Figure 6.

Enrichment of Transcription Factor Binding Sites Predicts Cell Type and Gene-Specific Patterns of Expression and Binding of Specific Transcription Factors in ID4-eGFP^Bright and ID4-eGFP^Dim Spermatogonia

(A) De novo motif discovery within promoters of genes most differentially expressed in ID4-eGFP^Bright and ID4-eGFP^Dim spermatogonia.

(B–D) Differential enrichment of transcription factor binding motifs within active, poised, or primed enhancers in ID4-eGFP^Bright (red) and ID4-eGFP^Dim (blue) spermatogonia, including active enhancers (B), poised enhancers (C), and primed enhancers (D).

(E) ChIP-qPCR confirms predicted binding patterns of specific transcription factors in ID4-eGFP^Bright and ID4-eGFP^Dim spermatogonia. FOXP1 preferentially binds to promoters of Egr1, Egr2, and Etv5 in ID4-eGFP^Bright spermatogonia. DMRTB1 preferentially binds to promoters of Syce2 and Sohlh2 in ID4-eGFP^Dim spermatogonia. LHX1 preferentially binds to the Cited2 gene promoter (Cited2.p), the Cited2 proximal enhancer (Cited2.e), and the Spry4 enhancer (Spry4.e).

(F) Immunochemistry staining (IHC) of marker proteins in seminiferous cords in whole-mount sections of P6 mouse testes. GFRA1 and FOXC2 localized specifically to ID4-eGFP^Bright SSCs, OCT4 appeared in both ID4-eGFP^Bright SSCs and progenitor-enriched ID4-eGFP^Dim spermatogonia but was more prominent in the latter, FOXO1 and DMRTB1 localized specifically in progenitor-enriched ID4-eGFP^Dim spermatogonia, DMRT1 appeared in ID4-eGFP^Dim spermatogonia and some Sertoli cells, but not in ID4-eGFP^Bright SSCs, and EGR1 and REST co-localized with both ID4-eGFP^Bright SSCs and ID4-eGFP^Dim progenitor/early differentiating spermatogonia as well as in some somatic Sertoli cells. , ID4-eGFP^Bright spermatogonium; , ID4-eGFP^Dim spermatogonium; , Sertoli cell. Scale bars, 20 μm.