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et al
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SUMOylation of Bonus, the
Drosophila
homolog of Transcription Intermediary
Factor 1, safeguards germline identity
by recruiting repressive chromatin
complexes to silence tissue-
specific genes
Baira Godneeva
1,2
, Maria Ninova
3
, Katalin Fejes-
Toth
1
, Alexei Aravin
1
*
1
California Institute of Technology, Division of Biology and Biological Engineering,
Pasadena, United States;
2
Institute of Gene Biology, Russian Academy of Sciences,
Moscow, Russian Federation;
3
University of California, Riverside, Riverside, United
States
Abstract
The conserved family of Transcription Intermediary Factors (TIF1) proteins consists
of key transcriptional regulators that control transcription of target genes by modulating chro-
matin state. Unlike mammals that have four TIF1 members,
Drosophila
only encodes one member
of the family, Bonus. Bonus has been implicated in embryonic development and organogenesis
and shown to regulate several signaling pathways, however, its targets and mechanism of action
remained poorly understood. We found that knockdown of Bonus in early oogenesis results in
severe defects in ovarian development and in ectopic expression of genes that are normally
repressed in the germline, demonstrating its essential function in the ovary. Recruitment of Bonus
to chromatin leads to silencing associated with accumulation of the repressive H3K9me3 mark. We
show that Bonus associates with the histone methyltransferase SetDB1 and the chromatin remod-
eler NuRD and depletion of either component releases Bonus-
induced repression. We further
established that Bonus is SUMOylated at a single site at its N-
terminus that is conserved among
insects and this modification is indispensable for Bonus’s repressive activity. SUMOylation influ-
ences Bonus’s subnuclear localization, its association with chromatin and interaction with SetDB1.
Finally, we showed that Bonus SUMOylation is mediated by the SUMO E3-
ligase Su(var)2–10,
revealing that although SUMOylation of TIF1 proteins is conserved between insects and mammals,
both the mechanism and specific site of modification is different in the two taxa. Together, our
work identified Bonus as a regulator of tissue-
specific gene expression and revealed the impor
-
tance of SUMOylation as a regulator of complex formation in the context of transcriptional
repression.
eLife assessment
This
important
study advances our knowledge of
Drosophila
Bonus, the sole ortholog of the
mammalian transcriptional regulator Tif1.
Solid
evidence, both in vivo and in vitro, shows how
SUMOylation controls the function of the Bonus protein and what the impact of SUMOylation on the
function of Bonus protein in the ovary is.
RESEARCH ARTICLE
*For correspondence:
aravin@caltech.edu
Competing interest:
The authors
declare that no competing
interests exist.
Funding:
See page 20
Preprint posted
15 April 2023
Sent for Review
12 July 2023
Reviewed preprint posted
05 September 2023
Reviewed preprint revised
15 November 2023
Version of Record published
24 November 2023
Reviewing Editor:
Claude
Desplan, New York University,
United States
Copyright Godneeva
et al
.
This article is distributed under
the terms of the Creative
Commons Attribution License,
which permits unrestricted use
and redistribution provided that
the original author and source
are credited.
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2 of 24
Introduction
Epigenetic regulation of gene expression is an essential mechanism that guides cell differentiation
during development. The post-
translational modifications of chromatin proteins act in combination
with various chromatin-
remodeling proteins to mediate changes in transcriptional activities and chro-
matin structure (reviewed in
Berger, 2007
;
Kouzarides, 2007
). TRIM/RBCC is an ancient protein
family characterized by the presence of an N-
terminal RING finger domain closely followed by one
or two B-
boxes and a coiled coil domain. Additional protein domains found at their C termini have
been used to classify TRIM proteins into subfamilies. The Transcriptional Intermediary Factor 1 (TIF1)
proteins present in Bilaterian species contain PHD and Bromo domains at their C-
terminus and belong
to Subfamily E according to
Marin, 2012
or structural class E according to
Ozato et al., 2008
. In
vertebrates this subfamily contains four proteins: TIF1
α
/TRIM24, TIF1
β
/TRIM28, TIF1
γ
/TRIM33, and
TIF1
δ
/TRIM66, while only one protein, Bonus (Bon), is present in
Drosophila
, making it an attractive
model to understand the conserved functions of TIF1 proteins.
Mammalian TIF1 proteins are chromatin-
associated factors that have been shown to play an essen-
tial role in transcription, cell differentiation, cell fate decisions, DNA repair, and mitosis (
Bai et al.,
2010
;
Cammas et al., 2004
;
Cammas et al., 2000
;
Kulkarni et al., 2013
;
Le Douarin et al., 1996
;
Nielsen et al., 1999
;
Sedgwick et al., 2013
). TIF1 proteins modulate the transcription of target
genes by binding to co-
regulators in the genome and controlling the chromatin state (
Khetchoumian
et al., 2004
;
Nielsen et al., 1999
;
Schultz et al., 2002
;
Schultz et al., 2001
;
Venturini et al., 1999
).
One of the best characterized TIF1 proteins, KAP-
1 (TIF1
β
), is the universal cofactor for the large
family of Krüppel-
associated box zinc-
finger proteins (KRAB-
ZFPs) composing one of the best-
studied
gene silencing systems in vertebrates (
Friedman et al., 1996
). Diverse KRAB-
ZFPs recognize specific
DNA sequences with the majority targeting endogenous retroviruses, ensuring their repression. After
target recognition by KRAB-
ZFPs, KAP-
1 suppresses target transcription with the help of the H3K9-
specific histone methyltransferase SetDB1, the H3K9me3 reader HP1, and the NuRD histone deacety-
lase complex (
Schultz et al., 2002
;
Schultz et al., 2001
).
The only member of the TIF1 subfamily in
Drosophila
, Bon was shown to be important in the devel-
opment of several organs and somatic tissues during embryogenesis and metamorphosis, including
the nervous system and the eye (
Allton et al., 2009
;
Beckstead et al., 2001
;
Ito et al., 2012
;
Kimura
et al., 2005
;
Salzberg et al., 1997
;
Zhao et al., 2023
). Bon has been shown to regulate the function
of different signaling pathways to drive developmental fate decisions, such as the ecdysone pathway
(
Beckstead et al., 2001
) and the Hippo pathway in the eye (
Zhao et al., 2023
). Bon can act as both
an Enhancer and a Suppressor of position-
effect variegation (
Beckstead et al., 2005
), suggesting that
it might play different roles that depend on specific interactors.
Many TRIM proteins from different subfamilies, including the mammalian TIF1
γ
/TRIM33, act as
ubiquitin ligases, suggesting that this was the ancient function of the family. On the other hand,
several members, including the mammalian KAP-
1 was shown to be active as E3 SUMO-
ligases.
Furthermore, SUMOylation plays an essential role in KAP-
1 function: KAP-
1 is SUMOylated through
its own activity and SUMOylation is required for its repressive function by facilitating recruitment of
the SetDB1 histone methyltransferase (
Ivanov et al., 2007
;
Lee et al., 2007
;
Li et al., 2007
;
Mascle
et al., 2007
). SUMO (small ubiquitin-
like modifier) is a small protein that is covalently conjugated to
lysine residues of substrates that can modify and enhance protein–protein interactions (
Gareau and
Lima, 2010
;
Jentsch and Psakhye, 2013
;
Martin et al., 2007
). SUMOylation has been implicated
in facilitating formation of protein complexes and condensates, especially in the nucleus, in different
contexts including DNA repair, transcriptional repression and formation of subnuclear structures,
and chromatin domains (reviewed in
Garvin and Morris, 2017
;
Gill, 2005
;
Verger et al., 2003
).
The SUMO conjugation cascade involves the E1-
activating enzyme, the E2-
conjugating enzyme, and
multiple E3-
ligases that interact with E2 and facilitate the transfer of SUMO to the final substrates
(
Gill, 2004
;
Johnson and Gupta, 2001
).
Here, we show that depletion of Bon in the female germline results in defective oogenesis and
female infertility. We found that Bon controls oogenesis through repression of ectopic gene expres-
sion indicating that it serves as a guardian of cell-
type identity. Mechanistically, we found that Bon
induces transcriptional repression through interaction with the dNuRD chromatin remodeler and the
SetDB1 histone methyltransferase. We show that Bon is SUMOylated at a single site at its N-
ter
-
minus and that this modification is essential for Bon-
induced transcriptional silencing. Furthermore,
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this modification is important for Bon subnuclear localization and chromatin association as well as
its interaction with SetDB1. The N-
terminal SUMOylation site is conserved in insect species, but not
in mammalian KAP-
1 where several SUMOylation sites were reported at the C-
terminal portion of
the protein. Finally, we established that Bon SUMOylation depends on a distinct SUMO E3-
ligase,
Su(var)2–10, in contrast to mammalian KAP-
1 that auto-
SUMOylates itself. Our results identify Bon
as a regulator of tissue-
specific gene expression and highlight the universal function of SUMOyla-
tion as a regulator of complex formation in the context of transcriptional repression. On the other
hand, our work suggests that SUMOylation of
Drosophila
Bon and mammalian KAP-
1 has evolved
independently and through distinct mechanisms revealing a remarkable case of parallel evolution in
insects and vertebrates.
Results
bon
knockdown in the female germline interferes with germline stem
cells function and leads to arrested oogenesis and sterility
According to FlyAtlas, the
bon
gene encodes a nuclear protein that is expressed throughout devel-
opment with high level of expression in several tissues including the brain, gut, and ovaries (FlyAtlas;
Chintapalli et al., 2007
). Immunostaining with antibodies against Bon revealed that it is expressed
in both the germline and somatic cells at all stages of oogenesis, starting from the germarium which
contains GSCs to late-
stage egg chambers where GSC-
derived nurse cells support maturing oocytes
(
Figure 1A
). While
bon
was shown to be required for metamorphosis and the development of the
nervous system (
Beckstead et al., 2001
;
Ito et al., 2012
), its function in the germline remained
unknown. To gain insights into the germline functions of Bon, we generated transgenic flies expressing
short hairpin RNAs (shRNAs) against
bon
under control of the UAS/Gal4 system and performed
germline-
specific RNAi knockdown (GLKD). Using the
maternal tubulin- Gal4 (MT- Gal4)
driver, we
found by RT-
qPCR (quantitative reverse transcription PCR) that two distinct shRNAs targeting
bon
led
to 75% and 88% reduction in ovarian Bon expression, respectively (
Figure 1B
). Because the
MT- Gal4
driver is active in the germline, but not in follicular cells, the actual knockdown efficiency of
bon
in
germ cells is even higher than what we detected from whole ovarian lysates. Indeed, immunofluores-
cence confirmed that Bon protein had been efficiently depleted from germline cells (
Figure 1C
). For
all subsequent experiments, we used the shRNA that resulted in higher knockdown efficiency.
To analyze the role of Bon throughout the developmental progression of the germline we combined
the
bon
shRNA construct with three different germline Gal4 drivers using different stage-
specific
promoters:
bam- Gal4
, which is expressed from cystoblasts to eight-
cell cysts;
MT- Gal4
, which drives
expression in germ cells starting in stage 2 of oogenesis, and
nos- Gal4
driver, which induces expres-
sion in two distinct stages, in GSCs and at late stages of oogenesis (
Figure 1—figure supplement
1A
;
Chen and McKearin, 2003
;
Van Doren et al., 1998
;
McKearin and Ohlstein, 1995
). Bon GLKD
driven by either
MT- Gal4
,
bam- Gal4
, or the double driver (
MT + bam
) did not result in significant
changes in ovarian morphology compared to controls (
Figure 1D
). Furthermore, such females laid
eggs and were fertile. Thus, germline depletion of Bon starting at the cystoblast stage does not lead
to morphological or obvious functional defects in oogenesis. In contrast, silencing of Bon beginning
in the GSCs by expressing the shBon using
nos- Gal4
driver induces visible morphological changes
with 34% of flies having only rudimentary ovaries lacking late stages of oogenesis and another ~39%
having one of the two ovaries rudimentary (
Figure 1E
). An even stronger phenotype was observed
upon GLKD using a double
nos + bam
driver which drives expression at all stages of oogenesis
(
Figure 1D, F
). 100% of such females displayed rudimentary ovaries and were completely sterile
(
Figure 1D, E
). Consistent with this, immunostaining for the germ cell marker Vasa demonstrates
that depletion of Bon results in partial loss of germ cells and arrested oogenesis as morphological
defects were accompanied by loss of vasa-
positive cells from the egg chambers (
Figure 1F
). The loss
of germ cells was further confirmed by the TUNEL assay which detects DNA fragmentation associated
with cell death (
Figure 1—figure supplement 1B
). Additionally, we proved the importance of Bon
in the
Drosophila
ovarian germline by using CRISPR/Cas9-
mediated mutagenesis. Transgenic flies
from the Heidelberg CRISPR Fly Design Library (
Port et al., 2020
) expressing sgRNAs targeting
bon
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bonus
A
control
shBonus1
shBonus2
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
Bonus RNA expr
ession
(relative to rp49)
Contr
ol
nos-Gal4>BonusKD
MT
-Gal4>BonusKD
bam-Gal4>BonusKD
bam+nos>BonusKD
bam+MT>BonusKD
Cont
rol
nos-Gal4>BonusKD
bam-Gal4>BonusKD
MT
-Gal4>Bon
usKD
bam+nos>BonusKD
bam+MT>BonusK
D
0
20
40
60
80
100
% of ovaries with observed
phenotype
Rudimentary
Normal
Hypomorphic
Phenotype
175
85
52
71
64
58
n=
MT
-Gal4>BonusKD
Contr
ol
BC
nos-Gal4>BonusKD
DE
Contr
ol
bam+nos>BonusKD
F
30 μm
30 μm
vasa
DAPI
mer
ge
normal
hypomorphic
rudimentary
Figure 1.
Germline expression of Bonus is required for oogenesis. (
A
) Bon is expressed throughout oogenesis. Stacked confocal image of wild-
type
Oregon-
R flies stained for Bon. (
B
) Bar graph shows the relative expression of Bon (normalized to rp49 level) in control and Bon-
depleted ovaries (RT-
qPCR, dots correspond to three independent biological replicates (n=3); error bars indicate st. dev.; p<0.001, two-
tailed Student’s t-
test). (
C
) Confocal
images of egg chambers from wild-
type Oregon-
R flies (control) and flies expressing
MT- Gal4
-
driven shRNA against Bon stained for Bon (scale bar:
Figure 1 continued on next page
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were crossed to
nos- Gal4;UAS- Cas9
to achieve germline-
specific knockout of
bon
. Almost 65% of
the female offspring with
nos- Cas9;sgRNA- bon
were sterile and had defects in ovarian morphology,
another 23% had one rudimentary ovary and only 12% showed normal phenotype (
Figure 1—figure
supplement 1C
). These results indicate that Bon function in the early stages of oogenesis, particularly
in GSCs, is essential for proper oogenesis.
To further analyze the role of Bon in the maintenance of GCSs and early oogenesis we used immu-
nofluorescence against the cytoskeletal protein
α
-spectrin, which marks the spectrosome, a spher
-
ical intracellular organelle present in GSCs and cystoblasts. At later stages, spectrosomes become
fusomes, branched structures that are localized in cytoplasmic bridges connecting differentiating
germ cells in the growing cysts. Thus, fusome formation is a hallmark of normal oogenesis progres-
sion. In ovarioles of control flies, we observed a normal germarium organization with two to three
spectrosome-
containing GSCs, and branched fusomes in germ cells at later stages. In contrast, germ
cells with normal fusomes were absent upon depletion of Bon using
nos- Gal4
. Instead, the germarium
of Bon-
depleted flies harbored several cells containing spherical spectrosomes, a hallmark of GSCs or
cystoblast-
like undifferentiated germ cells (
Figure 1—figure supplement 1D, E
). Overall, our results
indicate that loss of Bon in early germ cells interferes with maintenance of GSCs and arrests their
further differentiation.
Loss of Bonus triggers the ectopic expression of tissue-specific genes
in the ovary
To investigate the effect of Bon depletion on gene expression in the female germline, we performed
transcriptome profiling using RNA sequencing (RNA-
seq) analysis. We tested the effects of loss of Bon
in both early and later stages of oogenesis using the
nos- Gal4
or
MT- Gal4
driver to drive
bon
shRNA
expression, respectively. RNA-
seq libraries were prepared in triplicates and compared to respective
control libraries. As knockdown using the
nos- Gal4
driver causes early arrest of oogenesis and rudi-
mentary ovaries, while later-
stage knockdown with the
MT- gal4
driver yields normal ovaries, we used
different controls depending on the driver to assure that ovary size and cell composition of the Bon
GLKD and control are similar. For
nos- Gal4
we used ovaries from young (0- to 1-
day old) flies that
lack later stages of oogenesis and compared them to their age-
matched siblings that lack the shRNA,
and for
MT- gal4
we used 1- to 2-
old flies that express either shRNA against
bon
or the
white
gene,
which is not expressed in the germline. Thus, in both cases, GLKD and control flies had the same age
and similar ovary size. As the mammalian homolog of Bon, KAP-
1, plays a central role in repression
of many transposable elements (TEs) through its function as co-
repressor for multiple KRAB-
ZFPs that
recognize TEs sequences, we analyzed expression of both host genes and TEs.
Most TEs families were not affected by Bon depletion using either driver. Using the
nos- Gal4
-
driven shRNA, only 6 out of 207 (~3%) TE families present in the
Drosophila
genome significantly
increased their expression more than twofold (log
2
FC >1, and qval <0.05, LRT test (Likelihood Ratio
Test), Sleuth) (
Figure 2—figure supplement 1A
) and none showed strong (>sixfold) upregulation.
Similarly, depletion of Bon at later stages of oogenesis also did not lead to strong (>sixfold) change
in transposon expression (
Figure 2—figure supplement 1A
). This phenotype is in stark contrast to
the significant activation of many TE families when the main TE repression pathway in the ovary – the
piRNA pathway – is abolished, suggesting that Bon is not involved in the piRNA pathway and that
oogenesis defects observed upon Bon depletion likely have a different molecular basis.
In contrast to TEs, the protein-
coding transcriptome was severely disrupted upon Bon depletion –
differential gene expression analysis using Sleuth revealed many genes with altered steady-
state RNA
levels upon Bon GLKD at either stage. As expected, Bon was one of the most strongly downregulated
20 μm). (
D
) Bon depletion leads to rudimentary ovaries. Phase contrast images of dissected ovaries from flies of indicated genotypes. Wild-
type
Oregon-
R flies were used as control. (
E
) Top: phase contrast image of dissected ovaries with different phenotypes from flies with Bon GLKD driven by
nos- Gal4
. Bottom: graph showing the percentage of normal, hypomorphic, and rudimentary ovary phenotypes of indicated genotypes (
n
= 85, 175, 52,
71, 64, and 58, respectively). (
F
) Confocal images of whole ovaries from wild-
type Oregon-
R flies (control) and flies with Bon GLKD driven by
bam + nos
double driver stained for Vasa (red) and DAPI (4
,6-
diamidino-
2- phenylindole) (blue) (scale bar: 30 μm).
The online version of this article includes the following figure supplement(s) for figure 1:
Figure supplement 1.
An important function of Bonus in the early stages of oogenesis.
Figure 1 continued
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genes, showing ~sevenfold reduction and confirming the efficiency of KD and the validity of the RNA-
seq data (
Figure 2A
). Early Bon GLKD resulted in 694 differentially expressed genes (qval <0.05, LRT
test) (
Figure 2A
), of which 464 (~67%) and 28 (~4%) genes, respectively, increased and decreased their
expression more than twofold, while late Bon GLKD using
MT- Gal4
revealed 1769 genes that were
differentially expressed (qval <0.05, LRT test), with 231 genes (~13.6%) showing more than twofold
increase, while 72 genes (~4.2%) showed more than a twofold decrease in mRNA level (
Figure 2—
figure supplement 1B
). Interestingly, the sets of genes that change their expression upon Bon GLKD
at the early and late stages of oogenesis are quite different: only 51 genes were derepressed at both
stages of oogenesis, while the remaining genes that changed their expression were unique for one
or the other stage (
Figure 2—figure supplement 1C
). Overall, our results indicate that Bon plays an
important role in regulation of gene expression during oogenesis with distinct targets at different
stages.
To characterize Bon targets in oogenesis, we performed gene ontology (GO) analysis of genes
strongly upregulated upon
nos- Gal4
-
driven Bon GLKD (
n
= 464). GO analysis identified enrichment of
genes from 27 biological process in the set of Bon-
repressed genes (
Figure 2B
). These included terms
such as mesoderm development, myofibril assembly, sarcomere organization, hemolymph coagula-
tion, motor neuron axon guidance, and visceral muscle development, suggesting that Bon loss leads
to the ectopic ovarian activation of genes normally expressed in other tissues. To comprehensively
explore the specific expression patterns of the 464 Bon-
repressed genes, we used modENCODE RNA-
seq data from different tissues. This analysis revealed that many genes that are derepressed in the
ovary upon Bon GLKD are normally expressed in other tissues and have no (55%) or low (33%) expres-
sion in the ovary of wild-
type flies. Instead, many of these genes are predominantly expressed in the
head (50%), digestive system (38%), and central nervous system (28%) of wild-
type flies (
Figure 2C
).
We used RT-
qPCR and in situ hybridization chain reaction (HCR) for selected upregulated genes
including
rbp6
,
CG34353
, and
ple
, which are highly expressed in the head, and
pst
, which is highly
expressed in the gut, to confirm that germline depletion of Bon triggers their ectopic activation. No
signal for these genes was detected in wild-
type ovaries, while abundant
rbp6
,
CG34353
, and
pst
transcripts were identified in germ cells upon
MT- Gal4>Bon
GLKD (
Figure 2D- F
,
Figure 2—figure
supplement 1D- F
). Surprisingly, depletion of Bon in germ cells caused the appearance of
ple
tran-
scripts in somatic follicular cells that surround germline cells, suggesting that Bon depletion causes
activation of
ple
indirectly, through a process that involves signaling between the adjacent germline
and follicular cells (
Figure 2D
). Overall, our results indicate that in the ovary, Bon is required for
repression of genes that are typically expressed in non-
ovarian tissues.
Recruitment of Bonus to a genomic locus induces transcriptional
repression associated with accumulation of the H3K9me3 mark
Transcriptome profiling upon Bon germline depletion demonstrated global changes in steady-
state
RNA levels of hundreds of genes. As exemplified by the activation of the
ple
gene in the somatic
follicular cells some of these effects might be indirect and even mediated by intercellular signaling.
To test the ability of Bon to directly induce transcriptional silencing, we took advantage of a tethering
approach in which Bon is recruited to a reporter locus via binding to nascent transcripts (
Figure 3A
).
Tethering was achieved through fusion of Bon to the
λ
N RNA-
binding domain that has high affinity
for BoxB RNA hairpins encoded in the 3’UTR region of the reporter gene (
De Gregorio et al., 1999
).
λ
N-
eGFP-
Bon and the reporter were co-
expressed in the germline using the
MT- Gal4
driver; recruit-
ment of
λ
N-
eGFP was used as a control.
RT-
qPCR showed that tethering of Bon triggers ~22-
fold reporter repression (
Figure 3B
). Similar
results were obtained with a different reporter in another genomic location, indicating that recruitment
of Bon induces strong repression regardless of the genomic locus (
Figure 3—figure supplement 1A
).
ChIP-
qPCR analysis revealed that Bon recruitment results in a strong increase in the repressive H3K9
trimethylation (H3K9me3) chromatin mark, at the reporter locus (
Figure 3C
), suggesting that repres-
sion induced by Bon is mediated, at least in part, by the deposition of H3K9me3.
We also examined changes in H3K9me3 enrichment on genes upregulated upon Bon depletion
(Bon GLKD driven by
nos- Gal4
). Global ChIP-
seq analysis revealed that many Bon-
dependent genes
show low or no H3K9me3 signal in control ovaries and no change upon Bon depletion, hence might be
secondary targets (
Figure 3D
,
Figure 3—figure supplement 1B
). For instance, the gene
pst
despite
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A
3
4
log2FC(BonusKD/control)
3
4
log2F
C
(BonusKD/control
)
Gene expression, RNA-seq
(nos-Gal4)
0
10
20
30
40
50
60
70
80
90
100
% of genes
extremely hig
h
very high
high
moderate high
moderate
low
very low
no expression
Expression
imaginal disc
salivary gland
CNS
fat body
head
digestive syste
m
carcas
s
testis
ovary
C
Not significant
Down
Up
Expression
B
myofibril assembly
response to bacterium
oxidation-r
eduction pr
ocess
extracellular matrix or
ganization
hemolymph coagulation
response to fungus
insecticide catabolic pr
ocess
calcium ion transmembrane transport
regulation of membrane potential
ster
ol transport
larval visceral muscle development
bicarbonate transport
proteolysis
synaptic tar
get inhibition
antibacterial humoral r
esponse
regulation of intracellular pH
flight
defense r
esponse to bacterium
innate immune r
esponse
defense re
sponse to Gram-positive bacterium
!
"#
Fold enrichment
GO biological pr
ocesses
Numbe
r
of genes
10
20
30
immune re
sponse
motor neuro
n axon guidance
defense re
sponse
sar
comere
or
ganization
response to DDT
mesoderm development
transmembrane transport
0
1
2
3
4
5
-log10(FDR)
bonus
ple
D
Contr
ol
MT
-Gal4>BonusKD
E
Contr
ol
MT
-Gal4>BonusKD
bonus
rbp6
20 μm
20 μm
DAPI
bonus
rbp6
0.00
0.01
0.02
0.03
0.04
0.05
mRNA expression
(relative to rp49)
pl
e
rbp
6
BonusKD
Control
RT
-qPCR
F
Figure 2.
Bonus functions as a repressor of tissue-
specific genes in ovary. (
A
) Bon GLKD leads to misexpression of tissue-
specific genes in the ovary.
Volcano plot shows fold changes in genes expression upon Bon GLKD driven by
nos- Gal4
in the ovary as determined by RNA-
seq (
n
= 3). Siblings that
lack shRNA against Bon produced in the same cross were used as a control. Genes that change significantly (log
2
FC >1, qval <0.05, LRT test, sleuth;
Pimentel et al., 2017
) are highlighted. Genes
bon
,
pst
,
Rbp6
, and
ple
are labeled. Genes with infinite fold change values (zero counts in control
Figure 2 continued on next page
Research article
Chromosomes and Gene Expression | Genetics and Genomics
Godneeva
et al
. eLife 2023;12:RP89493. DOI: https://doi.org/10.7554/eLife.89493
8 of 24
being activated upon Bon GLKD displayed a low H3K9me3 signal (
Figure 3—figure supplement 1B
).
However, several Bon-
regulated genes are enriched in H3K9me3 mark in wild-
type ovaries including
in the proximity of the transcription start site (TSS) and show prominent loss of H3K9me3 upon Bon
depletion (
Figure 3D
). For example, gene
CG1572
, which was activated twofold upon Bon GLKD,
showed almost a twofold decrease in H3K9me3 level upstream of its TSS (
Figure 3E
). Independent
ChIP-
qPCR analysis of few Bon-
regulated genes such as
CG3191
and
Spn88Eb
also showed a slight
decrease in the repressive mark upon Bon depletion (
Figure 3F
).
Altogether, these data indicate that Bon recruitment to genomic targets induces transcriptional
repression associated with accumulation of the H3K9 trimethylation mark. However, repression of
many Bon-
regulated genes might be indirect and/or independent of H3K9me3.
Bonus interacts with dNuRD complex components Mi-2 and Rpd3, as
well as the histone methyltransferase SetDB1
Mammalian KAP-
1 was shown to associate with the NuRD histone deacetylase and chromatin-
remodeling complex and with the H3K9me3 writer SetDB1, and their interactions are important for
its function in transcriptional repression (
Schultz et al., 2002
;
Schultz et al., 2001
). In
Drosophila,
the
dNuRD complex mediates chromatin remodeling and histone deacetylation through dMi-
2 and Rpd3
(HDAC1 homolog), respectively (
Bouazoune and Brehm, 2006
;
Brehm et al., 2000
;
Kunert and
Brehm, 2009
;
De Rubertis et al., 1996
;
Tong et al., 1998
). To study whether dNuRD and SetDB1 are
required for Bon’s ability to trigger transcriptional repression in the
Drosophila
germline, we tested
reporter expression upon Bon tethering and concomitant knockdown of SetDB1 and dNuRD compo-
nents. GLKD of either Mi-
2 or SetDB1, but not Rpd3, inhibited silencing, indicating that Mi-
2 and
SetDB1 act downstream of Bon to induce repression (
Figure 4A
). Notably, we found that 29% of the
derepressed genes (135 out of the 464 genes) overlap with those upregulated in
nos- Gal4
- driven
SetDB1 GLKD, suggesting that Bon and SetDB1 co-
regulate many genes.
To explore physical interactions of Bon with components of the dNuRD and SetDB1 complexes,
we employed co-
immunoprecipitation assay using tagged proteins in S2 cells. We found that both
components of dNuRD, Mi-
2, and Rpd3, as well as SetDB1 co-
purify with Bon (
Figure 4B- D
,
Figure 4—
figure supplement 1A
). The interaction between Bon and Mi-
2 is mediated by the C-
terminus of Mi-
2
(
Figure 4D
), similar to interaction between Mi-
2
α
/CHD3 and KAP-
1 in mammals (
Schultz et al., 2001
).
In
Drosophila
Mi-
2 is found in two distinct complexes, the canonical dNuRD complex and the dMec
complex that contains the zinc-
finger protein Mep-
1 (
Kunert et al., 2009
). We did not detect an inter
-
action between Bon and Mep-
1 (
Figure 4—figure supplement 1B
), indicating that Bon interacts with
Mi-
2 in the context of the dNuRD complex but not dMec. Overall, our results indicate that the inter
-
actions between Bon and the NuRD and SetDB1 chromatin remodeler and modifying complexes are
ovaries) are not shown. (
B
) Bon represses genes with diverse functions. Bubble plot shows the analysis of gene ontology (GO) enrichment at the level of
biological processes (BP) for genes that are derepressed upon Bon GLKD driven by
nos- Gal4
(log
2
FC >1, qval <0.05, LRT test, sleuth;
Pimentel et al.,
2017
). Only GO terms above the established cut-
off criteria (p-
value <0.01 and >3 genes per group) are shown. BP are ranked by fold enrichment
values. The most significant processes are highlighted in purple, and the less significant in yellow according to log
10
(FDR) values. The bubbles size
reflects the number of genes, assigned to the GO BP terms. (
C
) Normal expression level of deregulated genes upon Bon GLKD in the tissues where
they are normally expressed indicates Bon-
mediated silencing of genes normally expressed in the head and digestive system. The graph shows the
percentage of derepressed genes upon Bon GLKD driven by
nos- Gal4
(log
2
FC >1, qval <0.05, LRT test, sleuth;
Pimentel et al., 2017
) with given
expression level in the indicated enriched tissues. Expression levels according RPKM values from modENCODE anatomy RNA-
seq dataset are no
expression (0–0), very low (1–3), low (4–10), moderate (11–25), moderate high (26–50), high (51–100), very high (101–1000), and extremely high (>1000). (
D
)
GLKD of Bon leads to
ple
expression in follicular cells. Confocal images of egg chambers show RNA in situ hybridization chain reaction (HCR) detecting
ple
and
bonus
mRNAs in flies with
MT- Gal4
>
Bon
GLKD and control siblings from the same cross that lack Bon shRNA (scale bar: 20 μm). (
E
) Bon
represses
rbp6
in the germline. Confocal images of egg chambers show RNA in situ HCR detecting
rbp6
and
bonus
mRNAs in flies with
MT- Gal4
>
Bon
GLKD and control siblings from the same cross that lack Bon shRNA (scale bar: 20 μm). (
F
) Bar graph shows the relative expression of
ple
and
rbp6
(normalized to rp49 level) in control and Bon-
depleted ovaries (RT-
qPCR, dots correspond to three independent biological replicates (n=3); error bars
indicate st. dev.).
The online version of this article includes the following figure supplement(s) for figure 2:
Figure supplement 1.
Depletion of Bon induces ectopic activation of non-
ovarian genes.
Figure 2 continued