OVO Positively Regulates Essential Maternal Pathways by Binding Near the Transcriptional Start Sites in the Drosophila Female Germline

Leif Benner author has email address
Savannah Muron
Jillian G. Gomez
Brian Oliver

Section of Developmental Genomics, Laboratory of Biochemistry and Genetics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA. 2
Department of Biology, Johns Hopkins University, Baltimore, MD, USA

https://doi.org/10.7554/eLife.94631.1

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Figures and data

Significantly Enriched OVO DNA Binding Motifs and OVO ChIP Attributes Genome-Wide.
A-C) Immunofluorescent staining of adult ovarioles of ovo^Cterm-3xFHA/ovo^Cterm-GFPfemales (20x, scale bar = 20 μm). Ovarioles were stained for Vas (purple) to label the germline, HA (cyan) to label OVO-HA, and GFP (yellow) to label OVO-GFP. The homozygous version of these alleles were used to ChIP OVO. D-G) Significantly enriched motifs found within overlapping OVO ChIP peaks. The percentage of OVO ChIP peaks each motif was found and their corresponding p-value are indicated to the right. H) OVO ChIP minus input control ChIP-seq read coverage density centered on each individual motif’s location. I) Relative distance of OVO ChIP peaks to gene level promoters, terminations sequences, and open reading frames genome-wide. J) OVO ChIP minus input control ChIP-seq read coverage density for genes containing significant OVO ChIP peaks and the corresponding OVO DNA binding motif. Genes are centered on the transcriptional start site. K, L) OVO ChIP minus input control ChIP-seq read coverage density and heatmap plots centered on the dominant significant ovary CAGE-seq TSSs overlapping or not overlapping OVO ChIP peaks.

OVO DNA Binding is Associated with Open Chromatin and Transcriptionally Active Histone Marks.
A, B) OVO ChIP minus input control, GSC and 32c ATAC-seq, GSC H3K27ac, H3K4me3, H3K27me3, H3K9me3, 8c NC H3K9me3, 32c NC H3K27ac, and H3K27me3 ChIP-seq read coverage density centered on each OVO peak maximum or individual motif’s location. C) Relative distance of OVO ChIP peaks to significantly called peaks for GSC and 32c ATAC-seq, GSC H3K27ac, H3K4me3, H3K27me3, H3K9me3, 8c NC H3K9me3, 32c NC H3K27ac, and H3K27me3 ChIP-seq genome-wide. D) Total number of significant peaks and the total number of overlapping peaks between OVO ChIP and GSC and 32c ATAC-seq, GSC H3K27ac, H3K4me3, H3K27me3, H3K9me3, 8c NC H3K9me3, 32c NC H3K27ac, and H3K27me3 ChIP-seq. Asterisk indicates significantly enriched overlap while hashtag indicates significantly depleted overlap between datasets.

OVO Bound Promoters are Enriched for INR, DPE, and MTE elements.
A) Histogram of the distance of in vivo and in vitro OVO DNA binding motifs within significant overlapping OVO ChIP peaks from the closest genes TSS. B-F) Histogram of the percent of promoters from tissue specific CAGE-seq analysis of common promoter motif elements centered on the dominant significant TSS.

Genes Bound by OVO Increase in Expression in the Presence of OVO Genome-Wide.
A-B) Immunofluorescent staining of adult ovarioles of the indicated genotypes (20x, scale bar = 20 μm). Ovarioles were stained for Vas (purple) to label the germline, α-Spectrin (yellow) to label dot spectrosome and fusomes, and DAPI (cyan) to label nuclei. Line indicates the dissection point for germarium through previtellogenic RNA-seq samples. C) MA plot of ovo^ΔBP/ovo^ovo-GAL4; UASp-3xFHA-OVO-B versus ovo^ΔBP/ovo^ovo-GAL4; UASp-GFP RNA-seq differential expression results. Purple dots indicate genes that significantly increased in expression and were not bound by OVO, cyan dots indicate genes that significantly increased in expression and were bound by OVO, yellow dots indicate genes that significantly decreased in gene expression and were not bound by OVO, blue dots indicate genes that significantly decreased in gene expression and were bound by OVO, and gray dots indicate genes that were not differentially expressed from our analysis. D) MA plot of ovo^ΔBP/ovo^ovo-GAL4; UASp-3xFHA-OVO-B versus ovo^ΔBP/ovo^ovo-GAL4; UASp-GFP RNA-seq differential expression results. Cyan dots indicate genes that significantly increased in expression and were found to be moderately expressed in 0-2 hour old embryos, yellow dots indicate genes that significantly decreased in gene expression and were found to be moderately expressed in 0-2 hour old embryos, purple dots indicate genes that were not differentially expressed and were found to be moderately expressed in 0-2 hour old embryos, and gray dots indicate genes that were not differentially expressed and were not found to be moderately expressed in 0-2 hour old embryos. E) Gene level read coverage heatmaps of OVO ChIP minus input, GSC and 32c ATAC-seq, GSC H3K27ac, H3K4me3, H3K27me3, H3K9me3, 8c NC H3K9me3, 32c NC H3K27ac, and H3K27me3 ChIP-seq, and ovo^ΔBP/ovo^ovo-GAL4; UASp-3xFHA-OVO-B minus ovo^ΔBP/ovo^ovo-GAL4; UASp-GFP RNA-seq for genes bound by OVO.

OVO ChIP-seq, ATAC/Histone ChIP-seq, RNA-seq, and DNA Binding Motifs at the ovo Locus.
ovo gene level read coverage tracks for OVO ChIP minus input, GSC and 32c ATAC-seq, GSC H3K27ac, H3K4me3, H3K27me3, H3K9me3, 8c NC H3K9me3, 32c NC H3K27ac, and H3K27me3 ChIP-seq, and ovo^ΔBP/ovo^ovo-GAL4; UASp-3xFHA-OVO-B minus ovo^ΔBP/ovo^ovo-GAL4; UASp-GFP RNA-seq. Red rectangles and black rectangles represent significant OVO DNA binding motifs and OVO ChIP peaks, respectively. Gene models are represented at bottom. Small rectangles represent untranslated regions, large rectangles represent translated regions. Arrows indicate transcriptional start sites.

OVO Binds and Significantly Increases the Expression of a Number of Genes Involved in Essential Maternal Processes.
A) Significantly enriched GO biological process terms for genes bound by OVO and significantly increase in expression in the presence of ectopic rescue OVO. GO terms are restricted to terms containing less than 125 associated genes. B-E) Example GO term gene level read coverage tracks for OVO ChIP minus input and ovo^ΔBP/ovo^ovo-GAL4; UASp-3xFHA-OVO-B minus ovo^ΔBP/ovo^ovo-GAL4; UASp-GFP. Red rectangles and black rectangles represent significant OVO DNA binding motifs and OVO ChIP peaks, respectively. Gene models are represented at bottom. Small rectangles represent untranslated regions, large rectangles represent translated regions. Arrows indicate transcriptional start sites.

Proposed Model for OVO Regulation in the Female Germline Throughout Development.
Embryonic pole cells, which are transcriptionally quiescent, contain maternally deposited nuclear OVO. As development ensues and embryonic germ cells become transcriptionally competent, maternal OVO binds to its target promoters but does not positively influence gene expression at this stage. Later in embryonic development, in the presence of other activating transcription factors, maternal OVO works in concert to positively influence gene expression, activating a number of essential germ cell-specific genes.

Significant OVO DNA Binding Motif Matches and da-GAL4 X UAS Transgene Viability.
A) Significant alignment of the in vivo OVO DNA binding ‘motif one’ and in vitro OVO DNA binding motif (OVO Garfinkel).

OVO ChIP-seq, ATAC/Histone ChIP-seq, RNA-seq, and DNA Binding Motifs at the otu Locus.
A) otu gene level read coverage tracks for OVO ChIP minus input, GSC and 32c ATAC-seq, GSC H3K27ac, H3K4me3, H3K27me3, H3K9me3, 8c NC H3K9me3, 32c NC H3K27ac, and H3K27me3 ChIP-seq, and ovo^ΔBP/ovo^ovo-GAL4; UASp-3xFHA-OVO-B minus ovo^ΔBP/ovo^ovo-GAL4; UASp-GFP RNA-seq. Red rectangles and black rectangles represent significant OVO DNA binding motifs and OVO ChIP peaks, respectively. Gene models are represented at bottom. Small rectangles represent untranslated regions, large rectangles represent translated regions. Arrows indicate transcriptional start sites.

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