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)

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... 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 synthesis (3, 5, 9, 16, 17) , major control of H1t expression is expected to occur at the transcriptional level. Because of its obvious sequence relationship to the common H1s, H1t probably originated as a somatically expressed, cell cycle regulated gene (or as a duplication of such a gene) that was captured by the male germ line. The first step in this proposed capture may have been a change in the promoter that allowed expression during the long G 2 period of the first meiotic division. Following up-regulation during male meiosis, a second change may have occurred to down-regulate H1t expression in somatic cells. Negative regulation plays an important role in eukaryotic gene expression (reviewed in Refs. 18 and 19), and this second change permitted H1t to evolve solely to fit the requirements of spermatogenesis. Changes in both positive and negative regulation may have developed over time to involve multiple reinforcing mechanisms. If H1t is indeed subject to negative regulation in somatic cells, elimination of this negative regulation should lead to an increased somatic activity of its promoter. Prior work suggests that such negative regulation must be controlled effectively by sequences in the near upstream region. Transgenic studies showed that expression of the natural rat H1t gene was directed exclusively to the male germ line by as little as 141 bp of 5Ј-flanking sequence (20). Further, with fusion genes stably transfected into mouse L cells, Kremer and Kistler (21) found that H1t-directed constructs were some 10-fold weaker than those with a somatic H1 promoter regardless of whether 174 bp or 2 kb of H1t upstream sequences were present. Because H1t and common H1 promoters share so many sequence elements, it seemed possible to identify the element(s) of the H1t promoter responsible for repression in somatic cells by systematically exchanging small regions between H1t and the somatic variant H1d (22) and then comparing activities of the constructs in transfected cells. In results presented here, we identified two regions of the H1t promoter that suppress H1d activity. The strongest inhibitory effect was localized to a C-rich sequence located between the TATA box and the cap site of H1t. Although this sequence element binds members of the Sp1 family of transcriptional regulators in vitro, the interaction is relatively weak, and other binding factors were identified by