Stonewall prevents expression of ectopic genes in the ovary and accumulates at insulator elements in D. melanogaster

Abstract

Germline stem cells (GSCs) are the progenitor cells of the germline for the lifetime of an animal. In Drosophila, these cells reside in a cellular niche that is required for both their maintenance (self-renewal) and differentiation (asymmetric division resulting in a daughter cell that differs from the GSC). The stem cell—daughter cell transition is tightly regulated by a number of processes, including an array of proteins required for genome stability. The germline stem-cell maintenance factor Stonewall (Stwl) associates with heterochromatin, but its molecular function is poorly understood. We performed RNA-Seq on stwl mutant ovaries and found significant derepression of many transposon families but not heterochromatic genes. We also discovered inappropriate expression of multiple classes of genes. Most prominent are testis-enriched genes, including the male germline sex-determination switch Phf7, the differentiation factor bgcn, and a large testis-specific gene cluster on chromosome 2, are upregulated or ectopically expressed in stwl mutant ovaries. Surprisingly, we also found that RNAi knockdown of stwl in somatic S2 cells results in ectopic expression of these testis genes. Using parallel ChIP-Seq and RNA-Seq experiments in S2 cells, we discovered that Stwl localizes upstream of transcription start sites and at heterochromatic sequences including repetitive sequences associated with telomeres. Stwl is also enriched at bgcn, suggesting that it directly regulates this essential differentiation factor. Finally, we identify Stwl binding motifs that are shared with known insulator binding proteins. We propose that Stwl affects gene regulation, including repression of male transcripts in the female germline, by binding insulators and establishing chromatin boundaries.

SafeguardingDrosophilafemale germ cell identity depends on an H3K9me3 mini domain guided by a ZAD zinc finger protein

“…STWL is a female-specific heterochromatin-associated protein that acts as a transcriptional repressor in vitro (Yi et al, 2009), associates with SETDB1 in yeast (Shokri et al, 2019), and localizes to the phf7 locus in S2 cells (Zinshteyn and Barbash, 2022). Importantly, loss of stwl in ovaries leads to the inappropriate expression of the testes phf7 transcript (Zinshteyn and Barbash, 2022). Together, these studies raise the possibility that stwl collaborates with IDC to silence phf7 .…”

“…One attractive contender is stonewall (stwl) . STWL is a female specific heterochromatin-associated protein that acts as a transcriptional repressor in vitro [66], associates with SETDB1 in yeast [67] and localizes to the phf7 locus in S2 cells [68]. Importantly, loss of stwl in ovaries leads to the inappropriate expression of the testis phf7 transcript [68].…”

Safeguarding Drosophila female germ cell identity depends on an H3K9me3 mini domain guided by a ZAD zinc finger protein

“…One attractive contender is stonewall (stwl). STWL is a heterochromatin-associated protein that acts as a transcriptional repressor in vitro [67], associates with SETDB1 in yeast [68] and localizes to the phf7 locus in S2 cells [69]. Importantly, loss of stwl in ovaries leads to the inappropriate expression of the testis phf7 transcript [69].…”

“…STWL is a heterochromatin-associated protein that acts as a transcriptional repressor in vitro [67], associates with SETDB1 in yeast [68] and localizes to the phf7 locus in S2 cells [69]. Importantly, loss of stwl in ovaries leads to the inappropriate expression of the testis phf7 transcript [69]. Future studies focused on the rules that govern female-specificity may reveal a general mechanism for context-dependent establishment of H3K9me3 silencing domains.…”

Genome organization regulates nuclear pore complex formation and promotes differentiation during Drosophila oogenesis

“…The transcription factor Stonewall (Stwl) is required for Drosophila oogenesis [49][50][51][52] , accumulates at insulator elements, and interacts with heterochromatin components such as HP1. [49][50][51] Intriguingly, loss of stwl phenocopies loss of SETDB1 or Nups, resulting in egg chambers that fail to grow, leading in sterility. 15,18,23,50,51,53 However, how Stwl regulates oogenesis was not fully known.…”