Binding of nuclear proteins to an upstream element involved in transcriptional regulation of the testis-specific histone H1t gene

The testis-specific histone Hit is synthesized during spermatogenesis exclu sively in late pachytene primary spermatocytes. Transcription of the Hit ge ne is repressed in every tissue except testis. Within the testis, transcrip tion is repressed during development before the spermatocyte stage and in l ater stages of germinal cell maturation. Mechanisms involved in transcripti onal repression of the H1t gene are unknown. To assess the contribution of upstream Hit promoter sequence to transcriptional silencing in nonexpressin g cells, Hit-promoted reporter vectors were constructed using pGL3 Basic. T ransient expression assays with these reporter vectors driven by H1t promot er deletions allowed us to identify a region from 948 to 780 bp upstream fr om the Hilt transcriptional initiation site that functions as a silencer. E xamination of nuclear protein binding to this DNA regulatory region by elec trophoretic mobility shift assays using extracts from C127I cells, rat test is, and pachytene spermatocytes revealed a low mobility band produced only by nuclear proteins derived from nonexpressing cells that may contain prote ins that repress Hit gene transcription. Published 1999 Wiley-Liss, Inc.(da gger)