**Background:** Spermatogonial stem cells (SSCs) sustain spermatogenesis throughout the male lifespan by balancing self-renewal and differentiation. Undifferentiated spermatogonia—comprising A~single~, A~paired~, and A~aligned~ spermatogonia—retain stem cell potential in vitro, but the mechanisms governing their fate decisions remain incompletely understood. The Polycomb group (PcG) proteins, particularly the Polycomb repressive complex 1 (PRC1), are epigenetic regulators essential for stem cell maintenance. Among PRC1 subunits, B cell-specific Moloney murine leukemia virus integration site 1 (BMI1, also called PCGF4) is predominantly expressed in undifferentiated spermatogonia and is known to regulate their proliferation and maintain male fertility. However, the molecular mechanisms by which BMI1 controls undifferentiated spermatogonia fate had not been fully elucidated.
**Methods:** Undifferentiated spermatogonia were isolated from testes of six-day-old ICR mice using enzymatic digestion and differential adherence selection, and their identity was confirmed by marker gene expression (ID4, PLZF, MVH) and transplantation assays showing homing and reconstitution capacity. BMI1 function was assessed by small interfering RNA (siRNA)-mediated knockdown. Cell proliferation was measured by CCK-8 assay, cell cycle distribution by flow cytometry with propidium iodide staining, and apoptosis by TUNEL staining. Transcriptome-wide changes were analyzed by RNA-seq (two biological replicates). Genome-wide binding of BMI1 and histone modifications (H2AK119ub1, H3K27me3, H3K27ac) were mapped by ChIP-seq (two biological replicates). BMI1-interacting proteins were identified by co-immunoprecipitation followed by mass spectrometry. Statistical comparisons used Student's t-test, with significance defined as p < 0.05.
**Key Results:** BMI1 knockdown resulted in smaller and fewer spermatogonial colonies, reduced proliferation from day 2 of culture, a significant increase in the percentage of cells in G0/G1 phase, a decrease in S phase cells, and increased apoptosis. RNA-seq identified 4,318 differentially expressed genes (2,338 upregulated, 1,980 downregulated) upon BMI1 knockdown. Downregulated genes included stemness-related transcription factors (Bcl6b, Lin28a, Id4), while upregulated genes included cell cycle inhibitors (Cdkn2b, Cdkn2c, Cdkn2d). KEGG analysis revealed enrichment of the PI3K-Akt signaling pathway among differentially expressed genes. BMI1 ChIP-seq identified 2,645 peaks corresponding to 1,135 genes, with 22.7% of binding regions near promoters (−2.5 kb to 0.5 kb around TSS), 35.2% in intergenic regions, 29.2% in introns, and 12.9% in exons. Combination analysis showed 146 BMI1-bound genes were upregulated and 113 were downregulated after knockdown. BMI1 repressed negative regulators of PI3K-Akt signaling (Tnc, Efna5, Osmr) via H2AK119ub1 and H3K27me3 deposition. Co-IP confirmed BMI1 assembles canonical PRC1 (cPRC1) through interaction with RNF2 and PHC1, with no association with ncPRC1-specific subunits RYBP or YAF2. Near 90% of BMI1 binding regions were marked by H3K27me3, and 44.8% by H2AK119ub1. BMI1 knockdown globally reduced both H2AK119ub1 and H3K27me3 levels. Additionally, 13.8% of BMI1 binding regions (n = 366) contained H3K27ac, an active histone mark, and BMI1 target genes with H3K27ac showed significantly higher expression. BMI1 positively regulated proliferation-related genes including Taf4 and Gdnf. Motif analysis identified SALL4 binding motifs among BMI1 ChIP-seq peaks, and Co-IP confirmed BMI1-SALL4 interaction. Of BMI1 and SALL4 co-bound regions, 95.93% (n = 400) were modified by H3K27me3, and 79.86% (n = 333) by both H2AK119ub1 and H3K27me3. Genes co-occupied by BMI1 and SALL4 were involved in cell differentiation (Sall4, Pax6, Lin28b, Dmrt3) and showed lower expression than BMI1-only targets. ChIP-qPCR in Sall4 knockdown cells showed significantly reduced BMI1 binding at co-targeted genomic regions.
**Clinical Implications:** This study reveals that BMI1 safeguards undifferentiated spermatogonia fate through dual mechanisms—repressing differentiation-related genes and negative regulators of proliferation via canonical PRC1 activity while activating self-renewal genes via H3K27ac. The identification of BMI1-SALL4 interaction as a recruitment mechanism for PRC1 to specific genomic loci provides insight into how epigenetic regulation directs germ cell fate decisions. These findings may inform therapeutic strategies for male infertility caused by spermatogonial stem cell dysfunction and contribute to understanding of epigenetic regulation in tissue-specific stem cells.