Citation:
Godneeva, B.; Fejes Tóth,
K.; Quan, B.; Chou, T.-F.; Aravin, A.A.
Impact of Germline Depletion of
Bonus on Chromatin State in
Drosophila
Ovaries.
Cells
2023
,
12
,
2629. https://doi.org/10.3390/
cells12222629
Academic Editor: Tao Liu
Received: 18 October 2023
Revised: 7 November 2023
Accepted: 13 November 2023
Published: 15 November 2023
Copyright:
© 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
cells
Article
Impact of Germline Depletion of Bonus on Chromatin State in
Drosophila
Ovaries
Baira Godneeva
1,2,
*, Katalin Fejes T
ó
th
1
, Baiyi Quan
1,3
, Tsui-Fen Chou
1,3
and Alexei A. Aravin
1,
*
1
Division of Biology and Biological Engineering, California Institute of Technology,
Pasadena, CA 91125, USA
2
Institute of Gene Biology, Russian Academy of Sciences, Moscow 119334, Russia
3
Proteome Exploration Laboratory, Beckman Institute, California Institute of Technology,
Pasadena, CA 91125, USA
*
Correspondence: bairagodneeva@gmail.com (B.G.); aaa@caltech.edu (A.A.A.)
Abstract:
Gene expression is controlled via complex regulatory mechanisms involving transcription
factors, chromatin modifications, and chromatin regulatory factors. Histone modifications, such as
H3K27me3, H3K9ac, and H3K27ac, play an important role in controlling chromatin accessibility
and transcriptional output. In vertebrates, the Transcriptional Intermediary Factor 1 (TIF1) family
of proteins play essential roles in transcription, cell differentiation, DNA repair, and mitosis. Our
study focused on Bonus, the sole member of the TIF1 family in
Drosophila
, to investigate its role in
organizing epigenetic modifications. Our findings demonstrated that depleting Bonus in ovaries
leads to a mild reduction in the H3K27me3 level over transposon regions and alters the distribution
of active H3K9ac marks on specific protein-coding genes. Additionally, through mass spectrometry
analysis, we identified novel interacting partners of Bonus in ovaries, such as PolQ, providing a
comprehensive understanding of the associated molecular pathways. Furthermore, our research
revealed Bonus’s interactions with the Polycomb Repressive Complex 2 and its co-purification with
select histone acetyltransferases, shedding light on the underlying mechanisms behind these changes
in chromatin modifications.
Keywords:
H3K9ac; H3K27ac; H3K27me3; transcription; Bonus; gene expression; chromatin
1. Introduction
Gene expression is a fundamental process that requires complex regulation and ex-
hibits significant differences across tissues and cell types. As cells develop into specific
types, they make important decisions that ultimately determine their unique cellular fates.
Such a cell-specific form of gene expression is regulated via the presence of specific tran-
scription factors, the dynamic landscape of chromatin modifications, and the activity of
chromatin-regulating factors. To achieve precise regulation, these regulatory factors often
work together. Histone marks are important regulators that determine chromatin accessi-
bility and influence transcriptional outcomes [
1
–
4
]. These modifications act as a molecular
code that is deciphered via specialized ‘reader’ proteins and associated complexes to
determine the transcriptional output and functional characteristics of genomic regions.
Histone acetylation, particularly H3K9ac and H3K27ac, is commonly associated with gene
activation and the establishment of an open chromatin structure conducive to transcrip-
tion [
5
–
7
]. In contrast, H3K27me3 is a hallmark of gene silencing and is closely associated
with facultative heterochromatin and transcriptional repression [
8
–
11
]. H3K27me3 is de-
posited via the Polycomb Repressive Complex 2 (PRC2) and distributed across extensive
genomic regions, referred to as Polycomb domains, to suppress cell type–specific expres-
sion
programs [12–15]
. The regulatory mechanisms that determine the precise targeting of
histone-modifying enzymes to genomic sites remain poorly understood. Transcriptional
Intermediary Factor 1 (TIF1) family proteins, known as chromatin-associated factors, have
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the ability to both activate and repress transcription by interacting with co-regulators
and controlling the chromatin state [
16
–
20
]. Bonus (Bon) is the sole member of the TIF1
family in
Drosophila
[
21
]. Bon encompasses all the conserved domains present in mam-
malian members of the TIF1 family, including an N-terminal RBCC motif (composed of
a RING domain, followed by two B-boxes, and a coiled coil domain) and a C-terminal
chromatin-binding unit comprising a PHD domain and a bromodomain [
17
,
21
–
23
]. Previ-
ous investigations have highlighted the important role of Bon in embryonic development
and organogenesis [
21
,
24
–
27
]. Our recent work unraveled Bon’s role as a regulator of
tissue-specific genes within the female germline, revealing the importance of its SUMO
modification in transcriptional repression [
28
]. In this study, we explored the role of Bon
in directing different activating and silencing epigenetic modifications. Specifically, we
examined the role of Bon in the distribution of the H3K27me3, H3K9ac, and H3K27ac
marks. We demonstrated that the depletion of Bon in the ovaries leads to changes in
the H3K27me3 landscape over transposon regions and an altered distribution of H3K9ac
marks across specific protein-coding genes. Furthermore, we identified novel interacting
partners of Bon, such as the PRC2 complex, PolQ, chromatin-binding proteins, and histone
acetyltransferases, providing mechanistic insight on how these changes in chromatin modi-
fications are achieved. Together, this study contributes to a deeper understanding of Bon’s
involvement in epigenetic regulatory mechanisms.
2. Materials and Methods
2.1. Drosophila Fly Stocks
All fly stocks and crosses were raised at 24
◦
C. Females were 0–1 days old and were
dissected right away. To obtain the fly lines with Bon knockdown, the short hairpin
sequences were ligated into the pValium20 vector, and then integrated into the attP2 landing
site (BDSC #8622). The UASp-
λ
N-GFP fly line control was previously described [
29
]. To
generate the UASp-
λ
N-GFP-Bonus fly line, full-length cDNA sequences of wild-type Bon
was cloned in vectors containing a miniwhite marker followed by the UASp promoter
sequence and
λ
N-GFP. Transgenic flies carrying these constructs were generated via phiC31
transformation by BestGene Inc and were integrated into the attP40 landing site (y1 w67c23;
P{CaryP}attP40). The expression of these constructs was driven via the maternal alpha-
tubulin67C-Gal4 (
MT-Gal4
) (BDSC #7063) and
nos-Gal4
(BDSC #4937) drivers.
2.2. S2 Cell Line
Drosophila
S2 cells (DGRC catalog #006) were cultured at 25
◦
C in Schneider ’s
Drosophila
medium supplemented with 10% heat-inactivated FBS and 1X penicillin–streptomycin.
2.3. Protein Co-Immunoprecipitation from S2 Cells
S2 cells were transfected with plasmids encoding GFP- and FLAG-tagged proteins
under the control of the actin promoter using TransIT-LT1 reagent (Mirus, Madison, WI,
USA). Around 35–40 h after transfection, cells were collected and resuspended in lysis
buffer (composed of 20 mM of Tris-HCl (pH 7.4), 150 mM of NaCl, 0.2% NP-40, 0.2% Triton-
X100,
5% glycerol
, 20 mM of N-Ethylmaleimide (NEM) (Sigma, St. Louis, MO, USA), and
complete protease inhibitor cocktail (Roche, Basel, Switzerland)). The cell lysate was incu-
bated on ice for 20 min, centrifuged, and the supernatant was subsequently collected. The
supernatant was incubated with magnetic agarose GFP-Trap beads (Chromotek, Planegg,
Germany) for 3 h at 4
◦
C with end-to-end rotation. The beads were washed four times
for 10 min with wash buffer (20 mM of Tris-HCl (pH 7.4), 0.1% NP40, and 150 mM of
NaCl) and boiled in 2
×
Laemmli buffer for 5 min at 95
◦
C. The eluate was used for Western
blot analysis.
2.4. Western Blotting
Proteins were separated via SDS-PAGE gel electrophoresis and transferred to a 0.45-
μ
m
nitrocellulose membrane (Bio-Rad, Hercules, CA, USA), according to standard procedures.
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The membrane was blocked with 0.2% I-block (Invitrogen, Carlsbad, CA, USA) in PBST
(PBS, 0.1% Tween-20) for 1 h. The membrane was incubated with primary antibodies
overnight at 4
◦
C, followed by 3
×
washes for
5 min
in PBST, and incubation with secondary
antibodies for 2 h at room temperature. The membrane was washed three times for
5 min
with PBST and then imaged with the Odyssey system (Li-Cor, Lincoln, NE, USA).
When the primary antibodies were HRP-conjugated, the membrane was washed
3
×
5 min
with PBST and incubated with the HRP substrate; an X-ray film developed on an X-ray
Film Processor (Konica Minolta, Tokyo, Japan). The following antibodies were used:
HRP-conjugated anti-FLAG (Sigma, A8592,
1 mg/mL
, dilution: 1:10,000), mouse anti-
FLAG (Sigma, F1804,
1 mg/mL
, dilution: 1:5000), rabbit polyclonal anti-GFP (dilution:
1:4000) [
29
], and IRDye anti-rabbit and anti-mouse secondary antibodies (Li-Cor, #925-68070
and #925-32211, 1 mg/mL, dilution: 1:10,000).
2.5. RNA Extraction and RNA-Seq Analysis
For RNA extraction, 10–20 pairs of dissected ovaries from lines with
BonusKD
driven
by
nos-Gal4
(and matched siblings that lack the shRNA as control) were homogenized in
TRIzol (Invitrogen); RNA was extracted, isopropanol precipitated, and treated with DNaseI
(Invitrogen), according to the manufacturer’s instructions.
For RT-qPCR, reverse transcription was performed using random hexamer oligonu-
cleotides with Superscript III Reverse Transcriptase (Invitrogen). qPCR was performed on
a Mastercycler
®
ep realplex PCR machine (Eppendorf, Hamburg, Germany). Three biological
replicates per genotype were used for all RT-qPCR experiments. Bon expression was normalized
to rp49 mRNA expression. The data were visualized using Python 3 via JupyterLab. The
following primers were used for qPCR analysis: bon-for: ACTTCTGGGTCTGACTGGCGAAG;
bon-rev: TCAACGCACCACGACGTGG; rp49-for: CCGCTTCAAGGGACAGTATCTG; and
rp49-rev: ATCTCGCCGCAGTAAACGC.
For RNA-seq libraries, PolyA+ selection was performed using a NEBNext Poly(A)
mRNA Magnetic Isolation Module (NEB, #E7490). RNA-seq libraries were made using the
NEBNext Ultra II Directional RNA Library Prep kit for Illumina (NEB, #E7760), accord-
ing to the manufacturer’s instructions. Libraries were sequenced on the Illumina HiSeq
2500 platform
. For RNA-seq coverage tracks, reads were first aligned to the
D. melanogaster
genome (dm6) using bowtie1 (v.1.2.2) allowing 2 mismatches and single mapping posi-
tions. Tracks were generated using the deepTools (v.3.5.1) bamCoverage function with
10 bp bin sizes.
2.6. ChIP-Seq
ChIP experiments were performed in two biological replicas for the H3K27ac and
H3K9ac marks and in one replica for the H3K27me3 and H3K9me3 marks, as previously de-
scribed [
30
]. In brief, 80–120 pairs of dissected ovaries were crosslinked with 1% formalde-
hyde in PBS for 10 min at room temperature, and then quenched with glycine (final
concentration 25 mM). Frozen ovaries were dounced in RIPA buffer and then sonicated
(Bioruptor sonicator) to a desired fragment size of 200–800 bp. Lysates were centrifuged
at 19,000
×
g
, and supernatants were collected. The supernatants were first precleared for
2 h
at 4
◦
C using Protein G Dynabeads (Invitrogen). Precleared samples were immunopre-
cipitated with anti-H3K27me3 (C36B11, Cell Signaling), anti-H3K9me3 (ab8898, abcam),
anti-H3K27ac (ab4729, abcam), or anti-H3K9ac antibodies (ab10812, abcam) for 3–5 h at
4
◦
C; then, 50
μ
L of Protein G Dynabeads were added, and the samples were further
incubated overnight at 4
◦
C. The beads were washed 3
×
10 min in LiCL buffer, followed by
proteinase K treatment for 2 h at 55
◦
C and then overnight at 65
◦
C. DNA was extracted via
standard phenol/chloroform extraction. ChIP-seq libraries were prepared using NEBNext
Ultra DNA Library Prep Kit Illumina and sequenced on the Illumina HiSeq 2500 platform
(SR 50 bp for H3K27me3 and H3K9me3; PE 50bp for the H3K27ac mark) and NovaSeq
6000 platform (PE150 for the H3K9ac mark). After removal of the adaptors, reads with a
minimal length of 18 nucleotides were aligned to the
D. melanogaster
genome (dm6) using
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bowtie1 (v.1.2.2) allowing 2 mismatches and single mapping positions. Protein-coding gene
annotations and their repetitive sequence annotations were obtained from the RefSeq and
RepeatMasker tables, respectively, retrieved from the UCSC genome browser. Genome cov-
erage tracks were generated using the deepTools (v.3.5.1) bamCoverage function with 10 bp
bin sizes. The ChIP signal was normalized to input counts by calculating the cpm (counts
per million) using the deepTools bamCompare function with 50 bp bin sizes (log2 values
ChIP/Input). Heatmaps and plot profiles were generated with deepTools plotHeatmap
and plotProfile using log2-normalized (ChIP/Input) BigWig files.
2.7. Liquid Chromatography–Mass Spectrometry (LS–MS)
All procedures were performed using MS-grade water, low binding plasticware, and
freshly prepared solutions. Dissected ovaries from 1–2-day old
Drosophila
flies expressing
UASp-
λ
N-GFP-Bonus or UASp-
λ
N-GFP (control) were used for MS. Frozen ovaries were
dounced in lysis buffer (20 mM of Tris-HCl (pH 7.4), 150 mM of NaCl, 10% glycerol, 0.5%
DMM (n-Dodecyl-
β
-D-maltoside), and 25 mM of NEM (N-Ethylmaleimide), supplied
with protease inhibitor). Lysates were centrifuged at 19,000
×
g
, and the supernatants
were collected. The supernatants were then precleared for 2 h at 4
◦
C using Protein G
Dynabeads (Invitrogen). Precleared samples were immunoprecipitated with anti-GFP
antibody for 4 h at 4
◦
C; then, 50
μ
L of Protein G Dynabeads were added, and the samples
were further incubated overnight at 4
◦
C. The beads were washed 5
×
10 min in lysis
buffer, and then 2
×
5 min in wash buffer (50 mM HEPES, pH 8.0). Then, samples were
eluted from the beads with 10 M urea for 15 min at 37
◦
C. After that, the samples were
diluted to 8M urea with wash buffer. After that, the eluates were incubated with 500 mM
of TCEP (Thermo Scientific, Waltham, MA, USA, 20490) for 20 min at 37
◦
C, and then with
500 mM of 2-chloroacetamide for 15 min at 37
◦
C; after that, the samples were incubated
with Endoproteinase LysC for 4 h at 37
◦
C. The samples were diluted to final 2 M urea
with wash buffer; after that, 100 mM of CaCl2 with trypsin (100 ng/
μ
L) was added for
overnight incubation at 37
◦
C. For desalting, C18 spin columns (Thermo Scientific, 89870)
were used, according to the manufacturer’s instructions. After elution, the samples were
freeze dried and submitted to the Caltech Proteome Exploration Laboratory for MS analysis.
Peptide samples were subjected to LC-MS analysis on an EASY-nLC 1200 (Thermo Fisher
Scientific) coupled to a Q Exactive HF Orbitrap mass spectrometer. Raw data files were
searched against a customized database (
Drosophila melanogaster
and bait proteins) using
the Proteome Discoverer 2.5 software based on the SEQUEST algorithm. The fragment
mass tolerance was set to 20 ppm. The maximum false peptide discovery rate was specified
as 0.01 using the Percolator Node validated by a
q
-value.
Gene ontology (GO) molecular function term enrichment analysis was performed on
proteins that were significantly enriched compared to the control (log2FC > 1, adjusted
p
-value < 0.05, Benjamini–Hochberg), using DAVID Bioinformatics Resources. The enriched
GO terms associated with 2 or less submitted genes were excluded. A significant threshold
was applied using a multiple testing correction (Fisher’s Exact test
p
-value < 0.01). Data
visualization was performed using standard plotting libraries using Python (version 3.9.15).
3. Results
3.1. Genome-Wide Analyses of H3K27me3 Distribution in Bonus-Depleted Ovaries
To gain insight into Bon’s role within the epigenetic landscape, we first performed
ChIP-seq analysis of genome-wide distribution of repressive H3K27me3 marks in Bon-
depleted ovaries. We used ovaries from flies expressing short hairpin RNAs against
bon
using a
nos-Gal4
driver to achieve the germline-specific knockdown of Bon (
BonusKD
). As
a control, we used ovaries from their siblings that lack the short hairpin. This approach,
utilizing siblings as controls, was chosen to minimize genetic variability. In a previous study,
we demonstrated the effectiveness of our knockdown system, showing an 88% reduction in
Bon gene expression via RT-qPCR and confirming this reduction in germ cells using confocal
imaging [
28
]. In our current analysis, we verified the efficiency of Bon knockdown via RT-
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qPCR, revealing an 85% reduction in ovarian Bon expression, consistent with our previous
findings (Figure 1A). The analysis of normalized H3K27me3 ChIP-seq signals
across
±
1 kb
of the transcription start sites (TSSs) and the transcription end site (TES) of all genes in
the
Drosophila
genome revealed distinct genic distributions, with H3K27me3 covering
the gene body in both the control and Bon-depleted ovaries (
Figure 1B and Figure S1A
).
Bon depletion does not alter the levels of the H3K27me3 mark over protein-coding genes,
including H3K27me3-decorated genes that become upregulated upon Bon knockdown, as
demonstrated by RNA-seq (Figure 1C). To validate our ChIP-seq analysis, we examined
the Hox gene clusters, well known for their spatial compartmentalization and significant
enrichment of the H3K27me3 mark [
31
,
32
]. The Antennapedia (Antp) Hox gene complex,
located on chromosome 3R in
Drosophila melanogaster
, displayed an unaltered and complete
coverage of H3K27me3, confirming the validity of our ChIP-seq analysis and the lack of an
effect following the germline-specific knockdown of Bon (Figure 1D). Therefore, our results
suggest that repression of Bon-regulated genes is independent of the H3K27me3 mark.
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depleted ovaries. We used ovaries from
fl
ies expressing short hairpin RNAs against
bon
using a
nos-Gal4
driver to achieve the germline-speci
fi
c knockdown of Bon (
BonusKD
). As
a control, we used ovaries from their siblings that lack the short hairpin. This approach,
utilizing siblings as controls, was chosen to minimize genetic variability. In a previous
study, we demonstrated the e
ff
ectiveness of our knockdown system, showing an 88% re-
duction in Bon gene expres
sion via RT-qPCR and con
fi
rming this reduction in germ cells
using confocal imaging [28]. In our current analysis, we veri
fi
ed the e
ffi
ciency of Bon
knockdown via RT-qPCR, revealing an 85%
reduction in ovarian Bon expression, con-
sistent with our previous
fi
ndings (Figure 1A). The analysis of normalized H3K27me3
ChIP-seq signals across ± 1kb of the transcri
ption start sites (TSSs) and the transcription
end site (TES) of all genes in the
Drosophila
genome revealed distinct genic distributions,
with H3K27me3 covering the gene body in both the control and Bon-depleted ovaries
(Figures 1B and S1A). Bon depletion does not
alter the levels of the H3K27me3 mark over
protein-coding genes, including H3K27me3-decorated genes that become upregulated
upon Bon knockdown, as demonstrated by RNA-seq (Figure 1C). To validate our ChIP-
seq analysis, we examined the Hox gene clus
ters, well known for their spatial compart-
mentalization and signi
fi
cant enrichment of the H3K27me3 mark [31,32]. The Anten-
napedia (Antp) Hox gene complex,
located on chromosome 3R in
Drosophila melanogaster
,
displayed an unaltered and complete coverage of H3K27me3, con
fi
rming the validity of
our ChIP-seq analysis and the lack of an e
ff
ect following the germline-speci
fi
c knockdown
of Bon (Figure 1D). Therefore, our results su
ggest that repression of Bon-regulated genes
is independent of the H3K27me3 mark.
Figure 1.
Distribution of the H3K27me3 mark in Bon-depleted ovaries.
(
A
) Bar graph shows the
relative expression of Bon (normalized to rp49 leve
l) in control and Bon-depleted ovaries (RT-qPCR;
dots correspond to 3 independent biological re
plicates; error bars indicate the st. dev.).
(
B
) Genome-
wide abundance of H3K27me3 over the gene body in control and Bon germline knockdown ovaries
Figure 1.
Distribution of the H3K27me3 mark in Bon-depleted ovaries. (
A
) Bar graph shows the
relative expression of Bon (normalized to rp49 level) in control and Bon-depleted ovaries (RT-qPCR;
dots correspond to 3 independent biological replicates; error bars indicate the st. dev.). (
B
) Genome-
wide abundance of H3K27me3 over the gene body in control and Bon germline knockdown ovaries
(
BonusKD
). The heatmap displays the distribution of H3K27me3
±
1 kb over the gene body in control
and
BonusKD
ovaries (input-normalized log2 values). (
C
) Heatmap showing H3K27me3 distribution
across Bon targets in control and
BonusKD
ovaries (input-normalized log2 values). (
D
) Distribution of
H3K27me3 over the Hox gene complex Antp. Tracks show the CPM-normalized coverage of ChIP-seq
data for the H3K27me3 mark in control and
BonusKD
ovaries (log2 values). Numbers show the
normalized log2 values of the ChIP/input signal (ChIP-seq) in a manually selected genomic location.
The bottom panel shows the structure of the gene; blue arrows indicate the direction
of transcription
.
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3.2. Depletion of Bonus in the Ovaries Affects the Distribution of the H3K27me3 Mark over
Transposon Regions
The PRC2 complex has been widely recognized for its role in depositing the H3K27me3
histone mark on protein-coding genes, while transposable elements (TEs) are repressed
via DNA methylation and/or the H3K9me3 mark. However, recent studies have revealed
that H3K27me3 is enriched over TE sequences across distantly related eukaryotes [
33
–
36
].
We consequently expanded our analysis to explore the impact of Bon knockdown on the
H3K27me3 profile of repetitive sequences. A closer examination of diverse repetitive
sequences unveiled that Bon depletion led to a reduction in H3K27me3 occupancy over
LTR and LINE retrotransposon sequences, as well as DNA transposons (Figure 2A). In
contrast, highly repeated satellites and simple repeats revealed no significant alterations
(Figure 2B). Additionally, when we compared the average H3K27me3 enrichment across
all LINE sequences in Bon-depleted ovaries and the control ovaries, we noted a reduction
in the H3K27me3 signal following Bon knockdown, unlike at satellites, where we did not
observe any significant changes (Figure 2C,D). For example, the region upstream of the
gene AGO3 that contains multiple LINE, LTR, and DNA transposons exhibited a significant
depletion of the H3K27me3 mark following Bon depletion (Figure 2E). It is noteworthy
that this region is also covered by the H3K9me3 repression mark, which remained largely
unchanged following Bon depletion. Thus, though Bon influences the accumulation of
H3K27me3 marks over transposon regions, the concurrent presence of H3K9me3 appears
to be sufficient to repress their transcription (Figure 2E). In comparison, the accumulation
of H3K27me3 at a distinct region enriched in simple repeats remained unaltered following
Bon knockdown (Figure 2F). Taken together, our results indicate that Bon is required for
the accumulation of the H3K27me3 mark across different classes of transposable elements,
but not simple repeats.
3.3. Modulation of the H3K27ac and H3K9ac Marks in Response to Bon Depletion
After exploring the effect of Bon on repressive chromatin marks, we studied the
acetylation of H3K27 and H3K9, which serve as catalysts for the establishment of open
chromatin and are associated with active transcription. H3K27ac has been uncovered
as a key player in cell identity control and a characteristic chromatin signature of active
enhancers [
37
,
38
], while the H3K9ac mark is considered a hallmark of active gene promot-
ers [
39
]. A genome-wide analysis revealed substantial occupancy of these activation marks
at TSS and TSS-proximal regions, aligning with their known presence in the proximity of
active promoters. Depleting Bon did not affect the overall distribution patterns of H3K27ac
and H3K9ac of the majority of TSSs genome-wide (Figure 3A and Figure S1B,C); however, a
subset of genes that become aberrantly expressed or silenced upon Bon depletion exhibited
a concomitant accumulation or loss of these marks, respectively. For example, Bon deple-
tion leads to ectopic activation of the
pst
gene and associated accumulation of the H3K27ac
mark near TSS-proximal regions (Figure 3B), while activated
CG6106
displays H3K9ac
accumulation at its TSS (Figure 3C). In contrast
Cyp6d4
, which experienced a stark decline in
expression upon Bon depletion, exhibited a notable decrease in H3K9ac levels (Figure 3D).
Overall, our results indicated that only a small fraction of Bon-regulated genes depend
on the H3K9ac or H3K27ac activation marks. Interestingly, a gain or loss of the H3K9
and H3K27 acetylation signals at promoters did not correlate with the fold change in the
corresponding genes’ expression level, pointing towards complex and context-dependent
regulatory relationships that may involve multiple pathways and mechanisms.
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Figure 2.
Di
ff
erential impact of Bonus depletion on H3K27m
e3 distribution over transposon regions.
(
A
) Heatmaps show H3K27me3 distribution at DNA
repeat elements (DNA), long interspersed nu-
clear elements (LINE), and long terminal repeat
elements (LTR) determined via RepeatMasker in
control and
BonusKD
ovaries. Bo
tt
om panels show the average pro
fi
les of H3K27me3 in control and
BonusKD
ovaries over indicated regions. (
B
) Heatmaps show H3K27me3 distribution at simple re-
peats (micro-satellites) and satellite repeats determined via RepeatMasker in control and
BonusKD
ovaries. Bo
tt
om panel shows the average pro
fi
les of H3K27me3 in control and
BonusKD
ovaries over
indicated regions. (
C
) Bar plot shows the average H3K27me3 enrichment at LINE sequences in con-
trol and
BonusKD
ovaries (averaged normalized log2 values of the ChIP/input signal). (
D
) Bar plot
shows the average H3K27me3 enrichment
at satellite sequences in control and
BonusKD
ovaries
(averaged normalized log2 values of the ChIP/input signal). (
E
) Example of a slight reduction in the
H3K27me3 mark over a transposon region located
upstream of the gene AGO3. Tracks show counts
per million (CPM)-normalized coverage of ChIP-seq data for H3K27me3 and H3K9me3 marks in
Figure 2.
Differential impact of Bonus depletion on H3K27me3 distribution over transposon regions.
(
A
) Heatmaps show H3K27me3 distribution at DNA repeat elements (DNA), long interspersed
nuclear elements (LINE), and long terminal repeat elements (LTR) determined via RepeatMasker
in control and
BonusKD
ovaries. Bottom panels show the average profiles of H3K27me3 in control
and
BonusKD
ovaries over indicated regions. (
B
) Heatmaps show H3K27me3 distribution at simple
repeats (micro-satellites) and satellite repeats determined via RepeatMasker in control and
BonusKD
ovaries. Bottom panel shows the average profiles of H3K27me3 in control and
BonusKD
ovaries
over indicated regions. (
C
) Bar plot shows the average H3K27me3 enrichment at LINE sequences in
control and
BonusKD
ovaries (averaged normalized log2 values of the ChIP/input signal). (
D
) Bar
plot shows the average H3K27me3 enrichment at satellite sequences in control and
BonusKD
ovaries
(averaged normalized log2 values of the ChIP/input signal). (
E
) Example of a slight reduction in the