Specific Binding of Nuclear Proteins to a Bifunctional Promoter Element Upstream of the H1/AC Box of the Testis-Specific Histone H1t Gene1

Abstract

The testis-specific histone H1t gene is transcribed exclusively in primary spermatocytes during spermatogenesis. Studies with transgenic mice show that 141 base pairs (bp) of the H1t proximal promoter accompanied with 800 bp of downstream sequence are sufficient for tissue-specific transcription. Nuclear proteins from testis and pachytene spermatocytes produce footprints spanning the region covering the repressor element (RE) from 100 to 125 nucleotides upstream of the H1t transcriptional initiation site. Only testis nuclear proteins bind to the 5'-end of the element and produce a unique, low-mobility complex in electrophoretic mobility shift assays. This testis complex is distinct from the complex formed by a repressor protein derived from several cell lines that binds to the 3'-end of the element. The testis complex band is formed when using nuclear proteins from primary spermatocytes, where the H1t gene is transcribed, and band intensity drops 70%-80% when using nuclear proteins from early spermatids, where H1t gene transcription ceases. Protein-DNA cross-linking experiments using testis nuclear proteins produce electrophoretic bands of 59, 52, and 50 kDa on SDS/PAGE gels.

Transcription factor RFX2 is abundant in rat testis and enriched in nuclei of primary spermatocytes where it appears to be required for transcription of the testis‐specific histone H1t gene

“…A change in binding from an RFX2 homodimer in primary spermatocytes to binding as a heterodimer with family members such as RFX1 in early spermatids may serve as a switch to downregulate H1t gene transcription. A similar mechanism may contribute to transcriptional repression of the H1t gene in other tissues [Clare et al, 1997; Wolfe et al, 1999; Singal et al, 2000; Wolfe and Grimes, 2003a,b]. Changes in the proteins that bind, changes in the binding affinities of the proteins, and changes in the interactions of these nuclear regulatory proteins with each other and with the H1t promoter in different cell types are currently being investigated to understand the regulation of transcription of this important testis‐specific linker histone gene.…”

Testis-specific transcriptional control

“…The 3V end of the element functions as a binding site for a transcriptional repressor in several cell lines but the 5V end of the element functions as a binding site for a transcriptional activator in primary spermatocytes (Wolfe and Grimes, 2003b). The repressor binding site is active in NIH-3T3 and GC-2spd cells, and we have determined that the repressor binding proteins differ significantly from those that bind to the GC-box 2 element downstream from the TATA box (Wolfe and Grimes, 2003b;Wolfe and Grimes, 2003a).…”

“…Nuclear proteins from these cells bind to the 5V end of the RE element and generate a DNase I footprint over the RE element when used in binding assays, but nuclear proteins from rat liver do not footprint this region. Thus, at least two proximal promoter elements bind nuclear proteins and contribute to inactivation of promoter activity: the 3V end of the bipartite RE element and the GC-box 2 located downstream from the TATA box (Wolfe and Grimes, 2003b;Wolfe and Grimes, 2003a).…”