GAF is essential for zygotic genome activation and chromatin accessibility in the early Drosophila embryo
- Department of Biomolecular Chemistry, University of Wisconsin School of Medicine and Public Health, United States
Figures
Figure 1 with 3 supplements
GAF binds thousands of loci throughout the MZT.
(A) Images of His2Av-RFP; sfGFP-GAF(N) embryos at the nuclear cycles (NC) indicated above. sfGFP-GAF(N) localizes to puncta during interphase and is retained on chromosome during mitosis. His2AvRFP …
Figure 1—figure supplement 1
N- and C- terminal sfGFP-tags label distinct GAF isoforms.
(A) Cartoon representation of the N-terminal sfGFP tag both GAF protein isoforms. (B) Cartoon representation of C-terminal sfGFP tag on the short GAF protein isoform. (C) Confocal images of living Hi…
Figure 1—figure supplement 2
GAF binds thousands of regions in the embyo and S2 cells.
(A) Heatmap of stage 5 GAF-sfGFP(C) ChIP, GAF-sfGFP(C) input, w1118 ChIP, and w1118 input peaks (n = 4175). Immunoprecipitation was performed with an anti-GFP antibody. (B) Heatmap of stage 3 …
Figure 1—figure supplement 3
GAF has tissue-specific binding.
(A) Heatmap of GAF ChIP-seq peaks in S2 cells (Fuda et al., 2015) and GAF-sfGFP(C) ChIP-seq peaks from stage 3 and stage 5 embryos (this work), excluding peaks that are shared in all three datasets. …
Figure 2 with 1 supplement
Embryos lacking maternal GAF die during early embryogenesis with nuclear and mitotic defects.
(A) Images of control (maternal genotype: His2Av-RFP; sfGFP-GAF(N)) and GAFdeGradFP (maternal genotype: His2Av-RFP/nos-deGradFP; sfGFP-GAF(N)) embryos at NC14, demonstrating loss of nuclear GFP …
Figure 2—figure supplement 1
sfGFP-GAF(N) and GAF-sfGF(C) are expressed at similar levels in the early embryo.
Boxplots showing the mean fluorescent intensity (arbitrary units, AU) of 10 nuclei in GAF-sfGFP(C) homozygous embryos (n = 27) and sfGFP-GAF(N) homozygous embryos (n = 26). The box indicates the …
Figure 3 with 3 supplements
GAF is required for zygotic genome activation.
(A) Volcano plot of transcripts mis-expressed in GAFdeGradFP embryos as compared to sfGFP-GAF(N) controls. Stage 5 GAF-sfGFP(C) ChIP-seq was used to identify GAF-bound target genes. (B) The …
Figure 3—figure supplement 1
RNA-seq replicates are reproducible.
(A) Pairwise scatterplots showing correlation between the three RNA-seq replicates for GAFdeGradFP and sfGFP-GAF(N) homozygous controls. Values are log2 (RPKM +1). Pearson correlation coefficients …
Figure 3—figure supplement 2
RNA-seq identifies genes mis-regulated in GAFdeGradFP embryos.
(A) MA plot of transcripts mis-expressed in GAFdeGradFP embryos as compared to sfGFP-GAF(N) homozygous controls. Stage 5 GAF-sfGFP(C) ChIP-seq was used to identify GAF-bound target genes. (B) log2 …
Figure 3—figure supplement 3
GAF regulates genes distinct from Zld and CLAMP.
(A) Overlap of transcripts down-regulated in GAFdeGradFP embryos, down-regulated in ZldCRY2 embryos at NC14 (McDaniel et al., 2019), and down-regulated in clamp-RNAi 2–4 hr embryos (Rieder et al., …
Figure 4 with 2 supplements
At the majority of loci, GAF and Zld bind chromatin independently.
(A) Overlap of Zld- and GAF-binding sites determined by GAF-sfGFP(C) stage 5 ChIP-seq and Zld NC14 ChIP-seq (Harrison et al., 2011). (B) Representative genome browser tracks of Zld and GAF ChIP-seq …
Figure 4—figure supplement 1
GAF and Zld bind to shared and unique regions of the genome.
(A) Heatmap of GAF-sfGFP(C) ChIP peaks at stage 5 and Zld ChIP peaks at NC14. Zld ChIP-seq from Harrison et al., 2011. (B) Heatmap of anti-GFP ChIP-seq peaks from GAF-sfGFP(C) homozygous stage 5 …
Figure 4—figure supplement 2
Independent chromatin binding by GAF and Zld.
(A) Heatmap of high confidence, anti-GFP ChIP-seq peaks from 2 to 2.25 hr AEL sfGFP-GAF(N) and zld-RNAi;sfGFP-GAF(N) embryos. The heatmap is divided into sites that have both GAF and Zld binding and …
Figure 5 with 1 supplement
GAF is required for chromatin accessibility.
(A) Volcano plot of regions that change in accessibility in GAFdeGradFP embryos as compared to sfGFP-GAF(N) controls, stage 5 GAF-sfGFP(C) ChIP-seq was used to identify GAF-bound target regions. (B) …
Figure 5—figure supplement 1
GAF is required for chromatin accessibility.
(A) Heatmap of Pearson correlation coefficients between ATAC-seq replicates for GAFdeGradFP and sfGPF-GAF(N) control embryos. (B) MA plot of regions that change in accessibility in GAFdeGradFP …
Figure 6 with 2 supplements
GAF and Zld independently shape chromatin accessibility over the MZT.
(A) Heatmaps of ChIP-seq and ATAC-seq data, as indicated above, for regions bound by both GAF and Zld and subdivided based on the change of accessibility in the absence of either factor. (B) Number …
Figure 6—figure supplement 1
A subset of regions bound by GAF, and not Zld, depend on GAF for accessibility.
Heatmaps of regions that are bound by GAF (excluding GAF and Zld co-bound sites) as determined by GAF-sfGFP(C) stage 5 ChIP-seq subdivided by whether or not these regions change in accessibility …
Figure 6—figure supplement 2
Regions that gain accessibility late during the MZT are accessible in GAFdeGradFP embryos used for ATAC-seq.
(A) Genome browser tracks showing a region that gains accessibility at NC13 that is maintained in GAFdeGradFP embryos (dark-blue shading) and a region that gains accessibility at stage 5, is bound …
Figure 7
Zld and GAF independently regulate embryonic reprogramming.
(A) During the minor wave of ZGA (NC10-13), Zld is the predominant factor required for driving expression of genes bound by Zld alone and genes bound by both Zld and GAF. (B) As the genome is more …
Videos
Video 1
Video of a GAFdeGradFP embryo going through several rounds of mitosis prior to gastrulation.
Nuclei are marked by His2Av-RFP.
Video 2
Video of a severely disordered GAFdeGradFP embryo going through several rounds of mitosis prior to gastrulation.
Nuclei are marked by His2Av-RFP.
Video 3
Video of a control (His2Av-RFP; sfGFP-GAF(N)) embryo going through several rounds of mitosis prior to gastrulation.
Nuclei are marked by His2Av-RFP.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (Drosophila melanogaster) | w1118 | Bloomington Drosophila Stock Center | BDSC:3605; FLYB:FBal0018186; RRID:BDSC_3605 | |
| Genetic reagent (D. melanogaster) | His2AV-RFP (II) | Bloomington Drosophila Stock Center | BDSC:23651; FLYB:FBti0077845; RRID:BDSC_23651 | |
| Genetic reagent (D. melanogaster) | mat-α-GAL4-VP16 | Bloomington Drosophila Stock Center | BDSC:7062; FLYB:FBti0016915; RRID:BDSC_7062 | |
| Genetic reagent (D. melanogaster) | UAS-shRNA-zld | Sun et al., 2015 DOI:10.1101/gr.192542.115 | ||
| Genetic reagent (D. melanogaster) | sfGFP-GAF(N) | This paper | Cas9 edited allele | |
| Genetic reagent (D. melanogaster) | GAF-sfGFP(C) | This paper | Cas9 edited allele | |
| Genetic reagent (D. melanogaster) | nos-deGradFP | This paper | Transgenic insertion into PBac{yellow[+]-attP-3B}VK00037 docking site (BDSC:9752) (FLYB: FBti0076455) NSlmb-vhhGFP4 amplified from BDSC:58740 | |
| Antibody | Anti-GFP (rabbit polyclonal) | Abcam | Cat# ab290 | ChIP (6 μg) WB (1:2000) |
| Antibody | Anti-Zld (rabbit polyclonal) | Harrison et al., 2010 DOI:10.1016/j.ydbio.2010.06.026 | ChIP (8 μg) WB (1:750) | |
| Antibody | Anti-alpha tubulin (mouse monoclonal) | Sigma-Aldrich | Cat# T6199 | WB (1:5000) |
| Antibody | Anti-rabbit IgG-HRP (Goat, secondary) | Bio-Rad | Cat#1706515 | WB (1:3000) |
| Antibody | Anti-mouse IgG-HRP (Goat, secondary) | Bio-Rad | Cat#1706516 | WB (1:3000) |
| cell line Mus musculus | H3.3-GFP | This paper | Cell line maintained in the lab of Peter Lewis | |
| software, algorithm | R | http://www.R-project.org | ||
| software, algorithm | bowtie 2 v2.3.5 | Langmead and Salzberg, 2012 | ||
| software, algorithm | Samtools v1.11 | http://www.htslib.org/ | ||
| software, algorithm | MACS v2 | Zhang et al., 2008 | ||
| software, algorithm | GenomicRanges R package | Lawrence et al., 2013 | ||
| software, algorithm | DeepTools | Ramírez et al., 2016 | ||
| software, algorithm | MEME-suite | Bailey et al., 2009 | ||
| software, algorithm | Gviz R package | Hahne and Ivanek, 2016 | ` | |
| software, algorithm | Subread (v1.6.4) | Liao et al., 2014 | ||
| software, algorithm | DESeq2 R package | Love et al., 2014 | ||
| software, algorithm | HISAT v2.1.0 | Kim et al., 2015 | ||
| software, algorithm | NGMerge | Gaspar, 2018 |
Additional files
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Supplementary file 1
Peaks called in ChIP-seq for GAF-sfGFP (C) in stage 3 and stage 5 hand-sorted embryos and sfGFP-GAF(N) 2–2.5 hr AEL embryos (as indicated in tabs).
Chromosome, start and end for each peak are provided as labelled.
- https://cdn.elifesciences.org/articles/66668/elife-66668-supp1-v2.xlsx
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Supplementary file 2
Differentially expressed genes identified by total RNA-seq in GAFdeGradFP embryos compared to controls.
Columns are defined in the first sheet and data are provided in the other sheet.
- https://cdn.elifesciences.org/articles/66668/elife-66668-supp2-v2.xlsx
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Supplementary file 3
Differential peaks identified in ATAC-seq of GAFdeGradFP embryos compared to controls.
Columns are defined in the first sheet and data are provided in the other sheet.
- https://cdn.elifesciences.org/articles/66668/elife-66668-supp3-v2.xlsx
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Supplementary file 4
Numbers for statistical analyses performed.
- https://cdn.elifesciences.org/articles/66668/elife-66668-supp4-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/66668/elife-66668-transrepform-v2.docx
