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The multifunctionality of genome is suggested at some loci in different species but not well understood. Here we identified a DES-K16 region in an intron of the Kctd16 gene as the chromatin highly marked with epigenetic modifications of both enhancers (H3K4me1 and H3K27ac) and silencers (H3K27me3) in mouse spermatocytes. In vitro reporter gene assay demonstrated that DES-K16 exhibited significant enhancer activity in spermatocyte-derived GC-2spd(ts) and hepatic tumor-derived Hepa1-6 cells, and a deletion of this sequence in GC-2spd(ts) cells resulted in a decrease and increase of Yipf5 and Kctd16 expression, respectively. This was consistent with increased and decreased expression of Yipf5 and Kctd16 , respectively, in primary spermatocytes during testis development. While known dual enhancer-silencers exert each activity in different tissues, our data suggest that DES-K16 functions as both enhancer and silencer in a single cell type, GC-2spd(ts) cells. This is the first report on a dual enhancer-silencer element which activates and suppresses gene expression in a single cell type. • DES-K16 was marked with H3K4me1, H3K27ac, and H3K27me3 in mouse spermatocytes. • DES-K16 exhibited enhancer activity by in vitro reporter gene assay. • A deletion of DES-K16 down- and up-regulated Yipf5 and Kctd16 genes in GC-2spd(ts). • Yipf5 and Kctd16 expression was correlated to DES-K16 activity in the testis. • DES-K16 is a dual enhancer-silencer functioning in a single cell type. [ABSTRACT FROM AUTHOR]