SALL4 and PLZF mediate genomic actions of GDNF signaling in spermatogonial stem cells

Spermatogonial stem cells (SSCs) maintain spermatogenesis throughout adulthood through balanced self-renewing and differentiating fate decisions, yet little is known about how these fate decisions are controlled. Glial cell-lined derived neurotrophic factor (GDNF) is an essential growth factor that promotes SSC survival and self-renewal through activation of signaling cascades which in turn leads to changes in expression of downstream genes. However, the transcriptional mechanisms mediating gene expression in response to GDNF was unknown. To potentially address this question we turned to the two transcription factors Sal-like 4 (SALL4) and zinc finger and BTB domain containing 16 (ZBTB16, aka: PLZF), which are known to be required for normal SSC self-renewal and differentiation. Little was known on how PLZF and SALL4 exert regulatory influence in SSCs so we performed a ChIP-Seq analysis in THY1+ spermatogonia, which are enriched for SSCs, to identify the binding repertoires of PLZF and SALL4. Subsequent gene ontology (GO) analysis of these binding repertoires identified an over-representation of GDNF-responsive genes. That led us to hypothesize that the changes in gene expression of GDNF-responsive genes was mediated by the binding and transcriptional regulation of PLZF and SALL4. Targeted knockdown of each transcription factor in THY1+ spermatogonia under steady state conditions revealed that SALL4 and PLZF are required to maintain mRNA levels of putative target genes, suggesting that transcription factor binding to these genes at the positions identified by ChIP-Seq activates their transcription. To confirm if the observed transcriptional response to GDNF was mediated by PLZF and SALL4 we evaluated THY1+ spermatogonia cultures deficient for PLZF and SALL4 and measured transcriptional response to GDNF stimulating conditions. Tandem knockdown and EU labeling experiments revealed that not all GDNF-responsive genes rely on PLZF or SALL4 to induce a response. SALL4 appeared to potentially have more of a role in mediating the response in GDNF-stimulated genes, like Egr3, while loss of Plzf suggested it potentially mediates the transcriptional response in GDNF-inhibited genes.