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Expression of the transcription factor ZBTB16 in porcine testes and molecular mechanisms for its regulation in porcine immature spermatogonia self-renewal [CUT&Tag]

ABSTRACT: ZBTB16, a transcription factor, plays a critical role in the self-renewal and differentiation of mouse primordial germ cells. However, the subcellular localization of ZBTB16 in the porcine testis and its molecular mechanism in regulating the self-renewal of immature porcine germ cells remain unclear. Using immunofluorescence co-staining, we examined the subcellular localization of ZBTB16 and its co-localization with molecular markers for immature porcine germ cells (DBA, ZBTB16, UCHL1) and the proliferation marker Ki67 in the testes of pigs at 7, 30, 70, and 90 days of age. The results showed that ZBTB16-positive cells in the four age groups of pig testes highly overlapped with SALL4/UCHL1-positive cells and partially overlapped with DBA-positive cells, indicating that ZBTB16 is predominantly expressed in immature porcine germ cells, including pig primordial germ cells, spermatogonial stem cells, and undifferentiated spermatogonia. Furthermore, the immunofluorescence co-staining of ZBTB16 and Ki67 revealed that ZBTB16-positive cells in pig testes at different developmental stages exhibited proliferative activity, but with significant fluctuations. RNA-seq analysis identified 5931 differentially expressed genes in ZBTB16-knockdown immature porcine germ cells, while CUT&Tag analysis identified 5409 ZBTB16 target genes, with 2084 of them overlapping with the differentially expressed genes. ZBTB16 preferentially bound to gene promoters. Knockdown of ZBTB16 expression resulted in the suppression of mRNA levels of target genes involved in the self-renewal of immature germ cells. Additionally, we found that ZBTB16 regulates the self-renewal of immature porcine germ cells through the PI3K/AKT/mTOR pathway. These findings reveal the expression pattern of ZBTB16 in the porcine testis and its regulation of target genes and pathways involved in the self-renewal of immature germ cells. This contributes to the understanding of the function and regulatory network of the transcription factor ZBTB16 in porcine germ cell development and spermatogenesis, and holds significant implications for unraveling the molecular mechanisms underlying porcine germ cell development and sperm production.

ORGANISM(S): Sus scrofa

PROVIDER: GSE271393 | GEO | 2025/06/30

REPOSITORIES: GEO

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Project description:Spermatogonial stem cells (SSCs) balance self-renewal versus differentiation/spermatogenesis to ensure continuous sperm production. Here, we uncover multiple roles for the transcription factor ZBTB16/PLZF in juvenile mouse undifferentiated spermatogonia (uSPG). ZBTB16 activates genes in uSPG promoting self-renewal and cell cycle progression (Ccnd1) to maintain uSPG and transit-amplifying states. Remarkably, in uSPG, ZBTB16, SALL4, SOX3 all co-localize at over 12,000 promoters regulating uSPG and meiosis. These regions also feature broad H3K4me3 and H3K27ac marks, DNA hypomethylation, and often CTCF binding. Hi-C analyses reveal robust promoter-promoter physical interactions, revealing a transcription factor and higher-order active chromatin interaction network within uSPG that poises meiotic promoters for subsequent activation. Conversely, these factors do not occupy germline-specific promoters driving spermiogenesis, which instead lack promoter-promoter physical interactions and bear DNA hypermethylation. Therefore, ZBTB16 ensures uSPG cell cycle progression and colocalizes with SALL4, SOX3 and often CTCF to establish a novel chromatin poising network.Spermatogonial stem cells (SSCs) balance self-renewal and differentiation to ensure continuous sperm production in the testis. The transcription factor Zbtb16 (PLZF) supports undifferentiated SSC maintenance through partly unknown mechanisms. We combined genomics (RNA-seq and ChIP-seq) and genetic approaches to reveal multiple functions of Zbtb16 in juvenile mouse SSCs. Zbtb16-bound loci show a striking correlation with active promoters bearing H3K4me3 and the activator Sall4. Zbtb16 activates genes that support SSC self-renewal and cell cycle progression (e.g., Ccnd1) that help maintain undifferentiated SSC pools, including both self-renewing SSCs and transit-amplifying progenitors. Zbtb16 also attenuates certain genes, including meiotic genes and specific retrotransposons that confer genome instability. Notably, Zbtb16 genome localization and its impact on the transcriptome are dynamic, displaying mesenchymal gene targets in vivo, which are not maintained in cultured SSCs. Our data reveal dynamic roles for Zbtb16 in ensuring SSC identity, amplification, and maintenance in vivo.

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Expression of the transcription factor ZBTB16 in porcine testes and molecular mechanisms for its regulation in porcine immature spermatogonia self-renewal [CUT&Tag]

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