Characterization of the Promoter Region of the Rat Testis-Specific Histone H1t Gene1

Abstract

Histone H1t is synthesized only in male germ cells during the late pachytene stage of meiosis and is retained in spermatids until the nucleus elongates. Transgenic experiments suggest that spermatocyte-directing sequences lie within 140 base pairs of the cap site. To study the mechanism of this specificity we compared the DNase I footprints made on the immediate promoter regions of H1t and H1d (a typical somatic H1) by testis and liver extracts and observed both common and differentially protected regions. The common footprints of H1t included an Sp1 consensus (GC box 1) and a CCAAT motif. Electrophoretic mobility shift assays (EMSA) identified ubiquitous binding factors for GC box 1 and a binding factor for the CCAAT element that we identified immunologically as H1TF2. H1t-specific footprints occurred over the palindrome CCTAGG and a GC-rich sequence downstream of the TATA box (GC box 2). EMSA analysis of the palindrome identified testis-specific as well as ubiquitous binding factors. UV irradiation of a palindrome-binding reaction generated a cross-linked doublet of about 50 kDa from both testis and liver. Protein factors that bound to the GC box 2 sequence were similar from testis and liver, and GC box 1 and an Sp1 consensus competed for them. In vitro transcription directed by H1t occurred at comparable levels in testis and liver extracts. The importance of both GC box 1 and CCAAT elements was demonstrated by deletion analysis and by oligonucleotide competition. No dependence on the H1t palindrome was observed for in vitro transcription.

A 74-bp Promoter of the Tnp2 Gene Confers Testis- and Spermatid-Specific Expression in Transgenic Mice

“…As described above (8) spermatidspecific transcription of mouse protamine 1 is induced by a 113 bp region from Ϫ150 to Ϫ37. For rat histone H1t gene, studies in transgenic mice revealed that the region from Ϫ141 to ϩ70 was sufficient to confer spermatocyte-specific expression of the gene (19). Thus, our data confirm the observation that genes with male germ cell specific expression tend be regulated by very small promotor regions.…”

The Testis-specific Histone H1t Gene Is Strongly Repressed by a G/C-rich Region Just Downstream of the TATA Box

“…In our earlier investigation, comparison of the H1t and H1d promoters by in vitro transcription did not demonstrate consistent differences using liver or testis extracts (39). We do not know why the strong inhibitory effect of H1t GC box 2 seen with transient transfections was not observed with in vitro transcription.…”

“…Promoter-An earlier study of the H1t promoter demonstrated that GC box 1 is a good binding site for Sp1-like factors, and a strong stimulatory site for in vitro transcription (39). Since GC box 2 is shown here to be an inhibitory site, we reinvestigated the activating role of GC box 1 in the context of a mutation that relieved inhibition at GC box 2.…”

“…1). In vitro analysis of the H1t promoter has shown that GC box 1 is of major importance for transcriptional activation (39). However, the role of GC box 1 has not been investigated in cell transfections.…”

“…A previous study showed that H1t GC box 2 was covered by a strong DNase I footprint when using nuclear extracts from rat organs (39). To identify specific binding proteins for this region we performed gel shift assays.…”