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

Abstract

H1t is a testis-specific histone 1 variant restricted to the male germ line and expressed only in pachytene spermatocytes. Understanding the regulation of the H1t gene is an interesting challenge as its promoter shares all of the recognized control elements of standard somatic H1 genes, yet H1t is not expressed in somatic or in early spermatogenic cells. To investigate the mechanism of this apparent repression, we exchanged three promoter subregions between H1t and a major somatic H1 gene (H1d) by introduction of suitable restriction sites just 5 of the TATA box and 3 of the conserved H1 AC box. Hybrid promoters were joined to a lacZ reporter gene and assayed by transient transfection in NIH3T3 fibroblasts. In this system the wild type H1d promoter was 20-fold stronger than the H1t promoter. Much of this difference in activity was traced to inhibitory sequences immediately downstream of the TATA box in H1t, although sequences upstream of the H1t AC box and within the H1t 5-untranslated region played some role as well. A series of deletions and short oligonucleotide mutations scanned across the region between the TATA box and cap site identified two tracts of C (GC box 2) as the inhibitory sequences. While both Sp1 and Sp3 bind to this region weakly in vitro, they are unlikely to be responsible for the inhibitory effect of GC box 2, and additional binding proteins (CTB-4 and CTB-5) were identified by electrophoretic mobility shift assays as better candidates for mediating the repressive effect. When repression of the H1t promoter was relieved by mutation of GC box 2, additional mutations introduced into GC box 1 upstream of the CAAT box led to a large decrease in activity, indicating that these two G/C-rich elements have opposite effects on promoter activity.Spermatogenesis is the only example of cellular development in mammals that involves expression of tissue-specific histone variants (1-3). H1t 1 is a testis-specific linker histone variant, appearing late in the prophase of meiosis I in pachytene spermatocytes, retained in early haploid cells, and lost from the nucleus prior to release of mature sperm (3-5). While the amino acid sequence of H1t has a number of novel features (6, 7), it is clearly related to the standard somatic H1 histone family (8). Isolation of the H1t gene from several mammals (9 -12) revealed that the promoter region also shows a surprising similarity to those of standard somatic H1 variants (13,14). Homologies include a TATA box, a CAAT box, a GC box, and an H1-specific AC motif within 100 nucleotides of the cap site as well as an inverted AC motif located farther upstream (15). Despite these shared regulatory elements, H1t expression differs almost completely from that of the common somatic H1 variants. Common variants are produced during S phase of the cell cycle to accommodate the duplication of the chromosomes (14), whereas H1t is expressed only in pachytene spermatocytes, well after completion of replicative DNA synthesis (4,5,16,17). As the presence of H1t mRNA correlates with H1t ...

Identification and characterization of a silencer regulatory element in the 3′-flanking region of the murine CD46 gene

“…To date, several genes showing testis-specific expression have been studied, including histone H1t [30,31], calmodulin gene II (CaMII) [32] and lactate dehydrogenase (LDHC) [33]. Transgenic studies showed that expression of the rat H1t gene was restricted (a) The presence of binding molecule(s) in the nuclear extract of L cells using CDPSE as probe.…”

“…Given the importance of promoters in the expression of a variety of testisspecific genes [30][31][32][33], we focused on the 5h-flanking region of murine CD46 to determine the regulatory mechanisms governing its expression. Primer-extension analysis with the primer pr-1, a 35-mer corresponding to a portion of the signal peptide of murine CD46 revealed a major transcription start point, at 171 bp upstream of the Met start codon (Figure 1a and 1b).…”

ldhc Expression in Non-germ Cell Nuclei Is Repressed by NF-I Binding

“…Sequences in this region have been described previously as negative regulators of testis-specific genes including the rat sperm H2B gene and the rat H1t genes (22,23). In these cases the protein binding these sites was not identified.…”

“…The testis-specific histone H1t gene is also expressed only in pachytene spermatocytes. A negative regulatory region designated GC box 2 lies just downstream of the TATA box and plays a major role in gene repression (23). The Sp1 proteins bind weakly to this element but are not involved in repression of this gene.…”

Control of male germ‐cell development in flowering plants

“…Regulation of germcell-specific genes via the suppression of their transcription in somatic cells is a recurring theme in mammalian systems. (62)(63)(64) For example, histone variants that are expressed during mammalian spermatogenesis are subject to negative regulation in somatic cells. (62)(63)(64) Significant numbers of genes appear to be expressed in the male germ line of flowering plants, and it is likely that some of these genes are regulated by a mechanism other than repressor-controlled negative regulation.…”