Integrated bioinformatics analysis of the transcription factor-mediated gene regulatory networks in the formation of spermatogonial stem cells

Background: In vitro induction of spermatogonial stem cells (SSCs) from embryonic stem cells (ESCs) provides a promising tool for the treatment of male infertility. A variety of molecules are involved in this complex process, which needs to be further clarified. Undoubtedly, the increased knowledge of SSC formation will be beneficial to facilitate the currently complex induction process. Methods: Based on ATAC-seq, DNase-seq, RNA-seq, and microarray data from GEO datasets, chromatin property data (ATAC-seq, DNase-seq) and gene expression data (RNA-seq, microarray data) were combined to search for SSC-specific transcription factors (TFs) and hub SSC-specific genes by using the WGCNA method. Then, we applied RNA-seq and microarray data screening for key SSC-specific TFs and constructed key SSC-specific TF-mediated gene regulatory networks (GRNs) using ChIP-seq data. Results: First, after analysis of the ATAC-seq and DNase-seq data of mouse ESCs, primordial germ cells (PGCs), and SSCs, 33 SSC-specific TFs and 958 targeting genes were obtained. RNA-seq and WGCNA revealed that the key modules (turquoise and red) were the most significantly related to 958 SSC-specific genes, and a total of 10 hub SSC-specific genes were identified. Next, when compared with the cell-specific TFs in human ESCs, PGCs, and SSCs, we obtained five overlapping SSC-specific TF motifs, including the NF1 family TF motifs (NFIA, NFIB, NFIC, and NFIX), GRE, Fox:Ebox, PGR, and ARE. Among these, Nfib and Nfix exhibited abnormally high expression levels relative to mouse ESCs and PGCs. Moreover, Nfib and Nfix were upregulated in the testis sample with impaired spermatogenesis when compared with the normal group. Finally, the ChIP-seq data results showed that NFIB most likely targeted the hub SSC-specific genes of the turquoise module (Rpl36al, Rps27, Rps21, Nedd8, and Sec61b) and the red module (Vcam1 and Ccl2). Conclusion: Our findings preliminarily revealed cell-specific TFs and cell-specific TF-mediated GRNs in the process of SSC formation. The hub SSC-specific genes and the key SSC-specific TFs were identified and suggested complex network regulation, which may play key roles in optimizing the induction efficiency of the differentiation of ESCs into SSCs in vitro.