Pervasive SUMOylation of heterochromatin and piRNA pathway proteins

Pervasive SUMOylation of heterochromatin and piRNA pathway

proteins

Maria Ninova

1,2

Hannah Holmes

Brett Lomenick

Katalin Fejes

, Alexei

Aravin

Department of Biochemistry

University of California Riverside

3401

Watkins Drive, Boyce Hall

, R

iverside, CA 92521, USA

Division of Biology and Biological Engineering

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

mall

biquitin

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.

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

and lineage

inappropriate genes.

However, the specific targets of SUMO and its mechanistic

role in different

ellular pathways

are still poorly understood.

Here

developed a proteomics

based

strategy to characterize the SUMOylated

proteome

Drosophila

that allowed us

to identify

~1500 SUMO

sites in 843 proteins

the

fly ovary.

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

, 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

Keywords

SUMO, pi

RNAs, heterochromatin, transposons, proteomics

, germline

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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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.

)

brief,

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

sufficient for SUMOylation

in vi

–

Nevertheless

, non

catalytic SUMO E3 ligases can facilitate Ubc9 or

enable substrate

specificity

and

seem to be required

for SUMOylation

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.

)

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.

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

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.

work

Drosophila

implicated the SUMO pathway in the regulation of heterochromatin

establishment

and

piRNA

mediated

transposon silencing

–

. In germ cells, Piwi clade proteins (Piwi,

Aub and Ago3

flies

) and Piwi

interacting small RNAs (piRNAs) cooperate in intimately linked processes

hat

ensure transcriptional and post

transcriptional transposon silencing and continuous production of

mature piRNAs

(reviewed in ref.

)

. 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

–

To date

, SUMOylation

is know

n to

participate

in the nuclear piRNA pathway

several

ways

First, t

he SUMO E3 ligase

Su(var)2

was

found

to interact with piRNA pathway and

heterochromatin

proteins

and

play an

essential role

the recruitment of the enzymatic complex SetDB1/Wde that deposits the silencing

epigenetic mark H3K9me3

, as well as with the MEP

1/Mi

2 chromatin remodeler complex

. 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

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

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

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

proteins specific

the

piRNA pathway

among SUMO targets

, including the central effector of

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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

(Spn

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

ues

toward

our understanding of the molecular

mechanisms of genome regulation

and transposon

control

Results

Establishing a system for

the

detection

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

peptides

from total protein lysates. Basic methods

for

the

enrichment of

SUMO

modified protein

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

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

(herein referred to as the SUMOylome)

Drosophila

adapted

approach

that

allows

stringent purification

enrichment

, and proteomic detection of

peptides containing

a modified SUMO

remnant

. This approach, originally developed

for the study of SUMOylation in

human cells

, employs

ectopically expressed

SUMO

protein

with

His

tag

and a

point mutation

(

R substitution

, herein

referred to as SUMO

) that

enables

trypsin cleavage

before

the C

terminal

motif

at the conjugation

site

(Fig

)

In a two

step purification process,

SUMO

modified proteins are

first

selected

based on

the His tag

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

then

trypsinized to

generat

a mixture of peptides including

branched

peptides

with

short

glycine

(diGly)

emnant

from

cleaved

SUMO

moiety.

These

diGly remnant

containing

peptides are

further

selected

from the total pool

by pulldown with

specific

antibody

purified

analyzed by mass

spectrometry

Of note

, Ubiquitin and other ubiquitin

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).

enable

this method for SUMOylation detection

Drosophila

tissues,

created a transgenic

line

that

carr

ies

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

)

and

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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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.

comparison,

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

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

several diGly sites on

SUMO itself

(Table

)

;

as s

ome

these sites can be detected in the negative control

this pattern is

indicative of

hybrid

SUMO

Ubiquitin chains.

To gain

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

identified the canonical consensus and inverse

consensus SUMOylation motifs (ψ

xE/D and E/Dx

ψ, respectively) at 45%

of the sites

(141 and 49)

this set (Fig

2E). ~10% of the sites have the motif ψK

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

n other systems, the SUMOylation consensus motif is not obligatory.

observations in human

cells

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

(Fig

2E).

Finally

, we assessed

the position of SUMOylation sites with respect to predicted structural features

based on deep learning

language models

including alpha sheets, beta helices and intrinsically disordered regions (IDR

). 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

(

Fig

). 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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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

different cellular

and

organismal

contexts.

Functional groups of SUMOylated proteins

investigate

the possible roles of SUMOylation in the ovary, we carried out functional

enrichment and int

eractome analys

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

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

Table S2

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

significantly

above

the background

(

ribosomal proteins

carry

57 out

of the

421

bona fide

SUMO sites

(13%)

versus

37 out

the

714 background diGly

sites(5

)

and could therefore represent genuine targets. Indeed, the

SUMOylation machinery has been implicated in ribosomal protein regulation

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.

)

. Notably,

SUMOylation is

widespread

among chromatin

associated proteins.

igh

confidence SUMOylation sites

can be found

several core histones

and

the

HP1 paralogs HP1b and HP1c

; a

weak

site

was

also

detected

the central heterochromatin effector

HP1a/Su(var)205

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

re terms relate

heterochromatin and transposon repressio

; for example,

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

asa

–

central component of

the

nuage compartment

where

piRNA

ogenesis and transposon RNAi

mediated cleavage take place

–

(Fig

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

igh confidence SUMOylation sites

are

present

in Piwi

as well as several others piRNA pathway proteins

including

Panoramix (Panx)

–

component of the so

called SFiNX/Pand

s/PICTS

–

complex required for H3K9me3 deposition

downstream of Piwi (also recently reported as SUMO target

in another study

)

Maelstrom (Mael)

–

essential for transcriptional repression and piRNA biogenesis from dual

stranded cluster

16,31

, and the

nuage component Spindle

E (SpnE

)

–

a germline

specific DEAD

box helicase essential for piRNA

production

–

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

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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localization of

the core nuage component

asa

, and the nuage

localized piRNA effector Aub, upon

SUMO depletion. To this end, we employed a previously characterized small hairpin RNA targeting

SUMO

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).

previ

ously reported, under normal conditions Aub and Vasa appear as a relatively smooth perinuclear

layer

(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

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

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

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

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

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

. 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

. 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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characterized UASp

controlled small hairpin RNA

(shRNA)

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

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

, 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.

explore this possibility

we decided to analyze

the SUM

Oylation of Panx, Spn

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

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

evidenced by the

severe phenotypes

of loss

function mutants of SUMO and SUMO

ligase

in various

systems

mechanistic

understanding of

SUMOylation however

has

remain

limited, partly

owing to the technical difficulties

detecting this modification

. Here,

we adapted the

diGly

remnant enrichment method for unbiased

discovery

of SUMO

modified proteins

with specific site predictions

Drosophila

. We note that while diGly

peptide enrich

ment improves the sensitivity and specificity of SUMOylated protein detection, as with other

bottom

proteomics methods

detection is

only possible for

sites

residing

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

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

large

SUMO

ylated

complement

that display

conserved

features of this modification, including

sequence and

structural biases

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

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

Western blotting (Fig. 5) demonstrated

characteristic “ladder” of multiple modified forms

These patterns are

indicative of

multi

SUMOyl

ation

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(when multiple residues in the same protein are SUMOylated)

, and possibly, concomitant

modification

SUMO and

other

protein modifiers

In addition

to multiple modifications at different

residues

studies

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

a complex regulatory

“code”

38,39

We detected s

everal diGly sites on the endogenous SUMO proteins in control samples (Table

), confirming the presence of hybrid SUMO

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

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’

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.

)

. 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

. 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.

Our data identifying dozens of

heterochromatin proteins as SUMOylation

targets

suggests that

heterochromatin

can be considered

nother

hotspot of group SUMOylation.

Moreover,

SUMO was

recently

shown

to affect

interconnected

repressive chromatin factors in mouse embryonic stem cells

inting to an evolutionary conserved role

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

More recently,

functional studies demonstrated that SUMO

is essential for

the

nuclear compartment of the piRNA pathway that enforces co

transcriptional silencing of transposon

the installation of the silencing mark H3K9me3

at target loci

. First, transcriptional silencing was shown

involve

Su(var)2

SUMO

dependent recruitment of chromatin modifying complexes downstream of

Piwi and

the

repressor Panx

12,14

. Additionally, transposon silencing

requires

a SUMO

mediated

interaction between Panx and t

he general heterochromatin factor Sov

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

belong to

spatially and

functionally distinct piRNA pathway compartments

such as the nuage

, and found

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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

ues. First, t

he SUMO

modified forms of all four

proteins

are

of relatively low

abundance and only detectable

highly sensitive antibodies, indicative of transient nature.

Dynamic

and reversible combinatorial modifications via SUMO and SUMO

Ubiquitin chains

can create unique

surfaces for multivalent

otein

interactions.

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.

)

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

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

is essential for H3K9me3

deposition at transposon targets

. The finding that Piwi, Panx, Spn

E or Mael SUMOylation d

o not

depend

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

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

, 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

the cytoplasmic piRNA pathway branch is further highlighted by the modification

of Spn

. Spn

E is a

putative RNA helicase which, despite unclear mechanistic role, is a well

established

nuage component essential fo

r piRNA biogenesis

–

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

Piwi

–

suggests

Panx

is modified in a process that

occurs

independently of

Piwi

mediated target

recognition.

Surprisingly, th

result

contrast

with recent data from

OSC cells

–

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

. 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

different stages

of oogenesis. A potential mechanistic difference in the nuclear piRNA pathway between germline and

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soma has critical implications to our un

derstanding of this process and will be important to address in the

future.

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

. Flies encoding shRNA against

su(var)2

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

–

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

. All procedures were performed using low binding plasticware

HPLC grade

water

, and

freshly prepared solutions.

Hand dissected ovaries from 600 2

day old

melanogaster

individuals

of smt3[04493]/CyO; {6xHis

smt3[T

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

™

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

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

GG) Kit (Cell

Signaling

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#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.

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

) were analyzed and figures were generated using custom R scripts

(

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

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

, or IUPred2 with default parameters

. To test whether the numbers of diGly sites

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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

, Egg, Wde and

asa

were retrieved from FlyBase and nodes present in the diGly SUMO dataset

were custom colored in CytoScape

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

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

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

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

or overnight at 4°C. The following antibodies were used for detection:

HRP

conjugated anti

Flag antibody (Sigma, A8592), rabbit anti

GFP (Abcam, a

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.

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

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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Ovaries were dissected in cold PBS and fixed in 4% formaldehyde in PBS

T (0.1% Tween

20 in

PBS) with end

end rotation for 2

minutes at room temperature, following by 3 washes with PB

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

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

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

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

. 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.

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

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

for sites with high and low

SUMO

control

intensity ratios

in a representative replicate

. Also see Fig. S1

. (Bottom) Distribution of

SUMO

Control intensity ratios of diGly sites

in each

experiment. C.

verlap of exact SUMO sites or

SUMO

modified genes

(

irrespective of exact sites

)

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

1 and +2 from

the

modified

lysine. F.

Localization of

sites with high or low intensity ratios in predicted structured or intrinsically

disordered

regions, compared to randomly sel

ected lysine

from the same protein over 1000 iterations. G.

Localization of

diGly

SUMO sites embedded in different motifs to IDRs.

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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:

bioRxiv preprint

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