Dataset Information

Identification of ID4-interacting proteins in cultured undifferentiated spermatogonia

ABSTRACT: ID4 belongs to a family of transcriptional regulators involved in stem cell function. ID family proteins act by binding and inhibiting activity of basic-HLH (bHLH) transcription factors, including E-proteins and class II factors. ID function is also mediated through interaction with non-bHLH proteins. Spermatogonial stem cells (SSCs) are found within a population of undifferentiated spermatogonia in the testis and are essential for the sustained production of sperm over the lifespan. It is reported that ID4 is involved in SSC maintenance but the downstream targets of ID4 in SSCs are unclear. To gain insight into the function of ID4 in SSCs we identified interacting partners in cultures of mouse undifferentiated spermatogonia by immunoprecipitation using a specific ID4 antibody and mass spectrometry analysis.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Type A Spermatogonium

SUBMITTER: Ralf Schittenhelm

LAB HEAD: Robin Hobbs

PROVIDER: PXD049346 | Pride | 2025-05-06

REPOSITORIES: Pride

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Publications

Functionally redundant roles of ID family proteins in spermatogonial stem cells.

La Hue M HM Chan Ai-Leen AL Hutchinson Ashlee M AM Su Bianka Y M BYM Rossello Fernando J FJ Schittenhelm Ralf B RB Hobbs Robin M RM

Stem cell reports 20240926 10

Spermatogonial stem cells (SSCs) are essential for sustained sperm production, but SSC regulatory mechanisms and markers remain poorly defined. Studies have suggested that the Id family transcriptional regulator Id4 is expressed in SSCs and involved in SSC maintenance. Here, we used reporter and knockout models to define the expression and function of Id4 in the adult male germline. Within the spermatogonial pool, Id4 reporter expression and inhibitor of DNA-binding 4 (ID4) protein are found thr ...[more]

PMID: 39332405

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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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