ALF (TFIIA␣/␤-like factor) is a germ cell-specific counterpart of the large (␣/␤) subunit of general transcription factor TFIIA. Here we isolated homologous GC-rich promoters from the mouse and human ALF genes and used promoter deletion analysis to identify sequences active in COS-7 and 293 cells. Further, bisulfite sequence analysis of the mouse ALF promoter showed that all 21 CpG dinucleotides between ؊179 and ؉207 were partially methylated in five somatic tissues, brain, heart, liver, lung,

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... nd muscle, and in epididymal spermatozoa from adult mice. In contrast, DNA from prepubertal mouse testis and from purified spermatocytes were unmethylated except at C ؉19 G and C ؉170 G. We also found that ALF expression correlates with a strong promoterproximal DNase I-hypersensitive site present in nuclei from testis but not from liver. Finally we show that in vitro methylation of the ALF promoter inhibits activity and that 5-aza-2-deoxycytidine treatment reactivates the endogenous ALF gene in a panel of seven different mouse and human somatic cell lines. Overall the results show that silencing in somatic cells is methylation-dependent and reversible and that a unique CpG-specific methylation pattern at the ALF promoter precedes expression in pachytene spermatocytes. This pattern is transient as remethylation of the ALF promoter in haploid germ cell DNA has occurred by the time spermatozoa are present in the epididymis.