
role in different
c
ellular pathways
are still poorly understood.
Here
,
we
developed a proteomics
-
based
strategy to characterize the SUMOylated
proteome
in
Drosophila
that allowed us
to identify
~1500 SUMO
sites in 843 proteins
in
the
fly ovary.
A
high
-
confidence
set of
SUMOy
lated proteins
is highly enriched in
factors involved in heterochromatin regulation and
several different aspects of
the piRNA pathway that
represses
transposon
s
, including piRNA biogenesis and function. Furthermore, we show that
SUMOylation of s
everal
piRNA pathway
proteins
occurs in a Piwi
-
dependent manner, indicating a
functional implication of this modification in the cellular response to transposon activity.
Together, these
data highlight the impact of SUMOylation on epigenetic regulation and
reveal
an unexpectedly broad role
of the SUMO pathway in the
cellular
defense against genomic parasites. Finally, this work provides a
valuable resource and a system that can be adapted to the study of
SUMOylation
in other
Drosophila
tissue
s
.
Keywords
SUMO, pi
RNAs, heterochromatin, transposons, proteomics
, germline
In
troduction
Post
-
translational modifications (PTM) impact a broad range of cellular functions such as protein
turnover, localization to different subcellular compartments, or specific interactions, and are utilized in the
regulation of various molecular pathways. PTMs
involve the covalent attachment of diverse moieties from
small chemical groups to entire modifier proteins to the main polypeptide chain. The
best
-
known
protein
modifier is ubiquitin
a ~9kDa unit that gets conjugated to target lysine side chains via an
isopeptide
bond between its C
-
terminal carboxyl group and the lysine epsilon amino group
.
Following the discovery
of Ubiquitin, other small proteins that can act as modifiers have emerged, including the small ubiquitin
-
.
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2
like modifier SUMO. SUMO is a ~11
kDa
protein
that shares structural and sequence homology
with
Ubiquitin and similarly gets covalently attached to target lysines.
However,
SUMO conjugation is
mediated by different enzymes and serves distinct and non
-
redundant functions
(reviewed in ref.
1
)
.
In
brief,
t
he SUMOylation cascade involves activation by a dedicated E1 heterodimer followed by transfer
to the SUMO E2 conjugating enzyme Ubc9. Ubc9 catalyzes SUMO transfer to the final acceptor
lysine
which often (but not always) resides within a consensus motif
ψKxE (ψ=hydrophobic aminoacid),
and
is
sufficient for SUMOylation
in vi
tr
o
2
4
.
Nevertheless
, non
-
catalytic SUMO E3 ligases can facilitate Ubc9 or
enable substrate
specificity
,
and
seem to be required
for SUMOylation
in
some
contexts
and perhaps for
non
-
consensus sites
in vivo
5,6
.
SUMO is primarily nuclear, and since its
discovery has emerged as an
important regulator of different nuclear processes
(reviewed in ref.
7
)
such as transcription factor activity,
DNA repair, rRNA biogenesis, chrom
osome organization and segregation. Mechanistically, SUMOylation
may lead to diverse consequences including changes in protein conformation or localization, masking or
competing with other PTMs, and most famously, regulating protein
-
protein interactions.
S
UMO
-
mediated
interactions typically involve an aliphatic stretch flanked by acidic amino acids (SUMO interacting motif,
SIM) in the partner protein and although
individually
weak in their nature, multivalent SUMO
-
SIM
interactions were proposed to stabilize
large molecular
complexes
8,9
and promote the formation of phase
-
separated compartments such as PML (promyelocytic leukemia protein) bodies
10
.
How
ever, despite
being implicated in a myriad of biological processes, our understanding of SUMO’s mechanistic role
within different molecular and cellular contexts is far from complete.
Previous
work
in
Drosophila
implicated the SUMO pathway in the regulation of heterochromatin
establishment
and
piRNA
-
mediated
transposon silencing
11
14
. In germ cells, Piwi clade proteins (Piwi,
Aub and Ago3
in
flies
) and Piwi
-
interacting small RNAs (piRNAs) cooperate in intimately linked processes
t
hat
ensure transcriptional and post
-
transcriptional transposon silencing and continuous production of
mature piRNAs
(reviewed in ref.
15
)
. Mature piRNA production and post
-
transcriptional cleavage of
transposon RNAs by Aub and Ago3 occur in a dedicated
perinuclear
structure, the nuage. Antisense
piRNAs produced in the nuage also become load
ed in Piwi, which then enters the nucleus
to find
transposon nascent RNA and
enforce co
-
transcriptional silencing at
target
loci
16
18
.
To date
, SUMOylation
is know
n to
participate
in the nuclear piRNA pathway
in
several
ways
:
First, t
he SUMO E3 ligase
Su(var)2
-
10
was
found
to interact with piRNA pathway and
heterochromatin
proteins
and
play an
essential role
in
the recruitment of the enzymatic complex SetDB1/Wde that deposits the silencing
epigenetic mark H3K9me3
12
, as well as with the MEP
-
1/Mi
-
2 chromatin remodeler complex
14
. Second,
SUMOylat
ion of
Panoramix (Panx)
a co
-
repressor required for H3K9me3 deposition downstream of
Piwi
was found to mediate its interaction with the
general heterochromatin effector Sov
13
.
In addition to
silencing of transposons
,
Su(var)2
-
10 and SUMO
were found to
control H3K9me3 deposition at
piRNA
-
independent
genomic loci such as developmentally silenced tissue
-
specific genes
19
.
The pervasive effect
of SUMO depletion on glob
al H3K9me3 levels at
diverse classes of
genomic targets suggests that
SUMOylation is a critical process in the regulation of repressive chromatin integrated within different
regulatory pathways. However, further understand
ing of
the role of SUMOylation on
chromatin
requires
knowledge of
the full spectrum of SUMO targets.
We developed a proteomics approach that enables the identification of SUMOylated proteins with
aminoacid
-
level site predictions from different tissues in the classic model for piRNA and he
terochromatin
studies
D
.
melanogaster
. Here, we report a comprehensive dataset of SUMO targets in the fly ovary,
which represents the most extensive analysis of the SUMOylated complement in
a metazoan
reproductive tissue to date.
Notably
, we identified strong enrichment of heterochromatin factors among
SUMOylated proteins, supporting the notion that SUMO plays a complex role in heterochromatin
regulation that extends to multiple targets and protein complexes. Moreover, we find a
striking
enrichment
of
proteins specific
to
the
piRNA pathway
among SUMO targets
, including the central effector of
.
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;
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3
epigenetic silencing by piRNAs, Piwi
, and several proteins that localize to the
nucleus and
nuage
. We
further validated the SUMOylation of selected p
iRNA pathway factors
:
Piwi, Panx, Sp
indle
-
E
(Spn
-
E)
and
Mael
strom (Mael)
in germ
cells and
showed
that th
e modification
of Mael and Spn
-
E, but not Panx, is
Piwi
-
dependent, indicative of
multiple
SUMO
roles
in distinct steps of transposon silencing. Altoget
her,
our findings point to a previously unappreciated multilayered role of SUMOylation in the piRNA pathway
and heterochromatin regulation that provide important c
l
ues
toward
our understanding of the molecular
mechanisms of genome regulation
and transposon
control
.
Results
Establishing a system for
the
detection
of
SUMOylated proteins in Drosophila
Proteome
-
wide studies of PTMs have benefited from the development of methods and reagents
that enable specific enrichment of modified
protein
s
or
peptides
from total protein lysates. Basic methods
for
the
enrichment of
SUMO
-
modified protein
s
involve pulldo
wn with
anti
-
SUMO
antibodies
. However,
this
approach
prohibits
the
use of
stringent washing conditions and is therefore prone to high background.
Furthermore,
endogenous SUMO does not have trypsin cleavage sites close to its C
-
terminus
, and
trypsin digesti
on of SUMOylated proteins generates large, branched peptides from modified regions
that
are incompatible with conventional bottom
-
up proteomics
(Fig
.
1A). Accordingly, specific modified
residues remain unknown and
SUMO
yl
ation events
can only be inferred indirectly from the abundance
of other peptides from
purified SUMOylated proteins
.
To overcome these obstacles and
obtain a
high
-
confidence
dataset of
the SUMO
-
modified
protei
n
s
(herein referred to as the SUMOylome)
in
Drosophila
,
we
adapted
an
approach
that
allows
stringent purification
,
enrichment
, and proteomic detection of
peptides containing
a modified SUMO
remnant
20
. This approach, originally developed
for the study of SUMOylation in
human cells
20
, employs
ectopically expressed
SUMO
protein
with
6x
His
-
tag
and a
point mutation
(
T>
R substitution
, herein
referred to as SUMO
-
TR
) that
enables
trypsin cleavage
before
the C
-
terminal
GG
motif
at the conjugation
site
(Fig
.
1A
,B
)
.
In a two
-
step purification process,
SUMO
-
modified proteins are
first
selected
based on
the His tag
by
nickel
affinity under denaturing conditions
,
which
eliminates the activity of SUMO proteases
and
most
background
from
noncovalently bound proteins
.
The resulting
SUMO
ylated protein
-
enriched
fraction
is
then
trypsinized to
generat
e
a mixture of peptides including
branched
peptides
with
short
di
-
glycine
(diGly)
r
emnant
from
cleaved
SUMO
-
TR
moiety.
These
diGly remnant
-
containing
peptides are
further
selected
from the total pool
by pulldown with
a
specific
antibody
,
purified
,
a
nd
analyzed by mass
spectrometry
.
Of note
, Ubiquitin and other ubiquitin
-
like
protein modifiers
naturally have an arginine
residue before the terminal diGly
motif
, therefore, any ubiquitinated protein that unspecifically co
-
purifies
with SUMOylated proteins
during the His
-
based enrichment
step
can generate background diGly
peptides. However, this background can be accounted for
through
negative control samples
from tissues
that
do not express SUMO
-
TR (Fig
.
1B).
To
enable
this method for SUMOylation detection
in
Drosophila
tissues,
we
created a transgenic
line
that
carr
ies
a
modified
copy of the
smt3
locus
(
which encodes the
single SUMO homolog in this species
)
where
the SUMO
coding sequence
has
a 6xHis N
-
terminal tag
,
T86
>
R substitution
before
the C
-
terminal GG motif
,
and a ~2.5kb upstream region containing the putative
endogenous promoter
.
The
His
-
tagged SUMO
-
TR protein was detectable by Western Blotting (Fig
.
1
C
)
and
i
mportantly, this transgene completely rescues the let
hality of
the
null allele
smt3
04493
, confirming that
it encodes a
fully
functional protein
.
Identification and characterization of the Drosophila ovarian SUMOylome
We used flies expressing SUMO
-
TR to optimize
the
two
-
step purification procedure
described
above (Fig.
1B)
and obtain SUMO
ylation site
-
derived peptides from ovarian tissue (See Materials and
.
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;
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Methods for details).
Following
this procedure, we performed 3 independent experiments, where each
replica
involv
ed parallel sample preparation from SUMO
-
TR ovaries and
wild
-
type
controls followed by
label
-
free
tandem mass spectrometry
(MS)
. Each sample yielded ~1000
-
2500 diGly sites (Fig
.
2A) and
altogether, we detected 3159 exact diGly sites mapping to the protein
products of 1295 genes.
In
comparison,
MS
analyses of input samples prior to diGly enrichment yielded less than 100 diGly sites
(data not shown), highlighting the importance of the diGly enrichment step
for
capturing SUMOylation
sites.
For each
diGly site,
we calculated the
normalized intensity
ratio
s
between SUMO
-
TR and control
samples. The ratios showed a prominent bimodal distribution: approximately half sites in each experiment
were detected exclusively in SUMO
-
TR samples, indicative of genuine SUMOylation
targets (
Fig
.
2B).
The diGly sites detecte
d with similar intensities in SUMO
-
TR and control samples or biased to control
samples likely originate from unspecifically bound ubiquitinated proteins.
Consistent with this,
motif
analysis showed that diGly sites detected in SUMO
-
TR samples are enriched
in the
canonical
SUMOylation motif, while diGly sites from the negative control
do not have
any
motif enrichment
(Fig
.
2B, Fig
.
S1).
The sets of predicted
bona fide
SUMO targets (SUMO
-
TR/Control intensity ratio >10) were highly
reproducible between the 3
experiments, with ~50% overlap on the specific site level, and a 75% overlap
on the target
protein
-
coding
gene level (Fig
.
2C).
Previously known SUMO
targets
including
RanGAP1,
PCNA, Su(var)2
-
10/dPIAS
are present in the high confidence set
.
Specific sites
that do not appear in all
experiments tend to have the lowest intensities (Fig
.
S2). Considering that SUMOylation
is often transient
and
affects a minuscule fraction of a given protein, it is likely that sensitivity is the major limitation to the
reproduci
bility of detected sites
between replicates
. About ~25% of the proteins
with diGly remnant
have
more than one
modification
site, with widely variable
distances
between
two
sites (Fig
.
2D). This pattern
could indicate that proteins can be alternatively SUMOylated at different residues, multi
-
SUMOylated, or
even SUMO
-
and Ubiquitin
-
modified at the same time.
As an aside
, we detect
ed
several diGly sites on
SUMO itself
(Table
S1
)
;
as s
ome
of
these sites can be detected in the negative control
,
this pattern is
indicative of
hybrid
SUMO
-
Ubiquitin chains.
To gain
a
further insight into the sequence features of the fly SUMOylome, we assessed the
presence of amino acid motifs in the conservati
ve set of 421
distinct
high
-
confidence
SUMOylation sites
detected in all 3 experiments. Sequence pattern search
es
identified the canonical consensus and inverse
consensus SUMOylation motifs (ψ
K
xE/D and E/Dx
K
ψ, respectively) at 45%
of the sites
(141 and 49)
in
this set (Fig
.
2E). ~10% of the sites have the motif ψK
K
E/D with the
modification
assigned to the second
lysine
a pattern that might arise
from
uncertainty in
diGly
position prediction in the case of neighboring
lysines.
The remaining
sites
were not enriched for any known motif
,
and
de novo
motif search via MoMo
did not identify any significant sequence bias around the central lysine (not shown), suggesting that as
i
n other systems, the SUMOylation consensus motif is not obligatory.
Like
observations in human
cells
17
,
consensus sites displayed a marked preference for I
soleucine and Valine as hydrophobic
residues
.
Additionally, a sizable fraction of the sites with strong SUMOylation consensus had a downstream Proline
an extended motif associated with SUMOylation/acetylation
switch
21
(Fig
.
2E).
Finally
, we assessed
the position of SUMOylation sites with respect to predicted structural features
based on deep learning
language models
22
including alpha sheets, beta helices and intrinsically disordered regions (IDR
s
). This
analysis demonstrated that SUMOylated sites are significantly overrepresented in IDRs and under
-
represented in structured regions c
ompared to randomly selected lysines
from
the same protein or
the
background diGly sites (TR
-
SUMO/control ratio < 3 in all replicates)
.
Similar results were obtained using
IDR predictions generated with IUPred2A
23
(
Fig
.
S3
). Notably, SUMOylation sites within canonical motifs
show
ed the strongest bias towards IDRs (Fig
.
2G).
Altogether, these results show that SUMOylation affects a large number of proteins in the
Drosophila
ovary, with the SUMO
ylome
displaying conserved sequence and structural features with other
.
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;
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5
organisms. Import
antly, the breadth of the SUMOylome, together with the observations that a substantial
fraction of SUMOylation sites do not have a specific association with
a motif
or structural features highlight
the need
for
unbiased
experimental approaches to identifying
SUMOylation targets
in
different cellular
and
organismal
contexts.
Functional groups of SUMOylated proteins
To
investigate
the possible roles of SUMOylation in the ovary, we carried out functional
enrichment and int
eractome analys
e
s for the conservative set of
proteins that carry
reproducible SUMO
sites in all experiments
,
i.e.
421 sites
(Fig. 2C)
in 292 proteins
,
using
g
ene ontology
(GO)
and STRING
(Fig. 3A,B,
Table S2
).
Results
showed that SUMOylation is common amo
ng specific
physically and
functionally linked
groups of ribosomal and nuclear proteins
(Fig. 3A
,B
,
Table S2
).
R
ibosomal proteins
are highly abundant and hence a
frequent
source of background in proteomics
. However,
the number of
reproducible diGly sites
mapping to
ribosomal proteins in the SUMO
-
TR samples
is
significantly
above
the background
(
ribosomal proteins
carry
57 out
of the
421
bona fide
SUMO sites
(13%)
,
versus
37 out
of
the
714 background diGly
sites(5
%)
)
and could therefore represent genuine targets. Indeed, the
SUMOylation machinery has been implicated in ribosomal protein regulation
24
.
Beyond the ribosome,
SUMO targets are common among proteins
participating in
rRNA processing
, mRNA splicing
, DNA
damage response and repair
, consistent with a conserved role of the SUMO pathway in these processes
from yeast to mammals
(reviewed in
ref.
7
)
. Notably,
SUMOylation is
widespread
among chromatin
-
associated proteins.
H
igh
-
confidence SUMOylation sites
can be found
in
several core histones
and
the
HP1 paralogs HP1b and HP1c
; a
weak
er
site
was
also
detected
in
the central heterochromatin effector
HP1a/Su(var)205
at
position homologous to the SUMO site in its mammalian homolog
HP1
ɑ/CBX5
(K32).
Strikingly
,
among the most enriched gene ontology terms in the SUMOylated set
a
re terms relate
d
to
heterochromatin and transposon repressio
n
; for example,
2
1
out of the 96 genes
associated with the
“heterochromatin organization” GO term, and 8 out of 25 genes
associated with the
“negative
regulation
of transposition” GO term are in the high confide
nce SUMO target set
.
To further examine the breadth of SUMOylation within heterochromatin
-
and piRNA pathway
-
associated factors
that control transposons in the ovary
, we mapped the presence of SUMOylation sites
among the physical interactors of the key heterochromatin effectors SetDB1/Egg, Wde and HP1a, as
well as nuclear piRNA pathway effector Piwi, and
V
asa
central component of
the
nuage compartment
where
piRNA
bi
ogenesis and transposon RNAi
-
mediated cleavage take place
25
27
(Fig
.
4
A
).
Several
HP1a interactors
are modified by SUMO
, including the split orthologs of the mammalian chromatin
remodeler ATRX dADD1 and XNP, pointing to a multifaceted role of SUMOylation in transcriptional
silencing and heterochromatin regulation.
Furthermore
,
several
h
igh confidence SUMOylation sites
are
present
in Piwi
,
as well as several others piRNA pathway proteins
including
Panoramix (Panx)
a
component of the so
-
called SFiNX/Pand
a
s/PICTS
28
30
complex required for H3K9me3 deposition
downstream of Piwi (also recently reported as SUMO target
in another study
13
)
,
Maelstrom (Mael)
essential for transcriptional repression and piRNA biogenesis from dual
-
stranded cluster
s
16,31
, and the
nuage component Spindle
-
E (SpnE
)
a germline
-
specific DEAD
-
box helicase essential for piRNA
production
32
35
.
Additionally, manual inspection of the data showed that several proteins involved in
piRNA biogenesis such as thoc7, CG13
741/Bootlegger and Hel25E can be SUMOylated (Table
S
1).
The
presence of SUMO at a wide range of proteins involved in piRNA biogenesis and function suggests that
this modification may regulate the cellular response to transposon activity at multiple levels
including but
not limited to previously identified SUMO functions.
Following the observation that several nuage
-
associated proteins are SUMO
-
modified, we
wondered if the SUMO pathway affects the cytoplasmic piRNA compartment
in addition to its previous
imp
lication in the nuclear piRNA pathway
13,19
. To explore this possibility, we
investigated the subcellular
.
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;
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localization of
the core nuage component
V
asa
, and the nuage
-
localized piRNA effector Aub, upon
SUMO depletion. To this end, we employed a previously characterized small hairpin RNA targeting
SUMO
12
under the control of maternal tubulin Gal4 driver
,
which
is active
from stage 2
-
3 of oogenesis
onward. In this condition,
oogenesis fails to complete with nurse cell nuclei collapsing around stage 7,
however,
prior to that
egg chambers
appear morphologically normal and can be analyzed (Fig. S4).
As
previ
ously reported, under normal conditions Aub and Vasa appear as a relatively smooth perinuclear
layer
36
(Fig. 4). However, SUMO knockdown
leads to
markedl
y disrupted Aub and Vasa localization at
the nuclear periphery and
dispersed granules throughout the cytoplasm
(Fig
.
4B).
Collectively, t
hese
results indicate that the SUMO pathway may
support
transposon silencing not only th
r
ough its
involvement
in heterochromatin formation but also through effects on the nuage compartment.
SUMOylation of piRNA pathway factors in the female germline
To further
understand
SUMO’s roles in the piRNA pathway, we
sought to validate the
SUMOylation of four essential piRNA pathway
proteins
Piwi, Panx, Spn
-
E and Mael
. As SUMOylation
typically affects only a small fraction of the total protein pool, and the detection of such species is limited
by antibody
availabili
ty and
affinities, we devised
a
sensitive system to analyze SUMOylation of proteins
of interest
in the germ cells of the ovary which express the full cytoplasmic and nuclear piRNA pathway.
Specifically
, we utilized UASp/Gal4 to express Flag
-
tagged SUMO and
GFP
-
tagged target protein
in
nurse cells from stage 2
-
3 of oogenesis
and later
using the maternal tubulin
-
Gal4 drive
r (except GFP
-
Piwi which was under the control of its native regulatory region).
In this system, the GFP tag and high
affinity anti
-
GFP nan
obody allows protein purification under stringent washing conditions, while the
sensitive and specific monoclonal anti
-
F
lag antibody maximizes detection of small amounts of SUMO
modified proteins by Western Blot
ting (WB)
.
Analyses of immunopurified GFP
-
Spn
-
E, Piwi, Panx and Mael from ovaries showed
Flag
-
SUMO
conjugated higher molecular weight forms consistent with SUMO modification
in all cases
(Fig
.
5A).
Note
that
free
Flag
-
tagged SUMO
migrates
at
about ~17 kDa
on WB
, sli
ghtly
higher
than its predicted
molecular weight of
~
11 kDa.
Each
target displayed multiple higher molecular weight bands, supporting
the presence of multiple SUMO moieties or mixtures of SUMO and other protein PTMs on the same
protein.
The migration patte
rn of Panx indicates the existence of mono
-
SUMOylated and an array of poly
-
modified forms. A similar pattern of multiple modified Panx species was reported
in another
recent
study
where authors used a
custom
-
made
antibody against the native protein
13
. The observed shift in molecular
weight for GFP
-
Piwi from ~120kDa to ~160kDa and above indicates that it carries two or more protein
modifiers, at least one of which is SUMO. Mael’s and SpnE’s SUMO
-
modified forms have molecular
weights of ~40
-
50 kDa great
er than their unmodified forms, also consistent with at least 2
-
3 modified
sites within the same protein.
Of note, the number of higher molecular weight bands in Mael and Panx
exceeds the number of predicted diGly sites. While our proteomics detection is l
imited to peptides within
a particular size range and thus is unlikely to cover all possible modified residues, we also cannot rule
out the existence of poly
-
SUMO or hybrid SUMO
-
Ubiquitin chains (see Discussion).
We also tested whether ectopically
over
expr
essed piRNA pathway
proteins
can become
SUMOylated in S2 cells, a somatic cell line from embryonic haematocyte origin which does not have an
active piRNA pathway. While SUMOylation was still detectable, its pattern was drastically different from
the charac
teristic “ladder” observed in the female germline
(Fig
.
S5)
. The tissue
-
specific patterns of Piwi,
Panx, Mael and SpnE SUMOylation therefore suggest that this modification is involved in specific
regulatory steps in the germline in the context of active pi
RNA pathway response to genomic parasites.
Previous work identified the SUMO E3 ligase Su(var)2
-
10 as an effector of transposon silencing
and heterochromatin establishment that acts downstream of Piwi
12
. To test if Su(var)2
-
10 regulates the
SUMOylation of
Mael, Panx, Spn
-
E or Piwi
, we in
duced its germline
-
specific knockdown via previously
.
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7
characterized UASp
-
controlled small hairpin RNA
(shRNA)
12
.
Su(var)2
-
10 depletion did not abolish the
SUMOylation of any of the investigated
proteins
, despite efficient knockdown evidenced by female
sterility and RT
-
qPCR (Fig
.
5B). In fact, we systematically observed (n=2
-
4 independent experiments for
each protein)
a slight increase of SUMOylated species upon Su(var)2
-
10 germline knockdown. Of these,
the most prominent change was seen for Panx, which displayed a marked increase of its poly
-
modified
forms and
decrease
of mono
-
SUMOylated form (Fig
.
5A, asterisk).
Thus
, it appears that
Su(var)2
-
10 is
not a SUMO E3 ligase for Piwi, Mael, Spn
-
E,
or
Panx, but the modification of these proteins
in germ cells
is regulated in a manner influenced by Su(var)2
-
10 loss.
As Su(var)2
-
10 loss leads to
strong
transposon upregulation
12
, the increased SUMOylation of
piRNA pathway proteins
upon Su(var)2
-
10 knockdown
may indicate
SUMO implication in protein
complexes
actively engaged in transposon response.
To
explore this possibility
,
we decided to analyze
the SUM
Oylation of Panx, Spn
-
E
,
and Mael upon
Piwi depletion
that disrupts the nuclear piRNA
-
dependent silencing machinery
. To this end, we utilized UASp
-
controlled shRNA against Piwi, which
induces efficient Piwi germline knockdown
(Fig
.
5B,C
).
Notably, Piwi depletion resulted in marked loss of
Mael and Spn
-
E SUMOylated forms
(Fig
.
5C), suggesting that the modification of both
protein
s in germ
cells depends on Piwi
. I
n contrast,
Piwi
germline
knockdown did not affect
the number and levels of
SUMO
-
modified Panx forms, indicating that Panx SUMOylation is governed by a distinct mechanism
independent of
the
piRNA
-
Piwi complex
in this cellular context
.
Altogether, t
hese results indicate that
SUMOylation of Mael and Spn
-
E
occur in a regulated manner
dow
nstream of Piwi
,
pointing to
novel and
multifaceted roles of SUMOylation in the piRNA pathway.
Discussion
The
SUMO pathway is
essential
for normal cell function
as
evidenced by the
severe phenotypes
of loss
-
of
-
function mutants of SUMO and SUMO
ligase
s
in various
systems
37
.
O
ur
mechanistic
understanding of
SUMOylation however
has
remain
ed
limited, partly
owing to the technical difficulties
of
detecting this modification
. Here,
we adapted the
diGly
remnant enrichment method for unbiased
discovery
of SUMO
-
modified proteins
with specific site predictions
in
Drosophila
. We note that while diGly
peptide enrich
ment improves the sensitivity and specificity of SUMOylated protein detection, as with other
bottom
-
up
proteomics methods
,
detection is
only possible for
sites
residing
in
protease
-
generated
peptides
within a specific
size
range
.
Also, exact site
assignment
might be imprecise
when two lysine
residues
are in
proximity
, and
in the event a protein simultaneously
carries
SUMO and
an
other
modifi
cation
that
leav
es a
diGly remnant
after trypsin cleavage
such as ubiquitin
(in
such cases, the target
protei
n would be enriched in the SUMOylated
pool but
detected diGly remnants might come
from
any
concomitant modification
).
Despite these limitations, the absolute identification of SUMO
protein targets
remains of high confidence, and predicted sites are informative to narrow down exact modified
residues
for
future mutational studies.
Altogether, our proteomics survey
of the
Drosophila
ovary
uncovered
a
large
SUMO
ylated
complement
that display
s
conserved
features of this modification, including
sequence and
structural biases
of
preferred SUMOylation sites, collective targeting of
proteins from the same
process/complex
, and
enrichment of SUMO targets among proteins involved in various aspects of nucleic
aci
d metabolism.
Notably, we
found
SUMO targets
with complex modification patterns
among
a variety
of
piRNA pathway proteins
functioning
in both transcriptional and post
-
transcriptional transposon
silencing, pointing to a multifaceted role of this modification in the cellular response to genomic parasites.
Complexity of the SUMO
-
modified proteome
Approximately a third of all SUMO target
s in our high confidence set contained two or more diGly
remnants,
and analysis of
selected protein
s in germ cells
by
Western blotting (Fig. 5) demonstrated
a
characteristic “ladder” of multiple modified forms
.
These patterns are
indicative of
multi
-
SUMOyl
ation
.
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8
(when multiple residues in the same protein are SUMOylated)
, and possibly, concomitant
modification
by
SUMO and
other
protein modifiers
.
In addition
to multiple modifications at different
residues
,
studies
in
yeast and mammalian
systems have identified a variety of
homotypic and heterotypic SUMO and
Ubiquitin
chains, as well as hybrid SUMO
-
Ubiquitin chains
that could
creat
e
a complex regulatory
“code”
38,39
.
We detected s
everal diGly sites on the endogenous SUMO proteins in control samples (Table
S
1
), confirming the presence of hybrid SUMO
-
U
biquitin chains in
various configurations in flies.
In vitro
studies and comparative genomics suggested that
SUMO
-
only chains
common in yeast, plants and
mammals
have been evolutionarily
lost in
flies
40
.
D
istinguish
ing
SUMO chains from SUMO
-
Ubiquitin
hybrid chains
is not possible with
our approach
. However, considering the
presence of diGly remnants at
syntenic positions to typical sites of SUMO
chain formation
in other species
, the existence of SUMO
chains
in vivo
merits future investigation.
Heterochromatin as a SUMOylation ‘hot
-
spot’
A
prominent
feature of the
identified SUMO targets is that they are often
found among physically
and functionally related proteins.
This is consistent with previous notions of an unusual property of
SUMOylation compared to other PTMs, namely, that SUMO ligases can modify entire groups of
physically interacting proteins at multi
ple and perhaps redundant sites
(reviewed in ref.
9
)
. Such group
SUMOylation is thought to create
multiple
SUMO
-
SIM interactions within large molecular complexes that
act synergistically to
facilitate their assembly and function
.
Previous examples of “SUMO hot spots”
include
DNA repair pathway effectors in yeast, ribosome biogenesis, or PML bodies
9
. The SUMO
pathway has long been linked to heterochromatin: SUMOylated histones, SUMO and SUMO ligases were
shown to localize to heterochromatic regions in various systems, and several essential hetero
chromatin
proteins including HP1, H3K9 methyltransferase and histone deacetylase complex components were
identified as SUMO targets or interactor
s (reviewed in ref.
41
).
Our data identifying dozens of
heterochromatin proteins as SUMOylation
targets
suggests that
heterochromatin
can be considered
a
nother
hotspot of group SUMOylation.
Moreover,
SUMO was
recently
shown
to affect
interconnected
repressive chromatin factors in mouse embryonic stem cells
42
,
po
inting to an evolutionary conserved role
of
SUMOylation
in the regulation of chromatin organization
.
Notably, SUMO polymers were shown to
drive phase separation in different subcellular contexts
10,43
.
In the future, it will be interesting to establish
the functional significance of the heterochromatic SUMO target spectr
a, particularly from the perspective
of the bio
physical properties of heterochromatin and
heterochromatin
-
related multi
-
subunit regulatory
complexes.
A multifaceted role of SUMO in the piRNA pathway
The discovery of a large set of SUMO
-
modified proteins among piRNA pathway factors paints a
complex picture of the roles of SUMOylation in the cellular response to transposons.
First hints on the
implication of SUMO in the piRNA pathway emerged from genetic screens to identify factors require
d for
transposon repression
11
.
More recently,
functional studies demonstrated that SUMO
is essential for
the
nuclear compartment of the piRNA pathway that enforces co
-
transcriptional silencing of transposon
s
by
the installation of the silencing mark H3K9me3
at target loci
. First, transcriptional silencing was shown
to
involve
Su(var)2
-
10
/
SUMO
-
dependent recruitment of chromatin modifying complexes downstream of
Piwi and
the
co
-
repressor Panx
12,14
. Additionally, transposon silencing
requires
a SUMO
-
mediated
interaction between Panx and t
he general heterochromatin factor Sov
13
.
The proteomic identification of
numerous general heterochromatin protei
ns as well as Panx itself as SUMO targets
emphasizes the
possibility that SUMOylation plays a multifaceted role in the transcriptional silencing of transposons by
piRNAs.
However
, in addition to that
, we also uncovered SUMO targets among
piRNA proteins
whi
ch
belong to
spatially and
functionally distinct piRNA pathway compartments
such as the nuage
, and found
.
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9
that SUMO depletion disrupts the nuage structure
, suggesting that SUMOylation has even broader
implications in
transposon silencing.
While
dissecting
the multiple roles of SUMOylation in the piRNA pathway will require extensive
future work, observations of the SUMOylation patterns of selected piRNA pathway proteins provide
several interesting c
l
ues. First, t
he SUMO
-
modified forms of all four
proteins
are
of relatively low
abundance and only detectable
by
highly sensitive antibodies, indicative of transient nature.
Dynamic
and reversible combinatorial modifications via SUMO and SUMO
-
Ubiquitin chains
can create unique
surfaces for multivalent
pr
otein
interactions.
C
omplex chains
were previously shown to act as signals in
the regulated recruitment and assembly of protein complexes involved in centromere organization and
DNA damage repair
(reviewed in ref.
38
)
.
It is possible
that analogous
ly,
complex SUMOylation patterns
may be involved in dynamically regulated steps of piRNA
biogenesis and function.
A second line of c
l
ues
comes from the different effects of Piwi and Su(var)2
-
10 germline knockdown on the SUMOylation of
the
examined piRNA pa
thway proteins. The SUMO E3 ligase Su(var)2
-
10
is essential for H3K9me3
deposition at transposon targets
12
. The finding that Piwi, Panx, Spn
-
E or Mael SUMOylation d
o not
depend
on
Su(var)2
-
10 is consistent with a model where Su(var)2
-
10 acts at downstream steps of
heterochromatin establishment, but SUMOylation
is also involved in upstr
eam or parallel processes
related to piRNA biogenesis and function. Moreover, as
the SUMO
-
modified forms of the four proteins
increase upon Su(var)2
-
10 knockdown, it is tempting to speculate that SUMOylation might be associated
with increased piRNA pathway
activity in response to the strong transposon upregulation that occurs
upon this genetic perturbation
. Nevertheless, it is important to consider that Su(var)2
-
10 depletion causes
substantial transcriptomic changes beyond transposon activation, including t
he up
-
and down
-
regulation
of hundreds of protein coding genes
12,19
, with possible indirect consequences on the SUMOylome.
Interestingly
, Piwi depletion has differential effects on Mael, Spn
-
E, and Panx SUMOylation
:
Mael
and Spn
-
E lose their SUMOylation in Piwi kn
ockdown germ cells, while Panx SUMOylation remains
unaffected (Fig
.
5C).
Previous studies recognized
Mael
as an essential factor for
transcriptional
repression at piRNA targets as well as piRNA
-
independent genomic loci
, although its
precise
mechanistic
rol
e has remained
unclear
16,31
. Considering th
e
nuclear
functions
of Mael, loss of its SUMOylation upon
Piwi knockdown
might reflect a regulatory step following
piRNA/Piwi recognizing their targets.
Nevertheless, Mael also localizes to the nuage
16
, and further work will be required to establish whether
Mael SUMOylation is related to its
roles in the nucleus
or a yet unknown function in the
nuage. A possible
role of SUMOylation i
n
the cytoplasmic piRNA pathway branch is further highlighted by the modification
of Spn
-
E
. Spn
-
E is a
putative RNA helicase which, despite unclear mechanistic role, is a well
-
established
nuage component essential fo
r piRNA biogenesis
32
35
.
S
ince Piwi
operates
in the nucleus, yet its loading
with piRNA occurs in the nuage
, the dependence of Spn
-
E SUMOylation on Piwi raises the intriguing
possibility that SUMO
-
med
iated interactions may be
involved
in certain aspects of piRNA biogenesis.
Future work combining our highly sensitive and robust germline SUMOylation assay with different genetic
perturbations and SUMOylation
-
deficient mutants promises to improve our under
standing of the precise
mechanisms that orchestrate the piRNA pathway in its different subcellular contexts.
Finally, the lack of effect of Piwi loss on
the SUMOylation of
Panx
one of the core components
of the RNA
-
binding SFiNX/Pandas/PITSC complex that is essential for H3K9me3
deposition
downstream
of
Piwi
suggests
Panx
is modified in a process that
occurs
independently of
Piwi
-
mediated target
recognition.
Surprisingly, th
is
result
contrast
s
with recent data from
OSC cells
a
n immortalized
cell
line
derived from the ovarian
somatic
follicle cells which expresses only the nuclear portion of the piRNA
pathway
where Panx SUMOylation was found to
completely
depend on Piwi
13
. While we cannot rule
out that residual levels of Piwi may be sufficient to maintain Panx SUMOylation in our system
, it is
also
possible that the regulation of Panx differs between the germline and soma, or even
at
different stages
of oogenesis. A potential mechanistic difference in the nuclear piRNA pathway between germline and
.
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;
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10
soma has critical implications to our un
derstanding of this process and will be important to address in the
future.
M
aterials and M
ethods
Drosophila stocks and husbandry
Drosophila lines encoding GFP
-
tagged Piwi under the native promoter (3rd chromosome), UASp
-
GFP
-
Panoramix,
-
Maelstrom, and
-
Spn
-
E, on the 2nd chromosome
, and small hairpin RNA against the
smt3
gene
, and GFP
-
tagged Aub under the control of the endogenous A
ub promoter
,
were previously
generated in Aravin
/Fejes Toth
lab
oratories
12,18,36
. Flies encoding UASp
-
driven HisFlag
-
tagged SUMO
on the 3rd chromosome were a gift from Courey Lab
oratory
44
. Flies encoding shRNA against
su(var)2
-
10
,
piwi
, and
white
, the maternal tubulin(Mt)
-
Gal4 driver, the iso
-
1 Celera sequencing strain, and
smt[04493] were obtained from the
Drosophila Bloomington stock center (#32956, #33724, #33623,
#7063, #2057, #11378). To
study
SUMOylation in
germ cells
, four transgenes including
UASp
-
HisFlag
-
SUMO, GFP
-
tagged target,
UASp
-
shRNA
and Mt
-
Gal4 driver
were combined by crossing balanced
parenta
l lines encoding GFP
-
tagged target and Mt
-
Gal4 driver, and
lines carrying
UASp
-
shRNA and
UASp
-
HisFlag
-
SUMO.
To generate SUMO
-
TR flies, the smt3 locus + ~2kb upstream region fragments
(chr2R:21704005
-
21717574, dm6 reference genome)
were amplified with the i
ntroduction of a 6xHis tag and a T86>R
mutation using Gibson assembly, subcloned into a phiC31 backbone based on the pCasper5 vector, and
integrated into the P{CaryP}attP2 locus at BestGene Inc.
All stocks were maintained at 25°C on standard molasses
-
base
d media. Experiments were
performed on samples from
3
5
-
day
old females maintained on
standard
media supplemented with yeast
for 2 days prior to dissections. Ovaries were manually dissected in cold PBS
,
flash frozen in liquid nitrogen
and stored at
-
80°C.
Proteomics sample preparation
The preparation of SUMO
-
derived GG
-
modified peptide samples was adapted from Impens et al.
with some modifications
20
. All procedures were performed using low binding plasticware
,
HPLC grade
water
, and
freshly prepared solutions.
Hand dissected ovaries from 600 2
-
5
-
day old
D
.
melanogaster
individuals
of smt3[04493]/CyO; {6xHis
-
smt3[T
86
R]}attP2 genotype (SUMO
-
TR) or iso
-
1 (Bloomington
#2057) (control) were used in each experiment. Frozen samples were imme
diately lysed in 2.5 mL
denaturing buffer (6M guanidium HCl, 10 mM Tris, 100 mM sodium phosphate buffer pH 8.0) using glass
Potter
-
Elvehjem tissue grinder. Lysates were cleared by centrifugation at 20,000rpm for 10 minutes at
4°C. Cleared lysates were trea
ted by 5mM tris(2
-
carboxyethyl)phosphine for 30 min at 37°C with gentle
rotation, followed by 10 mM N
-
ethylmaleimide (Sigma) for 30 min at room temperature, and finally, 10
mM Dithiothreitol (Sigma). Lysate volume was brought to 8 mL with lysis buffer and
imidazole was added
to 5 mM final concentration. Lysates were incubated with 1 mL HisPur
Ni
-
NTA Resin
(ThermoFisher)
overnight at 4°C with gentle rotation. After incubation, the Ni
-
NTA slurry was washed once with lysis
buffer supplemented with 10 mM imida
zole, once with wash buffer 1 (8M urea, 10 mM Tris, 100 mM
sodium phosphate (pH 8.0), 0.1% Triton X
-
100, 10 mM imidazole), and three times with wash buffer 2 (8
M urea, 10mM Tris, 1
mM sodium phosphate buffer pH
6.3, 0.1% Triton X
-
100, 10mM imidazole),
fol
lowed by two rounds of elution with elution buffer (300mM imidazole, sodium phosphate buffer pH 6.8)
for 2 hours at 4°C to a final eluate volume of 1.5 mL. The eluate volume was increased to 10 mL with
50
mM
ammonium bicarbonate and treated with sequencing
grade trypsin (Promega, V5111) at 1:50
trypsin:protein ratio overnight at 37°C with gentle agitation. The following steps were performed according
to the manual of the PTMScan® HS Ubiquitin/SUMO Remnant Motif (K
-
ε
-
GG) Kit (Cell
Signaling
.
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The copyright holder for this preprint
this version posted October 25, 2022.
;
https://doi.org/10.1101/2022.08.15.504007
doi:
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11
#59322). In brief
, trypsinized samples were acidified to pH 2
-
3 by the addition of
0.5 m
L 20%
trifluoroacetic acid (TFA), kept on ice for 15 mins, and centrifuged to remove potential precipitates.
Peptides were then purified on a C18 column according to the kit manual, and
lyophilized for >24hrs. Dry
peptides were then resuspended in an IAP buffer (Cell Signalling #59322) and incubated with anti
-
diGly
antibody
-
conjugated slurry (Cell
Signaling
#59322) at 4°C overnight, followed by two washes with IAP
buffer, and 3 washes wi
th HPLC
-
grade water. Finally, peptides were eluted with 0.15% TFA, purified
using
C18
StageTips, vacuum dried
, and submitted to
the Caltech Proteome Exploration Laboratory
for
Mass spectrometry analysis.
LC
-
MS/MS and raw data processing
Label
-
free
peptide
samples were subjected to LC
-
MS/MS analysis on an EASY
-
nLC 1200
(Thermo Fisher Scientific, San Jose, CA) coupled to a Q Exactive HF Orbitrap mass spectrometer
(Thermo Fisher Scientific, Bremen, Germany) equipped with a Nanospray Flex ion source. Sa
mples were
directly loaded onto an Aurora 25cm x 75μm ID, 1.6μm C18 column (Ion Opticks) heated to 50°C. The
peptides were separated with a 60 min gradient at a flow rate of 220 nL/min for the in
-
house packed
column or 350 nL/min for the Aurora column. The
gradient was as follows: 2
6% Solvent B (3.5 min), 6
-
25% B (42 min), 25
-
40% B (14.5 min), 40
-
98% B (1 min), and held at 100% B (12 min). Solvent A
consisted of 97.8 % H2O, 2% ACN, and 0.2% formic acid and solvent B consisted of 19.8% H2O, 80 %
ACN, and 0.
2% formic acid. The Q Exactive HF Orbitrap was operated in data dependent mode with the
Tune (version 2.7 SP1build 2659) instrument control software. Spray voltage was set to 1.5 kV, S
-
lens
RF level at 50, and heated capillary at 275°C. Full scan resolutio
n was set to 60,000 at m/z 200. Full
scan target was 3 × 106 with a maximum injection time of 15 ms. Mass range was set to 400−1650 m/z.
For data dependent MS2 scans the loop count was 12, AGC target was set at 1 × 105, and intensity
threshold was kept at
1 × 105. Isolation width was set at 1.2 m/z and a fixed first mass of 100 was used.
Normalized collision energy was set at 28. Peptide match was set to off, and isotope exclusion was on.
Data acquisition was controlled by Xcalibur (4.0.27.13), with ms1 dat
a acquisition in profile mode and
ms2 data acquisition in centroid mode.
Thermo raw files were processed and searched using MaxQuant (v. 1.6.10.43)
45,46
. Spectra were
searched against
D. melanogaster
UniProt entries plus the His
-
SUMO
-
TR sequence and a common
contaminant database. Trypsin was specified as the digestion enzyme and u
p to two missed cleavages
were allowed. False discovery rates were estimated using a target
-
decoy approach, where the decoy
database was generated by reversing the target database sequences. Protein, peptide, and PSM scores
were set to achieve a 1% FDR at
each level. Carbamidomethylation of cysteine was specified as a fixed
modification. Protein N
-
terminal acetylation, methionine oxidation, and diGly remnant on lysine were
specified as variable modifications with a maximum of 5 mods per peptide. Precursor m
ass tolerance
was 4.5 ppm after mass recalibration and fragment ion tolerance was 20 ppm. Search type was specified
as Standard with multiplicity of 1. Fast LFQ and normalization were used, and both re
-
quantify and match
-
between
-
runs were enabled.
Bioinfo
rmatics analysis of
SUMOylation sites
Summary tables of the normalized diGly sites output from MaxQuant (GlyGly (K)Sites.txt
, Table
S1
) were analyzed and figures were generated using custom R scripts
(
a
ssociated R markdown
notebooks
will be
available
from
GitHub
after peer review
)
.
In brief,
SUMO sites were considered sites
with MS intensity ratios in the SUMO
-
TR to corresponding control samples >10, and background diGly
sites were considered sites with
SUMO
-
TR/control ratios <3.
Motif searches were performed using
regular expressions and the MoMo suite
47
using a 11 aminoacid window centered on the predicted GG
-
modified lysine.
Protein structure predictions were performed using
the
‘bio_embeddings’ package
with
default parameters
22
, or IUPred2 with default parameters
23
. To test whether the numbers of diGly sites
.
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;
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falling within a specific structural region are different than expected by random chance, we created a
bootstrap distribution (n=1000) of the fraction of randomly selected lysines from the same set of proteins
falling within each region. Functional enrichm
ent analyses presented on Figure 3 were carried out using
the clusterProfiler R package with annotations from the OrgDb package (GO source date 2021
-
Sep13).
Semantic simplification was used to merge highly redundant terms.
We note that similar results were
obtained using TopGO, STRING and the bINGO Cytoscape plugin (not shown). STRING Interactions
were plotted using CytoScape at 0.6 confidence cutoff and nodes were grouped manually according to
association with specific GO categories
flagged by STRING annot
ations
. Interaction networks of Piwi,
HP1
a
, Egg, Wde and
V
asa
were retrieved from FlyBase and nodes present in the diGly SUMO dataset
were custom colored in CytoScape
48
.
Detection of prote
in SUMOylation by Immunoprecipitation (IP) and Western blotting
All samples were prepared in parallel with
respective
controls.
Hand dissected ovaries from 100
-
150 flies
(depending on the expressed protein)
of appropriate genotypes were lysed in RIPA buffe
r
supplemented with Complete Protease Inhibitor cocktail (Roche, 11836170001) and 20 mM NEM. Debris
were removed by centrifugation at 20,000 rpm for 10 minutes at 4°C. Cleared lysates were incubated
with GFP
-
Trap magnetic agarose beads (ChromoTek, gtma
-
20)
for 1
-
2 hrs with end
-
to
-
end rotation at
4°C. Beads were washed 5 times with “harsh” wash buffer (20 mM Tris pH 7.4, 0.5 M NaCl, 1% NP40
(Igepal), 0.5% Sodium deoxycholate, 1% SDS). Finally, samples were transferred to fresh tubes and
boiled in LDS sample
buffer (Invitrogen, NP0007) for 5 minutes at 95°C.
For optimal separation of high
molecular weight SUMOylated species, IP samples were analyzed on 3
8% tris
-
glycine gels (Invitrogen).
Additionally, input
samples
were separately analyzed at high
er
percent g
els to capture low molecular
weight free SUMO. After electrophoresis, proteins were transferred to 0.45μm nitrocellulose membrane
(BioRad) and transfer was verified by Ponceau S stain. Membranes were then blocked with 5% nonfat
milk in PBS
-
T(0.1% Tween in
PBS) for >30 min at
room temperature (RT)
, followed by incubation with
primary antibodies for 2 hrs at
RT
or overnight at 4°C. The following antibodies were used for detection:
HRP
-
conjugated anti
-
Flag antibody (Sigma, A8592), rabbit anti
-
GFP (Abcam, a
b
290), mouse anti
-
Tubulin (Sigma, T5168), mouse anti
-
Piwi (Santa Cruz, sc
-
390946), HRP
-
conjugated anti
-
mouse IgG (Cell
Signalling, 7076), HRP
-
conjugated anti
-
rabbit IgG (Cell Signalling, 7074), IRDye® 800CW anti
-
rabbit IgG
(Licor).
For SUMOylation analysis
in S2 cells, plasmids encoding GFP
-
tagged Piwi, Mael, Spn
-
E and
Panx and 3xFlag3xHA
-
tagged SUMO under the control of ubiquitin and actin promoter, respectively,
were co
-
transfected using TransIT
-
Insect Transfection reagent (Mirus, MIR6105). Cells were harv
ested
48 hrs post
-
transfection and samples were processed and analyzed following the procedure
described
above.
R
T
-
qPCR
RNA was extracted from 20
-
25 pairs of ovaries from appropriate genotypes using Trizol
(
Invitrogen, 15596018
) according to the manufactu
rer instructions. Purified RNA was treated with DNAse
and subjected to reverse transcription with the SuperScript IV with ezDNAse kit (Invitrogen, 11766050)
according to the manufacturer instructions. qPCR was performed using SybrGreen and the following
pr
imers: Su(var)2
-
10: CCAGCACAGGACGAACAGCCC and CGTGGAACTGGCGACGGCTT, Piwi:
CTGCTGATCTCCAAAAATAGGG and TCGCGTATAACTGCTCATGG; Rp49 (internal control):
CCGCTTCAAGGGACAGTATCTG and ATCTCGCCGCAGTAAACGC. Relative expression was
calculated with respect to the Rp49
expression using the delta Ct method.
Protein localization imaging
.
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available under a
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The copyright holder for this preprint
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;
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doi:
bioRxiv preprint
13
Ovaries were dissected in cold PBS and fixed in 4% formaldehyde in PBS
-
T (0.1% Tween
-
20 in
PBS) with end
-
to
-
end rotation for 2
0
minutes at room temperature, following by 3 washes with PB
S
-
T for
>10 minutes each.
For Vasa detection, f
ixed
ovaries were
incubated with blocking solution (0.3% Triton
-
X, 0.1% Tween
-
20, 3% Bovine serum albumin (BSA)) for 1 hour with end
-
to
-
end rotation
, followed by an
overnight incubation with 1:10
dilution of anti
-
vasa antibody (DSHB anti
-
vasa supernatant, AB_760351)
at 4°C
with rotation
. Next, ovaries were washed 3 times with washing solution 0.3% Triton
-
X, 0.1%
Tween
-
20 in PBS) for 10
minutes and incubated with 1:400 diluted AlexaFlour 594 seconda
ry antibody
(Invitrogen A
-
11007) in blocking solution for 2 hours at
RT
in the dark. Finally, ovaries were washed with
washing solution for 10 minutes 3 times, 1 μg/mL DAPI solution for 5 minutes, and a final rinse of 5
minutes.
For proteins GFP
-
Aub, fixed
ovaries were only stained with DAPI.
Samples were mounted on
glass slides in Vecta
s
hield
Antifade Mounting Medium (Vectorlabs, H
-
1200
-
10) and analyzed on an
Upright Zeiss LSM 880 Confocal Microscope at the UCR Microscopy and Imaging Facility. Final images
were processed and assembled with Fiji
49
. Shown are representative images of 10 or more analyzed
ovaries.
Fertility test
10 2
-
day old females of appropriate genotype and 5 wild type males were maintained at
standard
fly media for 3 and 5 days, and the numbers of viable adult offspring from each vial was manually counted.
Results are presented as viable offspring per day. Additionally, vials where females expressing shRNAs
against Piwi and Su(var)2
-
10 from di
fferent crosses were maintained prior dissection were inspected for
viable progeny to verify expected sterility phenotypes.
Data availability statement
The mass spectrometry raw data are deposited to the ProteomeXchance Consortium
(https://www.ebi.ac.uk/pride/) via the PRIDE repository with the dataset identifier
PXD037421. Reviewer
account details: Username: xxx Password: xxx.
Figure Legends
Figure 1.
P
roteomics approach for SUMO site detection.
A. Diagram of the peptides generated after
trypsin digestion of proteins modified by wild
-
type and T>R mutant SUMO. B. Diagram of the
experimental design and workflow for SUMOylation
-
derived diGly
-
modified peptide MS analysis. C.
Diagram of the recombinant SUMO construct used to generate SUMO
-
TR expressing
transgenic
flies.
D. Western blot of total ovary lysate confirming the expression of recombinant SUMO. Ovaries from flies
overexpressing 6xHisFl
ag
-
tagged SUMO are used as a positive control.
Figure 2.
Characteristics of the ovarian SUMOylome
.
A. Distribution of diGly site intensities in
different samples. B. (Top)
Amino acid frequencies
flanking predicted K
-
ε
-
GG
,
for sites with high and low
SUMO
-
TR
:
control
intensity ratios
in a representative replicate
. Also see Fig. S1
. (Bottom) Distribution of
SUMO
-
TR
:
Control intensity ratios of diGly sites
in each
experiment. C.
O
verlap of exact SUMO sites or
SUMO
-
modified genes
(
irrespective of exact sites
)
b
etween replicates
. D.
Numbers
of predicted SUMO
sites per protein
and distance between sites
for proteins with 2 sites
. E.
Sequence motifs at
421 high
confidence SUMO sites. The predicted
diGly
-
modified lysine is in bold. Bar graphs show the
counts of
topmost
frequent
aminoacids at positions
-
1 and +2, and
-
2,
-
1 and +2 from
the
modified
lysine. F.
Localization of
di
G
ly
sites with high or low intensity ratios in predicted structured or intrinsically
disordered
regions, compared to randomly sel
ected lysine
s
from the same protein over 1000 iterations. G.
Localization of
diGly
SUMO sites embedded in different motifs to IDRs.
.
CC-BY-NC-ND 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted October 25, 2022.
;
https://doi.org/10.1101/2022.08.15.504007
doi:
bioRxiv preprint
14
Figure 3.
Functional groups of SUMOylated proteins
.
A. Summary of Gene Ontology analysis. (Left)
Bar charts of the most enri
ched GO terms and corresponding gene numbers among the high confidence
SUMOylated targets after semantic simplification, ordered by adjusted p
-
value. (Right) Network diagram
of proteins annotated in the top 5 enriched Biological Process GO
categories
. B. S
TRING diagram of the
interactions between identified high confidence SUMOylation targets. Singl
e
nodes are not shown. Nodes
are
colored according to selected GO terms and manually grouped according to functional annotations.
Figure 4.
SUMO
targets
nuclear and cytoplasmic piRNA factors.
A.
N
etwork diagram show
ing
physical interacting partners of Piwi, HP1, SetDB1, Wde, and
V
asa proteins retrieved from FlyBase.
Nodes
are
colored according to
detection frequency of SUMOylation sites in the MS data.
Ad
ditional
interactions between partner nodes are not shown. B.
(Top) Diagram of an ovariole with indicated stages
where the maternal tubulin
-
Gal4
(MT
-
gal4)
driver
is active
; stages of imaging are highlighted
. (Bottom)
Confocal images
of
Vasa (immunostaining
) and Aub (GFP
-
tagged Aub)
localization
in
stage 5
-
7
egg
chambers
from flies expressing SUMO shRNA under the control of the MT
-
gal4 driver, or no driver control
siblings.
Figure 5.
SUMOylation of Piwi, Mael, Spn
-
E and Panx.
A. (Top) Diagrams
of
structural features,
consensus SUMOylation motifs
,
and
experimentally
detected diGly sites with TR
-
SUMO
:
Control
i
ntensity
ratios >10. Color indicate
s
diGly site
intensity
percentile
s
. (Bottom)
WB analysis of
target SUMOylation
in the female germline. Ovar
y lysates from flies carrying indicated transgenes (mtG4=maternal tubulin
Gal4 driver) were subjected to GFP
IP
followed by WB detection first with anti
-
Flag antibody, and after
stripping,
anti
-
GFP
antibody. Note that SUMOylated species are only detectable
by the highly sensitive
anti
-
Flag antibody, but not anti
-
GFP antibody. Lanes in Piwi and Spn
-
E gels were cropped
for
sample
order
consistency
. Images are representative of 3
-
4 independent experiments per target. B.
Efficiency of
su(var)2
-
10
and
piwi
germl
ine knockdown
estimated by fertility test (top) and
RT
-
qPCR (bottom). Results
are from samples from the same genetic crosses used for Western blotting in C
,
GFP
-
Panx.
C.
WB
of
target SUMOylation in the female germline upon
white
(control),
su(var)2
-
10
and
piwi
germline
knockdown. Images are representative of two independent experiments. Un.=unspecific band.
Acknowledgements
We thank former Caltech Protein Exploration Laboratory (PEL) members Dr. Michael Sweredosk
i and
Dr. Annie Moradian for their advice on diGly proteomics, and Corinne Karalun (
former
laboratory assistant
in MN lab
oratory
, UCR)
,
Hannah Ryon (
former
rotation s
tudent in KFT lab
oratory
, Caltech
)
, and Matea
Ibrahim (undergraduate students in UC Riverside)
for assistance with
Western Blotting, fly dissections
and genotyping
.
This work was supported by
grants from the NIH
(
K99/R00
HD099316
)
to
MN;
the NIH
(R01 GM097
363)
and
the Howard Hughes Medical Institute Faculty Scholar Award
to AAA, and the NIH
(R01
GM110217) and Ellison Medical Foundation Awards to KFT.
Authors contributions
MN, KFT
,
and AAA conceptualized the
proteomics
study. MN designed and
performed experiments, data
curation
,
and formal analysis, except LC
-
MS/MS runs and raw data processing which were performed at
the Caltech PEL facility by BL.
HH performed GFP
-
Aub localization experiments.
MN prepared figures
and drafted the manuscript. M
N and AAA edited the manuscript.
Declaration of interests
The authors declare no competing interests.
.
CC-BY-NC-ND 4.0 International license
available under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprint
this version posted October 25, 2022.
;
https://doi.org/10.1101/2022.08.15.504007
doi:
bioRxiv preprint
15
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