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

Differentiation of female germline stem cells into a mature oocyte includes the expression of RNAs and proteins that drive early embryonic development in Drosophila. We have little insight into what activates the expression of these maternal factors. One candidate is the zinc-finger protein OVO. OVO is required for female germline viability and has been shown to positively regulate its own expression, as well as a downstream target, ovarian tumor, by binding to the transcriptional start site (TSS). To find additional OVO targets in the female germline and further elucidate OVO’s role in oocyte development, we performed ChIP-seq to determine genome-wide OVO occupancy, as well as RNA-seq comparing hypomorphic and wild type rescue ovo alleles. OVO preferentially binds in close proximity to target TSSs genome-wide, is associated with open chromatin, transcriptionally active histone marks, and OVO-dependent expression. Motif enrichment analysis on OVO ChIP peaks identified a 5’-TAACNGT-3’ OVO DNA binding motif spatially enriched near TSSs. However, the OVO DNA binding motif does not exhibit precise motif spacing relative to the TSS characteristic of RNA Polymerase II complex binding core promoter elements. Integrated genomics analysis showed that 525 genes that are bound and increase in expression downstream of OVO are known to be essential maternally expressed genes. These include genes involved in anterior/posterior/germ plasm specification (bcd, exu, swa, osk, nos, aub, pgc, gcl), egg activation (png, plu, gnu, wisp, C(3)g, mtrm), translational regulation (cup, orb, bru1, me31B), and vitelline membrane formation (fs(1)N, fs(1)M3, clos). This suggests that OVO is a master transcriptional regulator of oocyte development and is responsible for the expression of structural components of the egg as well as maternally provided RNAs that are required for early embryonic development.

. G, H. GSC and 32c ATAC-seq and H3K27ac ChIP-seq minus input control read coverage density for genes bound by OVO over the TSS, gene body, closest TSS in intergenic space, closest TSS for all, or not bound.Plots are centered on the TSS.
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was not certified by peer review) is the author/funder.This article is a US Government work.It is not subject to copyright under 17 USC 105 The copyright holder for this preprint (  and is also made available for use under a CC0 license. was not certified by peer review) is the author/funder.This article is a US Government work.It is not subject to copyright under 17 USC 105 The

Figure S1 :
Figure S1: Significant OVO DNA Binding Motif.A) Cartoon of the transcripts expressed from the ovo locus based on Benner et al. 2023.Gray box represents endogenously generated deletions, orange triangle represents the location of the T2A-GAL4-3xSTOP insertion, purple triangles represent the location of small peptide insertions and yellow boxes represent the location of the shared C2H2 zincfingers.Small rectangles represent untranslated regions, large rectangles represent translated regions and arrows indicate TSSs.B) Significant alignment of the in vivo OVO DNA binding 'motif one' and in vitro OVO DNA binding motif (Lee and Garfinkel 2000).C-F) Significantly enriched DNA motifs within OVO ChIP peaks.C is a signficiant match for the DNA binding motif of GAF and CLAMP (Omelina et al. 2011;

OVO DNA Binding is Associated with Open Chromatin and Transcriptionally Active Histone Marks Across Variations of Gene Binding Patterns
. A-D) OVO ChIP minus input control, GSC H3K27ac, H3K4me3, H3K27me3, H3K9me3, 8c NC H3K9me3, 32c NC     H3K27ac, and H3K27me3ChIP-seq read coverage density centered on OVO peak maximums located within a significant OVO ChIP peak for genes bound by OVO over the TSS, gene body, closest TSS in intergenic space, or closest TSS for all.and is also made available for use under a CC0 license.wasnot certified by peer review) is the author/funder.This article is a US Government work.It is not subject to copyright under 17 USC 105 Benner et al 4/29/24 34 1125 and is also made available for use under a CC0 license.wasnotcertified by peer review) is the author/funder.This article is a US Government work.It is not subject to copyright under 17 USC 105The copyright holder for this preprint (which this version posted April 30, 2024.