1. Stem Cells and Regenerative Medicine
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Ovaries absent links dLsd1 to HP1a for local H3K4 demethylation required for heterochromatic gene silencing

  1. Fu Yang  Is a corresponding author
  2. Zhenghui Quan
  3. Huanwei Huang
  4. Minghui He
  5. Xicheng Liu
  6. Tao Cai
  7. Rongwen Xi  Is a corresponding author
  1. National Institute of Biological Sciences, China
  2. Tsinghua University, China
Research Communication
Cite this article as: eLife 2019;8:e40806 doi: 10.7554/eLife.40806
8 figures, 1 table, 1 data set and 2 additional files

Figures

Figure 1 with 2 supplements
Ova is a niche factor for GSCs and ovary development in Drosophila.

(a) Phase contrast images of dissected ovaries from flies of indicated genotypes. Scale bar, 500 μm. (b) A graph shows the total offspring number of indicated females (n = 20, 20, 16, 16, 20 respectively). (c) Representative image of germaria from indicated genotypes labeled by α-Spectrin (red), Vasa (green), and DAPI (blue). ova1/1 and ova1/4 ovaries have numerous spherical-shaped spectrosome-containing cells (tumorous) or are empty of germline cells (germless), indicated by lack of germline cell marker Vasa (green). A wild-type (WT) germarium is usually 2 GSCs localized to the anterior tip. Scale bar, 10 μm. (d) A graph shows the percentage of normal, germless, and tumorous germaria of indicated genotypes (n = 14, 20, 30, 117, 57 respectively). (e) c587ts > ova RNAi germarium accumulated GSC-like cells after shift to 29°C for 7 and 14 days. Scale bars, 10 μm. (f) Escort cell-specific expression of ova rescued oogenesis and GSC differentiation defect of ova1/4 females. Red, α-Spectrin; Green, Vasa (g) Quantification of GSC-like cell number in germaria of indicated genotypes (n = 25, 20, 30, 117, 57, 30, 21, 31, 23 respectively). (h) Confocal sections of germaria stained by indicated antibodies or reporter. Scale bars, 10 µm. (i) Quantitative results of pMad and Dad-lacZ positive cell numbers from germaria of indicated genotypes. Values are mean ± SEM.; n > 20. P values by two-tailed Student t-test.

https://doi.org/10.7554/eLife.40806.003
Figure 1—figure supplement 1
Ova is allelic to CG5694.

(a) Top, a schematic drawing of mapping results of ova1 allele using deficiency kit. Two small deficiencies, Df(2L)ED737 and Df(2L)BSC144, both failed to complement ova1 (Figure 1—figure supplement 1a). Bottom, analysis of the overlapping regions of these deficiencies implicated five candidate genes. Gray box, ova genomic rescue fragment. (b) DNA sequencing revealed a nucleotic deletion in the coding region of CG5694. (c) Schematic drawings of Ova, Drosophila Ewg-PA, and mouse NRF-1 PA proteins. Black box, Nrf1 DNA-binding domain. Grey box, Nrf1 activator-binding domain. (d) Multiple sequence alignments of Nrf1-binding domain of Ova, Ewg, and NRF-1 by Clustal Omega. (e) Schematic drawings of two CG5694/ova alleles, ova1, and ova4ova1 is a deletion allele generated using CRISPR-Cas9.

https://doi.org/10.7554/eLife.40806.004
Figure 1—figure supplement 2
Ova is not cell- autonomously required for GSC differentiation.

(a–d) Clonal analysis of wild-type and ova mutant GSCs. Anti-α-Spectrin staining was shown in red. The control clones marked by the absence of lacZ (anti-β-galactosidase, green) at 4 days or 14 days after clone induction (ACI) (a,b). The ova1 mutant clones at 4 days or 14 days ACI (c, d). Scale bar, 10 μm. (e) A ova1 germline clone with properly specified oocyte (anti-Orb, red). Scale bar, 20 μm. (f) A time course analysis of GSC maintenance rate in wild-type and ova mutant GSC clones. (g) Phase contrast images of germline-specific knock-down ova (ova GLKD) ovary. Scale bar, 500 μm. (h) Quantification of GSC-like cell number in ova GLKD germaria. Values are mean ± SEM.; n = 24.

https://doi.org/10.7554/eLife.40806.005
Ova acts downstream of Piwi genetically.

(a) Phase contrast images of dissected ovaries from flies of indicated genotypes. Scale bar, 500 µm. (b) A graph shows the percentages of germaria with rudimentary or rescued ovaries (n = 62, 51, 143, 198 respectively). (c) Confocal sections of piwi2/3 and ova-rescued ovaries. Arrow indicates the GSC-like tumor; asterisk indicates the developing germline cyst. Red, α-Spectrin. Scale bars, 10 µm. (d) Confocal sections of piwi RNAi and panx RNAi germaria. Red, α-Spectrin. Scale bars, 10 µm. (e) Quantification of GSC-like cell number in germaria of indicated genotypes. (n = 55, 43 respectively). f, qPCR result of TE levels in total ovarian RNA from indicated genotypes (normalized to actin5c). Values are means ± SEM.; n = 3. P values by two-tailed t-test (*, p<0.05; **, p<0.01; ***, p<0.001).

https://doi.org/10.7554/eLife.40806.006
Figure 3 with 3 supplements
Ova interacts with the heterochromatin machinery.

(a) Y2H assay for protein interaction between Ova and proteins as indicated. (b–c) Western blots showing reciprocal co-IP between Ova and HP1a, and between Ova and dLsd1. The RFP-HP1a transgene was driven by the endogenous promoter. The dLsd1-GFP transgene was driven by a ubiquitous promoter. The Flag-ova transgene was driven by nos-GAL4. (d) Heat map displaying steady state mRNA levels as reads per million (rpm) for the top 60 detected transposons in nosGAL4 driven ova-RNAi, EGFP-RNAi, and w1118 ovaries. The average of three replicates is shown. The most upregulated transposons are highlighted in bold. (e) Correlation scatter plot of log10 transposon mRNA-seq reads between ova GLKD and dLsd1 GLKD ovaries. R = 0.9463, p<2.2×10−16 by Pearson’s correlation coefficient. The most upregulated transposons in both genotypes are highlighted in red dots.

https://doi.org/10.7554/eLife.40806.007
Figure 3—figure supplement 1
Protein interaction mapping among components of heterochromatin machinery by Y2H assay.

Y2H assay to test the interaction between proteins that associated with Piwi/piRNAs complexes and proteins in the heterochromatin machinery. SD-WL, non-selective medium; SD-WLH and SD-WLHA, selective media. Ф, empty vector.

https://doi.org/10.7554/eLife.40806.008
Figure 3—figure supplement 2
Ova acts as a suppressor of position-effect variegation (PEV).

(a) Photographs showing representative eye pigments of adult females bearing X chromosome wm4h reporter plus wild-type and ova heterozygous alleles. (b) Photographs showing representative eye pigments of adult females bearing fourth chromosome PEV reporters plus heterozygous or trans-heterozygous of ova mutant alleles. (c) Quantitative measure of eye pigment levels of indicated genotypes by a spectrophotometer. Values are mean ± SEM.; n > 5. P values by two-tailed Student’s t-test.

https://doi.org/10.7554/eLife.40806.009
Figure 3—figure supplement 3
Ova is not required for piRNA biogenesis.

(a) A scatter plot shows gene expression (rpkm, reads per kilobase per million sequenced reads) comparison of protein-coding genes between w1118 and ova GLKD ovaries. R by Pearson’s correlation coefficient. (b) Top, normalized small RNA length profiles: miRNAs (gray columns, insets) and piRNAs/siRNAs (black or red columns) from ovaries with indicated genotypes. Bottom, length distribution and abundance of repeat-derived small RNAs (blue, sense; red, antisense). (c) GFP-Piwi immunostaining from ovaries of indicated genotypes. Scale bars, 10 μm. (d) qPCR result of ova mRNA levels in control and ova GLKD ovaries. Values are means ±SEM.; n = 4. P value by two-tailed Student t-test. (e) Heat map displaying repeat small RNA levels as reads per million (rpm) for individual transposon in ova GLKD and w1118 ovaries. (f) qPCR result of TE mRNA levels in w1118 and tjGAL4 >ova RNAi (shift to 29°C for 7 days) ovaries. ***, p<0.001 by two-tailed Student’s t-test. Values are means ± SEM.; n = 3.

https://doi.org/10.7554/eLife.40806.010
Figure 4 with 1 supplement
Ova acts as a protein adaptor to link dLsd1 with HP1a.

(a) Schematic drawings of full length and truncated forms of Ova. (b) Mapping the reciprocal-binding regions between HP1a and Ova by Y2H assay. (c) Mapping the reciprocal-binding regions between HP1a and dLsd1 by Y2H assay. (d) Ovaries from flies of indicated genotypes. Escort cell-specific expression of ova full length, ova250-486 or HP1a::dLsd1 rescued ova1/4 ovary defect. Scale bar, 500 µm. (e) A representative image of ova1/4 germarium rescued by escort cell-specific expression of HP1a::dLsd1. Red, α-spectrin; Blue, DAPI. Scale bar, 10 µm. (f) A graph shows the total offspring number of indicated females (n = 11, 23 respectively). (g) A graph shows fold changes of TEs in total ovarian RNA from indicated genotypes (normalized to actin5c). Values are means ± SEM.; n > 4. P values by two-tailed t-test.

https://doi.org/10.7554/eLife.40806.011
Figure 4—figure supplement 1
HP1a::dLsd1 expression rescues ova loss induced derepression of protein-coding genes.

Heat map showing relative z-score of mRNA-seq from indicated genotype ovaries. The top 39 upregulated genes in ova germline mutant ovaries are shown and the genes, which are rescued to wild-type levels by HP1a::dLsd1, are highlighted in red dashed box. Grey shadow, not significant.

https://doi.org/10.7554/eLife.40806.012
Figure 5 with 2 supplements
Ova regulates HP1a-induced local H3K4 demethylation.

(a) Graphs showing H3K4me2 and Pol II ChIP-seq profiles mapped to indicated transposon loci in control versus ova GLKD ovaries. Dashed boxes, enhancer regions of transposons. RPM, reads per million. Bin, 100 bp. (b) Quantitative comparison of H3K4me2 and Pol II densities in the indicated enhancer regions in 5a (dashed boxes). P values by two-tailed Student’s t-test. (c), Ovarioles with indicated genotypes expressing ubiquitous lacO-GFP reporter in the germline cells. GFP was visualized by antibody staining. Scale bar, 50 µm. (d) A graph showing the percentage of ovarioles of indicated genotypes with normal, reduced, or abolished GFP signals. (e) Quantitative RT-PCR results of GFP mRNA from ovaries of indicated genotypes. Values are means ±SEM.; n > 4. P values by two-tailed Student’s t-test. (f) Graphs showing normalized H3K4me2 density mapped to lacO-GFP reporter region from ovaries of indicated genotypes. Grey box, lacO-binding sites; Purple box, nanos promoter. (g) Graphs showing normalized H3K4me2 density mapped to lacO-terminator-GFP reporter region from ovaries of indicated genotypes. Blue box, VASA terminator. (h) Quantitative comparison of H3K4me2 density in regions indicated by the dashed boxes in f or g. P values by two-tailed Student’s t-test. (i) Quantitative RT-PCR results of GFP mRNA from lacO-GFP and lacO-terminator-GFP reporter ovaries. Values are means ± SEM.; n = 4. P value by Student t-test. (j) A schematic model for Ova function: Ova functions as a protein adaptor to link HP1a with dLsd1 for local H3K4 demethylation during HP1a-induced transcriptional gene silencing.

https://doi.org/10.7554/eLife.40806.013
Figure 5—figure supplement 1
Comparison of H3K4me2 density on all transposons in w1119 and ova GLKD ovaries.

Correlation scatter plot of log10 H3K4me2 ChIP-seq reads between w1118 and ova GLKD ovaries. The Het-A and TAHRE transposons are highlighted in red dots.

https://doi.org/10.7554/eLife.40806.014
Figure 5—figure supplement 2
Tethering Ova to DNA or RNA is unable to induce co-transcriptional gene silencing.

(a), A cartoon to describe the DNA tethering assay using the lacI and lacO binary system. (b, b’, c, c’) Confocal images of GFP expression in ovarioles of indicated genotypes. Green, anti-GFP staining. Red, anti-Armadillo. (d), A cartoon to describe the RNA tethering assay using λN and boxB binary system. (e, e’, f, f’) Confocal images of GFP expression in ovarioles of indicated genotypes. Scale bars, 50 μm.

https://doi.org/10.7554/eLife.40806.015
Author response image 1
dLsd1 and HP1a expression and subcellular localization in control and ova GLKD germaria
Author response image 2
qPCR to detect TE levels in controls and ova GLKD ovaries.
Author response image 3
H3K9me3 expression in control and ova GLKD germaria.

Tables

Key resources table
Reagent type (species) or
resource
DesignationSource or referenceIdentifiersAdditional information
Genetic reagent
(Drosophila
melanogaster)
ova[1]This paperSee Materials and
methods
Genetic reagent
(Drosophila
melanogaster)
ova[4]This paperSee Materials and
methods
Genetic reagent
(Drosophila
melanogaster)
c587-GAL4(Song et al., 2004)
(DOI: 10.1242/dev.01026)
RRID:BDSC_67747
Genetic reagent
(Drosophila
melanogaster)
Dad-lacZ(Tsuneizumi et al., 1997)
(DOI: 10.1038/39362)
RRID:DGGR_118114
Genetic reagent
(Drosophila
melanogaster)
bam-GFP(Chen and McKearin, 2003)RRID:DGGR_118177
Genetic reagent
(Drosophila
melanogaster)
piwi[2](Lin and Spradling, 1997)RRID:BDSC_43319
Genetic reagent
(Drosophila
melanogaster)
piwi[3](Lin and Spradling, 1997)RRID:BDSC_12225
Genetic reagent
(Drosophila
melanogaster)
GFP-piwiKatalin Toth
(California Institute of
Tchnology)
Genetic reagent
(Drosophila
melanogaster)
118E-10Lori Wallrath
(University of Iowa)
Genetic reagent
(Drosophila
melanogaster)
118E-15Lori Wallrath
(University of Iowa)
Genetic reagent
(Drosophila
melanogaster)
6 M-193Lori Wallrath
(University of Iowa)
Genetic reagent
(Drosophila
melanogaster)
39C.72Lori Wallrath
(University of Iowa)
Genetic reagent
(Drosophila
melanogaster)
dLsd1-GFPYu Yang (Institute of
Biophysics, Chinese
Academy of Science)
Genetic reagent
(Drosophila
melanogaster)
EGFP-RNAiBloomington Drosophila
Stock Center
(#41553)
Genetic reagent
(Drosophila
melanogaster)
RFP-HP1aBloomington Drosophila
Stock Center
(#30562)
Genetic reagent
(Drosophila
melanogaster)
UAS-Dcr2; nos-GAL4Bloomington Drosophila
Stock Center
(#25751)
Genetic reagent
(Drosophila
melanogaster)
tub-GAL4Bloomington Drosophila
Stock Center
(#5138)
Genetic reagent
(Drosophila
melanogaster)
tub-GAL80tsBloomington Drosophila
Stock Center
(#7016, #7018)
Genetic reagent
(Drosophila
melanogaster)
Df(2L)BSC144Bloomington Drosophila
Stock Center
(#9504)
Genetic reagent
(Drosophila
melanogaster)
attP2Bloomington Drosophila
Stock Center
(#25710)
Genetic reagent
(Drosophila
melanogaster)
In(1)wm4hKyoto Stock Center(#101652)
Genetic reagent
(Drosophila
melanogaster)
Df(2L)ED737Kyoto Stock Center(#150520)
Genetic reagent
(Drosophila
melanogaster)
ova-RNAiVienna Drosophila
Research Center
(#102156)
Genetic reagent
(Drosophila
melanogaster)
piwi-RNAiVienna Drosophila
Research Center
(#101658)
Genetic reagent
(Drosophila
melanogaster)
panx-RNAiVienna Drosophila
Research Center
(#102702)
Genetic reagent
(Drosophila
melanogaster)
EGFP-5xBoxBVienna Drosophila
Research Center
(#313408)
Genetic reagent
(Drosophila
melanogaster)
lacO-GFP-PiwiVienna Drosophila
Research Center
(#313394)
Genetic reagent
(Drosophila
melanogaster)
lacI-HP1a; lacO-
GFP-Piwi
Vienna Drosophila
Research Center
(#313409)
Genetic reagent
(Drosophila
melanogaster)
nos-Cas9Jianquan Ni
(Tsinghua University)
Genetic reagent
(Drosophila
melanogaster)
pCasper4-ova-gThis paperSee Materials and
methods
Genetic reagent
(Drosophila
melanogaster)
GFP-ovaThis paperSee Materials and
methods
Genetic reagent
(Drosophila
melanogaster)
UASP-ovaThis paperSee Materials and
methods
Genetic reagent
(Drosophila
melanogaster)
UASP-piwiThis paperSee Materials and
methods
Genetic reagent
(Drosophila
melanogaster)
UASP-ova1-249This paperSee Materials and
methods
Genetic reagent
(Drosophila
melanogaster)
UASP-ova250-486This paperSee Materials and
methods
Genetic reagent
(Drosophila
melanogaster)
UASP-ova388-623This paperSee Materials and
methods
Genetic reagent
(Drosophila
melanogaster)
UASP-ova1-388This paperSee Materials and
methods
Genetic reagent
(Drosophila
melanogaster)
UASP-HP1a::dLsd1This paperSee Materials and
methods
Genetic reagent
(Drosophila
melanogaster)
UASP-λN-ovaThis paperSee Materials and
methods
Genetic reagent
(Drosophila
melanogaster)
UASP-lacI-ovaThis paperSee Materials and
methods
Genetic reagent
(Drosophila
melanogaster)
lacO-terminator-GFP-PiwiThis paperSee Materials and
methods
Recombinant DNA
reagent
UASP-λNJulius Brennecke
(Institute of Molecular
Biotechnology)
Recombinant DNA
reagent
UASP-lacI
Recombinant DNA
reagent
lacO-GFP-Piwi
Recombinant DNA
reagent
pGBKT7Clontech (Cat#630443)
Recombinant DNA
reagent
pGADClontech (Cat#630442)
Antibodyrabbit polyclonal
anti-pMad
Ed Laufer (Columbia
Universtity Medical
Center)
RRID:AB_2617125IHC(1:1000)
Antibodyrabbit polyclonal
anti-β-galactosidase
MP Biologicals
(Cat#0855976)
RRID:AB_2687418IHC(1:3000)
Antibodymouse monoclonal
anti-α-Spectrin
Developmental Studies
Hybridoma Bank
IHC(1:50)
Antibodymouse monoclonal
anti-Tubulin
Developmental Studies
Hybridoma Bank
RRID:AB_1157911WB(1:2000)
Antibodyrabbit polyclonal
anti-mCherry
BioVision (cat#5993)RRID:AB_1975001WB (1:2000)
Antibodyrabbit polyclonal
anti-GFP
Life (cat#A11122)RRID:AB_221569IHC(1:1000)
WB(1:10000)
Antibodypolyclonal
anti-rabbit IgG-HRP
ZSJQ-BIO (cat#ZB2301)WB(1:10000)
Antibodyrabbit polyclonal
anti-H3K4me2
Abcam (cat#ab7766)RRID:AB_732924
Antibodymouse monoclonal
anti-RNA polymerase II
Abcam (cat#ab817)RRID:AB_306327
Antibodymouse monoclonal
anti-Flag
Sigma (cat#F1804)RRID:AB_439685IHC(1:300)
WB(1:6000)
Chemical
compound, drug
4’,6’-diamidino-2-
phenylindole
Sigma
(cat#10236276001)
Commercial
assay or kit
anti-Flag resinSigma (cat#A2220)RRID:AB_10063035
Commercial
assay or kit
GFP-Trap agaroseChromoteck
(cat#gta-10)
Commercial
assay or kit
RFP-Trap agaroseChromoteck
(cat#rta-10)
Commercial
assay or kit
Qiagen Plasmid
Midi Kit
Qiagen (#12145)
Commercial
assay or kit
Immobilon Western
Chemiluminescent
HRP Substrate Kit
Millipore
(cat#WBKLS0500)
Commercial
assay or kit
HiScript II Q RT
SuperMix
Vazyme Biotech
(cat#R223-01)
Commercial
assay or kit
ChamQ SYBR qPCR
master Mix
Vazyme Biotech
(cat#Q331)
Commercial
assay or kit
Oligo d(T)25 Magnetic
beads
NEB (cat#S1419S)
Commercial
assay or kit
NEBNext Ultra IIDNA
Library Prep Kits for
Illumina
NEB (cat# E7645S)
Commercial
assay or kit
VAHTS Small RNA
Library Prep Kit
for Illumina
Vazyme Biotech
(cat#NR801)
Commercial
assay or kit
VAHTS Universal DNA
Library Prep Kit
Vazyme Biotech
(cat#ND607)
Commercial
assay or kit
TruePrep Index KitVazyme Biotech
(cat#TD202)
Software,
algorithm
GraphPad PrismGraphPad Prism
(https://graphpad.com)
RRID:SCR_002798
Sequenced-based
reagent
RT-qPCR primersThis paperSee Supplementary file 1,
Table 2

Data availability

High-throughput sequence data GEO link: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE104925

The following data sets were generated
  1. 1
    NCBI
    1. H Huang
    (2018)
    Impact of CG5694/ova in Drosophila ovaries.

Additional files

Supplementary file 1

Table 1: Viability test of ova mutants. Table 2: Primers used in this study.

https://doi.org/10.7554/eLife.40806.016
Transparent reporting form
https://doi.org/10.7554/eLife.40806.017

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