Testis-Specific Expression of the Rat Histone H1t Gene in Transgenic Mice

Abstract

The testis-specific histone Hlt gene is transcribed only in testis. The appearance of testis-specific nuclear proteins that bind to a unique promoter sequence element designated Hlt/TE located between the H1/AC box and the H1/CCAAT box correlates with the onset of transcription of the Hlt gene during the meiotic cell cycle. In order to determine whether sequences flanking the rat Hlt gene are sufficient to confer tissue-specific expression in vivo, a 6859 bp EcoRI restriction fragment of genomic DNA containing the rat histone Hlt gene has been microinjected into mouse embryos. S1 nuclease protection analysis has shown that the descendants of the resulting transgenic mice express the rat gene in the proper tissue and at the proper meiotic cell cycle stage. Furthermore, when populations of mouse testis cells were prepared by centrifugal elutriation, only the fraction enriched in pachytene primary spermatocytes had a significant steady-state level of rat Hlt mRNA. Although the copy-number of the transgene was variable in these animals, rat Hlt mRNA levels in high copy-number animals never exceeded 2.6 times the level in normal rat testes. The appearance of appropriate meiotic cell cycle-specific transcription indicates the importance of the conserved promoter sequence elements between the two species.

Transcription factor RFX4 binding to the testis‐specific histone H1t promoter in spermatocytes may be important for regulation of H1t gene transcription during spermatogenesis

“…The H1t promoter contains a TATA-box, a CCAAT-box, and an AC-box that are conserved in most mammalian H1 family members [vanWert et al, 1995]. However, only the H1t promoter contains X-box sequences [Wolfe and Grimes, 2003;Wolfe et al, 2004].…”

“…Two X-box sequences that are located between the TATA-box and AC-box are critical for activation of the H1t promoter. When the rat H1t promoter region containing the two X-boxes was deleted in transgenic mouse experiments, the rat transgene was no longer expressed in mouse spermatocytes [vanWert et al, 1995]. Nuclear proteins extracted from rat pachytene primary spermatocytes and early spermatids bind tightly in these X-box sequences in EMSA [Wolfe et al, 2004].…”

“…See Figure 7 for a diagram of the testis-specific Histone H1t promoter. Studies in transgenic mice showed that mutation of the rat H1t X-boxes abrogated expression of the rat transgene [vanWert et al, 1995[vanWert et al, , 2008. When the X-box sequence was used as an affinity probe to purify binding proteins from testis nuclear extracts, the major protein identified was RFX2 [Wolfe et al, 2004], a protein of 692 amino acids (GenBank #XM_23677) with a mass of approximately 87 kDa.…”

Testis-specific transcriptional control

“…2). If the TE element is replaced with nonsense DNA sequence, the mutant rat gene is not expressed in transgenic mice (VanWert et al, 1995;VanWert et al, 1998). The 40 bp element contains three sub-elements (Fig.…”

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

“…Regulatory sequence elements that are necessary and sufficient for spermatocyte-specific expression of histone H1t are present within 2.5 kilobases (kb) of upstream DNA and 3.8 kb of downstream DNA flanking the H1t transcriptional start site [11]. The promoter, truncated to within 141 base pairs (bp) of the transcriptional start site in constructs containing the native gene and 800 bp of downstream sequence, maintains testis-specific activity in transgenic mice [12].…”