Tissue-specific gene expression is tightly regulated by various elements such as promoters, enhancers, and long noncoding RNAs (lncRNAs). In the present study, we identified a conserved noncoding sequence (CNS1) as a novel enhancer for the spermatocyte-specific mouse testicular cell adhesion molecule 1 (Tcam1) gene. CNS1 was located 3.4kb upstream of the Tcam1 gene and associated with histone H3K4 mono-methylation in testicular germ cells. By the in vitro reporter gene assay, CNS1 could enhance Tcam1 promoter activity only in GC-2spd(ts) cells, which were derived from mouse spermatocytes. When we integrated the 6.9-kb 5'-flanking sequence of Tcam1 with or without a deletion of CNS1 linked to the enhanced green fluorescent protein gene into the chromatin of GC-2spd(ts) cells, CNS1 significantly enhanced Tcam1 promoter activity. These results indicate that CNS1 could function as a spermatocyte-specific enhancer. Interestingly, CNS1 also showed high bidirectional promoter activity in the reporter assay, and consistent with this, the Smarcd2 gene and lncRNA, designated lncRNA-Tcam1, were transcribed from adjacent regions of CNS1. While Smarcd2 was ubiquitously expressed, lncRNA-Tcam1 expression was restricted to testicular germ cells, although this lncRNA did not participate in Tcam1 activation. Ubiquitous Smarcd2 expression was correlated to CpG hypo-methylation of CNS1 and partially controlled by Sp1. However, for lncRNA-Tcam1 transcription, the strong association with histone acetylation and histone H3K4 tri-methylation also appeared to be required. The present data suggest that CNS1 is a spermatocyte-specific enhancer for the Tcam1 gene and a bidirectional promoter of Smarcd2 and lncRNA-Tcam1.
Keywords: GC-2spd(ts); Tcam1; conserved noncoding sequence; enhancer; long noncoding RNA.
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