Fig. 2: Testis-specific expression of Ccer1 requires demethylation of CGIs.

a Western blot analysis of the CCER1 protein in different tissues of adult mice. β-actin was used as a protein loading control. CCER1 protein is approximately 55KD. b Western blot analysis of the CCER1 protein in mouse testis tissue lysates at different time points (postnatal day, PD) in postnatal development. c Immunofluorescence staining with an anti-CCER1 antibody (red, C-terminal) and PNA (green, peanut agglutinin, a sperm acrosome marker) in adult testes. Nuclear DNA was counterstained with DAPI. Scale bar: 5 μm. d Immunofluorescence staining with anti-CCER1 (red) and PNA (green) in stage I–XII in adult testes. Nuclear DNA was counterstained with DAPI. Scale bar: 2 μm. (UP); We drew the schematic during spermatogenesis according to the schematic pattern outlined by Russell L et al.⁴⁰ and labelled the expression of CCER1 (red) in the schematic pattern (Down). e Immunofluorescence staining shows that the localization of CCER1 (red) was immiscible with H3k9me3⁺ heterochromatin (green) and DAPI-stained nuclear regions (blue). Scale bar: 1 μm. f In silico sequence analysis of the human and mouse Ccer1 genes showing the discovery of multiple CpG islands (highlighted in red) in promoter and coding regions. g Demethylation of CpG islands was found in late spermatogenic cells (PAC pachytene spermatocytes, RS round spermatid, ES elongating spermatid) in the mouse testis but not in other tissues or spermatogonia (SPA). h Methylation of CpG islands in other tissues is much higher than in human testis. i Schematic of the human CCER1 CpG island linked to the dual-luciferase (Luc) reporter system. Before transfection, Plasmid CpG-CMV/EF1(upper) and CpG-CMV/EF1+CpG island (bottom) were treated by methyltransferase without S-adenosylmethionine (SAM, the substrates for methyltransferase; M.SssI+without SAM) or methyltransferase with SAM (M.SssI + SAM), respectively. j Effect of CpG islands on CMV/EF1 promoter-driven luciferase expression levels in transfected HEK293T cells. (n = 4 replicate wells for CpG-CMV/EF1 plasmid and n = 5 replicate wells for CpG-CMV/EF1+CpG island plasmid). Two-sided student’s t-test. Error bars, mean ± SD. P = 1.95 × 10⁻³. Source data are provided as a Source Data file.