Drosophila P75 safeguards oogenesis by preventing H3K9me2 spreading

Kun Dou^{a, b
, ,},
Yanchao Liu^{c, 1},
Yingpei Zhang^{c, 1},
Chenhui Wang^a,
Ying Huang^{c, d
, ,},
ZZ Zhao Zhang^{a, e, f
, ,}

a.
Department of Embryology, Carnegie Institution for Science, Baltimore, MD, 21218, USA
b.
School of Life Science and Technology, ShanghaiTech University, Shanghai 201210, China
c.
State Key Laboratory of Molecular Biology, Shanghai Key Laboratory of Molecular Andrology, Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Shanghai 200031, China
d.
State Key Laboratory of Oncogenes and Related Genes, Shanghai Key Laboratory of Biliary Tract Disease Research, Shanghai Research Center of Biliary Tract Disease, Department of General Surgery, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200092, China
e.
Department of Pharmacology and Cancer Biology, Duke University School of Medicine, Durham, NC, 27710, USA
f.
Regeneration Next, Duke University, Durham, NC, 27710, USA

More Information

Corresponding author: E-mail address: doukun@shanghaitech.edu.cn (Kun Dou); E-mail address: huangying@xinhuamed.com.cn (Ying Huang); E-mail address: z.z@duke.edu (ZZ Zhao Zhang)

These authors contributed equally to this work.

摘要

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Abstract: Serving as a host factor for human immunodeficiency virus (HIV) integration, LEDGF/p75 has been under extensive study as a potential target for therapy. However, as a highly conserved protein, its physiological function remains to be thoroughly elucidated. Here, we characterize the molecular function of dP75, the Drosophila homolog of LEDGF/p75, during oogenesis. dP75 binds to transcriptionally active chromatin with its PWWP domain. The C-terminus integrase-binding domain–containing region of dP75 physically interacts with the histone kinase Jil-1 and stabilizes it in vivo. Together with Jil-1, dP75 prevents the spreading of the heterochromatin mark-H3K9me2-onto genes required for oogenesis and piRNA production. Without dP75, ectopical silencing of these genes disrupts oogenesis, activates transposons, and causes animal sterility. We propose that dP75, the homolog of an HIV host factor in Drosophila, partners with and stabilizes Jil-1 to ensure gene expression during oogenesis by preventing ectopic heterochromatin spreading.
- LEDGF/p75 /
- Jil-1 /
- H3K9me2
These authors contributed equally to this work.
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Fig. 1. CG7946/dP75, the LEDGF/p75 homolog in Drosophila, is essential for female fertility. A: dP75 resides on the right arm of chromosome 3, encoding a protein with PWWP and integrase-binding domain (IBD). Two sgRNAs used to generate dP75 mutants are labeled. B: Characterization of dP75 mutants. Left panel: Western blot of ovarian lysate using polyclonal antibody against dP75. The bright band between 50 kDa and 75 kDa corresponds to the predicted size of dP75 protein. Right panel: immunostaining with dP75 antibody in wild-type and dP75-mutant ovaries. C: Fertility of females over time. X-axis shows time (in days) since the hatch ratio experiment starts. Reintroducing dP75 transgene driven by actin-Gal4 into dP75 mutant rescued the sterility phenotype. D: Fertility of females with dP75 depletion either in germ cells or in somatic follicle cells. shRNA against mCherry was used as a control. The hatch ratio was counted as the average hatch ratio during the 6 days of egg-hatching experiment, and error bars = standard deviation.

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Fig. 2. dP75 binds to actively transcribed chromatin regions in vivo. A: dP75 expression pattern as examined by V5 antibody staining in ovaries with V5 knocked in at endogenous locus of dP75. B: Validation of V5 tag knockin at endogenous dP75 locus by Western blot. Western blot was carried out using anti-V5 antibody. After V5 detection, the same membrane was used for Western blot of internal control (using anti-Tubulin antibody). Carcass, fly body with ovaries ectomised. C: V5 ChIP-Seq and RNA-Seq signal across a 300-kb representative genome region on chromosome 2. V5 ChIP-Seq performed in ovaries without V5 tag serves as negative control. D: Heatmap for V5-dP75 ChIP-Seq signal on genes and their mRNA levels in wild-type fly ovaries. Each line from the heatmap represents the coding region of one gene plus 3 kb upstream and 5 kb downstream of the coding region. E: Isothermal titration calorimetry (ITC) measurement of the binding affinity of PWWP domain to different oligos (upper panel) and various histone peptides (lower panel). ITC measurements were performed in duplicates. Binding isotherms and standard deviation were derived from nonlinear fitting using Origin 8.0 (MicroCal, Inc, USA). The initial data point was routinely deleted. The ITC data were fit to a one-site binding model in 1:1 binding mode.

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Fig. 3. dP75 interacts with Jil-1 both in vivo and in vitro. A: Identification of dP75 interacting proteins by affinity purification and mass spectrometric analysis. The number of peptides identified from mass spectrometry for dP75 and Jil-1 in each sample is listed. B: dP75 and Jil-1 colocalize in fly ovaries. Immunostaining of fly ovaries were carried out using both V5 antibody (mouse) and Jil-1 antibody (chicken). At least two replicates were performed, and the same results were reached. C: Interaction measurement between dP75 and full-length or truncated versions of Jil-1 by yeast two-hybrid assay. Interaction strengths are indicated by β-gal activity. At least three independent clones were selected. Jil-1CI fragment exhibits the strongest interaction with full-length dP75. D: Isothermal titration calorimetry (ITC) to measure the interaction between dP75 and wild-type or mutated Jil-1 peptide (984-1004 fragment). Mutation of the conserved amino acids in the FYGF region of Jil-1 peptide severely attenuates its binding affinity with dP75. E: Graphic illustration for the interacting regions between dP75 and Jil-1.

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Fig. 4. dP75 is required for stabilizing Jil-1 protein. A: Immunostaining of Jil-1 in the ovary from either wild-type or dP75 mutants. Jil-1 shows robust nucleus-localized signal in wild-type flies, while these signals are nearly absent in dP75 mutant. B: Strategy for mosaic analysis in (C). C: Clonal analysis of Jil-1 expression. dP75-mutant clones were generated through strategy shown in (B) and were subjected to immunostaining with Jil-1 antibody seven days after clone induction. dP75-mutant clones are marked by the absence of GFP and outlined with dashed lines. The detailed method for clone generation is introduced in Materials and methods. D: Immunostaining of transgenic Jil-1 protein in either control or in ovaries with dP75 depleted in germ cells. UAS-Flag:Myc:Jil-1 driven by germline-specific Gal4 shows robust expression in wild-type ovaries but is undetectable when dP75 is depleted. E: Western blot for transgenic Jil-1 protein in control and dP75-depleted ovaries. F: RNA-Seq data show that the abundance of Jil-1 mRNAs is not affected in dP75 mutants.

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Fig. 5. dP75 restrains H3K9me2 from spreading on chromosome arms. A: Coimmunostaining of dP75 and H3K9me2 in wild-type or dP75-mutant ovaries. Three replicates were carried out, and consistent patterns were observed in stage 6 to stage 8 egg chambers in each genotype. Representative pictures are shown here. B: Density of H3K9me2 ChIP-Seq signal across chromosome arms in wild-type or dP75-mutant ovaries. The same sequencing depth (mapped reads) was used for analysis of the two samples, and the density of H3K9me2 ChIP-Seq reads was calculated with Rstudio. C: Expression of genes measured by RNA-Seq. Gene expression was calculated as FPKM, and both the x-axis and y-axis are in log₂ scale. D: Browser view of RNA-Seq, H3K9me2 occupancy, and dP75 ChIP-Seq signals for the top decreased genes in dP75 mutants. Loss of dP75 leads to increase of H3K9me2 deposition and gene silencing.

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Fig. 6. dP75 and Jil-1 function together to ensure oogenesis. A: Expression of genes measured by RNA-Seq. Gene expression was calculated as FPKM, and both the x-axis and y-axis are in log₂ scale. B: dP75 and Jil-1 protect common targets for transcription. C: DAPI staining shows similar defects of germ cells by either depleting of dP75 or Jil-1 in germ cells. Upper panel: arrow heads point the nurse cells failed to pass the five-blob configuration. Percentage of each panel: 100% in GLD of mCherry (normal morphology); 57% in GLD of Jil-1 (abnormal morphology); 71% in GLD of dP75 (abnormal morphology). Egg chambers of stage 7 to stage 9 were observed for quantification. Lower panel: oocyte DNA failed to compact into karyosome (pointed by arrow head). Percentage of each panel: 98% in GLD of mCherry (normal morphology), 34% in GLD of Jil-1 (abnormal morphology), 27% in GLD of dP75 (abnormal morphology). Egg chambers of stage 7 to stage 8 were observed for quantification.

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Fig. 7. dP75 is required for transposon silencing during oogenesis. A: RNA-Seq, V5:dP75 ChIP-Seq, and H3K9me2 ChIP-Seq signals across the boYb gene, which encodes a piRNA pathway component for transposon silencing. dP75 protects boYb from H3K9me2 spreading. B: The change in transposon expression, relative to control, was compared for dP75 mutants. Transposon expression was calculated as FPKM, and both the x-axis and y-axis are in log2 scale. C: RNA FISH to detect the expression and localization of transposon transcripts. D: Graphic model to depict the function of dP75 during oogenesis. dP75 physically interacts with Jil-1, antagonizing the spreading of H3K9me2. Upon loss of dP75, Jil-1 protein is unstable and leads to H3K9me2 deposition on genes that are required for ensuring oogenesis and transposon silencing.

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Table 1. Reagents, fly strains and resources used in this study.

Reagent/resource	Source	Identifier
Antibodies
Anti-V5 tag mouse monoclonal antibody	Thermo Fisher Scientific, USA	CAT#: R960-25
Anti-V5 tag antibody (agarose)	Abcam, USA	CAT#: Ab1229
Rabbit polyclonal anti-dP75	This study	N/A
Chicken anti-Jil-1	Johansen lab	N/A
Mouse monoclonal anti-H3K9me2-ChIP grade	Abcam	CAT#: Ab1220
Mouse monoclonal anti-Flag M2 antibody	Sigma chemical, USA	CAT#: F3165-.2MG
Anti-alpha-tubulin antibody	DSHB, USA	12G10
Alexa 488 Donkey anti-Mouse	Thermo Fisher Scientific	CAT#: A-21202
Alexa 568 Goat anti-Mouse	Thermo Fisher Scientific	CAT#: A-11004
Alexa 488 Goat anti-Chicken	Thermo Fisher Scientific	CAT#: A-11039
Alexa 568 Goat anti-Rabbit	Thermo Fisher Scientific	CAT#: A-11011
Amersham ECL Mouse IgG, HRP-linked whole Ab	GE Healthcare Life Science, USA	NXA931-1ML
Amersham ECL Rabbit IgG, HRP-linked whole Ab	GE Healthcare Life Science	NA934-1ML
Experimental m odels: o rganisms/s trains
D. melanogaster: w; Act5c-Gal4/CyO	Zhao Zhang lab	N/A
D. melanogaster: P(otu-Gal4::VP16.R)1 w[∗]; P(Gal4-nos.NGT)40; P(Gal4::VP16-nos.UTR)CG6325[MVD1]	Bloomington Drosophila Stock Center, USA	CAT#: 31777
D. melanogaster: jil-1-sh (attP2)	Bloomington Drosophila Stock Center	57293
D. melanogaster: mcherry-sh (attP2)	Bloomington Drosophila Stock Center	35785
D. melanogaster: dP75-sh (attP40)	Bloomington Drosophila Stock Center	55274
D. melanogaster: V5::dP75 (at its endogenous locus)	This study	N/A
D. melanogaster: UASp-FlagMyc-dP75 (attP40)	This study	N/A
D. melanogaster: UASp-FlagMyc-Jil-1 (attP2)	This study	N/A
D. melanogaster: dP75-sg1/TM6c, sb	This study	N/A
D. melanogaster: dP75-sg2/TM6c, sb	This study	N/A
Competent cells
Top10 one shot kit	Life Technologies, USA	C404006
BL21 (DE3)	NEB, USA	C2527H
Critical commercial assay
Vectashield Mounting Medium	VECTOR LABORATORIES INC MS	CAT#: 101098-042
V5 peptide	Abcam	Ab15829
EDTA-free complete protease inhibitor	Roche, USA	CAT#: 4693159001
pENTR™/D-TOPO® Cloning Kit	Thermo Fisher Scientific	K240020
Gateway™ LR Clonase™ II Enzyme mix	Thermo Fisher Scientific	11791020
Deposited data
Illumina sequencing raw data (FASTQ)	This study	PRJNA589981
Oligonucleotides
Forward primer to validate V5 knockin: ACC CTC TTC TTG GTC TAG ATA	This study	N/A
Reverse primer to validate V5 knockin: AGC ATC TCG TTC CGG ATG TT	This study	N/A
Forward primer to sequence V5 knockin fly: ATA CGA GCA CTG CTA GCG C	This study	N/A
Forward primer to validate dP75 knockout: ACC AAA CGA GCT AGA AGT CAA	This study	N/A
Reverse primer to validate dP75 knockout: ATC GAT GGC CTC GAT GAA CT	This study	N/A
Forward primer to generate dP75 transgene: CAC CAT GGG TAA GGA AAA GGC GGC	This study	N/A
Reverse primer to generate dP75 transgene: TTA CTG TGG AGC CGA TCC G	This study	N/A
Forward primer to generate jil-1 transgene: CAC CAT GAG TCG CTT GCA AAA GCA A	This study	N/A
Reverse primer to generate jil-1 transgene: TCA TTG GAA CTG ATA AAG TTG AC	This study	N/A
Software and algorithms
piPipes	Han etal. (2015)	N/A
EDTA, edthylenediaminetetraacetic acid.

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