Target gene regulation by PLZF in mouse undifferentiated spermatogonia

Spermatogonial stem cells (SSCs) are adult stem cells, which through their balanced self-renewal and differentiation maintain continuous spermatogenesis. SSC fate decisions are thought to rely upon a combination of extrinsic signals from the stem cell niche as well as intrinsic capacity to execute self-renewal and differentiation programs. PLZF is a transcription factor which is primarily restricted to undifferentiated spermatogonia (i.e., A_single, A_paired, and A_aligned), which includes SSCs, and is required for SSC maintenance. We hypothesized that PLZF may regulate cohorts of genes that are responsible for executing SSC renewal and differentiation fate decisions. Thus, we performed PLZF ChIP-seq using cultured THY1+ mouse spermatogonia, which contain SSCs, to identify the PLZF binging sites throughout the mouse genome in these cells. This approach revealed 3075 PLZF binding sites which mapped to 4266 genes based upon proximity (within 10kb of the transcriptional start site and transcript terminus). Gene ontology (GO) analysis revealed ubiquitination genes, including Uchl1 (a gene co-expressed with PLZF and known to be essential for SSC function), were over-represented among putative PLZF targets. Therefore, I hypothesized that Uchl1 is activated by PLZF in SSCs and tested this hypothesis in Chapter 2. However, transient transfection reporter gene studies using the Uchl1 promoter and PLZF expression construct did not show PLZF-dependent Uchl1 promoter activity in C18-4 and GC-1 cells. One possible explanation for this outcome was that the assignment of PLZF binding sites to particular genes, which was based upon proximity, is too arbitrary to accurately predict regulatory targets of PLZF. Therefore, I hypothesized that PLZF-bound genes which are differentially-expressed between THY1+/- testis cells, which differentially express PLZF, are the most likely regulatory targets of PLZF. In Chapter 3, I found that 2908 of the putative PLZF target genes (68%) are not expressed in either cell population. However, 600 (20%) of these genes are active later in spermatogenesis, supporting a potential role for PLZF in silencing later spermatogenic genes in undifferentiated spermatogonia. Meanwhile, 327 (24%) of the expressed genes were up/down-regulated 2-fold or greater in THY1+ cells (including Uchl1), suggesting they are actively regulated by PLZF in undifferentiated spermatogonia. Thus, the results of these studies highlight the need to examine putative transcription factor-gene regulatory targets identified by ChIP-seq very carefully. Overall, these studies demonstrate genes potentially activated or repressed by PLZF in undifferentiated spermatogonia, including SSCs, and set the stage for future experiments which will examine the functional implications of PLZF-driven gene expression in SSCs.